Effects of fibrin on the integration hydroxyapatite coating implants

合集下载

The effects of baclofen

trigger reflexive pharyngealswallow was similar in the two groups (Rapid: Healthy0.4_+0.03SE, GERD 0.32_+0.04; Slow: Healthy 0.9-+0.06. GERO 1.2_+0.17, p ns). In subjects with GERD in whom PUCR was present, the threshold volume to trigger PUCR during slow pharyngeal water injection was higher compared to normal volunteers (p = O.01; table). Conclusions: Pharyngo-UES Contractile reflex is adversely affected in the presence of GERD. This finding may have implications in the development of supraesophagealcomplications in patients with reflux disease. ThresholdVolume to trigger Pharyngo-UESContractileReflex(ml mean±SE) Healthy Subjects Rapid In.kdon Slow Injec~on 0.15-1-0.03 0.3_+0.03" Subjects with GERD Rapid Injection Slow Injection 0.22~0.06 0.20.09"

Effect of Surfactants on the Interfacial

Effect of Surfactants on the Interfacial Tension and Emulsion Formation between Water and Carbon Dioxide Sandro R.P.da Rocha,Kristi L.Harrison,and Keith P.Johnston*Department of Chemical Engineering,University of Texas,Austin,Texas78712Received July8,1998.In Final Form:October7,1998 The lowering of the interfacial tension(γ)between water and carbon dioxide by various classes of surfactants is reported and used to interpret complementary measurements of the capacity,stability,and average drop size of water-in-CO2emulsions.γis lowered from∼20to∼2mN/m for the best poly(propylene oxide)-b-poly(ethylene oxide)-b-poly(propylene oxide)(PPO-b-PEO-b-PPO)and PEO-b-PPO-b-PEO Pluronic triblock copolymers,1.4mN/m for a poly(butylene oxide)-b-PEO copolymer,0.8mN/m for a perfluoropolyether (PFPE)ammonium carboxylate and0.2mN/m for PDMS24-g-EO22.The hydrophilic-CO2-philic balance (HCB)of the triblock Pluronic and PDMS-g-PEO-PPO surfactants is characterized by the CO2-to-water distribution coefficient and“V-shaped”plots of logγvs wt%EO.A minimum inγis observed for the optimum HCB.As the CO2-philicity of the surfactant tail is increased,the molecular weight of the hydrophilic segment increases for an optimum HCB.The stronger interactions on both sides of the interface lead to a lowerγ.Consequently,more water was emulsified for the PDMS-based copolymers than either the PPO-or PBO-based copolymers.IntroductionSupercritical fluid(SCF)carbon dioxide(T c)31°C,P c )73.8bar)is an environmentally benign alternative to organic solvents for waste minimization.It is nontoxic, nonflammable,and inexpensive.However,because of its very low dielectric constant, ,and polarizability per volume,R/v,CO2is a poor solvent for most nonvolatile lipophilic and hydrophilic solutes.1It may be considered a third type of condensed phase,different from lipophilic and hydrophilic phases.Consequently,it is possible to disperse either lipophilic or hydrophilic phases into CO2, in the form of microemulsions,emulsions,and latexes, given an appropriate surfactant.Because of the low values of and R/v for CO2,the most CO2-philic types of functional groups have low cohesive energy densities,e.g.,fluoro-carbons,fluoroethers,and siloxanes.2-6The solvent strength of carbon dioxide may be understood by the fact that the solubility of a polymer in carbon dioxide is highly correlated with the surface tension of the pure polymer melt.7For example,poly(fluoroacrylates)with low surface tensions of10-15mN/m are highly soluble,whereas poly-(dimethylsiloxanes)with surface tensions of20mN/m are moderately soluble,and hydrocarbon polymers with higher surface tensions show very low solubility.For nonpolar or slightly polar polymers,the surface tension is a measure of the van der Waals forces and is related to the cohesive energy density.Because R/v is so small for CO2,polymers with low cohesive densities and surface tensions are the most soluble.The first generation of research involving surfactants in SCFs addressed reverse micelles and water-in-SCF microemulsions,for fluids such as ethane and propane8,9 as reviewed recently.10,11Microemulsions are thermody-namically stable and optically transparent,with typical droplet diameters of about2-10nm.The mechanistic insight gained from these studies of phase equilibria, interfacial curvature,and droplet interactions in a su-percritical fluid is directly applicable to carbon dioxide. Attempts to form water-in-CO2(w/c)microemulsions have been elusive.6,12,13For PFPE COO-NH4+w/c microemul-sions,FTIR,UV-visible absorbance,fluorescence,and electron paramagnetic resonance(EPR)experiments have demonstrated the existence of an aqueous domain in CO2 with a polarity approaching that of bulk water,14as has also been shown by small-angle neutron scattering (SANS).15Organic-in-CO2microemulsions have also been formed for600molecular weight poly(ethylene glycol) (PEG600)and for polystyrene oligomers.16,17In many previous studies,surfactant activity in CO2has been characterized in terms of water uptake into a CO2 microemulsion.Since the results were negative most of the time,it has been difficult to determine how to design surfactants to the water-CO2interface.A more direct property,such as the interfacial tension,is needed to understand the activity of surfactants at various interfaces containing carbon dioxide.In SCF systems,only a few studies have measured the interfacial tension(γ)even for simple binary systems(1)O’Shea,K.;Kirmse,K.;Fox,M.A.;Johnston,K.P.J.Phys.Chem. 1991,95,7863.(2)McHugh,M.A.;Krukonis,V.J.Supercritical Fluid Extraction: Priciples and Practice,2nd ed.;Butterworth:Stonham,MA,1994.(3)Hoefling,T.A.;Newman,D.A.;Enick,R.M.;Beckman,E.J.J. Supercrit.Fluids1993,6,165-171.(4)Newman,D.A.;Hoefling,T.A.;Beitle,R.R.;Beckman,E.J.; Enick,R.M.J.Supercrit.Fluids1993,6,205-210.(5)DeSimone,J.M.;Guan,Z.;Elsbernd,C.S.Science1992,257, 945.(6)Harrison,K.;Goveas,J.;Johnston,K.P.;O’Rear,ngmuir 1994,10,3536.(7)O’Neill,M.L.;Cao,Q.;Fang,M.;Johnston,K.P.;Wilkinson,S. P.;Smith,C.D.;Kerschner,J.;Jureller,S.Ind.Chem.Eng.Res.1998, 37,3067-3079.(8)Fulton,J.L.;Smith,R.D.J.Phys.Chem.1988,92,2903-2907.(9)Johnston,K.P.;McFann,G.;Lemert,R.M.Am.Chem.Soc.Symp. Ser.1989,406,140-164.(10)Bartscherer,K.A.;Minier,M.;Renon,H.Fluid Phase Equilib. 1995,107,93-150.(11)McFann,G.J.;Johnston,K.P.In Microemulsions:Fundamental and Applied Aspects;Kumar,P.,Ed.;Dekker:New York,1998;Vol.in press.(12)Iezzi,A.;Enick,R.;Brady,J.Am.Chem.Soc.Symp.Ser.1989, No.406,122-139.(13)Consani,K.A.;Smith,R.D.J.Supercrit.Fluids1990,3,51-65.(14)Johnston,K.P.;Harrison,K.L.;Clarke,M.J.;Howdle,S.M.; Heitz,M.P.;Bright,F.V.;Carlier,C.;Randolph,T.W.Science1996, 271,624-626.(15)Zielinski,R.G.;Kline,S.R.;Kaler,E.W.;Rosov,ngmuir 1997,13,3934-3937.419Langmuir1999,15,419-428including carbon dioxide and a liquid phase.18-20None of these studies included a surfactant.Surfactants have been studied for the generation of CO2foams in water21typically for water-soluble surfactants.The effects of various surfactants on theγbetween supercritical CO2and PEG (600MW)were reported recently.16At276bar,the addition of1%PFPE COO-NH4+reducesγfrom3.2to2.1mN/m, and the interfacial area of the surfactant is437Å2/ molecule.Interfacial tension measurements have also been made between poly(2-ethylhexyl acrylate)(PEHA)and CO222and styrene oligomers and CO2.23As is well-known for water-in-oil(w/o)emulsions and microemulsions,the phase behavior,γ,and curvature are interrelated,as shown in Figure1.24A minimum inγis observed at the phase inversion point where the system is balanced with respect to the partitioning of the surfactant between the phases.25,26Upon change of any of the formulation variables away from this point,for example,the temperature or the hydrophilicity/hydro-phobicity ratio(in our case the hydrophilic/CO2-philic ratio),the surfactant will migrate toward one of the phases. This phase usually becomes the external phase,according to the Bancroft rule.27Unlike the case for conventional solvents,a small change in pressure or temperature can have a large influence on the density and thus on the solvent strength of a supercritical fluid.By“tuning”the interactions between the surfactant tail and the solvent,it becomes possible to manipulate the phase behavior,and therefore the activity of the surfactant at the interface and curvature,and also the extension of the surfactant tails.As an example of pressure tuning,a water-in-propane microemulsion is inverted to a propane-in-water microemulsion by varying the pressure by50bar in the C12EO6/brine/propane system, at constant temperature.28This system undergoes a phase inversion density,by analogy with the phase inversion temperature,for conventional systems.If the density is changed so that the surfactant prefers either phase over the other,the surfactant is less interfacially active and γincreases.16,22,23The objective of this study is to achieve a fundamental understanding of the lowering of the water-CO2inter-facial tension by different classes of surfactants and to use this knowledge to explain the formation and stability of water-in-CO2(w/c)emulsions.The surfactants include PFPE COO-NH4+,Pluronic R(PPO-b-PEO-b-PPO)and Pluronic L(PEO-b-PPO-b-PEO)triblock copolymers,poly-(butylene oxide-b-ethylene oxide)(PBO-b-PEO),and poly-(dimethylsiloxane)(PDMS)copolymers with PEO-PPO grafts(PDMS-g-PEO-PPO).Fromγmeasurements ver-sus concentration,the adsorption is investigated for PFPE COO-NH4+and used to determine the critical micro-emulsion concentration.For the PPO-and PDMS-based surfactants,the concept of a hydrophilic-CO2-philic bal-ance(HCB)is introduced by relatingγand the distribution coefficient of the surfactant to the EO fraction(see Figure 1).To understand howγand the HCB influence colloid stability,we chose to study w/c emulsions in contrast to previous studies of microemulsions,since so few of these surfactants form microemulsions.Emulsions are ther-modynamically unstable,but may be kinetically stable, with droplets from100nm to several micrometers in diameter.The presence of the surfactant at the interface lowers theγand thus the Laplace pressure,reducing the energy necessary to deform the interface.29The emulsions may be stabilized against flocculation due to van der Waals forces by steric stabilization,as has been analyzed theoretically,30-33and/or Marangoni stresses,due to gradients in interfacial tension at the interface.To characterize emulsion capacity,stability,and the average droplet size of the emulsions,an in-situ turbidity technique has been applied in addition to visual observations.The ability to design surfactants for the interface between CO2 and an aqueous phase based upon knowledge of the relationship between colloid formation and stability,phase behavior,andγis of interest for a wide variety of heterogeneous reactions and separation processes in CO2. Examples include dry cleaning,extraction with micro-(16)Harrison,K.L.;Johnston,K.P.;Sanchez,ngmuir1996, 12,2637-2644.(17)McClain,J.B.;Betts,D.E.;Canelas,D.A.;Samulski,E.T.; DeSimone,J.M.;Londono,J.D.;Cochran,H.D.;Wignall,G.D.;Chillura-Martino,D.;Triolo,R.Science1996,274,2049.(18)Heurer,G.Ph.D.Thesis,The University of Texas at Austin, 1957.(19)Chun,B.-S.;Wilkinson,G.T.Ind.Eng.Chem.Res.1995,34, 4371-4377.(20)Schiemann,H.;Wiedner,E.;Peter,S.J.Supercrit.Fluids1993, 6,181-189.(21)Lee,H.O.;Heller,J.P.;Hoefer,A.M.W.SPE Reservoir Eng. 1991,11,421-428.(22)O’Neill,M.;Yates,M.Z.;Harrison,K.L.;Johnston,P.K.;Canelas,D.A.;Betts,D.E.;DeSimone,J.M.;Wilkinson,S.P.Macromolecules1997,30,5050-5059.(23)Harrison,K.L.;da Rocha,S.R.P.;Yates,M.Z.;Johnston,K. P.;Canelas,D.;DeSimone,ngmuir1998,14,6855-6863.(24)Aveyard,R.;Binks,B.P.;Clark,S.;Fletcher,P.D.I.J.Chem. Technol.Biotechnol.1990,48,161-171.(25)Bourrel,M.;Schechter,R.S.Microemulsions and Related Systems:Formulation,Solvency and Physical Properties;Marcel(27)Ruckentein,ngmuir1996,12,6351-6353.(28)McFann,G.J.;Johnston,ngmuir1993,9,2942.(29)Walstra,P.Chem.Eng.Sci.1993,48,333-349.(30)Peck,D.G.;Johnston,K.P.Macromolecules1993,26,1537.(31)Meredith,J.C.;Johnston,K.P.Macromolecules1998,31,5507-5555.(32)Meredith,J.C.;Sanchez,I.C.;Johnston,K.P.;Pablo,J.J.d.Figure1.Schematic representation of phase behavior andinterfacial tension for mixtures of water,CO2,and nonionicsurfactants as a function of formulation variables.420Langmuir,Vol.15,No.2,1999da Rocha et al.emulsions and emulsions,phase transfer reactions,34,35and emulsion polymerization.36Experimental SectionMaterials.All of the surfactants were used as received,unless indicated.The CF 3O(CF 2CF(CF 3)O)∼3CF 2COO -NH 4+(PFPE COO -NH 4+),a gift from A.Chittofrati,37was stored in a desiccator.The single tail Krytox-sulfate,R -COOCH 2CH 2OSO 3--Na +,where R )CF 3(CF 2CF(CF 3)O)n CF 2CF 2-,and the triple tail Krytox-sorbitol surfactants were synthesized by E.Singley and Dr.E.J.Beckman at the University of Pittsburgh.38Pluronic L,PEO-b -PPO-b -PEO (PEO -PPO -PEO),and Pluronic R,PPO-b -PEO-b -PPO (PPO -PEO -PPO),surfactants were a gift from BASF.The block copolymer PEO-b -PBO (EO 15-BO 12,SAM185)(where the subscripts indicate the number of repeat units of each moiety)was provided by Pittsburgh Paint and Glass.The surfactant (CH 3)3SiO[Si(CH 3)2O]20[Si(CH 3)(R)]2OSi(CH 3)3,with graft R )(CH 2)3O(C 2H 4O)∼11H,(PDMS 24-g -EO 22),M w ∼2600,was a gift synthesized by Unilever.7SILWET L-7500(M w )3000),(CH 3)3SiO(Si(CH 3)2O)x (Si(CH 3)(R))y OSi(CH 3)3,with R )(CH 2)3O-(C 3H 6O)n Bu (PDMS 11-g -PO 39),with n ,x ,and y not specified,and SILWET L-7622(M w )10000),with a similar backbone,but R )(CH 2)3O(C 2H 4O)m Me (PDMS 105-g -EO 68),were provided by OSi Specialties,Inc.ABIL B 8851(M w ∼6000),(CH 3)3SiO(Si-(CH 3)2O)22(Si(CH 3)(R)O)4Si(CH 3)3,with R )(CH 2)3O(C 2H 4O)∼17-(C 3H 6O)∼4H (PDMS 28-g -EO 67-PO 17),and ABIL B 88184(M w ∼13000),(CH 3)3SiO(Si(CH 3)2O)73(Si(CH 3)(R)O)4Si(CH 3)3,with R ∼(CH 2)3O(C 2H 4O)∼32(C 3H 6O)∼7H (PDMS 79-g -EO 126-PO 28)were obtained from Goldschmidt AG.PDMS homopolymer with a M w of 13000was synthesized by J.M.DeSimone at U.N.Carolina.Poly(ethylene glycol)with a molecular weight of 600was obtained from Polysciences,Inc.Poly(butylene glycol)monoether,composed of an ethylene oxide backbone with an ethyl side group (PBO,800g/mol)was supplied by Air Products.Poly(propylene glycol)(1025g/mol)was obtained from Polysciences,Inc.,and used as received.Deionized water (NANOpureII;Barnstead)and instrument grade carbon dioxide (99.99%)were used for all experiments.Phase Behavior.Phase boundaries were determined in the variable-volume view cell as described in further detail else-where.7For a given weight of surfactant and CO 2,the pressure of the system was increased until a single phase was observed in the view cell.The pressure was then decreased slowly until the solution became slightly turbid.The pressure was then increased again,and the process was repeated.The pressure where the system became turbid was classified as the cloud point pressure.The pressure and temperature were measured to (0.2bar and (0.1°C,respectively.Interfacial Tension Measurements.The tandem variable-volume pendant drop tensiometer described previously 16was used to measure the interfacial tension between CO and water (γ).The apparatus consisted of two variable volume view cells (the drop phase cell and the measurement cell (continuous phase cell)),an optical rail for proper alignment,a light source,a video camera,and a computer.The drop phase cell contained water saturated with an excess amount of pure CO 2,and the continuous phase cell contained CO 2and surfactant (if present).In this configuration,the surfactant only has to diffuse short distances in the small volume of the droplet phase.Pendant drops were formed on the end of a stainless steel or PEEK capillary tube with an inside diameter ranging from 0.01to 0.03in.Once a suitable drop was formed,the six-port switching valve connecting the two cells was closed and timing of the drop age was started.Several images were recorded as a function of drop age.Images of the drop were obtained in a tagged imagefile format (TIFF)and the edge of the drop was extracted from data at various global threshold values using a C ++program.From the shape of the interface,the γmay be obtained from the Laplace equationwhere ∆P is the pressure differential across the interface,R 0is the radius of curvature at the apex of the drop,and z is the vertical distance from the apex.A set of three first-order differential equations was used to express Laplace’s equation,and a computer program 39,40was used to solve for γ.The density difference between the two phases was calculated by using an equation of state for pure CO 241and steam tables for pure water.The aqueous phase density was assumed to change less than 0.0025g/cm 3for the concentrations of surfactant studied.Emulsion Formation,Stability,and Average Droplet Size Estimation.Figure 2shows a schematic representation of the experimental apparatus,similar to a previous version,for turbidimetric measurement and visual observation of emulsion formation and stability.22The system consists of a 28-mL variable-volume view cell,an optical cell (0.1cm path length)which was mounted in a spectrophotometer (Cary 3E UV -vis),a high-pressure reciprocating pump (minipump with a flow rate of 8-80mL/min),and a manual pressure generator (High-Pressure Equip.,model 87-6-5).A six-port switching valve (Valco Instru-ments Co.,Inc.)with an external sampling loop was used to add water to the system.The pressure was monitored to (0.2bar with a strain gauge pressure transducer (Sensotec),and the temperature was controlled to within (0.1°C.Surfactant was initially loaded into the view cell,and the desired amount of CO 2was added with the pressure generator.The pressure was increased,and the system equilibrated at the desired T ,for ∼2h,by using a magnetic stir bar.The cloud point of the surfactant was obtained as described above.The solution was then recirculated,and deionized water was injected into the system via the 150-µL sample loop in the switching valve.The solution was sheared through a 130µm i.d.×50mm long stainless steel capillary tube upstream of the optical cell.Emulsion formation and stability were characterized based upon turbidity measurements versus time (t )at a constant wavelength (λ)650nm)and also visual observation.The turbidity is a measure of the reduction in transmitted intensity,τ)(1/l )ln(I 0/I ),where l is the path length and I 0and I are the incident and transmitted intensities,respectively.After the injection of each increment of water,the emulsion was stirred and recirculated for ∼20min (approximate time required for the absorbance to reach a maximum value).Immediately after recirculation and stirring were stopped,τmeasurements started.The stability was assessed from τas a function of t ,while the(34)Jacobson,G.B.;Lee,C.T.;daRocha,S.R.P.;Johnston,.Chem.,in press.(35)Jacobson,G.B.;Lee,C.T.;Johnston,.Chem.,in press.(36)Adamsky,F.A.;Beckman,E.J.Macromolecules 1994,27,312-314.(37)Chittofrati,A.;Lenti,D.;Sanguineti,A.;Visca,M.;Gambi,C.M.C.;Senatra,D.;Zhou,Z.Prog.Colloid Polym.Sci.1989,79,218-(39)Jennings,J.W.;Pallas,ngmuir 1988,4,959-967.Figure 2.Apparatus for emulsion formation and turbidimetry measurement.∆P )2γ/R 0+(∆F )gz(1)Surfactant Effect on Interfacial Tension Langmuir,Vol.15,No.2,1999421effective average droplet size was determined fromτversusλ.For a monodisperse system of nonabsorbing spheres in theabsence of multiple scatteringτis given byτ)3K*φ/2D,42where φis the dispersed phase volume fraction,D is the droplet diameter, and K*is the scattering coefficient.According to Mie theory,Κ*is a complex function of R(R∼D/λ,whereλis the wavelengthof the incident light)and m the ratio of the refractive indices ofthe dispersed and continuous phases.The refractive indices wereapproximated by those of the pure components,water(1.333)and CO2.43By evaluation of turbidities at two wavelengths,theaverage droplet size can be determined by an iteration proce-dure.44Results and DiscussionInterfacial Tension of the CO2-Water Binary System.The interfacial tension between pure CO2and water is shown in Figure3for two temperatures as a function of pressure,along with the data of Heurer18and Chun and Wilkinson.19Our interfacial tensions were measured1h after drop formation.Theγvalues obtained by Chun and Wilkinson19were measured with the capillary rise technique.Whereas local equilibrium was achieved within the capillary tube,the entire system was not at equilibrium.Heurer used the pendant drop technique; however,the values reported were obtained from the drop profile within10s of drop formation.Therefore,the lower values ofγin the present study suggest a closer approach to true equilibrium.A simple physical picture may be used to explain the behavior for most of the pressure range studied.16At pressures below70bar,γdecreases with increasing pressure.The cohesive energy density or free energy density of CO2is well below that of water at all pressures. The density and free energy density of CO2change over a wide range with pressure,whereas the values for essentially incompressible water are constant.As the density of the CO2phase increases,its free energy density becomes closer to that of water,andγdecreases.At low pressures where the density and free energy density change a great deal with pressure,the decrease inγis pronounced.At high pressures,where CO2is more “liquidlike”,it is much less compressible and the decrease inγwith pressure is small.For the CO2-PEG600interface,γwas predicted quantitatively with a gradientmodel and the lattice fluid equation of state.16The latticefluid model is less applicable for water due to thecomplexities resulting from hydrogen bonding and car-bonic acid formation.A cusp in the curve ofγversus pressure is observed attemperatures and pressures near the critical point of CO2.The region of the cusp inγshifts to slightly higherpressures as the temperature is increased above the criticaltemperature of CO2.For supercritical temperatures,themagnitude of the cusp increases as the temperature isdecreased toward the critical temperature.At25,1935,and38°C,the cusp in the interfacial tension is verynoticeable,while it becomes small at45°C and is notvisible at71°C.18The following argument explains how the cusp is relatedto the large compressibility of CO2.An upward pointingcusp has been observed for the surface excess of ethyleneon graphitized carbon black.45The excess adsorption canbe defined in terms of the density of the bulk phase andthe density of the interfacial region46where F(z)is the molar density of the fluid at a distancez from the surface.At pressures below the critical pressureregion,F(z)can be much larger than F,due to attractionof solvent to the surface,leading to a largeΓex.At higherpressures,the bulk fluid is much denser,so that thedifference between F(z)and F is much smaller resultingin a smallerΓex.As temperature increases above thecritical temperature of the solvent,the tendency of thesurface to raise F(z)to“liquidlike”densities diminishesandΓex decreases.Similar arguments apply to theadsorption of CO2at the water-CO2interface.TheenhancedΓex is manifested as the downward cusp inγ.Inboth examples,the cusps become broader and shift tohigher pressures at higher temperatures.Similar behavioris observed for peaks in plots of the isothermal compress-ibility of pure CO2versus pressure at constant temper-ature.To put the above results in perspective,new interfacialtension data are shown for the PEG600-CO2interface tocomplement earlier data16only at45°C(Figure4).Thevalues ofγfor the water-CO2interface are considerablylarger than those for the PEG600-CO2,PS(M n)1850),23CO2-PEHA(M n)32k)interfaces.22This result is dueprimarily to the much larger surface tension of water,∼72mN/m,versus that of PEG,∼35mN/m,and PEHA, 30mN/m.However,it is interesting thatγbetween CO2and water at high pressures,20mN/m,is below that forwater-hydrocarbon interfaces.For heptane and octane,the hydrocarbon-waterγis about50mN/m.This lower γis consistent with the higher miscibility between CO2 and water47versus hydrocarbons and water.The stronger interactions between CO2and water versus hydrocarbons and water are due to the small size of CO2which causes a smaller penalty in hydrophobic hydration,CO2’s quad-rupole moment,and,finally,Lewis and Bronsted acid-base interactions.Over the entire pressure range for PEG600-CO2at25and45°C,the interfacial tension decreased monotonicallywith increasing pressure,unlike the case for CO2-water(42)Yang,K.C.;Hogg,R.Anal.Chem.1979,51,758-763.(43)Burns,R.C.;Graham,C.;Weller,A.R.M.Mol.Phys.1986,59,(45)Findenegg,G.H.In Fundamentals of Adsorption;Myers,A.L., Belfort,G.,Eds.;Engineering Foundation:New York,1983;p207.Figure3.Interfacial tension at the CO2-water interface asa function of pressure at various temperatures.Γex≡∫(F(z)-F bulk)d z(2) 422Langmuir,Vol.15,No.2,1999da Rocha et al.at 35°C.The lack of a dip near the critical pressure may be due to the much lower compressibility at 25and 45°C versus 35°C.This contrast in behavior may also be due to a difference in the density gradient and thickness in the interfacial region for the two systems,for example,greater miscibility for the CO 2-PEG600system.Interfacial Tension:PFPE Ammonium Carboxy-late.The addition of small amounts of PFPE COO -NH 4+decreases γsubstantially as shown at 45°C and 276bar in Figure 5.As the concentration is raised above 0.03%surfactant,a discontinuity is observed,and the magnitude of the slope becomes much smaller.Because it has been shown that w/c microemulsions are formed in this system,14the discontinuity can be attributed to a critical microemulsion concentration (c µc)for the PFPE COO --NH 4+surfactant,as has been done for oil -water inter-faces.24At concentrations above the c µc,the less negative slope is caused by the addition of surfactant primarily to adsorption at the pendant drop interface,the change in γis reduced.The adsorption obtained from the Gibbs’adsorption equationfor the PFPE COO -NH 4+surfactant was 1.77×10-10mol/cm 2,which corresponds to a surface coverage of ∼100Å2/molecule.Such a high surface coverage is sufficient for the formation of microemulsions.A comparable value of ∼140Å2/molecule was measured by Eastoe et al.48at 500bar and 25°C for the hybrid hydrocarbon -fluorocarbon C 7F 15CH(OSO 3-Na +)C 7H 15surfactant in CO 2.This value was determined by assuming that all the surfactant is adsorbed at the interface of spherical droplets of 25Å2radius,as measured by SANS,with a polydispersity of ∼0.2.The substantial reduction in γand relatively high surfactant adsorption explain why it was possible to form a w/c microemulsion with PFPE COO -NH 4+.The same surfactant had an absorption of 400Å2/molecule at the CO 2-PEG interface.16Phase behavior studies indicated that PEG-in-CO 2microemulsions are also formed with this surfactant,but the nature of the core has not been characterized.16Interfacial Tension:Fluoroether Sulfate and Sorbitol Surfactant.The phase behavior of fluoroether sulfates and fluoroether sorbitols was measured by Singley et al.38for various molecular weights of single-,twin-,and triple-tailed surfactants.The surfactants were soluble in CO 2at 33°C and moderate pressure (<300bar).The sorbitol surfactants were found to be more soluble in CO 2than the sulfate ones,as expected due to the low solubilities of ions in CO 2,because of its low dielectric constant.The results showed that branching depresses the cloud point curve of a surfactant until the solubility becomes domi-nated by the overall molecular weight.These surfactants were used to form CO 2-in-water and middle-phase emul-sions with excess CO 2and water.38The interfacial tension was measured at the water -CO 2interface for the single-tailed M w 2500sulfate and the triple-tailed (7500g/mol total)sorbitol surfactants.Our measured cloud point for the 1.4%(w/w)CO 2sorbitol surfactant was 215.6bar at 45°C.For 0.56%sulfate surfactant,it was 139.8bar at 45°C.The sulfate surfactant did not lower the interfacial tension significantly over the pressure range of 180-283bar 45°C at a concentration of 0.56%.The interfacial tension was difficult to determine accurately,because bubbles and possibly surfactant precipitate appeared on the surface of the pendant drop within 15min of drop formation.The interfacial tension was estimated to be ∼15mN/m by using manual edge detection of the pendant drop.For the sorbitol surfactant,the interfacial tension decreased to ∼5.5mN/m at 276bar and 45°C with a concentration of 1.4%.Relative to other surfactants reported in this study,these surfactants were less successful in lowering the interfacial tension.Interfacial Tension:PPO -PEO -PPO,PEO -PPO -PEO,and PBO -PEO Surfactants.Block co-polymers containing CO 2-philic and hydrophilic (CO 2-phobic)functional groups may be designed to be active at the CO 2-water interface.In this section,the CO 2-philic blocks are poly(propylene oxide)and poly(butylene oxide),while the CO 2-phobic block is poly(ethylene oxide).TheFigure 4.Interfacial tension for the PEG600-CO 2interface at varioustemperatures.Figure 5.Interfacial tension for the water -CO 2-PFPE COO -NH 4+system at 45°C and 276bar.The dotted line is used to determine the surfactant adsorption via the Gibbs adsorption equation.A discontinuity is present at the critical micromemulsion concentration.Γ2)-1RT (d γd ln c 2)T ,P(3)Surfactant Effect on Interfacial Tension Langmuir,Vol.15,No.2,1999423。

