Harringtonine_HNMR_22863_MedChemExpress
非酒精性脂肪性肝病代谢组学研究进展
机制尚未完全明确，１９９８ 年Ｄａｙ 等［１２］提出“二次打击”学说。 开。同时ＮＡＦＬＤ 肝硬化患者与酒精性肝硬化患者也可有效区
随后Ｔｉｌｇ 等［１３ －１４］提出“多重平行打击”理论，包括遗传因素、 分开（ＡＵＣ ＝０． ８３）。他们认为此方法可作为区分ＮＡＦＬＤ 纤维
ＩＲ、氧化应激、脂毒性、慢性炎症、纤维化、免疫和肠道菌群等， 化程度及诊断的无创生物标志物，且可以显著减少对肝活检的
黄酯和１３ － ｃｉｓＲＡ 呈正相关。他们在人类组织中首次检测到 验证；单不饱和ＴＡＧ 的增加可能是ＮＡＦＬＤ 和ＣＨＢ 患者ＮＡＳＨ
ａｔＲＡ 的活性代谢物４ － ｏｘｏ － ａｔＲＡ，表明这种类维生素Ａ 可能 的特异性标志物。
有助于人体类维生素Ａ 的信号传导。肝脏维生素Ａ 的稳态平 ２． ３ 代谢组学对ＮＡＦＬＤ 药物作用与疗效研究的推动作用
录组学、蛋白质组学为代表的系统生物学技术提供了新的技术 展的新学科，代谢组学较为全面的展示了机体的代谢结果，为
与思路。区别于其他组学技术，以内源性小分子代谢物为研究 临床医学提供了新的技术和方法。
对象的代谢组学可以很好的揭示机体变化的最终代谢结果。因 ２ 非酒精性脂肪性肝病（ＮＡＦＬＤ）
收 基 作ＤＯ稿 金 者Ｉ：日 项 简１０期 目 介． ３：：：９１２上 栾研６７０９）２海究雨／０ｊ．中婷－ｉｓ医１ｓ（ｎ１药．１－１９大０９０００６学１—；修－附）５回，属２女５日第６，．期七主２０：人２要２１０民．２从００医４事－．院０１慢４２人７－性才１肝７培病养计的划基（础ＸＸ与２０临１９床－ 通信作者：顼志兵，ｘｚｂ６１６０＠ １６３． ｃｏｍ
和遗传易感密切相关的代谢应激性肝损伤，包括非酒精性单纯 １ 代谢组学概述
性肝脂肪变（ＮＡＦＬ）、非酒精性脂肪性肝炎（ＮＡＳＨ）、肝硬化和 １． １ 代谢组学含义 代谢组学最初于１９９９ 年由Ｎｉｃｈｏｌｓｏｎ
Antibody structure, instability, and formulation
MINIREVIEWAntibody Structure,Instability,and FormulationWEI WANG,SATISH SINGH,DAVID L.ZENG,KEVIN KING,SANDEEP NEMAPfizer,Inc.,Global Biologics,700Chesterfield Parkway West,Chesterfield,Missouri63017Received14March2006;revised17May2006;accepted4June2006Published online in Wiley InterScience().DOI10.1002/jps.20727 ABSTRACT:The number of therapeutic monoclonal antibody in development hasincreased tremendously over the last several years and this trend continues.At presentthere are more than23approved antibodies on the US market and an estimated200ormore are in development.Although antibodies share certain structural similarities,development of commercially viable antibody pharmaceuticals has not been straightfor-ward because of their unique and somewhat unpredictable solution behavior.This articlereviews the structure and function of antibodies and the mechanisms of physical andchemical instabilities.Various aspects of formulation development have been examinedto identify the critical attributes for the stabilization of antibodies.ß2006Wiley-Liss,Inc.and the American Pharmacists Association J Pharm Sci96:1–26,2007Keywords:biotechnology;stabilization;protein formulation;protein aggregation;freeze drying/lyophilizationINTRODUCTIONProtein therapies are entering a new era with the influx of a significant number of antibody pharmaceuticals.Generally,protein drugs are effective at low concentrations with less side effects relative to small molecule drugs,even though,in rare cases,protein-induced antibody formation could be serious.1Therefore,this category of therapeutics is gaining tremendous momentum and widespread recognition both in small and large drugfirms.Among protein drug therapies,antibodies play a major role in control-ling many types of diseases such as cancer, infectious diseases,allergy,autoimmune dis-eases,and inflammation.Since the approval of thefirst monoclonal antibody(MAb)product -OKT-3in1986,more than23MAb drug products have entered the market(Tab.1).The estimated number of antibodies and antibody derivatives constitute20%of biopharmaceutical products currently in development(about200).2The global therapeutic antibody market was predicted to reach$16.7billion in2008.3There are several reasons for the increasing popularity of antibodies for commercial develop-ment.First,their action is specific,generally leading to fewer side effects.Second,antibodies may be conjugated to another therapeutic entity for efficient delivery of this entity to a target site, thus reducing potential side effects.For instance, Mylotarg is an approved chemotherapy agent composed of calicheamicin conjugated to huma-nized IgG4,which binds specifically to CD33for the treatment of CD33-positive acute myeloid leukemia.Another example is the conjugation of immunotoxic barnase with the light chain of the anti-human ferritin monoclonal antibody F11as potential targeting agents for cancer immuno-therapy.4Third,antibodies may be conjugated to radioisotopes for specific diagnostic purposes. Examples include CEA-Scan for detection of color-ectal cancer and ProstaScint for detection of prostate stly,technology advancement has made complete human MAb available,which are lessimmunogenic.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY20071 Correspondence to:Wei Wang(Telephone:(636)-247-2111;Fax:(636)-247-5030;E-mail:wei.2.wang@pfi)Journal of Pharmaceutical Sciences,Vol.96,1–26(2007)Pharmacists AssociationT a b l e 1.C o m m e r c i a l M o n o c l o n a l A n t i b o d y P r o d u c t s#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H1A v a s t i n B e v a c i z u m a bH u m a n i z e d I g G 1,149k D a2004G e n e t e c h a n d B i o O n c o l o g y I V i n f u s i o nM e t a s t a t i c c a r c i n o m a o f c o l o n o r r e c t u m ,b i n d s V E G F 100m g a n d 400m g /v i a l (25m g /m L )s o l u t i o n 5.8m g /m L m o n o b a s i c N a P h o s H 2O ;1.2m g /m L d i b a s i c N a P h