基于组分区分的亚热带湿地松人工林土壤呼吸对氮添加的响应

ｃａｒｂｏｎ ( Ｃ) ｂｕｄｇｅｔａｎｄＣｓｅｑｕｅｓｔｒａｔｉｏｎｉｎｔｈｅｔｅｒｒｅｓｔｒｉａｌｅｃｏｓｙｓｔｅｍ. ＴａｋｉｎｇａｓｕｂｔｒｏｐｉｃａｌＰｉｎｕｓｅｌｌｉｏｔｔｉｉＰｌａｎｔａｔｉｏｎｉｎＣｈｉｎａａｓｔｈｅｒｅｓｅａｒｃｈａｖａｉｌａｂｉｌｉｔｙｗａｓｃｏｎｄｕｃｔｅｄｂｙｆｉｅｌｄｓｉｍｕｌａｔｉｏｎｃｏｎｔｒｏｌｅｘｐｅｒｉｍｅｎｔｂａｓｅｄｏｎｔｈｅｄｉｓｔｉｎｃｔｉｏｎｏｆｄｉｆｆｅｒｅｎｔｃｏｍｐｏｎｅｎｔｓｏｆｓｏｉｌｒｅｓｐｉｒａｔｉｏｎꎬ ａｎｄａ
ＸＩＡＯＳｈｅｎｇｓｈｅｎｇ１ꎬ２ ꎬ ＷＡＮＧＪｉａ２ ꎬ ＳＨＩＺｈｅｎｇ２ ꎬ ＺＨＡＯＪｉａｄｉｎｇ１ꎬ２ ꎬ ＴＡＮＧＣｈｏｎｇｊｕｎ１ꎬ２
Ａｂｓｔｒａｃｔ: Ｔｈｅｉｎｃｒｅａｓｅｏｆｎｉｔｒｏｇｅｎ ( Ｎ) ｄｅｐｏｓｉｔｉｏｎｗｏｕｌｄｏｂｖｉｏｕｓｌｙｄｉｓｔｕｒｂｔｈｅｓｏｉｌｒｅｓｐｉｒａｔｉｏｎꎬ ｆｕｒｔｈｅｒｍａｋｅｉｍｐｏｒｔａｎｔｉｎｆｌｕｅｎｃｅｓｏｎｔｈｅ
收稿日期: ２０１７￣１０￣１７㊀㊀㊀修订日期: ２０１８￣０３￣２０
ｒｅｓｐｉｒａｔｉｏｎｗｅｒｅ３�� ９１ꎬ ２�� ３０ａｎｄ１�� ７３ μｍｏｌ∕(ｍ２�� ｓ)ꎬ ｒｅｓｐｅｃｔｉｖｅｌｙｕｎｄｅｒＣＫ (０)ꎬ ＬＮｔｒｅａｔｍｅｎｔ (６０ｋｇ∕(ｈｍ２�� ａ)) ａｎｄＨＮｔｒｅａｔｍｅｎｔ (１２０
ｏｂｊｅｃｔꎬ ａｑｕａｎｔｉｔａｔｉｖｅｓｔｕｄｙａｂｏｕｔｔｈｅｒｅｓｐｏｎｓｅｓｏｆｔｈｅｒｏｏｔａｕｔｏｔｒｏｐｈｉｃｒｅｓｐｉｒａｔｉｏｎꎬ ｍｉｃｒｏｂｉａｌｈｅｔｅｒｏｔｒｏｐｈｉｃｒｅｓｐｉｒａｔｉｏｎｔｏｔｈｅｖａｒｙｉｎｇＮｐｒｅｌｉｍｉｎａｒｙｄｉｓｃｕｓｓｉｏｎｏｎｔｈｅｂｉｏｇｅｏｃｈｅｍｉｓｔｒｙａｎｄｍｉｃｒｏｂｉｏｌｏｇｉｃａｌｍｅｃｈａｎｉｓｍｏｆｔｈｅｒｅｓｐｏｎｓｅｗｅｒｅａｌｓｏｍａｄｅ. Ｒｅｓｕｌｔｓｓｈｏｗｅｄｔｈａｔ: (１) Ｔｈｅｄｙｎａｍｉｃｃｈａｒａｃｔｅｒｉｓｔｉｃｓｏｆｔｈｅｔｏｔａｌｓｏｉｌｒｅｓｐｉｒａｔｉｏｎꎬ ｒｏｏｔｒｅｓｐｉｒａｔｉｏｎａｎｄｍｉｃｒｏｂｉａｌｒｅｓｐｉｒａｔｉｏｎｄｉｓｐｌａｙｅｄｏｂｖｉｏｕｓｓｉｎｇｌｅｐｅａｋｃｕｒｖｅｉｎｂｏｔｈ２０１５ａｎｄ２０１６ꎬ ｗｉｔｈｔｈｅｍａｘｉｍｕｍｒｅｓｐｉｒａｔｉｏｎｒａｔｅｓｏｂｓｅｒｖｅｄｉｎＪｕｌｙｏｒＡｕｇｕｓｔ. ＳｉｍｕｌａｔｅｄＮｄｅｐｏｓｉｔｉｏｎｈａｄｎｏｓｉｇｎｉｆｉｃａｎｔｉｎｆｌｕｅｎｃｅｏｎｔｈｅｓｅａｓｏｎａｌｐａｔｔｅｒｎｏｆｓｏｉｌｒｅｓｐｉｒａｔｉｏｎｒａｔｅｓ. (２) Ｔｈｅａｎｎｕａｌａｖｅｒａｇｅｒａｔｅｓｏｆｔｈｅｔｏｔａｌｓｏｉｌｒｅｓｐｉｒａｔｉｏｎꎬ ｒｏｏｔｒｅｓｐｉｒａｔｉｏｎａｎｄｍｉｃｒｏｂｉａｌ

非诺贝特对胰岛素抵抗大鼠血清脂联素及肿瘤坏死因子_的影响

2009年6月第6卷第18期·论著·中国医药导报CHINA MEDICAL HERALD胰岛素抵抗（insulin resistance ，IR ）是指胰岛素执行其正常生物作用的效应不足，表现为外周组织尤其是肌肉、脂肪组织对葡萄糖的利用障碍。

非诺贝特是氯贝丁酸类血脂调节药，调脂的同时还可以改善IR ，但其机制尚不完全清楚。

本研究通过检测非诺贝特对高脂饮食诱导的IR 大鼠脂联素和肿瘤坏死因子α（TNF-α）表达的影响，探讨非诺贝特改善IR 的分子机制。

1材料与方法1.1材料1.1.1实验动物健康雄性Wistar 大鼠36只，体重140~200g ，由中国医科大学实验动物中心提供。

1.1.2主要试剂胰岛素注射液（上海第一生化药业有限公司），微粒化非诺贝特（200mg/粒，法国利博福尼公司），大鼠脂联素ELISA 试剂盒（美国Chemicon 公司），大鼠TNF-αELISA试剂盒（晶美公司）。

1.2动物模型的建立及分组36只大鼠适应性喂养1周后随机分为两组：普通饮食组（NC 组）12只，给予普通标准大鼠饲料；高脂饮食组（高脂组）24只，给予自行配制的高脂饲料（热量组成：碳水化合物20%，脂肪59%，蛋白质21%）。

大鼠分笼饲养，自由光照，自由摄取食物和水。

6周后将高脂组大鼠随机分为非诺贝特组（F 组）和高脂对照组（HF 组），每组12只，继续给予高脂饮食，同时分别给予非诺贝特30mg/（kg ·d ）和蒸馏水灌胃4周。

NC 组继续给予普通饮食并用蒸馏水灌胃4周。

1.3高胰岛素-正常葡萄糖钳夹技术评价模型大鼠的IR实验结束时，全部大鼠禁食12h 过夜。

每组大鼠随机选取4只做高胰岛素-正常葡萄糖钳夹实验：大鼠麻醉后仰卧位固定，行颈部正中切口分离右颈静脉和左颈动脉，分别用于输注胰岛素、葡萄糖和肝素盐水抗凝以及采动脉血标本。

术非诺贝特对胰岛素抵抗大鼠血清脂联素及肿瘤坏死因子-α的影响李杰，赵卫华，胡健*（中国医科大学附属第一医院心内科，辽宁沈阳110001）[摘要]目的：观察非诺贝特对胰岛素抵抗（IR ）大鼠血清脂联素、肿瘤坏死因子-α（TNF-α）的影响，探讨非诺贝特改善IR 的机制。

二氧化碳浓度增高对作物影响研究方法_FACE_的问题与讨论

中国农业大学学报　2008,13(3):23228Journal of China Agricultural University二氧化碳浓度增高对作物影响研究方法(FACE)的问题与讨论谢立勇1,2　马占云2　高西宁1　仝乘风2　林而达2(11沈阳农业大学农学院,沈阳110161;2.中国农业科学院农业环境与可持续发展研究所,北京100081)摘　要　综述了全球范围内CO2浓度增高对作物影响的主要研究方法,讨论了开放式CO2富集系统(Free2air CO2Enrichment,FACE)的原理结构特点、先进性和不足。

文章认为,FACE系统是当前研究CO2肥效作用最接近自然状况的模拟系统,对研究作物生理生态、品质与环境关系等问题具有重要的适用性和先进性。

但该系统也存在着CO2浓度与自然变幅不吻合、与温度等环境因子不匹配等人为调控的局限性。

作者结合近年来对FACE 系统和半开放式梯度实验系统(CO22Temperature Gradient Chambers,CT GC)的使用和调试,对2个系统做了比较。

建议FACE系统与CT GC系统配合使用,更利于解决模拟环境真实性,提高实验精度;在FACE系统中,建议把CO2的目标浓度改为幅度值以解决与自然变幅相符合的问题。

在研究对象方面,建议开展作物世代研究和环境综合研究,以求精确阐明CO2浓度增高对作物影响机理与影响程度,为准确判断气候变化背景下粮食生产和粮食安全提供科学依据。

关键词　CO2浓度增高;作物生育;研究方法;开放式CO2富集系统(FACE)中图分类号　S16215 文章编号　100724333(2008)0320023206 文献标志码　AFACE and its limitations on researches of impactsof elevated CO2on crop sXIE Li2yong1,2,MA Zhan2yun2,G AO X i2ning1,TONG Cheng2feng2,LI N Er2da2(1.Colle ge of Agronomy,Shenyang Agricultural University,Shenyang110161,China;2.Institute of Environment and Sustaina ble Development in Agriculture,Chines e Aca demy ofAgricultural Sciences,Beijing100081,China)Abstract　Main study methods of the imp acts of elevate d CO2on crops in the global were reviewe d.The mechanism and structure of Free-air CO2enrichment(FACE)system was illuminate d.The advanta ges and dis a dvanta ges of FACE system were dis cuss ed.In general,FACE is most approaching to natural environment and it is most effective for studying the relationship between eco-physiological characters of crops and environment.But FACE als o has s ome dis a dvantages,s uch as CO2concentration increasing s uddenly and not fit natural variation,not match to temp erature change.According to a s eries of res earches in recent years,the authors put forward to s ome s uggestions for impro2 ving FACE,namely,applying to CTGC with FACE together,a djusting target CO2concentration as a range,developing crop generation res earch and inte grate d environment factor res earch.Key words　elevated CO2;growth and development o f crops;research methods;free2air CO2enrichment(F ACE)收稿日期:2008204228基金项目:中国博士后科学基金(20060390547);中澳国际合作项目(00xx-0506-Norton);国家科技支撑计划“十一五”重大项目(2007BAC03A06)作者简介:谢立勇,博士(后),副教授,主要从事气候变化与农业可持续发展研究,E2mail:xly0910@;林而达,研究员,通讯作者,主要从事气候变化与农业相互影响的研究,E2mail:lined@中国农业大学学报2008年第13卷　1　研究背景与进展工业革命以来,大气中CO 2浓度迅速增高已得到证明和公认。

Lehninger Principles of Biochemistry 习题答案chapter 19

Electrons generally fall in energy through the chain - from complexes I and II to complex IV O2
NADH (reductant) + H+ + ? O 2 (oxidant) NAD+ + H2O
3
Oxidative Phosphorylation
2
Electron transfer from NADH to O2 involves multisubunit inner membrane complexes I, III, & IV, plus coenzyme Q and cytochrome c. Within each complex, electrons pass sequentially through a series of electron carriers.
4
Structure of mitochondrial cytochrome c
Heme is covalently linked to the protein via S atoms
Iron-sulfur centers (Fe-S)
- electron transfer proteins may contain multiple ironsulfur centers. - transfer only one electron even if they contain two or more iron atoms, because of the close proximity of the iron atoms.
• contains all of TCA cycle enzymes {except, succinate