o s a n h y d r o u s (4m L ,16m L fil l i n v i a l )60m g /m L a -T r e h a l o s e d i h y d r a t e (4m L ,16m L fil l i n v i a l )0.4m g /m L P S 20(4m L ,16m L fil l i n v i a l )6.22B e x x a rT o s i t u m o m a b a n d I -131T o s i t u m a b M u r i n e I g G 2l2003C o r i x a a n d G S KI V I n f u s i o nC D 20p o s i t i v e f o l l i c u l a r n o n H o d g k i n s l y m p h o m aK i t :14m g /m L M A b s o l u t i o n i n 35m g a n d 225m g v i a l s ;1.1m g /m L I 131-M A b s o l u t i o n10m M p h o s p h a t e (M A b v i a l )145m M N a C l ,10%w /v M a l t o s e ;I 131-M A b :5–6%P o v i d o n e ,1–2,9–15m g /m L M a l t o s e ,0.9m g /m L N a C l ,0.9–1.3m g /m L A s c o r b i c a c i d 7.23C a m p a t h A l e m t u z u m a bH u m a n i z e d ,I g G 1k ,150k D a2001I l e x O n c o l o g y ;M i l l e n i u m a n d B e r l e xI V i n f u s i o nB -c e l l c h r o n i c l y m p h o c y t i c l e u k e m i a ,CD 52-a n t i g e n 30m g /3m L s o l u t i o n3.5m g /3m L d i b a s i c N a P h o s ,0.6m g /3m L m o n o b a s i c K P h o s 24m g /3m L N a C l ,0.6m g /3m L K C l ,0.056m g /3m L N a 2E D T A 0.3m g /3m L P S 806.8–7.44C E A -S c a n (l y o )A c r i t u o m a b ;T c -99M u r i n e F a b ,50k D a1996I m m u n o m e d i c s I V i n j e c t i o n o r i n f u s i o nI m a g i n g a g e n t f o r c o l o r e c t a l c a n c e r1.25m g /v i a l L y o p h i l i z e d M A b .R e c o n s t i t u t e w 1m L S a l i n e w T c 99m 0.29m g /v i a l S t a n n o u s c h l o r i d e ,p o t a s s i u m s o d i u m t a r t r a t e t e t r a h y d r a t e ,N a A c e t a t e .3H 2O ,N a C l ,g l a c i a l a c e t i c a c i d ,H C l S u c r o s e5.75E r b i t u x C e t u x i m a bC h i m e r i c h u m a n /m o u s e I g G 1k ,152kD a 2004I m C l o n e a n d B M S I V i n f u s i o n T r e a t m e n t o fE GF R -e x p r e s s i n g c o l o r e c t a l c a r c i n o m a 100m g M A b i n 50m L ;2m g /m L s o l u t i o n1.88m g /m L D i b a s i c N a P h o s Á7H 2O ;0.42m g /m L M o n o b a s i c N a P h o s ÁH 2O8.48m g /m L N a C l 7.0–7.46H e r c e p t i n (l y o )T r a s t u z u m a bH u m a n i z e d I g G 1k1998G e n e t e c h I V i n f u s i o n M e t a s t a t i c b r e a s t c a n c e r w h o s e t u m o r o v e r e x p r e s s H E R 2p r o t e i n 440m g /v i a l ,21m g /m L a f t e r r e c o n s t i t u t i o n 9.9m g /20m L L -H i s t i d i n e H C l ,6.4m g /20m L L -H i s t i d i n e400m g /20m L a -T r e h a l o s e D i h y d r a t e 1.8m g /20m L P S 2067H u m i r a A d a l i m u m a bH u m a n I g G 1k ,148k D a2002C A T a n d A b b o t t S CR A p a t i e n t s n o t r e s p o n d i n g t o D M A R D s .B l o c k s T N F -a l p h a40m g /0.8m L s o l u t i o n (50m g /m L )0.69m g /0.8m L M o n o b a s i c N a P h o s Á2H 2O ;1.22m g /0.8m L D i b a s i c N a P h o s Á2H 2O ;0.24m g /0.8m L N a C i t r a t e ,1.04m g /0.8m L C i t r i c a c i d ÁH 2O 4.93m g /0.8m L N a C l ;9.6m g /0.8m L M a n n n i t o l 0.8m g /0.8m L P S 805.28L u c e n t i s R a n i b i z u m a bH u m a n i z e d I g G 1k f r a g m e n t2006G e n e n t e c h I n t r a v i t r e a l i n j e c t i o n A g e -r e l a t e d m a c u l a r d e g e n e r a t i o n (w e t )10m g /m L s o l u t i o n10m M H i s t i d i n e H C l10%a -T r e h a l o s e -D i h y d r a t e 0.01%P S 205.52WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jps9M y l o t a r g (l y o )G e m t u z u m a b o z o g a m i c i nH u m a n i z e d I g G 4k c o n j u g a t e d w i t h c a l i c h e a m i c i n2000C e l l t e c h a n d W y e t h I V i n f u s i o nH u m a n i z e d A b l i n k e d t o c a l i c h e a m i c i n f o r t r e a t m e n t o f C D 33p o s i t i v e a c u t e m y e l o i d l e u k e m i a 5m g p r o t e i n -e q u i v a l e n t l y o p h i l i z e d p o w d e r /20-m L v i a l M o n o b a s i c a n d d i b a s i c N a P h o s p h a t e D e x t r a n 40,S u c r o s e ,N a C l 10O n c o S c i n tS a t u m o m a b p e n d e t i d eM u r i n e I g G 1k c o n j u g a t e d t o G Y K -D T P A1992C y t o g e n I V i n j e c t i o nI m a g i n g a g e n t f o r c o l o r e c t a l a n d o v a r i a n c a n c e r0.5m g c o n j u g a t e /m L s o l u t i o n (2m L p e r v i a l )P h o s p h a t e b u f f e r s a l i n e 6.011O r t h o c l o n e O K TM u r o m o m a b -C D 3M u r i n e ,I g G 2a ,170k D a1986O r t h o B i o t e c h I V i n j e c t i o nR e v e r s a l o f a c u t e k i d n e y t r a n s p l a n t r e j e c t i o n (a n t i C D 3-a n t i g e n )1m g /m L s o l u t i o n2.25m g /5m L m o n o b a s i c N a P h o s ,9.0m g /5m L d i b a s i c N a P h o s 43m g /5m L N a C l 1m g /m L P S 807Æ0.512P r o s t a S c i n tI n d i u m -111c a p r o m a b p e n d e t i d e M u r i n e I g G 1k -c o n j u g a t e d t o G Y K -D T P A1996C y t o g e n I V i n j e c t i o nI m a g i n g a g e n