大豆凝集素对肉鸡脂肪代谢和脂肪生成基因表达的影响

Effects of soy-lecithin on lipid metabolism and hepatic expression oflipogenic genes in broiler chickensJin Huang a,b ,Dandan Yang a ,Shudong Gao c ,Tian Wang a,⁎aCollege of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,ChinabKey Laboratory of Animal Physiology &Biochemistry,Nanjing Agricultural University,Nanjing 210095,ChinacEastocean oils &grains industries (Zhangjiagang)Co.,Ltd.,Zhangjiagang,215634,ChinaReceived 16June 2007;received in revised form 4January 2008;accepted 13January 2008AbstractThe purpose of this experiment was to determine the effects of soy-lecithin on the lipid metabolism and expression of lipogenic genes in the liver of broiler chickens.Arbor Acres (AA)chicks growing from 1to 42days of age were randomly divided into 4groups and fed corn –soybean basal diets containing 0%soy-lecithin (control),0.5%soy-lecithin (SL1),1%soy-lecithin (SL2),2%soy-lecithin (SL3),respectively.At the end of experiment,samples of serum were taken for analyses of metabolites/hormones and liver tissue was collected to quantify expression of selected genes.The abdominal fat was removed and samples of thigh muscle and breast muscle also were collected.The hepatic expression of the genes encoding malic enzyme (ME),fatty acid synthase (FAS),acetyl-CoA carboxylase (ACC),sterol regulatory element binding protein-1(SREBP-1),stearoyl-CoA (Δ9)desaturase 1(SCD1)and liver fatty acid binding protein (L-FABP)were determined with reverse transcription-polymerase chain reaction (RT-PCR)using SYBR green as a flourophore monitored in a real time mode.The study showed that the percentage of abdominal fat and liver fat were not significantly affected by soy-lecithin (P N 0.05).SL2group had the highest percentage of thigh muscle fat compared with other groups (P b 0.05),but soy-lecithin had no significant effect on the percentage of breast muscle fat and the width of inter-muscular fat (P N 0.05).Serum concentration of total serum cholesterol (TC)and low-density lipoprotein cholesterol (LDL-C)were reduced by soy-lecithin,whereas high-density lipoprotein cholesterol (HDL-C)and triglyceride (TG)were improved (P b 0.05).The thyroid-stimulating hormone (TSH)and insulin (INS)were elevated in SL3group (P b 0.05).Furthermore,abundance of ME,FAS,ACC,SREBP-1,L-FABP and SCD1mRNA were greater (P b 0.05)in SL3group.The results indicate that soy-lecithin alters the serum hormone levels and affects hepatic gene expression and thereby regulates fat metabolism of broilers.©2008Elsevier B.V .All rights reserved.Keywords:Soy-lecithin;Lipid metabolism;Gene expression;Liver;Chickens1.IntroductionAmong the ingredients used in poultry diets,fats and oils are the most concentrated sources of energy (Blanch et al.,1996),therefore,fats and oils are usually added to broiler diet as dietary energy-yielding ingredients to im-prove productivity.But fats utilization as energy source isAvailable online at Livestock Science 118(2008)53–60/locate/livsciCorresponding author.College of Animal Science and Technology,Nanjing Agricultural University,Nanjing,China.Tel.:+862584395106;fax:+862584395314.E-mail address:twang18@ (T.Wang).1871-1413/$-see front matter ©2008Elsevier B.V .All rights reserved.doi:10.1016/j.livsci.2008.01.014limited by young birds because of the lack of several digestion enzymes.Fats are not efficiently used until lipase activity reached its maximum level(Krogdahl and Sell,1989).Soy-lecithin is a by-product from the processing of soybean oil that,apart from being a source of energy,also serves as an emulsifier and has the potential to facilitate fat absorption(Lechowski et al.,1999). Studies have shown that dietary supplementation of bile salts improves emulsion formation and fat digestibility in chickens(Kussaibati et al.,1982;Polin et al.,1980a).In avian species,lipogenesis takes place primarily in the liver and the liver account for95%of the de novo fatty acid synthesis and there is apparently a general assumption that almost all the fat that accumulates in broiler adipose tissue is synthesized in the liver or is derived from the diet(Harry et al.,1992).The liver is the most important organ for the intermediary metabolism of lipids and energy and hence,regulation of hepatic gene expression may play a central role in the adaptive response to altered digestion by changing the capacity of enzymes in relevant metabolic pathways(Theil and Lauridsen,2007).The development of adipose tissue depends on the availability of plasma triglycerides that are hydrolyzed prior to their uptake by adipocytes and de novo synthesized fatty acids.They are supplied specifically to adipocytes by specific lipoprotein classes (Amal et al.,2002).Dietary phospholipids of soybean may affect hepatic triglyceride synthesis and in turn modify serum lipid profiles(Ristic et al.,2003).Thus,to determine the effects of soy-lecithin on lipid metabolism,we investigated changes in the levels of serum lipid profiles,key metabolic hormones and the expression of selected hepatic lipogenic genes.2.Materials and methods2.1.Animals and dietsTwo hundred and forty1-day-old male Arbor Acres(AA) chicks obtained from a local commercial hatchery(Hewei, Anhui,China)were randomly assigned to4treatment groups consisting of10replicates of6birds.The average initial body weight did not differ among the four groups.Chicken were fed corn–soybean basal diets and supplemented with0%soy-lecithin(control),0.5%soy-lecithin(SL1),1%soy-lecithin (SL2),2%soy-lecithin(SL3),respectively.The percentage of all other major ingredients remained similar across treatments.The diet was formulated to meet the nutrient requirements of the broiler(NRC,1994).The birds were fed a starter diet until21d of age followed by a grower diet from21to42d(Table1).Birds were allowed to consume both feed and water on an ad libitum basis and housed(six per cage)in an environmentally controlled room maintained at34–36°C during1to14days and thenTable1Formulation and analyzed content of diets1–21days21–42daysItem Cont.SL1SL2SL3Cont.SL1SL2SL3 Ingredient(%)Maize65.963.962.762.47169.868.768.6 Soybean meal28.528.728.92922.923.123.323.3 Fishmeal22222222 Soy-lecithin–0.5 1.0 2.0–0.5 1.0 2.0 Limestone powder 1.28 1.59 2.03 1.25 1.12 1.49 1.98 1.12 Dicalcium phosphate 1.74 1.73 1.79 1.77 1.5 1.62 1.53 1.5 Methionine0.200.200.200.200.140.140.140.14 Premixa a11111111 Salt0.380.380.380.380.340.350.350.34 Total100100100100100100100100 Analyzed content bME(MJ/kg)12.0012.0012.0012.2612.3012.3012.3012.55 Crude protein(%)20.4420.1320.4120.3218.1518.1818.318.17 Lysine(%) 1.10 1.09 1.13 1.080.940.960.950.95 Methionine+cystine(%)0.800.810.790.820.730.710.700.74 Calcium(%)0.98 1.01 1.02 1.010.890.900.850.90 Total phosphorus(%)0.700.720.690.700.620.650.660.64 Control=basal diet;SL1=basal diet with0.5%soy-lecithin;SL2=basal diet with1%soy-lecithin;SL3=basal diet with2%soy-lecithin.a Provided per kg of diet:iron,60mg;copper,7.5mg;zinc,65mg;manganese,110mg;iodine,1.1mg;selenium,0.4mg;Bacitracin Zinc, 30mg;Vitamin A,4500IU;Vitamin D3,1000IU;Vitamin E,20mg;Vitamin K,1.3mg;Vitamin B1,2.2mg;Vitamin B2,10mg;Vitamin B3, 10mg;choline chloride,400mg;Vitamin B5,50mg;Vitamin B6,4mg;Biotin,0.04mg;Vitamin B11,1mg;Vitamin B12,1.013mg.b ME(metabolic energy)was calculated from the composition of diets,others were analyzed content of diets.54J.Huang et al./Livestock Science118(2008)53–60reduced progressively to 26°C at the end of experiment.The light regimen was a 12-h light –dark cycle (06:00–18:00h light).2.2.Experimental procedureThe last day of the experiment,all chickens were fasted for 12h with free access to water before experimentation.The morning after,one chicken was then randomly selected from each pen replicate (40chickens,totally),weighed and killed by exsanguina-tion.Individual blood samples were taken and separated by centrifugation at 3000×g for 15min and at 4°C.Serum samples were frozen at −20°C for further analysis.Liver samples were removed and immediately frozen in liquid nitrogen and stored in a freezer at −70°C for subsequent extraction of total RNA.Thigh muscle and breast muscle were removed and stored at −20°C for the analysis of fat content.The abdominal fat was removed and weighed and the thickness of subcutaneous fat and the width of inter-muscular fat were measured using sliding caliper.2.3.Lipid extractionThe lipid of thigh and breast muscle was extracted according to the method of Folch et al.(1956).Meat samples (5g ±0.01)were homogenized with 100ml of chloroform –methanol (2:1,v/v)solution for 2min,filtered,placed in separator funnels and mixed with 20ml saline solution (0.88%KCl).After separation in two phases,the methanol aqueous fraction (top layer)was discarded,whereas the lipid chloro-form fraction (bottom layer)was washed with distilled water/methanol (1:1,v/v)and evaporated under nitrogen (N 2)flow.2.4.Serum measurementTotal serum cholesterol (TC),triglyceride (TG),high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cho-lesterol (LDL-C),free fatty acid (FFA)and serum glucose (SG)were measured by colorimetric enzymatic methods usingTable 2Sequences of PCR primers Gene a Accession number b Primer sequence (5′⟶3′)Orientation Product size (bp)ACC J03541TCTCGCTTTATTATTGGTT Forward 312CATTGTTGGCTATCAGGAC Reverse FAS J04485TGAAGGACCTTATCGCATTGC Forward 195GCATGGGAAGCATTTTGTTGT Reverse ME AF408407GCTGCAATTGGTGGTGCTTForward 106ACTCTGCTTTGCTGGTAGGATTG Reverse SREBP-1AY029224GCAGAAGAGCAAGTCCCTCAA Forward 104TCGGCATCTCCATCACCTC Reverse SCD1X60465TCCCTTCTGCAAAGATCCAG Forward 402AGCACAGCAACACCACTGAG Reverse FABP AF380999GAGCTCCAGTCCCATGAAAA Forward 202TCAGCAGCTCCATCTCACAC Reverse β-actinL08165TGCGTGACATCAAGGAGAAG Forward 300TGCCAGGGTACATTGTGGTAReverseaACC =acetyl-CoA carboxylase;FAS =fatty acid synthase;ME =malic enzyme;SREBP-1=sterol regulatory element binding protein-1;SCD1=stearoyl-CoA (Δ9)desaturase 1;L-FABP =fatty acid binding protein (liver).bGenbank accession number.Table 3Body composition of broilers fed with different level of lecithin after 42days ItemControl SL1SL2SL3Body (kg) 1.94±0.06a 1.85±0.04ab 1.96±0.06a 1.71±0.04b Liver (g)43.6±1.742.0±2.242.6±1.242.9±1.03Liver (%body weight) 2.19±0.53B 2.18±0.78B 2.19±1.1B 2.53±0.88A Abdominal fat pad (g)27.3±2.57ab 33.2±2.6a 28.0±3.3ab 22.7±1.6b Abdominal fat pad (%body weight) 1.40±0.11 1.72±0.17 1.46±0.18 1.40±0.06Subcutaneous adipose tissue (mm)0.82±0.03b 0.93±0.04a 0.86±0.04ab 0.90±0.04ab Inter-muscular adipose tissue (mm) 1.00±0.03 1.06±0.06 1.02±0.02 1.00±0.02Percentage of breast muscle fat (%) 4.54±1.13 4.41±0.8 3.48±0.87 4.00±0.87Percentage of thigh muscle fat (%)5.35±2.02b4.83±0.59b12.77±2.01a7.89±0.78bControl =basal diet;SL1=basal diet with 0.5%soy-lecithin;SL2=basal diet with 1%soy-lecithin;SL3=basal diet with 2%soy-lecithin.Values are means±S.E.n =10.Values in a row not sharing a superscripts are different at P b 0.05(small letter)or P b 0.01(capital letter).55J.Huang et al./Livestock Science 118(2008)53–60commercial kits purchased from Nanjing Jiancheng Bioengi-neering Institute (Nanjing,China).Triiodothyronine (T3),thyroxine (T4),thyroid-stimulating hormone (TSH)and insulin (INS)measured with the RIA kits provided by Beijing North Institute of Biotechnology (Beijing,China).2.5.Real-time quantitative PCR analysis of gene expression Total RNA was isolated using the TRIzol reagent (Takara,Japan)according to the manufacturer's protocol.The RNA integrity was assessed via agarose gel electrophoresis and RNA concentration and purity was determined spectrophotometri-cally using A 260and A 280measurements in a photometer (Eppendorf Biophotometer).Ratios of absorption (260/280nm)of all preparations were between 1.8and 2.0.Reverse transcription (RT)reactions (25μl)consisted of 2μg total RNA,100U of M-MLV reverse transcriptase (Promega,Belgium),40U of recombinat RNAsin ribonuclease inhibitor (Promega,Belgium),0.8mmol/l dNTP (Promega,Belgium),and 1μg random primers (Promega,Belgium)in distilled water and buffer supplied by the manufacturer.After incubation (37°C,60min),the mixture was heated (95°C,5min).Polymerase chain reaction (PCR)was performed in 25μl containing 2.5μl of the RT reaction products,12.5μl SYBR Real-time PCR Master Mix (TOYOBO,Life Science Depart-ment,Osaka,Japan),0.1–0.2mmol/l of each gene specific primer and β-actin,the internal standard (Table 2).The expression of β-actin had no significant difference among the four groups.Thermal cycling parameters were as follows:1cycle 95°C for 5min,and then 40cycles at 95°C for 30s,58–60°C for 30s,72°C for 20s on an STRATAGENE MX3000P ™Sequence Detection System (MXpro ™QPCR software).Fluorescence data was collected in the latter stage by recording SYBR incorporation into amplified DNA.Fluor-escent data were used to derive the C (t )or the PCR cycle at a threshold which is noted as the first significant deviation in fluorescence from a base line value.Analyses were performed in duplicate.The resultant value was expressed relative to β-actin (control gene).Results (fold changes)were expressed as 2−ΔΔC (t )with ΔΔC (t )=[C (t )ij −C (t )β-actinj]−[C (t )i1−C (t )β-actin1],where C (t )ij and C (t )β-actin j are the C (t )for gene i and for β-actin in a pool or a sample (named j )and where C (t )i 1and C (t )β-actin1are the C (t )in target gene and in house keeping gene in the control group,respectively.2.6.Data analysisData were analyzed by one-way ANOV A using the general linear models (GLM)procedure of SPSS 11.5software.Significant differences among individual group means were determined with Duncan's multiple range test option of the GLM procedure of SPSS software.Pearson correlation coefficients for the interrelationship of selected variables were determined using Bivariate Procedure of SPSS software.Values were expressed as mean±SE .3.Results and discussionThis study compared the effects of lecithin on the circulating levels of lipid profiles and keymetabolicFig.1.Serum concentrations of TC,TG,HDL-C,LDL-C in chickens fed diets containing 0%(Control),0.5%(SL1),1%(SL2)or 2%(SL3)of soy-lecithin in diets after 42days.V alues are means±SE,n =10.Means without a common letter are significantly different,P b 0.05.Control =basal diet;SL1=basal diet with 0.5%soy-lecithin;SL2=basal diet with 1%soy-lecithin;SL3=basal diet with 2%soy-lecithin.Table 4Serum parameters in broilers fed with different level of lecithin after 42days Item Control SL1SL2SL3T3(nmol/l) 1.46±0.17a 0.89±0.21b 1.26±0.19ab 1.44±0.16ab T4(nmol/l)33.4±827.0±1443.2±1033.9±5TSH (IU/l) 4.68±0.41a 5.42±0.05ab 4.64±0.44a 6.30±0.36b INS (m IU/l)0.12±0.00a 0.18±0.01a 0.20±0.01a 0.25±0.01b SG (mmol/l)7.90±0.887.71±0.30 6.73±0.3 6.69±0.55FFA (μEq/l)645±127656±57488±7563±111Control =basal diet;SL1=basal diet with 0.5%soy-lecithin;SL2=basal diet with 1%soy-lecithin;SL3=basal diet with 2%soy-lecithin.Values are means±S.E.n =10.V alues in a row not sharing a superscripts are different at P b 0.05.56J.Huang et al./Livestock Science 118(2008)53–60hormones and the expression of selected lipogenic genes in AA broiler chickens.Fats and oils are important dietary ingredients due to their high energy value,and their fatty acid pattern is to a great extent reflected in that of animal products (Duran-Montg et al.,2007).But the utilization of dietary fat is limited in young broilers because of the lack of several digestion enzymes (Krogdahl and Sell,1989).Phospholipids are known to have surface active properties.They are important in the emulsification of lipids and may influence the absorption of fatty acids in the small intestine (Jenkins et al.,1989).Fig.2.Hepatic ME (A),FAS (B),ACC (C),SCD1(D),SREBP-1(E)and L-FABP (F)gene expression in chickens fed diets containing 0%(Control),0.5%(SL1),1%(SL2)or 2%(SL3)of soy-lecithin in diets after 42days.RNA molecules extracted from liver were reversely transcribed to cDNA and analyzed by quantitative Real-time RT-PCR.For comparison between different samples,the ME,FAS,ACC,SCD1,SREBP-1and L-FABP transcript level of each sample was normalized for the β-actin level and expressed as a multiple of expression level of control,respectively.Values are means±SE,n =6.Means without a common letter are significantly different at P b 0.05(small letter).57J.Huang et al./Livestock Science 118(2008)53–603.1.Body compositionThe body weight of broilers in SL3group were de-creased(P b0.05)after42days intake of soy-lecithin (Table3).Soybean lecithin did not influence the liver weight of6-week-old broilers and the relative abdominal adipose tissue(%of body weight).By contrast,the relative proportion of liver(%of body weight)was significantly increased(P b0.01)and abdominal adipose tissue was decreased(P b0.05).The content of breast muscle fat and the width of inter-muscular adipose tissue was not affected by the addition of soy-lecithin(P N0.05) while the subcutaneous adipose tissue was improved in SL1group and the percentage of thigh muscle fat was significantly improved in SL2group(P b0.05).The relative weight of liver can be improved by soy-lecithin in broilers,and this is consistent with what was observed in a previous study(Wang et al.,1999).In avian species, lipogenesis takes place primarily in the liver(Leveille et al.,1975).Soy-lecithin can improve the relative weight of liver and maybe correlated with the enhanced lipid metabolism in liver.3.2.Serum parametersConcentrations of plasma lipids and lipoproteins are indicative of the metabolic regulations in a steady state and,especially,of the basal adjustment of fatty acid circulation between the adipose tissue and the liver (Amal et al.,2002).Several studies have indicated that lecithin,a phosphatidyl choline containing phospholi-pid,has hypocholesterolemic properties(Thomas et al., 1998).Effects of soy-lecithin on serum concentrations of lipoproteins for all broilers after42days are shown in Fig.1.In SL3group,feeding soy-lecithin resulted in a lower serum concentration of TC(P=0.079)and LDL-C(P=0.071),and it is in agreement with previous works (Jones et al.,1992;Thomas et al.,1998;Tompkins and Parkin,1980),whereas serum concentrations of HDL-C and TG were increased.It is not clear through which mechanism soy-lecithin induces its plasma cholesterol-lowering effects.Possibly,this might be brought about by inhibiting the absorption of cholesterol in the small intestine as suggested by Iwata et al.(1992)and Spilburg et al.(2003).In the present study,the circulating levels of TSH and INS were increased when crude soybean lecithin was included in the diets of broilers in a proportion of2% (Table4).No significant differences were observed on the serum SG,FFA and T4except that the concentration of T3in SL1group was decreased compared with other groups(P b0.05).Key plasma metabolic hormones (insulin,glucagon and T3)are important factors that determine the level of hepatic lipogenesis in birds (Hillgartner et al.,1995).3.3.Hepatic lipogenic gene expressionSoybean lecithin affected the expression of hepatic genes involved in lipid metabolism,particularly those genes regulating lipogenesis including acetyl-CoA car-boxylase(ACC),malic enzyme(ME),fatty acid synthase (FAS)and stearoyl-CoA(Δ9)desaturase1(SCD1) (P b0.05)(Fig.2).In the present study,soybean lecithin caused the increases of ME,FAS,ACC and SCD1mRNA levels in a dose-dependent manner(Table5).In addition, expression of liver fatty acid binding protein(L-FABP), which is involved in lipid transport,and the transcriptionTable5Correlations between diet soybean lecithin levels and the expression of genes related to lipogenesis in broiler chickens aVariable b Lecithin ME FAS ACC SCD1SREBP-1FABP Lecithin1ME0.702(0.002)1FAS0.6330.807(0.002)(0.000)1ACC0.6360.7440.890(0.002)(0.001)(0.000)1SCD10.6290.5440.4420.708(0.002)(0.029)(0.058)(0.001)1SREBP-10.5660.7050.5790.7110.609(0.005)(0.002)(0.007)(0.000)(0.003)1FABP0.5000.2500.4750.2750.3190.357(0.015)(0.351)(0.034)(0.241)(0.170)(0.103)1a Pearson's correlation coefficients(r)shown with P values in parentheses.b ACC=acetyl-CoA carboxylase;FAS=fatty acid synthase;ME=malic enzyme;SREBP-1=sterol regulatory element binding protein-1;SCD1= stearoyl-CoA(Δ9)desaturase1.L-FABP=fatty acid binding protein(liver).58J.Huang et al./Livestock Science118(2008)53–60factor sterol regulatory element binding protein-1 (SREBP-1)were influenced in a similar manner.In general,lipogenic gene expression levels relative toβ-actin were increased significantly with inclusion of lecithin in the diets(P b0.05).Some of the highest correlations were found among lipogenic genes linked in the lipogenisis such as ME,FAS,ACC and SREBP-1. This undoubtedly reflects the role of this key transcription factor in coordinating hepatic lipogenesis.The expression of a number of lipogenic enzyme genes such as FAS,ME, ACC,SCD1is directly influenced by SREBP-1(Richards et al.,2003).In the present study,the expression of SREBP-1was closely correlated with the expression of FAS genes,and this is consistent with Gondret et al. (2007)who observed that the relative distribution of ADD-1/SREBP-1mRNA between adipose tissue and liver closely parallels that of FAS expression in various species including rodents(rabbit),monogastric mammals (pig),and birds(chicken).In birds,the accumulation of lipid in extra hepatic tissues results to a large extent from the combined effects of hepatic lipogenesis and lipopro-tein production.Therefore,plasma triglyceride levels are dependent on the level of hepatic lipogenesis.In the present experiment,soybean lecithin increased serum levels of INS and TSH and up-regulated the expression of lipogenic genes(P b0.05).Nutritional(energy)status and the subsequent responses of key plasma metabolic hormones(insulin, glucagon and T3)are important factors that determine the level of hepatic lipogenesis in birds.Hepatic lipogenesis is highly responsive to changes in the diet(Hillgartner et al., 1995).In the present study,the food consumption of broilers fed with2%soybean lecithin was lower than other groups as was observed by our previous work (Huang et al.,2007)and(Azman and Ciftci,2004).Gene expression may be regulated anywhere from transcription to the actual enzyme protein.Soybean lecithin is a well known emulsifier to promote the apparent digestibility of dietary fat in diets fed to chicks(Polin,1980b).The up-regulated expression of lipogenic genes may be related to the increased lipid metabolism in the live of broilers.In conclusion,soybean lecithin alters serum lipids profiles and some key metabolic hormones and hepatic lipogenic gene expressions in broilers.The results im-plied that soy-lecithin could regulate fat metabolism of broilers by altering the hormone levels and lipogenic gene expressions in broiler chickens.AcknowledgementsThis work was supported by the National Basic Research Program of China,Project No.2004CB117500.The authors are grateful to Pro.R.Q.Zhao,associate Pro.Y.M.Zhou.Thanks are also extended to L.N.Wang, S.Wei,Y.Hu,H.T.Fan for their kind help.ReferencesAmal,M.,Michel,L.,Solang,G.,Maryline,K.,2002.Effect of dietary fats on hepatic lipid metabolism in the growing p.Biochem.Physiol.,Part B132,473–483.Azman,M.A.,Ciftci,M.,2004.Effects of replacing dietary fat with lecithin on broiler chicken zootechnical performance.Revue Med.Vet.155,445–448.Blanch,A.,Barroeta,A.,Baucells,M.,1996.Utilization of different fats and oils by adult chickens as a source of energy,lipid and fatty acids.Anim.Feed Sci.Tech.61,335–342.Duran-Montg,P.,Lizardo,R.,Torrallardona,D.,Esteve-Garcia,E., 2007.Fat and fatty acid digestibility of different fat sources in growing pigs.Livest.Sci.109,66–69.Folch,J.,Lees,M.,Sloane,G.H.,1956.A simple method for the isolation and purification of total lipides from aninal tissues.Biol.Chem.226,497–509.Gondret,F.,Ferre,P.,Dugail,I.,2007.ADD-1/SREBP-1is a major determinant of tissue differential lipogenic capacity in mammalian and avian species.Lipid Res.42,106–113.Harry,D.G.,Kunda,G.,Dawn,W.,Simon,C.,1992.Adipose tissue lipogenesis and fat deposition in leaner broiler chickens.Nutr.122, 363–368.Hillgartner,F.,Salati,L.,Goodridge,A.,1995.Physiological and molecular mechanisms involved in nutritional regulation of fatty acid synthesis.Physiol.Rev.75,47–76.Iwata,T.,Hoshi,S.,Takehisa,F.,Tsutsumi,K.,Furukawa,Y.,Kimura, S.,1992.The effect of dietary safflower phospholipid and soybean phospholipid on plasma and liver lipids in rats fed a hypercho-lesterolemic diet.J.Nutr.Sci.Vitaminol(Tokyo)38(5),471–479. Jenkins,T.C.,Gimenez,T.,Cross, D.L.,1989.Influence of phospholipids on ruminal fermentation in vitro and on nutrient digestion and serum lipids in sheep.J.Anim.Sci.67,529–537. Huang,J.,Yang,D.,Wang,T.,2007.Effects of replacing soy-oil with soy-lecithin on growth performance,nutrient utilization and serum parameters of broilers fed corn-based n-Aust J.Anim.Sci.12,1880–1886.Jones,D.B.,Hancock,J.D.,Harmon,D.L.,Walker,C.E.,1992.Effects of exogenous emulsifiers and fat sources on nutrient digestibility, serum lipids,and growth performance in weanling pigs.J.Anim.Sci.70,3473–3482.Krogdahl,A.,Sell,J.L.,1989.Influence of age on lipase,amylase,and protease activities in pancreatic tissue and intestinal contents of young turkeys.Poult.Sci.68,1561–1568.Kussaibati,R.,Guillaume,J.,Leclercq,B.,1982.The effects of age, dietary fat and bile salts,and feeding rate on apparent and true metabolisable energy values in chickens.Br.Poult.Sci.23,393–403. Lechowski,R.,Bielecki,W.,Sawosz,E.,Krawiec,M.,Klucinski,W., 1999.The effect of lecithin supplementation on the biochemical profile and morphological changes in the liver of rats fed different animal mun.23,1–14.Leveille,G.A.,Romsos,D.R.,Yeh,Y.,O'Hea,E.K.,1975.Lipid biosynthesis in the chick.A consideration of site of synthesis, influence of diet and possible regulatory mechanisms.Poult.Sci.54,1075–1093.NRC,1994.Nutrient Requirements of Poultry.National Academy Press,Washington,A.59J.Huang et al./Livestock Science118(2008)53–60Polin,D.,Wing,T.,Ki,P.,Pell,K.E.,1980a.The effect of bile acids and lipase on absorption of tallow in young chicks.Poult.Sci.59, 2738–2743.Polin,D.,1980b.Increased absorption of tallow with lecithin.Poult.Sci.59,1652.Richards,M.P.,Poch,S.M.,Craig,N.C.,Rosebrough,R.W.,2003.Feed restriction significantly alters lipogenic gene expression in broiler breeder chickens.Nutr.133,707–715.Ristic,M.D.,Ristic,V.,Tepsic,V.,2003.Effect of soybean Leci-Vita product on serum lipids and fatty acid composition in patients with elevated serum cholesterol and triglyceride levels.Nutr.Res.23, 465–477.Wang,R.,Li,D.,Yang,W.,Gao,Y.,1999.Effects of soybean lecithin on broiler performance.Feed Indus.20,8–10.Spilburg,C.A.,Goldberg,A.C.,McGill,J.B.,Stenson,W.F.,Racette, S.B.,Bateman,J.,McPherson,T.B.,Ostlund,R.E.,2003.Fat-freefoods supplemented with soy stanol–lecithin powder reduce cholesterol absorption and LDL cholesterol.Am.Diet Assoc.103,577–581.Theil,P.K.,Lauridsen,C.,2007.Interactions between dietary fatty acids and hepatic gene expression in livers of pigs during the weaning period.Livest.Sci.108,26–29.Thomas,A.W.,Craig,M.M.,Robert,J.N.,1998.Soy lecithin reduces plasma lipoprotein cholesterol and early atherogenesis in hypercho-lesterolemic monkeys and hamsters:beyond linoleate.Atherosclerosis 140,147–153.Tompkins,R.K.,Parkin,L.G.,1980.Effects of long-term ingestion of soya phospholipids on serum lipids in humans.Am J.Surg.140, 360–364.60J.Huang et al./Livestock Science118(2008)53–60。

核酸适配体ppt课件

核酸适配体的应用
核酸适配体在分析化学中的应用核酸适配体与疾病诊断和新药研发
核酸适配体在分析化学中的应用
靶物质的分析与检测
该方面应用的基本思路是将各种报告基团，如荧光试剂，定点标记在aptamer核苷酸上，然后在一定条件下，使aptamer与靶物质发生相互作用，再通过对报告基团的信号检测实现对靶物质的定性检测或定量分析。
核酸适配体的筛选策略
核酸适配体的体外筛选是利用 SELEX 技术（ Systematic Evolution of Ligands by Exponential Enrichment) ）来完成的， SELEX是指数富集配体系统进化的简称，它的基本原理就是就是利用分子生物基(aptamer) ，经反复扩增、筛选数个循环，即可使与该靶分子特异结合的寡核苷酸序列得到富集。
Aptamer巨大的应用前景
APTAMERS: AN EMERGING CLASS OF THERAPEUTICS（.Annu. Rev. Med. 2005. 56:555–83）
Analytical Applications of Aptamers(Andrew D. Ellington .Annual Review of Analytical Chemistry (2008). Volume 1, Jul 2008)
基于适配体的生物传感器是用适配体作为识别元件来特异性地检测其相应的靶物质。
目前适配体生物传感器（Aptasensors）尚处于起步阶段。 Chunyan Yao等将IgE适配体固定在石英晶体微平衡生物传感器阵列，建立了适配体压电石英生物传感器模型，用于特异性检测标准溶液和人血清中的IgE。该方法最低可在标准和人血清溶液中分别检测出2.5-200 ug/L的IgE，整个检测时间只需15 min，而且固定在金膜表面的适配子在反复洗脱后并不影响其灵敏度。

固定效应不收敛的原因

固定效应不收敛的原因（中英文版）英文文档：Fixed effects models are commonly used in panel data analysis to account for time-invariant individual or entity-specific characteristics.However, one potential issue with fixed effects models is the problem of non-convergence.This occurs when the estimators of the model fail to converge to the true parameters as the sample size increases.There are several reasons why fixed effects models may experience non-convergence.One reason is that the fixed effects estimator is based on the first-differences of the data, which can lead to serial correlation.This means that the errors in the model are not independently and identically distributed, which can cause the estimators to be inefficient and non-convergent.Another reason for non-convergence is that the fixed effects estimator can be sensitive to the inclusion of certain variables.If there are omitted variable bias or measurement error in the included variables, the fixed effects estimator may not converge to the true parameters.Additionally, the problem of non-convergence can also be exacerbated by the presence of perfect multicollinearity among the fixed effects.This occurs when there is a perfect linear relationship between two or more of the fixed effects, which can cause the estimators to be non-convergent.To address the issue of non-convergence in fixed effects models, researchers can consider using alternative methods such as the random effects model or the generalized method of moments.Alternatively, researchers can try to increase the sample size or improve the quality of the data to ensure convergence of the estimators.中文文档：固定效应模型常用于面板数据分析，以考虑时间不变的个人或实体特定特征。

兴奋收缩耦联和心力衰竭的治疗

● In cardiac myocytes, the release of Ca2+ from the sarcoplasmic reticulum is induced by Ca2+ influx into the cell through voltage-gated calcium channels.
Bers DM. Nature, 2002, 415(6868): 198-205.
Excitation
The cardiac action potential
● A notable difference between skeletal and cardiac myocytes is how each elevates the myoplasmic Ca2+ to induce contraction.
Inotropic Agents for HF
Inotropic Agents and β-blocker
● Digitalis ● Phosphodiesterase inhibitor ● β- adrenoceptor blocker
Digitalis (﹥200 years)
Digilis purpurea Purple foxglove
TOPICS
Excitation-contraction (EC) coupling
➢ Excitation ➢ Calcium Cycling ➢ Contraction
Alterations of E-C coupling in HF Inotropic agents for HF
Excitation-contraction coupling
Jeffery D Molkentin. Nature Medicine 11, 1284 - 1285 (2005)

英语国际会议PPT课件

6
Materials and Methods
Patients
Materials and Methods
Cytokine assessment by ELISA
Western blot
Cell isolation and culture
Statistical analysis
7
Methods
IFN-c-induced protein of 10-kDa (IP-10)/CXCL10
we studied the effects of α-toxin on Th1- and Th2related chemokines in macrophages from patients with AD and psoriasis where the intrinsic abnormal and different chemokines production profile is well defined.
13
Figure 3 Punch biopsies (3 mm) from healthy individuals were left either unstimulated (A) or stimulated with a-toxin (100 ng/ ml) (B) or IFN-c (100 ng/ml) (C) for 24 h at 37C. 5-lm parafﬁn sections were stained for CXCL10 along with appropriate isotype as well as CD68.
16
Low effect of a-toxin on CXCL10 induction (Th1-related chemokine) in macrophages from patients with AD