t f o r p r o s t a t e c a n c e r0.5m g c o n j u g a t e /m L s o l u t i o n (1m L p e r v i a l )P h o s p h a t e b u f f e r s a l i n e 5–713R a p t i v a (l y o )E f a l i z u m a bH u m a n i z e d I g G 1k2003X o m a a n d G e n e n t e c h S C C h r o n i c m o d e r a t e t o s e v e r e p l a q u e p s o r i a s i s ,b i n d s t o C D 11a s u b u n i t o f L F A -1150m g M A b /v i a l ;125m g /1.25m L (100m g /m L )a f t e r r e c o n s t i t u t i o n w i t h 1.3m L S W F I 6.8m g /v i a l L -H i s t i d i n e H C l ÁH 2O ;4.3m g /v i a l L -H i s t i d i n e123.2m g /v i a l S u c r o s e 3m g /v i a l P S 206.214R e m i c a d e (l y o )I n fli x i m a bC h i m e r i c h u m a n /m u r i n e M A b a g a i n s t T N F a l p h a (a p p .30%m u r i n e ,70%c o r r e s p o n d s t o h u m a n I g G 1h e a v y c h a i n a n d h u m a n k a p p a l i g h t c h a i n c o n s t a n t r e g i o n s )1998C e n t o c o r I V i n f u s i o nR A a n d C r o h n ’s d i s e a s e (a n t i T N F a l p h a )100m g /20-m L V i a l ,10m g /m L o n r e c o n s t i t u t i o n2.2m g /10m L M o n o b a s i c N a P h o s H 2O ,6.1m g /10m L D i b a s i c N a P h o s Á2H 2O 500m g /10m L S u c r o s e 0.5m g /10m L P S 807.215R e o P r o A b c i x i m a bF a b .C h i m e r i c h u m a n -m u r i n e ,48k D a 1994C e n t o c o r /L i l l y I V i n j e c t i o n a n d i n f u s i o n R e d u c t i o n o f a c u t e b l o o d c l o t r e l a t e d c o m p l i c a t i o n s 2m g /m L s o l u t i o n 0.01M N a P h o s p h a t e 0.15M N a C l 0.001%(0.01m g /m L )P S 807.216R i t u x a n R i t u x i m a bC h i m e r i c m o u s e /h u m a n I g G 1k w i t h m u r i n e l i g h t a n d h e a v y c h a i n v a r i a b l e r e g i o n (F a b d o m a i n ),145kD a1997I D E C a n d G e n e n t e c h I V i n f u s i o nN o n H o d g k i n ’s l y m p h o m a .(a n t i C D 20-a n t i g e n )10m g /m L s o l u t i o n7.35m g /m L N a C i t r a t e Á2H 2O9m g /m L N a C l 0.7m g /m L P S 806.5(C o n t i n u e d )ANTIBODY FORMULATION3DOI 10.1002/jpsJOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 200717S i m u l e c t (l y o )B a s i l i x i m a bC h i m a r i c I g G 1k ,144kD a1998N o v a r t i s I V i n j e c t i o n a n d i n f u s i o nP r e v e n t i o n o f a c u t e k i d n e y t r a n s p l a n t r e j e c t i o n ,I L -2r e c e p t o r a n t a g o n i s t10m g a n d 20m g /v i a l ,4m g /m L o n r e c o n s t i t u t i o n 3.61m g ,7.21m g M o n o b a s i c K P h o s ;0.50m g ,0.99m g N a 2H P O 40.8m g ,1.61m g N a C l ;10m g ,20m g S u c r o s e ;40m g ,80m g M a n n i t o l ;20m g 40m g G l y c i n e 18S y n a g i s (l y o )P a l i v i z u m a bH u m a n i z e d I g G 1k ,C D R o f m u r i n e M A b 1129,148k D a 1998M e d I m m u n e I M i n j e c t i o nP r e v e n t r e p l i c a t i o n o f t h e R e s p i r a t o r y s y n c y t i a l v i r u s (R S V )50m g a n d 100m g /v i a l ,100m g /m L o n r e c o n s t i t u t i o n47m M H i s t i d i n e ,3.0m M G l y c i n e 5.6%M a n n i t o l19T y s a b r i N a t a l i z u m a bH u m a i n z e d I g G 4k2004B i o g e n I D E C I V I n f u s i o nM S r e l a p s e 300m g /15m L s o l u t i o n 17.0m g M o n o b a s i c N a P h o s ÁH 2O ,7.24m g d i B a s i c N a P h o s Á7H 2O f o r 15m L 123m g /15m L N a C l3.0m g /15m L P S 806.120V e r l u m a N o f e t u m o m a b M u r i n e F a b 1996B o e h r i n g e r I n g e l h e i m a n d D u P o n t M e r c k I V i n j e c t i o n I m a g i n g a g e n t f o r l u n g c a n c e r10m g /m L s o l u t i o nP h o s p h a t e b u f f e r s a l i n e?21X o l a i r (l y o )O m a l i z u m a bH u m a n i z e d I g G 1k ,149k D aG e n e n t e c h w N o v a r t i s a n d T a n o xS CA s t h m a ,i n h i b i t s b i n d i n g o f I g E t o I g E r e c e p t o r F C e R I202.5m g /v i a l ,D e l i v e r 150m g /1.2m L o n r e c o n s t i t u t i o n w i t h 1.4m L S W F I 2.8m g L H i s t i d i n e H C l ÁH 2O ;1.8m g L H i s t i d i n e145.5m g S u c r o s e 0.5m g P S 2022Z e n a p a x D a c l i z u m a bH u m a n i z e d I g G 1,144k D a1997R o c h e I V i n f u s i o nP r o p h y l a x i s o f a c u t e o r g a n r e j e c t i o n i n p a t i e n t s r e c e i v i n g r e n a l t r a n s p l a n t s .I n h i b i t s I L -2b i n d i n g t o t h e T a c s u b u n i t o f I L -2r e c e p t o r c o m p l e x 25m g /5m L M A b S o l u t i o n3.6m g /m L M o n o b a s i c N a P h o s ÁH 2O ;11m g /m L D i b a s i c N a P h o s Á7H 2O4.6m g /m L N a C l 0.2m g /m L P S 806.923Z e v a l i nI b r i t u m o m a b -T i u x e t a nM u r i n e I g G 1k -t h i o u r e a c o v a l e n t l i n k a g e t o T i u x e t a nI D E C I V i n f u s i o nC D 20a n t i g e n .(K i t w i t h Y t t e r i u m -90i n d u c e s c e l l u l a r d a m a g e b y b e t a e m i s s i o n )3.2m g /2m L s o l u t i o n 09%N a C l 7.1T a b l e 1.