朱章志运用扶正祛邪法论治糖尿病经验

ʌ临证验案ɔ朱章志运用扶正祛邪法论治糖尿病经验❋曾绘域1,朱章志2ә,周㊀海3,陈㊀珺3,张文婧3(1.深圳市中西医结合医院,广东深圳㊀518104;2.广州中医药大学第一附属医院,广州㊀510405;3.广州中医药大学,广州㊀510405)㊀㊀摘要:糖尿病属于中医学消渴病范畴,以往医家多认为其病机为阴虚燥热,治疗以滋阴清热为法㊂朱章志教授通过长期的临床观察与实践,立足于张仲景保胃气,扶阳气的理论,认为糖尿病的病机为正虚邪滞,即太阴虚损㊁阳气不足㊁收敛不及,寒㊁水㊁湿之邪阻滞阳气运行通道㊂治疗上不囿陈法,以扶正祛邪为大法,通过固护太阴㊁扶助阳气㊁收敛阳气,祛除寒水湿之邪,恢复阳气运行之通畅,使阳气功能复常㊁运行有序,为糖尿病的治疗提供临床新思路㊂㊀㊀关键词:扶正祛邪;糖尿病;朱章志㊀㊀中图分类号:R587.1㊀㊀文献标识码:A㊀㊀文章编号:1006-3250(2021)01-0149-03Discussion on ZHU Zhang-zhi's Experience in Treating Diabetes Mellitus by Using The Method of Reinforcing The Healthy Qi and Eliminating The Pathogenic FactorsZENG Hui-yu 1,ZHU Zhang-zhi 2ә,ZHOU Hai 3,CHEN Jun 3,ZHANG Wen-jing 3(1.Shenzhen Hospital of Integrated traditional Chinese and Western Medicine,Guangdong,Shenzhen 518104,China;2.The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,China;3.Guangzhou University of Chinese Medicine,Guangzhou 510405,China)㊀㊀Abstract :Diabetes mellitus belongs to the category of "xiao ke"in traditional Chinese medicine.Doctors used to think that its pathogenesis was Yin deficiency and dryness heat ,and the treatment was nourishing Yin and clearing heat.Through long-term clinical observation and practice ,and based on ZHANG Zhong-jing's theory of protecting stomach Qi and supporting Yng Q ,professor ZHU Zhang-zhi believes that the pathogenesis of diabetes is deficiency of healthy Qi and stagnation of pathogen.Because of the deficiency of greater Yin and Yang Qi ,and the lack of convergence ,the cold ,water and dampness block the operational channel of Yang Qi.The treatment of diabetes mellitus should be based on reinforcing the healthy Qi and eliminating the pathogenic factors.By strengthening Taiyin ,supporting Yang Qi ,astringent Yang Qi ,dispelling the evil of cold ,water and dampness ,we can restore the smooth operation of Yang Qi ,restore the function of Yang Qi to normal and operate orderly ,which provides a new clinical method for the treatment of diabetes mellitus.㊀㊀Key words :Reinforcing the healthy Qi and eliminating the pathogenic factors ;Diabetes mellitus ;ZHU Zhang-zhi❋基金项目:国家自然科学基金资助项目(81873190)-降糖三黄片在糖脂毒性所致胰岛β细胞损伤的自噬调控作用作者简介:曾绘域(1990-),女,广东云浮人,住院医师,硕士研究生,从事六经辨治内分泌疾病的临床与研究㊂ә通讯作者:朱章志(1963-),男,湖南衡阳人,主任医师,博士研究生导师,从事六经辨治内分泌疾病的临床与研究,Tel :************,E-mail :zhuangi@ ㊂㊀㊀随着人口老龄化和生活方式的改变,我国糖尿病的患病率呈上升趋势,2013年我国18岁以上人群糖尿病患病率为10.4%[1]㊂中医药在延缓糖尿病的进展及防治其并发症方面具有一定优势[2-4]㊂糖尿病属于中医学消渴病范畴,以往医家多认为其病机为阴虚燥热,治疗以滋阴清热为法,但疗效尚不能令人满意㊂朱章志教授通过长期的临床观察与实践,认为正虚邪滞乃糖尿病病机之核心,采用扶正祛邪法治之屡获奇效㊂1㊀正虚邪滞之糖尿病病机‘素问㊃经脉别论篇“曰: 饮入于胃,游溢精气,上输于脾,脾气散精水精四布,五经并行㊂食物入胃,经脾胃运化化生精气,然后输布全身㊂糖尿病患者常嗜食肥甘,起居无常,烦劳紧张,致太阴虚损,正气内虚,阳气戕伐,津液代谢异常,而生寒水湿之邪㊂寒㊁水㊁湿之邪气作为阴邪,又可阻滞阳气运行之通道㊂阳气运行通道不畅,不能敷布温煦四肢,可见手足逆冷;阳气运行受阻,又可出现郁而化热之象㊂因此朱章志认为,疗糖尿病的关键在于恢复阳气运行之通畅,根据糖尿病正虚邪滞的病机,治疗以扶正祛邪为法,顾护太阴㊁扶助阳气㊁收敛阳气,祛除寒水湿之邪,使阳气功能复常则行有序㊂2㊀运用扶正祛邪法治疗糖尿病2.1㊀扶正2.1.1㊀固护中气,扶助阳气㊀张仲景遣方用药常体现保胃气之思想[5],如桂枝汤中配伍生姜㊁大枣㊁炙甘草,发汗祛邪不忘顾护中气;又如白虎汤中加梗米㊁炙甘草以和中益胃,又可防止石膏㊁知母大寒伤中㊂有胃气则生,无胃气则死 ,故扶正之要以保胃气为先㊂朱章志认为,阳气在人体的生命活动中占主导9412021年1月第27卷第1期January 2021Vol.27.No.1㊀㊀㊀㊀㊀㊀中国中医基础医学杂志Journal of Basic Chinese Medicine地位㊂‘素问㊃生气通天论篇“曰: 阳气者若天与日,失其所则折寿而不彰是故阳因而上,卫外者也㊂ ‘黄帝内经“把阳气比作太阳,阳气运行失常可致短寿㊂阳气具有抵御外邪㊁护卫生命㊁维持机体生命活动的作用,津液的气化㊁血液的运行均需阳气的温煦与推动㊂因此,在人体的阴阳平衡中阳气起着主导作用㊂朱章志认为,正气虚衰㊁太阴虚损㊁阳气不足是糖尿病发生发展之根本原因,因此扶正首当固护中气,扶助阳气 ,故常以附子理中汤为底方,固护中宫㊂太阴脾土居中央,犹如足球比赛之中场,能联系前锋与后卫进可攻退可守,进可充养肺卫之气抵御外邪,退可顾护少阴以防寒邪内陷㊂‘四圣心源㊃卷二太阴湿土“提到: 湿者,太阴土气之所化也故胃家之燥,不敌脾家之湿,病则土燥者少而土湿者多也㊂[6] 阴脾土易挟寒湿,附子理中汤功善固护中气㊁温补脾阳而散寒湿,为治疗太阴阳虚寒湿之要方㊂方中附子辛温大热,补坎中真阳,又能散寒湿,荡去群阴;干姜去脏腑沉寒痼冷,温暖脾土,复兴火种;人参被誉为百草之王能大补元气,为扶正固本之极品;白术味苦性温,功善健脾燥湿,乃扶植太阴之要药;炙甘草善益气补中,调和药性,诸药合用以收培补中阳㊁散寒除湿之效㊂若其人神疲懒言,气虚较甚,在附子理中汤的基础上可重用红参㊁北芪以大补元气,健脾益气;若其人四肢不温㊁肢体困重㊁寒湿较重者,可加重附子㊁干姜之量,并加细辛㊁吴茱萸以散久寒;若其人口干口苦㊁舌苔黄腻㊁大便黏滞不爽兼夹湿热之象,可仿当归拈痛汤之意,加茵陈㊁当归㊁黄芩以利湿清热㊂2.1.2㊀收敛阳气,阳密乃固㊀朱章志认为, 阴可理解为阳气的收敛㊁收藏状态,糖尿病阴虚燥热之象乃阳气不足㊁收敛不及㊁升发太过所致[7]㊂‘素问㊃生气通天论篇“提到: 阳气者,烦劳则张㊂现代人起居无节,以妄为常,阳气因而不能潜藏,常常浮越于外容易出现假热之象,医者不察,妄投清热泻火之品,实乃雪上加霜㊂凡阴阳之要,阳密乃固 ,收敛阳气即是扶正,犹如太极之能收能放,收敛是为了聚集能量,阳气固密,正气才能强盛,方能更好的制敌㊂朱章志常用砂仁㊁肉桂㊁白芍㊁山萸肉㊁泽泻等药物收敛阳气㊂砂仁辛温,既能宣太阴之寒湿,又能纳气归肾,使阳气收敛于少阴,少火生气㊂‘本草经疏“提到: 缩砂蜜,辛能散,又能润辛以润肾,故使气下行气下则气得归元㊂[8] 肉桂引火归原,导浮越之阳气归于命门,益火消阴㊂若患者出现咽痛㊁牙龈肿痛㊁痤疮等阳气不敛㊁虚火上冲之象,常用砂仁㊁肉桂以收敛阳气,纳气归肾,引火归原㊂白芍味酸能敛,敛降甲木胆火,使相火归位㊂‘本草求真“曰: 气之盛者,必赖酸为之收,故白芍号为敛肝之液,收肝之气,而令气不妄行也㊂[9] 朱章志常使用白芍以补肝之体㊁助肝之用,收敛肝气,肝平则郁气自除,火热自消㊂山萸肉秘精气㊁敛阳气,使龙雷之火归于水中㊂朱章志常用山萸肉收敛正气,遇汗出多者,常重用以固涩敛汗㊂泽泻能泻能降,能入肾泻浊,开气化之源,泻浊以利扶正,又能降气而引火下行㊂朱章志常用泽泻打通西方潜藏之要塞[10],在温阳之品中加入泽泻,利于阳气潜藏,使孤阳有归㊂2.1.3㊀填补阴精,以滋化源㊀‘素问㊃金匮真言论篇“提到: 夫精者,身之本也㊂精是人体生命活动的物质基础,能化气生髓,濡养脏腑㊂人体之精禀受于父母,又由后天水谷之精不断充养,归藏于肾中㊂孤阴不生,独阳不长 ,无阳则阴无以生,无阴则阳无以化㊂肾乃水火之脏,阴精充足才能涵养坎中真火,使真阳固密于内,化生正气㊂朱章志常在秋冬之季嘱糖尿病患者进补阿胶等血肉有情之品填补肾精㊂肾主封藏,秋冬进补使肾精充养,以滋阳气化生之源㊂阿胶用黄酒烊化,既能祛除阿胶之腥,又能借黄酒通行之性解阿胶滋腻碍胃之弊,每日少量服用,以有形之精难以速生,填补肾精以缓补为要㊂除此之外,遣方用药时亦会注意顾护阴精,在使用温阳药的同时常常配伍山萸肉㊁白芍等养阴药,以防温燥伤阴之弊㊂2.2㊀祛邪2.2.1㊀外散寒水以运太阳㊀太阳为开 ,太阳乃三阳之表,巨阳也,其性开泄以应天,为祛邪之重要通道㊂在运气里,太阳在天为寒,在地为水,合而为太阳寒水㊂张仲景太阳病篇研究的是水循环过程,治太阳就是治水[11]㊂寒㊁水之邪闭郁在表,气血运行不畅,可见肌肤麻木不仁㊂邪气滞留太阳,阻碍阳气运行,当因势利导㊁开太阳之表以发汗,外散寒㊁水之邪㊂糖尿病患者正气亏虚为本,祛邪不能伤正,朱章志临床常用桂枝麻黄各半汤小发其汗,使玄府开张,邪有出路㊂桂枝麻黄各半汤乃发汗轻剂,为桂枝汤与麻黄汤相合而得,其中麻黄㊁桂枝㊁生姜㊁北杏发散宣肺以开皮毛,芍药㊁大枣㊁炙甘草酸甘化阴以益营,诸药相合,刚柔相济,祛邪而不伤正㊂邪去正安,阳气运行通畅,水液代谢复常则阳气自充,而无寒水之扰㊂若寒邪较重可用三拗汤,此为麻黄汤去桂枝而成,功善开宣肺气,疏散风寒,因去辛温之桂枝发汗力不及麻黄汤,祛邪而不伤正㊂2.2.2㊀下利水湿以健少阴㊀少阴乃水火交会之脏,元气之根,人身立命之本㊂‘医理真传“提到: 坎中真阳,一名龙雷火,发而为病,一名元阳外越,一名孤阳上浮,一名虚火上冲㊂此际之龙,乃初生之龙,不能飞腾而兴云布雨,惟潜于渊中,以水为家,以水为性,遂安其在下之位㊂水盛一分龙亦盛一分,水高一尺龙亦高一尺,是龙之因水盛而游 [12]㊂阴盛051中国中医基础医学杂志Journal of Basic Chinese Medicine㊀㊀㊀㊀㊀㊀2021年1月第27卷第1期January2021Vol.27.No.1则阳衰,水湿之邪泛滥,则龙雷之火因而飞越在外㊂叶天士深谙张仲景之理,提到通阳不在温,而在利小便 [10,13],通过利小便的方法,使水湿之邪从下而解,阳气运行通道无水湿之邪阻碍,则阳气无需温养而自通,水盛得除则真龙亦安其位㊂朱章志常用五苓散㊁真武汤下利水湿,以复阳气之通达,少阴之健运㊂五苓散具有通阳化气利水之效,治疗膀胱气化不利形成的蓄水证㊂方中猪苓㊁茯苓㊁泽泻导水湿之邪下行;白术健脾燥湿,杜绝生湿之源;桂枝助膀胱气化,通阳化气行水又通气于表,使全身在表之湿邪皆得解,五药合用,膀胱气化复常,水道通调使小便得利,水湿得出㊂真武汤为治疗少阴阳虚㊁水气泛滥之主方,方中附子振奋少阴阳气,使水有所主;白术㊁茯苓健脾制水;生姜助附子敷布阳气,宣散水气;芍药利小便,制附㊁姜之燥,五味相合共奏温阳利水之功㊂2.2.3㊀开郁逐寒以畅厥阴㊀肝为将军之官,肝气主动主升发,能统帅兵马,捍卫君主㊂厥阴肝经,体阴用阳,内寄相火,相火敷布阳气,祛阴除寒,是祛邪的先锋主力军㊂朱章志常用吴茱萸汤祛除厥阴肝经之寒邪,恢复肝经阳气之运行㊂方中吴茱萸辛苦而温,芳香而燥,‘本草汇言“曰: 开郁化滞,逐冷降气之药 [14],能温胃暖肝,降浊阴止呕逆,为治疗肝寒之要药㊂配以生姜温胃散寒,佐以人参㊁大枣健脾益气补虚,全方散寒与降逆并施,共奏暖肝温胃㊁降逆止呕之效㊂‘素问㊃至真要大论篇“说: 帝曰:厥阴何也?岐伯曰:两阴交尽也㊂物极必反,重阴必阳㊂厥阴为阴尽阳生之脏,足厥阴肝经与足少阳胆经互为表里,若出现肝气不疏㊁枢机不利㊁气郁化火,朱章志常用小柴胡汤和畅枢机,开郁以复气机调达㊂方中柴胡疏泄肝胆之气;黄芩清泄胆火,一疏一清,气郁通达,火郁得发;生姜㊁半夏和胃降逆;人参㊁大枣㊁炙甘草固护中宫,全方寒温并用㊁补泻兼施,以复厥阴疏泄之职,使气机和畅㊁阳气运行有序㊂3㊀典型病案患者杨某,女,65岁,2017年3月10日初诊:2型糖尿病病史6年余,症见疲乏,双下肢轻度浮肿,下肢冰凉,背部易汗出,口苦口干,偶有腰膝酸软,纳眠可,二便调,舌淡暗,苔黄腻,脉沉细㊂辅助检查示糖化血红蛋白10.8%,空腹血糖14.59mmol/L,总胆固醇6.38mmol/L,甘油三酯3.66mmol/L,低密度脂蛋白胆固醇4.34mmol/L㊂西医诊断2型糖尿病㊁高脂血症,治疗给予门冬胰岛素30(早餐前22u晚餐前20u)控制血糖,阿托伐他汀钙片(20mg, qn)调脂㊂中医诊断消渴病,少阴阳虚寒湿证㊂患者少阴阳气衰微不足以养神,固见疲乏;腰为肾之府,少阴阳虚则见腰膝酸软,阳虚寒盛则见下肢冰凉;背部正中乃督脉运行之所,阳气虚衰无以固摄则见背部汗出;少阴阳虚不能主水,寒水泛滥则见双下肢浮肿;水湿内停有郁而化热之象,则见口苦口干㊁舌苔黄腻㊁舌淡暗,脉沉细为少阴阳虚寒湿之征,治以温阳散寒㊁利水除湿为法㊂方以扶正祛邪方合当归拈痛汤加减:炮附片10g(先煎1h),红参10g (另炖),干姜15g,白术30g,炙甘草15g,桂枝12 g,麻黄8g,生姜30g,猪苓10g,泽泻30g,茯苓30 g,白芍30g,酒萸肉45g,当归15g,茵陈10g,5剂水煎服,2d1剂,水煎至250ml,饭后分2次服用,次日复煎㊂方中以附子理中汤为底方温补中焦,散寒除湿;加桂枝㊁麻黄使寒湿之邪从皮毛而解;加五苓散通阳化气,使湿邪从下而出;生姜散寒除湿;白芍㊁酒萸肉收敛阳气,以助正气祛邪;当归活血利水;茵陈清热利湿㊂2017年3月24日二诊:患者双下肢浮肿减轻,疲乏较前好转,无口干口苦,无腰膝酸软,仍觉下肢冰凉,背部仍有汗出,动则尤甚,大便每日二行,质偏烂,舌淡暗,苔白腻,脉细㊂患者大便质烂,乃邪有出路,导水湿之邪从大便而解㊂患者无口干口苦,舌苔由黄腻转为白腻,知湿郁化热之象已除,遂去茵陈㊂仍觉下肢冰凉乃内有久寒,加制吴茱萸12g以散沉寒痼冷;上方加酒萸肉至60g以加强收敛阳气㊁固摄敛汗之效,加黄芪60g以健脾益气敛汗;加砂仁6g(后下)㊁肉桂3g(焗服)以加强收敛阳气㊁扶助正气之效,7剂水煎服,服法同前㊂2017年4月7日三诊:患者背部汗出减少,下肢转温,余症皆除,大便每日二行质软,舌淡红,苔薄白,脉细较前有力,继续服二诊方药5剂㊂后给予附子理中丸(每次8粒,每日3次)服用1个月巩固疗效㊂2017年11月17日复诊:患者上述症状皆除,纳眠可,二便调㊂复查糖化血红蛋白6.8%,空腹血糖6.5mmol/L,总胆固醇5.14mmol/L,甘油三酯1.65 mmol/L,低密度脂蛋白胆固醇2.43mmol/L㊂4㊀结语以往医家多以滋阴清热为法治疗糖尿病,通过长期的临床实践,朱章志不囿陈法,根据糖尿病患者当前之病因病机特点,运用扶正祛邪法治疗糖尿病,通过顾护太阴㊁扶助阳气㊁收敛阳气,祛除寒水湿之邪气,恢复阳气运行之通畅,为糖尿病的治疗提供新思路㊂参考文献:[1]㊀WANG L GAO-P-ZHANG-M,et al.Prevalence and EthnicPattern of Diabetes and Prediabetes in China in2013[J].JAMA,2017,317(24):2515-2523.[2]㊀谭宏韬,刘树林,朱章志,等. 首辨阴阳,再辨六经论治惠州地区2型糖尿病的临床观察[J].中华中医药杂志,2018,33(9):4240-4244.(下转第181页)offspring of sleep-deprived mice[J].Psychoneuroendocrinology,2009,35(5):775-784.[9]㊀覃甘梅,覃骊兰.心肾不交型失眠动物模型研究进展[J].中华中医药杂志,2018,33(1):229-231.[10]㊀吕志平,刘承才.肝郁致瘀机理探讨[J].中医杂志,2000,41(6):367-368.[11]㊀游秋云,王平,田代志,等.老年肝郁失眠证候大鼠模型的建立和评价[J].中国实验方剂学杂志,2013,19(2):222-225. [12]㊀唐仕欢,杨洪军,黄璐琦. 以方测证方法应用的反思[J].中国中西医结合杂志,2007,27(3):259-262.[13]㊀卢岩,刘振华,于晓华,等.疏肝调神针法针刺对睡眠剥夺模型大鼠神经递质的影响[J].山东中医杂志,2017,36(4):322-325. [14]㊀YANG CR,SEAMANS JK,GORELOVA N.Developing aneuronal model for the pathophysiology of schizophrenia based onthe nature of electrophysiological actions of dopamine in theprefrontal cortex[J].Neuropsychopharmacology,1999,21(2):161-194.[15]㊀何林熹,诸毅晖,杨翠花,等.失眠肝郁化火证大鼠模型的建立及其评价[J].中华中医药杂志,2018,33(9):3890-3894. [16]㊀李越峰,徐富菊,张泽国,等.四逆散对大鼠睡眠时相影响的实验研究[J].中国临床药理学杂志,2014,30(10):936-938. [17]㊀张晓婷,刘文超,刘俊昌,等.电击法建立SD大鼠焦虑型心理应激-失眠模型的研究[J].现代中西医结合杂志,2018,27(30):3316-3319.[18]㊀钱伯初,史红,郑晓亮.新的失眠动物模型研究概述[J].中国新药杂志,2008,17(1):1-4.[19]㊀朱洁,申国明,汪远金,等.肝郁证失眠大鼠模型的建立与评价[J].中医杂志,2011,52(8):689-692.[20]㊀刘倩,李蜀平,廖磊,等.调和肝脾方治疗失眠的实验研究[J].北京中医药,2018,37(8):768-770.[21]㊀全世建,焦蒙蒙,黑赏艳,等.交泰丸对睡眠剥夺大鼠下丘脑Orexin A及γ-氨基丁酸的影响[J].广州中医药大学学报,2015,32(1):103-105.[22]㊀KOBAN M,SWINSON KL.Chronic REM-sleep deprivation ofrats elevates metabolic rate and increases UCP1gene expressionin brown adipose tissue[J].Am J Physiol Endocrinol Metab,2005,289(1):68-74.[23]㊀赵俊云,杨晓敏,胡秀华,等.失眠动物模型HPA轴和表观遗传修饰的变化及交泰丸的干预作用[J].中医药学报,2018,46(4):19-21.[24]㊀GORGULU Y,CALIYURT O.Rapid antidepressant effects ofsleep deprivation therapy correlates with serum BDNF changes inmajor depression[J].Brain Res Bull,2009,80(3):158-162.[25]㊀BENCA RM,PETERSON MJ.Insomnia and depression[J].Sleep Med,2008,9(1):S3-S9.[26]㊀郜红利,涂星,卢映,等.心肾不交所致失眠大鼠模型[J].中成药,2014,36(6):1138-1141.[27]㊀杨钰涵,孙雨,王珺,等.中医病证相符的大鼠心肾不交失眠模型的建立及其血清代谢组学研究[J].中国中药杂志,2020,45(2):383-390.[28]㊀石皓月,鲁艺,李钰昕,等.中药治疗对氯苯丙氨酸失眠模型大鼠影响的基础研究进展[J].中国医药导报,2018,15(11):33-36.[29]㊀全世建,何树茂,钱莉莉.交泰丸交通心肾治疗失眠作用机理研究[J].辽宁中医药大学学报,2011,13(8):12-14. [30]㊀GULEC M,OZKOL H,SELVI Y,et al.Oxidative stress inpatients with primary insomnia[J].Pro NeuropsychopharmacolBiol Psychiatry,2012,37(2):247-251.[31]㊀ZHANG H,CAO D,CUI W,et al.Molecular bases ofthioredoxin and thioredoxin reductase-mediated prooxidant actionsof(-)-epigallocatechin-3-gallate[J].Free Radic Biol Med,2010,49(12):2010-2018.[32]㊀谢光璟,刘源才,胡辉,等.基于Trx系统介导的抗氧化应激探讨天王补心方对失眠模型大鼠的干预作用[J].时珍国医国药,2019,30(4):805-808.[33]㊀黄攀攀,王平,李贵海,等.老年阴虚失眠动物模型的建立与评价[J].中华中医药学刊,2010,28(8):1719-1723.[34]㊀XIONG L,HUANG XJ,SONG PX.The experiment ofstudymodel of Deficiency of yin Insomnia by Yangyin anshenkoufuye[J].Chin J Pract Chin Mod Med,2005,18(18):1187-1188.[35]㊀韦祎,唐汉庆,李克明,等.脾阳虚证失眠大鼠模型的建立和附子理中汤的干预效应[J].中国实验方剂学杂志,2013,19(16):289-292.[36]㊀王志鹏.桂枝甘草龙骨牡蛎汤对阳虚证失眠大鼠脑内5-HT㊁NE含量的影响[D].南京:东南大学,2015.[37]㊀MURRAY NM,BUCHANAN GF,RICHERSON GB.InsomniaCaused by Serotonin Depletion is Due to Hypothermia[J].Sleep,2015,38(12):1985-1993.[38]㊀宋亚刚,李艳,崔琳琳,等.中医药病证结合动物模型的现代应用研究及思考[J].中草药,2019,50(16):3971-3978. [39]㊀李晓娟,白晓晖,陈家旭,等.中医动物模型研制方法及展望[J].中华中医药杂志,2014,29(7):2263-2266.[40]㊀刘臻,谢晨,赵娜,等.失眠动物模型的制作与评价[J].中医学报,2013,28(12):1846-1848.收稿日期:2020-05-18(上接第151页)[3]㊀司芹芹,牛晓红,杨海卿,等.温阳益气养阴活血方治疗2型糖尿病肾病的临床疗效[J].中华中医药学刊,2018,36(3):703-705.[4]㊀郭仪,石岩.中药复方治疗糖尿病大血管病变临床疗效及对血糖㊁血脂影响的系统评价[J].中华中医药学刊,2017,35(6):1369-1375.[5]㊀方春平,刘步平,朱章志.‘伤寒论“中胃气思想在病脉辨证中的运用[J].浙江中医药大学学报,2014,38(8):948-950.[6]㊀黄元御.四圣心源[M].北京:中国中医药出版社,2009:24.[7]㊀朱章志,林明欣,樊毓运.立足阳主阴从浅析糖尿病的中医治疗[J].江苏中医药,2011,43(4):7-8.[8]㊀缪希雍.本草经疏[M].北京:中国医药科技出版社,2011:56.[9]㊀黄宫绣.本草求真[M].北京:中国中医药出版社,2008:132.[10]㊀林明欣,裴倩,朱章志.浅析通阳不在温,而在利小便 [J].中医杂志,2011,52(19):1705-1706.[11]㊀刘力红.思考中医[M].桂林:广西师范大学出版社,2006:457.[12]㊀郑寿全.医理真传[M].北京:中国中医药出版社,2008:3.[13]㊀刘涛,张毅,李娟,等.结合‘伤寒论“探讨通阳不在温而在利小便 [J].中国中医药信息杂志,2017,24(9):106-107.[14]㊀倪朱谟.本草汇言[M].北京:中医古籍出版社,2010:87.收稿日期:2020-04-27。