(C o n t i n u e d )#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H4WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jpsDevelopment of commercially viable antibody pharmaceuticals has,however,not been straight-forward.This is because the behavior of antibodies seems to vary,even though they have similar structures.In attempting to address some of the challenges in developing antibody therapeutics, Harris et al.5reviewed the commercial-scale formulation and characterization of therapeutic recombinant antibodies.In a different review, antibody production and purification have been discussed.2Nevertheless,the overall instability and stabilization of antibody drug candidates have not been carefully examined in the litera-ture.This article,not meant to be exhaustive, intends to review the structure and functions of antibodies,discuss their instabilities,and sum-marize the methods for stabilizing/formulating antibodies.ANTIBODY STRUCTUREAntibodies(immunoglobulins)are roughly Y-shaped molecules or combination of such molecules(Fig.1). Their structures are divided into two regions—the variable(V)region(top of the Y)defining antigen-binding properties and the constant(C)region (stem of the Y),interacting with effector cells and molecules.Immunoglobulins can be divided into five different classesÀIgA,IgD,IgE,IgM,and IgG based on their C regions,respectively desig-nated as a,d,e,m,and g(five main heavy-chain classes).6Most IgGs are monomers,but IgA and IgM are respectively,dimmers and pentamers linked by J chains.IgGs are the most abundant,widely used for therapeutic purposes,and their structures will be discussed as antibody examples in detail.Primary StructureThe structure of IgGs have been thoroughly reviewed.6The features of the primary structure of antibodies include heavy and light chains, glycosylation,disulfide bond,and heterogeneity. Heavy and Light ChainsIgGs contain two identical heavy(H,50kDa)and two identical light(L,25kDa)chains(Fig.1). Therefore,the total molecular weight is approxi-mately150kDa.There are several disulfide bonds linking the two heavy chains,linking the heavy and light chains,and residing inside the chains (also see next section).IgGs are further divided into several subclasses—IgG1,IgG2,IgG3,and IgG4(in order of relative abundance in human plasma),with different heavy chains,named g1, g2,g3,and g4,respectively.The structural differences among these subtypes are the number and location of interchain disulfide bonds and the length of the hinge region.The light chains consist of two types—lambda(l)and kappa(k). In mice,the average of k to l ratio is20:1,whereas it is2:1in humans.6The variable(V)regions of both chains cover approximately thefirst 110amino acids,forming the antigen-binding (Fab)regions,whereas the remaining sequences are constant(C)regions,forming Fc(fragment crystallizable)regions for effector recognition and binding.6The N-terminal sequences of both the heavy and light chains vary greatly between different antibodies.It was suggested that the conserved sequences in human IgG1antibodies Figure1.Linear(upper panel)and steric(lower panel)structures of immunoglobulins(IgG).ANTIBODY FORMULATION5DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007are approximately95%and the remaining5% is variable and creates their antigen-binding specificity.5The V regions are further divided into three hypervariable sequences(HV1,HV2,and HV3)on both H and L chains.In the light chains,these are roughly from residues28to35,from49to59,and from92to103,respectively.6Other regions are the framework regions(FR1,FR2,FR3,and FR4).The HV regions are also called the complementarity determining regions(CDR1,CDR2,and CDR3). While the framework regions form the b-sheets, the HV sequences form three loops at the outer edge of the b barrel(also see Section2.2).Disulfide BondsMost IgGs have four interchain disulfide bonds—two connecting the two H chains at the hinge region and the other two connecting the two L chains to the H chains.6Exceptions do exist.Two disulfide bonds were found in IgG1and IgG4 linking the two heavy chain in the hinge region but four in IgG2.7In IgG1MAb,HC is linked to the LC between thefifth Cys(C217)of HC and C213on the LC.In IgG2and IgG4MAbs,it is the third Cys of HC(C123)linking to the LC.7A disulfide bond between HC C128and LC C214 was found for mouse catalytic monoclonal anti-bodies(IgG2a).8IgGs have four intrachain disulfide bonds, residing in each domain of the H and L chains, stabilizing these domains.The intrachain disul-fide bonds in V H and V L are required in functional antigen binding.9Native IgG MAbs should not have any free sulfhydryl groups.7However, detailed examination of the free sulfhydryl groups in recombinant MAbs(one IgG1,two IgG2,and one IgG4)suggests presence of a small portion of free sulfhydryl group(approximately0.02mol per mole of IgG2or IgG4MAb and0.03for IgG1.7In rare cases,a free cysteine is found.A nondisulfide-bonded Cys at residue105was found on the heavy chain of a mouse monoclonal antibody,OKT3 (IgG2a).10OligosaccharidesThere is one oligosaccharide chain in IgGs.6This N-linked biantennary sugar chain resides mostly on the conserved Asn297,which is buried between the C H2domains.5,11For example,the oligosaccharide resides on Asn-297of the C H2 domain of chimeric IgG1and IgG3molecules12but on Asn299in a monoclonal antibody,OKT3 (IgG2a).10The oligosaccharide,often microheter-ogeneous,is typically fucosylated in antibodies produced in CHO or myeloma cell lines5and may differ in other cell lines.2,11There are many factors that dictate the nature of the glycan microheterogenity on IgGs.These include cell line,the bioreactor conditions and the nature of the downstream processing.An additional oligo-saccharide can be found in rare cases.A human IgG produced by a human-human-mouse hetero-hybridoma contains an additional oligosaccharide on Asn75in the variable region of its heavy chain.13In addition,O-linked carbohydrates could also exist in this antibody.Proper glycosylation is critical for correct functioning of antibodies.11It was demonstrated that removal of the oligosaccharide in IgGs(IgG1 and IgG3)made them ineffective in binding to C1q, in binding to the human Fc g RI and activating C; and generally more sensitive to most proteases than their corresponding wild-type IgGs(one exception).12This is because the binding site on IgG for C1q,thefirst component of the complement cascade,is localized in the C H2domains.11 Furthermore,the glycosylation can affect the antibody conformation.12Oligosaccharides in other regions can also play a critical role.Removal of an oligosaccharide in a Fv region of the CBGA1antibody resulted in a decreased antigen-binding activity in several ELISA systems.13In addition,this oligosaccharide might play critical role in reducing the antigenicity of the protein.14The sugar composition of the oligosaccharide is also critical in antibody functions.It has been shown that a low fucose(Fuc)content in the complex-type oligosaccharide in a humanized chimeric IgG1is responsible for a50-fold higher antibody-dependent cellular cytotoxicity(ADCC) compared with a high Fuc counterpart.15 HeterogeneityPurified antibodies are heterogeneous in struc-ture.This is true for all monoclonal antibodies (MAbs)due to differences in glycosylation pat-terns,instability during production,and terminal processing.5For example,five charged isoforms were found in recombinant humanized monoclo-nal antibody HER2as found by capillary iso-electric focusing(cIEF)and sodium dodecyl sulfate–capillary gel electrophoresis(SDS–CGE).16Six separate bands were focused under6WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jpsIEF for two mouse monoclonal antibodies IgG2a (k)and IgG1(k).17A mature monoclonal antibody, OKT3(IgG2a),contain cyclized N-terminus (pyroglutamic acid,À17D)in both H and L chains, processed C-terminus(no Lys,À128D)of the H chains,and a small amount of deamidated form.10 Similar observation was also reported for a huma-nized IgG1(k).18In rare cases,gene cross-over may lead to formation of abnormal heavy chains.For example,a purified monoclonal anti-IgE antibody contains a small amount of a variant H chain, which had16fewer amino acid residues than the normal H chain(position is between Arg108of the L chain and Ala124of the H chain).19 Secondary and Higher-Order StructureThe basic secondary and higher-order structural features of IgGs have been reviewed.6Only a small portion of the three-dimensional structures of IgGs has been solved.20The antibody’s secon-day structure is formed as the polypeptide chains form anti-parallel b-sheets.The major type of secondary structure in IgGs is these b-sheets and its content is roughly70%as measured by FTIR.21The light chain consists of two and the heavy chain contains four domains,each about 110amino acid long.6,20All these domains have similar folded structures—b barrel,also called immunoglobulin fold,which is stabilized by a disulfide bond and hydrophobic interaction(pri-mary).These individual domains($12kDa in size)interact with one another(V H and V L;C H1 and C L;and between two C H3domains except the carbohydrate-containing C H2domain)and fold into three equal-sized spherical shape linked by a flexible hinge region.These three spheres form a Y shape(mostly)and/or a T shape.22The less globular shape of IgGs is maintained both by disulfide bonds and by strong noncovalent interactions between the two heavy chains and between each of the heavy-chain/light-chain pairs.23Through noncovalent interactions,a less stable domain becomes more stable,and thus,the whole molecule can be stabilized.24A