PHt伤口愈合

Up-Regulation of Keratinocyte Growth Factor and Receptor:A Possible Mechanism of Action of Phenytoin in Wound HealingSwarga Jyoti Das and Irwin Olsen 1Department of Periodontology,Eastman Dental Institute for Oral Health Care Sciences,University College London,University of London,256Gray’s Inn Road,London WC1X 8LD,United KingdomReceived March 7,2001A number of studies suggest that keratinocyte growth factor (KGF)plays a major part in reepitheli-alisation after injury,via binding to the speciﬁc KGF receptor (KGFR).Several pharmacological agents,in-cluding the anti-epileptic drug phenytoin (PHT),have been widely used clinically to promote wound healing.Although the mechanism of action of PHT in this pro-cess is still not well understood,it is possible that the activity of PHT in wound healing is mediated via KGF and the KGFR.In the present study,using the enzyme-linked immunosorbant assay and ﬂow cytometry we have shown that PHT increases KGF secretion and KGFR expression by more than 150%in gingival ﬁbro-blasts and epithelial cells,respectively.Moreover,semi-quantitative reverse transcriptase-polymerase chain reaction analysis showed that PHT also mark-edly increased both KGF and KGFR gene transcrip-tion by these cells.Our ﬁndings thus suggest that the wound healing activity of PHT in vivo may be medi-ated,at least partly,via KGF and its receptor.©2001Academic PressKey Words:ELISA;FGF;ﬂow cytometry;KGF;KGFR;phenytoin;regeneration;RT-PCR;wound healing.Wound healing is a complex process involving cell migration,proliferation,differentiation,and the for-mation of new extracellular matrix (1).Many of these events are controlled by growth factors and are medi-ated by the transduction of intracellular signals follow-ing the binding of the factors to speciﬁc transmem-brane tyrosine kinase receptors.The role of growth factors speciﬁc for epithelial cells is,for example,ofmajor importance in the regrowth of damaged epithe-lial tissues,which occurs rapidly after injury (2,3).Keratinocyte growth factor (KGF)is the seventh member of the ﬁbroblast growth factor (FGF)family and has 30to 45%homology with the other members of the FGF family.It is synthesised and secreted by mes-enchymal cells such as ﬁbroblasts and is a potent mi-togen for a wide variety of epithelial cells (4).KGF acts via the KGF receptor (KGFR),which is a splice variant of the FGF receptor-2/bek gene that binds both KGF and acidic FGF with high afﬁnity and basic FGF with lower afﬁnity (5).However,KGFR is expressed only by epithelial cells,whereas FGF receptor-2is expressed by several different cell types (6).There is substantial evidence that KGF is involved in the wound healing process since KGF transcripts have been reported to be markedly up-regulated within 24h of injury (7)and an enhanced rate of epithelial regeneration has been observed following topical appli-cation of recombinant KGF (8,9).In addition,a rela-tively lower level of this growth factor has been ob-served in wound tissues in certain conditions associated with impaired wound healing (10,11).Se-vere delay in wound reepithelialisation in transgenic KGFR-knockout mice also suggests the importance of KGFR signalling in wound healing processes (12).Wound healing has also been shown to be en-hanced by a number of pharmacological agents,in-cluding phenytoin (PHT),a potent anti-epileptic drug.Accelerated repair of periodontal wounds with reduced postsurgical inﬂammation and pain was ﬁrst noted with PHT in 1958(13)and,subsequently,in the healing of dental extraction sockets (14).Favourable results have also been reported with top-ical application of PHT in the treatment of various types of ulcers,burns,abscess cavities,and surgical wounds (15,16).In addition,PHT has been used to treat lichen planus and discoid lupus erythematosus,although with variable success (17).1To whom correspondence and reprint requests should be ad-dressed.Department of Periodontology,Room RL 16Levy Wing,Eastman Dental Institute for Oral Health Care Sciences,University College London,University of London,256Gray’s Inn Road,London WC1X 8LD,UK.Fax:0044(0)207915-1254.E-mail:iolsen@.Biochemical and Biophysical Research Communications 282,875–881(2001)doi:10.1006/bbrc.2001.4621,available online at onDespite numerous clinical trials and case studies, however,the mechanism of action of PHT in wound healing is still not well understood.Since KGF and KGFR have been shown to play a pivotal part in tissue repair processes,it is possible that the action of PHT is mediated at least partly via this growth factor and its receptor.In the present study we have therefore exam-ined the effects of PHT on the expression of KGF and KGFR and on the activity of their corresponding genes in gingival cells in vitro,as a model of the wound healing effects of PHT in vivo.MATERIALS AND METHODSPreparation of PHT.PHT(Sigma,Poole,UK)was dissolved in 100%ethanol(BDH,Laboratory supplies,Poole,UK)to obtain a stock solution of5mg/ml.It was used atﬁxed concentration of5and 10␮g/ml in the experiments described below.The sameﬁnal con-centration of ethanol was used in both the control and PHT-containing media.Cell culture.Normal gingival tissue samples were obtained un-der the Eastman Dental Institute and Hospital Joint Ethics and Research Committee.Conﬂuent cultures of gingivalﬁbroblast(GF) cells were obtained by incubating biopsy samples of normal gingiva (NG)fromﬁve healthy individuals(three males and two females;age range30to40years,mean age35Ϯ7.1),as previously described (18).The cells were detached from the monolayer by treating them with0.25%trypsin in1mM ethylenediaminetetraacetic acid(EDTA) (Gibco Life Technologies Ltd.,Paisley,UK),collected by centrifuga-tion and serially passaged in25cm2tissue cultureﬂasks(Falcon, Becton Dickinson Labware;Cowley,UK).The number and viability of the cells were determined by trypan blue staining at the time of each passage.Cells at the3rd–5th passages were used in this study. Gingival epithelial cells(GE)were grown from the epithelium, which was separated mechanically from the connective tissue of the excised normal gingiva.The epithelial tissue was cut into1mm3 pieces and cultured at37°C in a humidiﬁed atmosphere of air with 5%CO2in Keratinocyte Basal Medium-2(KBM-2)containing whole bovine pituitary extract,recombinant human epidermal growth fac-tor(rh EGF),insulin,hydrocortisone,transferrin,epinephrine,and gentamycin(BioWhittaker;Wokingham,UK).When individual col-onies of the outgrowth of the GE cells reached an average size of between50to200cells,they were detached using trypsin-EDTA and serially passaged.The viability of the GE cells was conﬁrmed by trypan blue exclusion at the time of passage.The purity of the GE cell culture was ascertained by positive staining of the cells with an antibody(clone MNF116)against cytokeratins5,6,8,and17(Dako Ltd.,High Wycombe,UK)(data not shown).Cells at the2nd and3rd passages were used in this study.Treatment ofﬁbroblasts with PHT:KGF secretion.GF cells were seeded at a density of2ϫ104into24-well tissue culture plates (Falcon)and incubated until they become conﬂuent.The medium was then replaced with fresh medium containing1%FCS for48h, after which the medium was replaced with fresh1%FCS-containing medium in the absence and presence of5and10␮g/ml of PHT.These concentrations were selected on the basis of posttherapeutic serum levels(19).A sample of the supernatant was removed from each of three replicate wells on each of4subsequent days of incubation, centrifuged at13,000rpm for1min and stored atϪ20°C.The amount of KGF secreted by the cells into the culture medium was measured by ELISA,as described previously(20).Brieﬂy,96-well microtitre plates(Immulon4)(Dynex Technologies,Inc.,Chan-tilly,VA)were coated with5␮g/ml of a mouse monoclonal antibody (mAb)against human KGF(preparation number IG4)(Amgen Inc., Thousand Oaks,CA)in carbonate-bicarbonate buffer(pH9.6)and incubated at4°C overnight.The wells were washed three times with phosphate buffer saline(PBS)containing0.1%Tween20(PBS-T) (BDH)and nonspeciﬁc protein binding sites were blocked using200␮l of PBS containing1%(w/v)bovine serum albumin(BSA)(BDH) for1h at room temperature.After washing,100␮l of the culturesupernatants to be tested were added to three replicate wells and incubated for2h at room temperature.The washing step was re-peated and biotinylated mAb against human KGF(preparation num-ber A1)(Amgen),diluted to20ng/ml in PBS containing0.2%BSA and0.02%Tween20,was added for30min at room temperature. The plates were washed again and the binding of the biotinylated mAb was detected by incubation with a1:1000dilution of extravidin-peroxidase(Sigma),for30min at room temperature,followed by incubation with100␮l of3,3Ј,5,5Ј-tetramethyl-benzidine(ICN Bio-medicals,Costa Mesa,CA)in0.1M citrate-phosphate buffer(pH5.0) containing0.03%hydrogen peroxide(Sigma)for30min at37°C.The reaction was terminated by the addition of50␮l of2M H2SO4and the absorbance at450nm measured spectrophotometrically(Titer-tek Multiskan Plus,Helsinki,Finland).The concentration of KGF in the supernatant was calculated from a standard curve constructed by using between0.31and5.00ng/ml of recombinant human KGF (Amgen)in complete medium containing1%FCS.The results shown are derived from the average of three replicate wells for each time point in each experiment.Flow cytometry(FCM)analysis of KGFR expression by GE cells. GE cells were grown in25cm2tissue cultureﬂasks to80%conﬂuency in KBM-2and then for48h in the absence of rh EGF.The medium was then replaced by fresh medium without EGF incubated in the absence and presence of10␮g/ml of PHT for3days and the cells prepared for FCM as described previously(21).Brieﬂy,the cells were detached using20mM EDTA in PBS,centrifuged andﬁxed with3% paraformaldehyde.Aliquots of105cells were stained with an afﬁnity-puriﬁed rabbit polyclonal antibody against human KGFR/ FGFR-2(C-17)(Santa Cruz Biotechnology,CA)for1h at room temperature and washed with PBS containing saponin,to permea-bilise the cells,and sodium azide(wash buffer).They were then incubated withﬂuorescein isothiocyanate(FITC)-labelled swine anti-rabbit antibody(Dako).Cells incubated with only secondary antibody were used as negative control.The averageﬂuorescence intensity(AFI)was measured using a FACScanﬂow cytometer(Bec-ton Dickinson,Oxford,UK).The experiments were carried out twice using each of theﬁve cell lines.Extraction of RNA and RT-PCR analysis of KGF and KGFR.The GF and GE cells were incubated in the absence and presence of PHT for3days,as described above.The cells were harvested using trypsin-EDTA and total RNA isolated as described previously(22). The RNA was measured by absorbance at260nm using an Ultrospec 2000spectrophotometer(Pharmacia Biotech,Cambridge,UK).Pro-tein contamination of the RNA preparations was assessed by mea-suring the absorbance at280nm.Theﬁrst strand of cDNA was synthesized from the total RNA using an oligonucleotide(oligo dT)primer(Promega,Madison,WI) and cloned Moloney murine leukaemia virus reverse transcriptase (Stratagene,Cambridge,UK).The resulting cDNA prepared from RNA extracted from the GF and GE cells were ampliﬁed using primer pairs speciﬁc for the KGF and KGFR genes,respectively.The primer pair used to amplifying KGF cDNA had the sequences[tct gtc gaa cac agt ggt acc t]and[gtg tgt cca ttt agt tga tgc at]for the forward and reverse direction,respectively,generating an expected PCR product of266bp(23).The primer pair used for ampliﬁcation of KGFR cDNA was[tcg ggg ata aat agt ttc caa tg]and[ctg ttt tgg cag gac agt ga]for the forward and reverse direction,respectively,and generates a PCR product of141bp(6).In addition,the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase(GAPDH)was am-pliﬁed at the same time and under the same PCR conditions as those for KGF and KGFR.The expression of the GAPDH gene is consid-ered to be constitutive and was used in this study as an internal standard for estimating the relative level of KGF and KGFR mRNA.The primer sequences used for ampliﬁcation of the GAPDH cDNA,as used in previous study,was [cca ccc atg gca tcc cat ggc a]and [ctg gac ggc agg tca ggt cca cc]for the forward and reverse direction,respec-tively,generating a product of 600bp (23).All three pairs of primers used in this study were obtained from Amersham Pharmacia Biotech (Amersham,UK).All PCR reactions were carried out within a linear range (25and 30cycles for KGF and KGFR,respectively),as initially determined by PCR ampliﬁcation in 5-cycle steps,from 15to 40cycles (data not shown).The ampliﬁcation programme consisted of initial denatur-ation at 96°C for 3min,followed by repeated cycles of denaturation at 95°C for 30s,annealing at 60°C for 30s,extension at 72°C for 90s,and ﬁnal extension at 72°C for 10min.The ampliﬁed PCR products were identiﬁed by comparing the electrophoretic mobility of the predicted product with that of a 100bp DNA size marker (Helena Biosciences Ltd.,Sunderland,UK).The PCR reaction was repeated twice for each sample with different RNA from each separate tissue culture experiment.To determine the relative amount of KGF mRNA transcripts present in each sample,the intensities of the KGF and GAPDH PCR bands in the same lane of each gel were measured using the Scion Image Software Programme (Scion Corporation,Frederick,MD).The intensity of the ampliﬁed GAPDH product was taken as the baseline value and the ratio of the KGF band to the GAPDH band intensity in each sample was calculated.These ratios were used as a measure of the relative expression of KGF mRNA in each of the samples,which is shown as a KGF expression index (KFI):KFI ϭintensity of KGF band/intensity of GAPDH band ϫ100.Further,the KFI of each PHT-treated cell sample was compared with the KFI of the control cells and expressed as the KFI relative to the control.The relative amount of KGFR transcripts in each sample was measured in a similar way as that of the KGF transcripts and calculated as the KGFR expression index (KRI).The KRI of each PHT-treated cell sample was compared with the KRI of the control cells and expressed as the KRI relative to the control.DNA sequencing.The ampliﬁed PCR products were puriﬁed from the agarose gels by using the QIAquick gel extraction kit (QIAGEN Ltd.,West Sussex,UK)and ampliﬁed using the forward primer of each of the primer pairs used to amplify KGF and KGFR cDNA and the DNA sequencing kit (Applied Biosystem,Warrington,UK).The ampliﬁed product was then analysed by capillary electrophoresis using the automatic ABI PRISM DNA sequencing analysis software (310Genetic analyzer)(Perkin Elmer,Cambridge,UK).The nucle-otide sequence obtained was compared with the sequence submittedoriginally for the KGF (Accession No.M25295)(24)and KGFR gene (Accession No.M80634)(6).Statistical analysis.The data were analysed by using a paired two-tailed Student’s t test.Values of P Յ0.05were considered as statistically signiﬁcant.RESULTSCellular reaction to PHT.The characteristic elon-gated,spindle-shaped morphology of GF cells was maintained even after incubation for 7days in the presence of 5and 10␮g/ml of PHT.In addition,when the cells were stained with trypan blue to evaluate their viability,no deleterious effect of the drug was detectable (data not shown).PHT was also observed to be similarly nontoxic and to have no apparently detri-mental effect on the GE cells.Effect of PHT on KGF secretion.ELISA assay of the culture media of the control and PHT-treated GF cells was carried out to determine the effects of PHT on KGF secretion during 4days of culture,as shown in Fig.1.Relatively low levels of KGF were present in the su-pernatants of the control cultures (average of 1.18Ϯ0.09ng/ml;range 0.78to 1.47ng/ml).In marked con-trast,much greater amounts of KGF were secreted into the culture media of cells incubated with PHT,reach-ing maximum levels on day 3with both drug concen-trations (1.95Ϯ0.33ng/ml;range 1.48to 1.99ng/ml and 2.66Ϯ0.39ng/ml;range 1.97to 2.66ng/ml,with 5␮g/ml and 10␮g/ml of PHT,respectively).A greater amount of KGF secretion was observed at all time periods at the concentration of 10␮g/ml compared with 5␮g/ml of PHT.The amount of KGF secretion induced by PHT,rel-ative to the control cultures,is shown in Table 1.The maximum relative levels of KGF secretion were on day 1,and were 1.9and 2.5times greater than the control level with 5and 10␮g/ml of PHT,respectively.Effect of PHT on KGF gene transcription.In order to determine whether the elevated levels of KGF secre-TABLE 1Effects of PHT on KGF Secretion by GF Cells in VitroIncubation time(days)Relative level of KGF secretionPHT (5␮g/ml)PHT (10␮g/ml)1 1.9(0.33–2.49) 2.5*(0.41–3.31)2 1.8(0.44–3.28) 2.3(1.09–3.49)3 1.3(0.86–2.91) 1.8(1.21–4.35)41.4(0.45–3.08)1.8(0.79–4.90)Note.The values shown are the relative average amounts of KGF secretion ϮSEM in the PHT-treated cultures compared with the control cultures,deﬁned as 1.0.The range of secretion levels is shown in brackets,and the maximum relative secretion in the PHT-treated cultures is shown in bold type.*Statistically signiﬁcant difference compared with controls (P Ͻ0.05).FIG.1.Effects of PHT on KGF secretion by GF cells.Cultures of normal gingival ﬁbroblasts were incubated in the absence (white bars)and presence of 5␮g/ml (black bars)and 10␮g/ml (grey bars)of PHT.The data shown is the average of triplicate assays from ten separate experiments ϮSEM (vertical lines).tion induced by PHT in vitro was associated with in-creased transcription of the respective gene,semi-quantitative PCR was carried out using GAPDH as the internal control,as described under Materials and Methods.Figure 2shows a representative gel and the corresponding image analysis proﬁles of the RT-PCR products of GF cells cultured in the absence and pres-ence of PHT.All the samples showed the single band corresponding to the predicted molecular size of the GAPDH gene sequence (600bp),conﬁrming that the RNA extracted from the samples was intact and not degraded.In addition to this GAPDH band,the band corresponding to the KGF gene sequence (266bp)was observed in the control as well as the PHT-treated cells.The surface areas of the histograms obtained from image analysis of the GAPDH and KGF band intensities were considered to be equivalent to the lev-els of the respective mRNAs in the samples.The rela-tive expression of KGF gene transcripts,deﬁned as the KFI,was calculated from these histogram proﬁles.The KFI values obtained in this representative experiment were 19for the control cells and 28and 47for the cells treated with 5and 10␮g/ml of PHT,respectively (1.5and 2.4times the control level,respectively).The overall average KFI for the ﬁve control cell line in duplicate experiments was found to be 17Ϯ3(range 6to 34),whereas cells incubated with 5and 10␮g/ml of PHT produced KFI values of 26Ϯ6(range 11to 58)and 34Ϯ8(range 14to 75),respectively.Thus,incu-bation of GF cells in the presence of 5and 10␮g/ml of PHT up-regulated the activity of the KGF gene by approximately 150and 200%,respectively,and these differences were statistically signiﬁcant (P Ͻ0.05).Effect of PHT on KGFR expression.To examine the effect of PHT on KGFR expression,the GE cells were immunostained with antibody speciﬁc for FGFreceptor-2and analysed by FCM,as described under Materials and Methods.Representative ﬂuorescence pro-ﬁles of KGFR expression are shown in Fig.3and dem-onstrate that the relative level of this antigen in the PHT-treated cells was 180%higher than in the control cells (AFI values of 202and 115,respectively).In ten experiments using ﬁve cell lines in duplicate,the average relative level of KGFR expression in the PHT-treated cells was calculated to be 161Ϯ22(range 66to 200)using 10␮g/ml of PHT,which was 1.5times the control level (AFI of 100Ϯ15;range 49to 139).This difference was found to be statistically signiﬁcant (P Ͻ0.05).Effect of PHT on KGFR gene transcription.A rep-resentative PCR gel of KGFR and the image analysis proﬁles are shown in Fig. 4.Integrity of the RNA samples was shown by the presence of the single band of the GAPDH gene sequence (600bp).A band corre-sponding to the KGFR gene sequence (141bp)was also observed in both the control and PHT-treated cells.The image analysis proﬁles of the relative intensities of the GAPDH and KGFR bands,expressed as the KRI,indicated that the KRI value in the PHT-treated cells was higher than in the control cells (64and 43,respectively).The overall KRI of ten experiments using ﬁve cell lines in duplicate was found to be 42Ϯ6(range 19to 70)with 10␮g/ml of PHT,whereas it was 28Ϯ4(range 16to 43)in the control cells.Thus,incubation of the GE cells in the presence of PHT up-regulated the activity of the KGFR gene by approximately 150%,although this difference was not statistically signiﬁcant (P Ͼ0.05).Speciﬁcity of PCR products.The nucleotide se-quences obtained from the DNA sequencing PCR of KGF and KGFR were identical to the previously re-ported sequences for KGF (22)and KGFR (6),thusFIG.2.Effects of PHT on KGF gene transcription in GF cells.A representative experiment showing the RT-PCR analysis of the GF cells cultured in the absence and presence of 5and 10␮g/ml of PHT for 3days.Each panel shows the electrophoretic mobility of the RT-PCR products and the corresponding image analysis of these bands:A,control cultures;B and C,GF cells incubated with 5and 10␮g/ml of PHT,respectively.Note the presence of GAPDH bands (600bp)of similar intensity in all three panels and increasing amounts of the KGF gene product (266bp)in panels B and C.Numbers in brackets are the KFI values in this experiment,which are 19,21,and 47for the control,5and 10␮g/ml of PHT,respectively.conﬁrming the speciﬁcity of the PCR products and also the primer pairs used to amplify the KGF and KGFR mRNA in this study (data not shown).DISCUSSIONWound healing is a complex series of cellular and molecular events which include inﬂammation,cell mi-gration,angiogenesis,matrix remodelling,and reepi-thelialisation.These entail the coordinated functions of many types of cell,including keratinocytes,ﬁbroblasts,endothelial,and inﬂammatory cells,and are closely regulated by growth factors and cytokines.Reepitheli-alisation,involving keratinocyte migration and prolif-eration,is considered to be one of the major features of wound healing.KGF has been shown to enhance the migration and proliferation of keratinocytes (4,25)and is also involved in matrix remodelling (26).It is notable that KGF is involved not only in reepithelialisation during wound healing,but also in destruction of reac-tive oxygen species in keratinocytes by induction of glutathione peroxidase,thereby protecting the epider-mis from oxidative injury due to free radicals (27).The cellular events that occur during wound healing are also modulated by various pharmacological agents,including PHT.PHT is a widely used anti-epileptic drug,although it has a wide range of pharmacologic effects in addition to its anti-epileptic activity.Its pri-mary site of action appears to be within the motor cortex,where the spread of seizure activity is inhibited by a membrane potential-dependent blockade of Na ϩchannels and perhaps presynaptic Ca ϩchannels (28).However,PHT was also found to have a secondary therapeutic role in promoting the wound healing which accompanies the occurrence of gingival hyperplasia that affects approximately 50%of patients taking this drug (29).Subsequently,a number of clinical trials have provided evidence of an increased rate of repair with reduced oedema and inﬂammation and enhanced formation of granulation tissue following the topical application of PHT to cutaneous wounds (13,14,16,17).Since KGF and KGFR signalling events havebeenFIG.3.Effects of PHT on KGFR expression by GE cells.A repre-sentative experiment showing FCM analysis of the ﬂuorescence proﬁles of 10,000cells derived from a control and 10␮g/ml of PHT-treated cultures.A solid vertical line is drawn arbitrarily on the histograms to indicate the mean AFI value of the KGFR positive cells in the control culture (115).Note that the proﬁle of PHT-treated cells has shifted to right side of the vertical line,indicating a higher level of KGFR expres-sion in PHT-treated cells (AFI of 202).The dashed lines represent the proﬁles of the negative control cells in correspondingexperiment.FIG.4.Effects of PHT on KGFR mRNA transcripts in GE cells.Representative experiments of normal GE cells cultured in the absence and presence of PHT.Each panel shows the electrophoretic mobility of the RT-PCR products and the corresponding image analysis of these bands:A,control cultures;B,PHT-treated cultures.Note the presence of GAPDH bands (600bp)of similar intensity in both panels.The KRI values obtained from this representative experiment are 43and 64for the control and PHT-treated cells,respectively.strongly implicated in the remodelling of damaged ep-ithelial tissue in vivo(7–9),in the present study we have examined whether PHT up-regulates this growth factor and its receptor in vitro.Ourﬁndings demonstrate,for theﬁrst time,that PHT signiﬁcantly increases KGF secretion and KGF gene transcription by GF cells.The reason for the wide variation which we found in KGF response is unclear, but may be related to the differential uptake,metabo-lism and binding of PHT to gingival cells obtained from different individuals(15).Although the precise effects of PHT on these cells has yet to be delineated,the up-regulation of KGF by PHT may be mediated via other growth factors,inﬂammatory mediators,hor-mones,and hormone antagonists which have been shown to regulate KGF expression in vitro and during wound healing in vivo(11,30–35).It is notable that recent studies have identiﬁed an-other member of the FGF family having a high degree of structural homology with KGF.However,unlike the original KGF studied here(KGF-1),this new growth factor,KGF-2,is mitogenic forﬁbroblasts as well as epithelial cells and has been shown to be involved in wound healing processes in vivo(36).Since the PCR primers and antibodies used in this study are speciﬁc for KGF-1,further studies are required to determine whether KGF-2is also modulated by PHT.KGFR has also been implicated in wound reepithe-lialisation,by signal transduction following the bind-ing of KGF(6).In addition,severe delay in the wound healing process has been reported in transgenic KGFR knockout mice(12).In the present study we used FCM to determine whether PHT also affected the expression of the KGFR.Although this technique measures rela-tive antigen levels,it is nevertheless proportional to the actual number of antigen molecules present in or on the cells(37).Moreover,the polyclonal antibody used for immunostaining of the GE cells was directed against the intracellular domain of the FGF receptor-2 which is present in the KGFR splice variant expressed by epithelial cells only(6).Furthermore,the GE cells were conﬁrmed to be of epithelial lineage by positive staining for cytokeratins,as noted under Materials and Methods.However,their characteristic slow growth and short life span in primary culture limited the use of more than one concentration of PHT for measuring the KGFR response,in contrast to the KGF response by the more rapidly-growing and longer-lived GF cells. Ourﬁndings have demonstrated that relatively higher KGFR levels are expressed by the PHT-treated cells compared with the control GE cells,thereby providing increased KGFR availability to form biologically active complexes with KGF which could enhance reepitheli-alisation of PHT-treated wounds in vivo.As with KGF, however,while the precise mechanisms involved in the regulation of KGFR have yet to be identiﬁed,the ex-pression of this receptor is clearly subject to the inﬂu-ence of growth factors and inﬂammatory cytokines which are expressed during the wound healing process (38).In conclusion,our study has shown that PHT in-duces both KGF and the KGFR at the protein level and also up-regulates the activity of the corresponding genes in vitro,suggesting the possibility that the action of PHT in wound healing in vivo may also be mediated at least partly via KGF and the KGFR. ACKNOWLEDGMENTSThe authors are grateful to the staff of the Periodontology Clinic, Eastman Dental Hospital,London,for generously providing redun-dant surgical materials.We also thank Amgen,Inc.(Thousand Oaks, CA)for providing the recombinant human KGF and mAbs against human KGF to carry out these experiments.S.J.Das acknowledges The Association of Commonwealth Universities for a Commonwealth Scholarship.REFERENCES1.Clark,R. A. F.(1993)Basics of cutaneous wound repair.J.Dermatol.Surg.Oncol.19,693–706.2.Risau,W.(1990)Angiogenic growth factors.Prog.Growth FactorRes.2,71–79.3.Pierce,G.F.,and Mustoe,T.A.(1995)Pharmacological enhance-ment of wound healing.Annu.Rev.Med.46,467–481.4.Rubin,J.S.,Osada,H.,Finch,P.W.,Taylor,W.G.,Rudikoff,S.,and Aaronson,S.A.(1989)Puriﬁcation and characterisation of a newly identiﬁed growth factor speciﬁc for epithelial cells.Proc.A86,802–806.5.Ornitz,D.M.,Xu,J.,Colvin,J.S.,McEwan,D.G.,MacArthur,C.A.,Coulier,F.,Gao,G.,and Goldfarb,M.(1996)Receptorspeciﬁcity of theﬁbroblast growth factor family.J.Biol.Chem.271,15292–15297.6.Miki,T.,Bottaro,D.P.,Fleming,T.P.,Smith,C.L.,Burgess,W.H.,Chan,AM-L.,and Aaronson,S.A.(1991)Determination of ligand-binding speciﬁcity by alternative splicing:Two distinct growth factor receptors encoded by a single gene.Proc.Natl.A89,246–250.7.Werner,S.,Peters,K.G.,Longaker,M.T.,Fuller-Pace, F.,Banda,M.J.,and Williams,L.T.(1992)Large induction of keratinocyte growth factor expression in the dermis during wound A89,6896–6900.8.Pierce,G.F.,Yanagihara,D.,Klopchin,K.,Danilenko,D.M.,Hsu,E.,Kennery,W.C.,and Morris,C.F.(1994)Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor.J.Exp.Med.179,831–840.9.Staiano-Coico,L.,Krueger,G.J.,Rubin,J.S.,D’limi,D.,Vallat,L.,Valentino,L.,Fahey,T.,III,Hawes,A.,Kingston,G.,and Madden,M.R.(1993)Human keratinocyte growth factor effects in a porcine model of epidermal wound healing.J.Exp.Med.178, 865–878.10.Werner,S.,Breeden,M.,Hubner,G.,Greenhalgh,D.G.,andLongaker,M.T.(1994)Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse.J.Invest.Dermatol.103,469–473.11.Brauchle,M.,Fassler,R.,and Werner,S.(1995)Suppression ofkeratinocyte growth factor expression by glucocorticoids in vitro and during wound healing.J.Invest.Dermatol.105,579–584.12.Werner,S.,Smola,H.,Liao,X.,Longaker,M.T.,Kreig,T.,Hofschneider,P.H.,and Williams,L.T.(1994)The function of。