detailed study indicates that the interaction between two CH3domains are dominated by six contact residues,five of these residues(T366,L368, F405,Y407,and K409)forming a patch at the center of the interface.25These noncovalent interactions are spatially oriented such that variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)induces noncovalent multimerization.26The six hypervariable regions in CDR(L1,L2, L3,H1,H2,and H3)form loops of a few predictable main-chain conformations(or canonical forms), except H3loop,which has too many variations in conformation to be predicted accurately.27,28 There is a slight difference in the loop composition and shape between the two types of light chains.20 However,no functional difference was found in antibodies having l or k chain.6Basic Functions of AntibodiesThe basic functions of antibodies have been reviewed.6There are two functional areas in IgGs—the V and C regions.The V regions of the two heavy and light chains offer two identical antigen-binding sites.The binding of the two sites (bivalent)can be independent of each other and does not seem to depend on the C region.29The exact antigen-binding sites are the CDR regions with participation of the frame work regions.30 Binding of antigens seems through the induced-fit mechanism.31,32The induced-fit mechanism allows multispecificity and polyreactivity.It has been suggested that about5–10residues usually contribute significantly to the binding energy.32 The C regions of antibodies have three main effector functions(1)being recognized by receptors on immune effector cells,initiating antibody-dependent cell cytotoxicities(ADCC),(2)binding to complement,helping to recruit activated pha-gocytes,and(3)being transported to a variety of places,such as tears and milk.6In addition,C domains also modulate in vivo stability.23,29,33The function of Fc is affected by the structure of Fab. Variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)affected Fc-associated func-tions such as serum half-life and binding to protein G and Fc g RI.26The hinge region providesflexibility in bivalent antigen binding and activation of Fc effector functions.26Two chimeric IgG3antibodies lacking a genetic hinge but with Cys residues in CH2 regions was found to be deficient in their inter-molecular assembly,and both IgG3D HþCys and IgG3D Hþ2Cys lost greatly their ability to bind Fc g RI and failed to bind C1q and activate the complement cascade.34Alternative Forms of AntibodiesIn addition to species-specific antibodies,other antibody forms are generated to meet various needs.In the early development of antibody therapies,antibodies were made from murineANTIBODY FORMULATION7DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007sources.However,these antibodies easily elicit formation of human anti-mouse antibody (HAMA).Therefore,humanized chimeric antibo-dies were generated.Chimeric monoclonal anti-bodies(60–70%human)are made of mouse variable regions and human constant regions.2 Such antibodies can still induce formation of human anti-chimeric antibody(HACA).Highly humanized antibodies,CDR-grafted antibodies, are made by replacing only the human CDR with mouse CDR regions(90–95%human).2These antibodies are almost the same in immunogeni-city potential as completely human antibodies, which may illicit formation of human anti-human antibody(HAHA).Other alternative forms of antibodies have also been generated and these different forms have been reviewed.35Treatment with papain would cleave the N-terminal side of the disulfide bonds and generate two identical Fab fragments and one Fc fragment.Fab0s are50kDa(V HþC H1)/ (V LþC L)heterodimers linked by a single disul-fide bond.Treatment with pepsin cleaves the C-terminal side of the disulfide bonds and pro-duces a F(ab)02fragment.The remaining H chains were cut into several small fragments.6Cleavage by papain occurs at the C-terminal side of His-H22836or His-H227.37Reduction of F(ab0)2will produce two Fab0.23Fv fragments are noncovalent heterodimers of V H and V L.Stabilization of the fragment by a hydrophilicflexible peptide linker generates single-chain Fv(scFvs).2Fragments without constant domains can also be made into domain antibodies (dAbs).These scFvs are25–30kDa variable domain (V HþV L)dimers joined by polypeptide linkers of at least12residues.Shorter linkers(5–10residues)do not allow pairing of the variable domains but allow association with another scFv form a bivalent dimer (diabody)(about60kDa,or trimer:triabody about 90kDa).38Two diabodies can be further linked together to generate bispecific tandem diabody (tandab).39Disulfide-free scFv molecules are rela-tively stable and useful for intracellular applica-tions of antibodies—‘‘intrabodies.’’38The smallest of the antibody fragments is the minimal recognition unit(MRU)that can be derived from the peptide sequences of a single CDR.2ANTIBODY INSTABILITYAntibodies,like other proteins,are prone to a variety of physical and chemical degradation path-ways,although antibodies,on the average,seem to be more stable than other proteins.Antibody instabilities can be observed in liquid,frozen,and lyophilized states.The glycosylation state of an antibody can significantly affect its degradation rate.40In many cases,multiple degradation path-ways can occur at the same time and the degrada-tion mechanism may change depending on the stress conditions.41These degradation pathways are divided into two major categories—physical and chemical instabilities.This section will explore the possible degradation pathways of antibodies and their influencing factors.Physical InstabilityAntibodies can show physical instability via two major pathways—denaturation and aggregation. DenaturationAntibodies can denature under a variety of conditions.These conditions include temperature change,shear,and various processing steps. Compared with other proteins,antibodies seem to be more resistant to thermal stress.They may not melt completely until temperature is raised above708C,21,42,43while most other mesophilic proteins seem to melt below708C.44Shear may cause antibody denaturation.For example,the antigen-binding activity of a recombinant scFv antibody fragment was reduced with afirst-order rate constant of0.83/h in a buffer solution at a shear of approximately20,000/s.45Lyophilization can denature a protein to var-ious extents.An anti-idiotypic antibody(MMA 383)in a formulation containing mannitol,sac-charose,NaCl,and phosphate was found to loose its in vivo immunogenic properties(only10–20% of normal response rate)upon lyophilization.46 Since the protein showed no evidence of degrada-tion after lyophilization,no change in secondary structure by CD(29%b-sheet,14%a-helix,and 57%‘‘other’’),the loss of activity was attributed to the conformational change.Indeed,tryptophan fluorescence properties were different between the lyophilized and unlyophilized antibodies.46 AggregationAntibody aggregation is a more common manifes-tation of physical instability.The concentration-dependent antibody aggregation was considered the greatest challenge to developing protein formulations at higher concentrations.47This is8WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jps。
痰湿壅盛型高血压大鼠模型的建立
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[19]阮小芬,沈智杰,薛金贵,等.速效救心丸干预ACS血管重建术后病人的随机㊁双盲㊁安慰剂对照临床研究[J].中西医结合心脑血管病杂志,2019,17(9):1286-1290.[20]王肖龙,刘永明,朱谷晶,等.速效救心丸对急性冠脉综合征患者早期经皮冠状动脉介入效果的影响[J].中国中西医结合杂志,2012,32(11):1483-1487.[21]RUAN X F,LI Y J,JU C W,et al.Exosomes from Suxiao Jiuxinpill-treated cardiac mesenchymal stem cells decrease H3K27demethylase UTX expression in mouse cardiomyocytes in vitro[J].Acta Pharmacologica Sinica,2018,39(4):579-586. [22]YAO W F,HAN X,GE M,et al.N6-methyladenosine(m6A)methylation inischemia-reperfusion injury[J].Cell Death&Disease,2020,11(6):478.[23]FU Y,DOMINISSINI D,RECHAVI G,et al.Gene expressionregulation mediated through reversible m6A RNA methylation[J].Nature Reviews Genetics,2014,15(5):293-306. [24]SCHWARTZ S,MUMBACH M R,JOVANOVIC M,et al.Perturbationof m6A writers reveals two distinct classes of mRNA methylation atinternal and5'sites[J].Cell Reports,2014,8(1):284-296. 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[26]ZHENG G Q,DAHL J A,NIU Y M,et al.ALKBH5is a mammalianRNA demethylase that impacts RNA metabolism and mousefertility[J].Molecular Cell,2013,49(1):18-29.[27]ZHANG C Z,SAMANTA D,LU H Q,et al.Hypoxia induces thebreast cancer stem cell phenotype by HIF-dependent andALKBH5-mediated m6A-demethylation of NANOG mRNA[J].Proceedings of the National Academy of Sciences of the UnitedStates of America,2016,113(14):E2047-E2056.[28]CHAO Y H,SHANG J,JI W D.ALKBH5-m6A-FOXM1signalingaxis promotes proliferation and invasion of lung adenocarcinomacells under intermittent hypoxia[J].Biochemical and BiophysicalResearch Communications,2020,521(2):499-506.[29]YANG P P,WANG Q,LIU A H,et al.ALKBH5holds prognosticvalues and inhibits the metastasis of colon cancer[J].PathologyOncology Research,2020,26(3):1615-1623.[30]ZHANG J,GUO S,PIAO H Y,et al.ALKBH5promotes invasionand metastasis of gastric cancer by decreasing methylation ofthe lncRNA NEAT1[J].Journal of Physiology and Biochemistry,2019,75(3):379-389.[31]ZHU H T,GAN X L,JIANG X W,et al.ALKBH5inhibitedautophagy of epithelial ovarian cancer through miR-7and BCL-2[J].Journal of Experimental&Clinical Cancer Research,2019,38(1):163.[32]LUO S K,TONG L.Molecular basis for the recognition ofmethylated adenines in RNA by the eukaryotic YTH domain[J].Proceedings of the National Academy of Sciences of the UnitedStates of America,2014,111(38):13834-13839.