马兰戈尼效应在有机半导体成膜中的应用

物理化学学报Acta Phys. -Chim. Sin. 2019,35 (11), 1183–1185 1183[Recommendation] doi:10.3866/PKU.WHXB201906062 马兰戈尼效应在有机半导体成膜中的应用吴凯北京大学化学与分子工程学院，北京 100871Application of Marangoni Effect in the Fabrication of Organic Semiconductor FilmsWU KaiCollege of Chemistry and Molecular Engineering, Peking University, Beijing 100871, P. R. China.Email: kaiwu@.Published online: June 27, 2019.1 背景介绍以有机发光二极管、有机太阳能电池和有机场效应晶体管为主的有机半导体薄膜器件，因在便携、可穿戴电子器件领域有巨大的应用前景，而逐渐成为无机半导体器件的一个重要补充。

溶液法生长有机半导体薄膜工艺更使有机半导体器件的低成本、可在柔性基底上大面积制备的优点得到体现，因而受到广泛关注。

但是，溶液法制备工艺也存在着诸多不足，比如被称为“咖啡环”效应的溶剂自然挥发造成的薄膜不均匀性现象等1。

针对薄膜的不均匀性，出现了表面修饰、使用混合溶剂和特殊结构等改进方法2，而在成膜溶液中构筑马兰戈尼流就是其中的一种3。

清华大学化学系董桂芳副教授课题组采用基于马兰戈尼与咖啡环效应协同作用的方法，利用浸润提拉及氧等离子体选区处理工艺，生长出图案化的C8-BTBT薄膜，通过对混合溶剂比例以及溶液浓度等参数的优化，得到高性能薄膜及基于此薄膜的有机场效应晶体管。

该工作已在物理化学学报上在线发表(doi：10.3866/PKU.WHXB201901056) 4。

2 研究亮点(1)在提拉法中，利用具有高沸点、低表面张力的甲苯与具有低沸点、高表面张力的四氯化碳两种溶剂，按比例混合，在成膜溶液中形成马兰戈尼流，有效降低“咖啡环”效应，提高薄膜均匀性。

异丙酚自身给药对大鼠伏隔核内ERK表达的影响

异丙酚自身给药对大鼠伏隔核内ERK表达的影响王本福;张富强;连庆泉;杨博;孙安娜;黄茜茜;金胜威;李军;赖苗军;周文华;刘惠芬【期刊名称】《医学研究杂志》【年(卷),期】2011(40)11【摘要】Objective To investigate the effect of propofol maintained self - administration on the expression of p - ERK in nucleus accumbens of rats. Methods Rats were divided into 3 groups (n =6) : intralipid control group and propofol groups (PI =1. 00mg/kg, P2 = 1.70mg/kg). To assess the psychic dependence of propofol, the technique of intravenous self - administration by rats was utilized. The expression of p - ERK/ERK in Nac was detected by Western blotting. Results All the rats trained with propofol at dose 1. 00mg/kg and 1.7mg/kg showed self - administration behavior. Compared to PI group, the number of active nose -poke response and injections of P2 group increased significantly (P <0. 01). Further, changed of propofol, the expression of p - ERK/ERK in nucleus accumbens was dose - dependent increased (P < 0.01). Conclusion The data suggested that propofol - maintained self - administration increase the expression of p - ERK in Nac, and the ERK signal Pathway maybe involve in psychic dependence of propofol.%目的观察异丙酚静脉自身给药对大鼠伏隔核内p - ERK表达的影响.方法SD大鼠随机分为3组(n=6):对照组(C组)、1.00mg/kg异丙酚组(P1组)、1.70mg/kg异丙酚组(P2组).用静脉自身给药法建立异丙酚精神依赖模型,Western blotting法检测大鼠伏隔核内p- ERK和ERK的变化.结果 P1组、P2组的异丙酚可以诱发大鼠建立静脉自身给药行为.与P1组相比,P2组大鼠有效鼻触(P＜0.01)和注射次数(P＜0.01)明显增加.随着异丙酚剂量的增加,大鼠伏隔核内p- ERK/ERK的表达明显增加(P＜0.01).结论异丙酚静脉自身给药增加了大鼠伏隔核内p- ERK的表达,ERK信号转导通路可能参与了异丙酚的精神依赖性.【总页数】3页(P39-41)【作者】王本福;张富强;连庆泉;杨博;孙安娜;黄茜茜;金胜威;李军;赖苗军;周文华;刘惠芬【作者单位】325027 温州医学院附属二院麻醉科、麻醉神经内分泌研究所;315010 宁波,微循环与莨菪类药研究所;325027 温州医学院附属二院麻醉科、麻醉神经内分泌研究所;325027 温州医学院附属二院麻醉科、麻醉神经内分泌研究所;315010 宁波,微循环与莨菪类药研究所;325027 温州医学院附属二院麻醉科、麻醉神经内分泌研究所;315010 宁波,微循环与莨菪类药研究所;315010 宁波,微循环与莨菪类药研究所;315010 宁波,微循环与莨菪类药研究所;315010 宁波,微循环与莨菪类药研究所;315010 宁波,微循环与莨菪类药研究所【正文语种】中文【相关文献】1.有氧游泳运动对吗啡精神依赖大鼠伏隔核中内阿片肽表达水平的影响 [J], 于动震;苏彦炬;柳新海;霍少华2.有氧游泳运动对吗啡精神依赖大鼠伏隔核中内阿片肽表达水平的影响 [J], 于动震;苏彦炬;柳新海;霍少华3.毁损伏隔核对吗啡给药大鼠条件性位置偏爱与脑功能的影响 [J], 邵云;魏孝琴;薛一雪4.吗啡急性给药对大鼠伏隔核中神经甾体水平的影响 [J], 吴红海;王娜;侯艳宁5.安君宁对吗啡依赖大鼠伏隔核内前阿黑皮素原表达的影响 [J], 谌红献;郝伟;刘铁桥;王绪轶;向小军;蔡溢因版权原因，仅展示原文概要，查看原文内容请购买。

1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。

Experimental Animal Science
Effects of fibrin on the integration hydroxyapatite coating implants: experimental study in a rabbit model
M. MORALES 1, R. NAVARRO2'3, M. ALMENARA2, J. M. MEDINA2, C. MELIAN 1 and C. GUTIERREZ 1
103
growth and the speed with which it occurs, since this has a beneficial effect on the longevity of the prosthesis (ENGH et al., 1987; KEAVENYand BARTEL, 1995). Hydroxyapatite (HAP) ceramic coatings are bioactive coatings which have demonstrated their ability to increase the development and the extension of bone (GEESINK al., et 1987) in non-cemented endoprostheses, achieving a strong bond in a relatively short period of time (GEESINK et al., 1987; SOBALLE et al., 1993), even under load (SOBALLE et al., 1990). The addition of certain substances to the HAP in order to increase osteoblastic activity and promote osteogenesis is currently under study (ONO et al., 1990; SATO et al., 1991; NAKAMURAet al., 1998). Following in this direction, the angiogenic and fibrogenic activity shown by the fibrin adhesive (SAF - Tissucol Inmuno ) may indicate the ability to increase osteoinduction. However, the influence of SAF as stimulant to osteogenesis is subject to debate. Some authors have found a beneficial effect of fibrin in wound repair (Albrektsson et al., 1982), in the repair of bone defects in Plastic and Reconstructive maxillofacial surgery in humans (BONUCCI et al., 1997; FORTUNATO et al., 1997), in the fixation of osteochondral fractures in dog femurs (KELLERet al., 1985), and in accelerating the repair process and new bone formation in bone defects of the proximal metaphysis of rats (ONO et al., 1990) and rabbits (SATo et al., 1991; NAKAMURAet al., 1998). Also, Ig~ANIAet al. (1998) reported benefitious effects when fibrin was added to coral implants compared to coral alone group in an experimental study carried out in rabbits. However, other authors have not found any stimulatory effect of fibrin and have observed a higher soft tissue development with a reduction in the bonding to bone in ceramic implants used to fill femoral defects in rabbits (RECK and BERNAL-SPREKELSEN, 1989). Another study found no significant increase in the blood supply nor in bone formation in a standardised defect of both tibiae filled with autologous graft material from the iliac crest in dogs (LuCH7 et al., 1986). Another study domonstrate a reduction in heterotopic osteoinduction in the abdominal musculature of rats (PINHOLTet al., 1992). At the current knowlegde, fibrin used alone in the fractures produces a retard allograft fusion mass formation (JARZEM et al., 1996); fibrin used as a carrier of inductive cytokines produces a higher yields of new bone (SATo et al., 1991); and fibrin used in a mixture containing apatite-wollastonite and glass ceramic granules (ONo et al., 1990), heterologous cancellous transplants (Bosh et al., 1980) or hydroxyapatite implants (BoNuccI et al., 1997) produced also a higher osteoinductive properties. However, the precise role that fibrin plays in these process is unclear. Thus, the purpose of this study was to evaluate the effects of the addition of SAF to HAP-coated titanium (Ti) implants on osteogenesis using radiographical and histo-morphometrical evaluations in a controlled rabbit model.
Key words: Hydroxyapatite, fibrin, rabbit model, coatting implants.
Introduction
Bone ingrowth in non-cemented implants takes place in humans and small animals. However, analysis of retrieved implants has shown great variation in the degree of bone growth, varying in porous coatings from 10% (COLLIER et al., 1988) to 50% (PIDHORZ et al., 1993). Thus, irrespective of the influence of certain variables (implant design, age, metabolic state of the bone, etc.), research into biological fixation seems to be focussing on a number of agents which, when added to the surface of the implant, favour bone
Summary
The purpose of this study was to investigate the effects of the addition of fibrin (SAF) to titanium alloy implants coated with hydroxyapatite (HAP) on osteogenesis in rabbits. A titanium (Ti) alloy implant was inserted into the femoral neck of twenty-four adult rabbits. Six rabbits were included on each of the following groups: Tile, HAP-coated Ti module with added fibrin glue and Ti module also with added fibrin glue. After seven weeks, bone growth was examined radiographically and by histo-morphometry. The SAF/HAP mixture did caused to a significant increase in bone growth compared to the other groups. The addition of fibrin did not result in an increase in new-bone growth and increase the formation of fibroustissue in contact with the implant. We concluded that SAF did not demonstrate osteoinductive properties.