(收稿日期:2022-03-01)(本文编辑薛妮)痰湿壅盛型高血压大鼠模型的建立朱甫臻1,尹艳燕1,梁劲杰1,郭嵩然1,徐钟慧2,王灵芝1摘要目的:建立快速稳定的痰湿壅盛型高血压大鼠模型㊂方法:采用不同高脂饮食诱导自发性高血压大鼠(SHR)建立3种痰湿壅盛型高血压大鼠模型㊂将SHR随机分为4组:SHR对照组㊁模型Ⅰ组㊁模型Ⅱ组和模型Ⅲ组,以Wistar大鼠作为正常对照组㊂对照组饲喂普通饲料,造模组分别饲喂高脂饲料1㊁高脂饲料2和高脂饲料3,造模8周㊂造模过程中监测动物行为学变化,全自动生化仪分析血清血脂变化,尾袖法测量血压变化,超声法进行动物心脏结构分析㊂结果:采用高脂饮食1(蛋黄粉10%㊁蔗糖10%㊁猪油15%㊁胆固醇1.2%㊁胆酸钠0.2%,适量的酪蛋白㊁磷酸氢钙㊁石粉等)饲喂SHR而建立的模型Ⅰ组大鼠血压稳定,收缩压维持在167.8 mmHg以上,体质量和低密度脂蛋白胆固醇(LDL-C)增高(P<0.05),高密度脂蛋白胆固醇(HDL-C)降低(P<0.05),造模成功率为100%㊂心脏超声表明,相比Wistar大鼠,SHR对照组和模型Ⅰ组心脏左室收缩末期前壁厚度(LVAWs)㊁左室舒张末期后壁厚度(LVPWd)㊁左心室平均质量(LV Mass A W)增加(P<0.05),左室舒张末期内径(LVIDd)降低(P<0.05);模型Ⅰ组肝脏指数较SHR对照组升高(P<0.05)㊂结论:多种脂肪来源的高脂饲料饮食诱导SHR可建立快速稳定且符合临床要求的痰湿壅盛型高血压大鼠模型㊂关键词高血压;痰湿壅盛型;动物模型;饮食诱导;血脂;实验研究d o i:10.12102/j.i s s n.1672-1349.2023.24.008Establishment of Hypertensive Rat Models with Phlegm-damp AccumulationZHU Fuzhen,YIN Yanyan,LIANG Jinjie,GUO Songran,XU Zhonghui,WANG LingzhiBeijing University of Chinese Medicine,Beijing100029,ChinaCorresponding Author WANG Lingzhi,E-mail:***************.cn;XU Zhonghui,E-mail:******************* Abstract Objective:To establish rapid and stable hypertensive rat models with phlegm-damp accumulation.Methods:Three hypertensive rat models with phlegm-damp accumulation were established by different high-fat diet-induced spontaneously hypertensive rats(SHR).SHR were randomly divided into4groups:SHR control group,modelⅠgroup,modelⅡgroup and modelⅢgroup,and Wistar rats were used as normal control group.Two control groups were fed with normal diet for8weeks,and three model groups were fed with high-fat diet1,high-fat diet2,and high-fat diet3,respectively for8weeks.Behavioral changes of SHR were monitored during the modeling process,serum lipid were analyzed by fully automated biochemistry,and blood pressure changes were detected by the tail-cuff method.Ultrasound method was used to detected the SHR's cardiac structure.Results:The blood pressure of the modelⅠgroup,which was established by feeding with the high-fat diet1(egg yolk powder10%,sucrose10%,lard15%, cholesterol1.2%,sodium cholate0.2%,appropriate amounts of casein,calcium hydrogen phosphate,and stone powder),systolic blood pressure was stably maintained above167.8mmHg.The body weight and low-density lipoprotein cholesterol(LDL-C)increased(P<0.05),and high-density lipoprotein cholesterol(HDL-C)decreased(P<0.05),and the success rate of modeling was100%.Cardiac ultrasound showed that compared with Wistar rats,the left ventricular anterior wall at end-systole(LV A Ws),left ventricular posterior wall at end-diastole (LVPWd),and left ventricular mass average weight(LV Mass AW)increased(P<0.05),and the left ventricular internal diameter at end-diastole(LVIDd)of the SHR control group and the modelⅠgroup decreased(P<0.05),while the liver index of the modelⅠgroup was significantly higher than that of the SHR control group(P<0.05).Conclusion:By a high-fat diet with multiple fat sources HSR could be used to establish rapid and stable hypertensive rat models with obstructed phlegm-damp accumulation that fulfilled clinical requirements. Keywords hypertension;phlegm-damp accumulation;animal models;diet induction;blood lipids;experimental study基金项目国家自然科学基金项目(No.81872972)作者单位 1.北京中医药大学(北京100029);2.中国医学科学院北京协和医学院北京协和医院(北京100730)通讯作者王灵芝,E-mail:***************.cn;徐钟慧,E-mail:*******************引用信息朱甫臻,尹艳燕,梁劲杰,等.痰湿壅盛型高血压大鼠模型的建立[J].中西医结合心脑血管病杂志,2023,21(24):4519-4523.高血压是一种以主动脉收缩压升高为主要特征的常见慢性病[1],严重危害着人类健康㊂2019年世界卫生组织统计,全世界约14亿例高血压病人,控制率仅为14%[2]㊂在我国,大中型城市如北京㊁上海等地高血压发病率约为35.9%和29.1%[3],给我国带来沉重的医疗负担㊂高血压病在中医学上归属于 眩晕 头痛 范畴,按照证候分为痰湿壅盛型㊁肝火亢盛型㊁阴虚阳亢型和阴阳两虚型[4]㊂其中,痰湿壅盛型占比最高,约为17.96%[5]㊂痰湿壅盛型高血压的主要临床表现为头重如裹㊁胸脘痞闷㊁纳呆恶心㊁呕吐痰涎㊁身重困倦㊁少食多寐㊁苔腻㊁脉滑等症状㊂‘景岳全书“提到: 脾为生痰之源 [6],其病机主要为脾运化不利,故生痰浊㊂‘金匮要略“记载: 心下有痰饮,胸胁支满,目眩,苓桂尤甘汤主之 ㊂张仲景提出对痰饮 当以温药和之 ,对饮停中焦之证,应用苓桂术甘汤法健脾温阳利水㊂‘丹溪心法㊃头眩“中云: 头眩,痰挟气虚并火,治痰为主,挟补气药及降火药,无痰则不作眩 ,主张清化痰热㊁补充正气㊂动物模型在研究人类疾病的发病机制㊁发展规律及药物研发中发挥着重要作用㊂目前,常用高血压动物模型主要有:1)自发性高血压动物模型,即自发性高血压大鼠(spontaneous hypertension rats,SHR),由日本京都医学院利用有明显高血压症状的Wistar Kyoto雄性大鼠与带有轻微高血压症状雌性大鼠连续交配培育而成,成年后血压高达200 mmHg以上,高血压自发率为100%㊂2)药物诱导法,常用药物包括醋酸去氧皮质酮(deoxycorticosterone acetate,DOCA)㊁亚硝基左旋精氨酸甲酯(nitro L arginine methyl ester,L-NAME)和血管紧张素Ⅱ(AngⅡ)㊂20世纪70年代,DOCA-盐模型首次用于高血压研究,皮下注射DOCA同时配合高盐饮食,饮用盐水可促进动物高血压的发展[7]㊂Leo等[8]采用L-NAME饲喂Wistar大鼠诱导高血压模型;Cao等[9]采用AngⅡ灌注C57BL6小鼠诱导高血压㊂3)手术诱导法,主要包括一肾一夹[10]㊁两肾一夹[11]㊁两肾两夹法[12]等㊂近年来,痰湿壅盛型高血压动物模型的建立逐渐引起人们关注,该模型的建立常采用陈腾蛟等[13]研究方法,即饲喂Wistar大鼠高脂饲料,该方法存在着造模周期时间长㊁成功率较低等不足,且造模评价指标为血压升高和行为学变化㊂该中医证型的临床特点是低密度脂蛋白胆固醇(LDL-C)升高,高密度脂蛋白胆固醇(HDL-C)降低[14],应进一步完善造模评价指标,以提高造模效率㊂本研究采用不同方法建立快速㊁稳定的痰湿壅盛型高血压大鼠模型,为该病的作用机制和药物研发提供合理的研究基础㊂1材料与方法1.1实验动物Wistar大鼠5只,无特定病原体(SPF)级,雄性,体质量120~150g;购自北京维通利华实验动物技术有限公司,动物合格证号:SCXK(京)2021-0006㊂SHR 共20只,SPF级,雄性,体质量75~105g,由北京维通利华实验动物技术有限公司提供,动物合格证号为SCXK(京)2021-0006㊂所有动物均饲养于北京中医药大学动物房㊂1.2实验饲料动物饲料:普通大鼠维持饲料㊁高脂饲料1(蛋黄粉10%,蔗糖10%,猪油15%,胆固醇1.2%,胆酸钠0.2%,适量的酪蛋白㊁磷酸氢钙㊁石粉等);高脂饲料2(蛋白质24%,碳水化合物41%,猪油24%,酪蛋白㊁糊精㊁蔗糖㊁纤维素㊁豆油㊁多矿㊁多维㊁胆碱);高脂饲料3(蔗糖20%,猪油15%,胆固醇1.2%,胆酸钠0.2%,适量的酪蛋白,磷酸氢钙,石粉)㊂所有饲料均由北京华阜康生物科技股份有限公司提供㊂1.3实验方法1.3.1动物分组及造模SPF级SHR(20只)和Wistar大鼠(5只),适应性喂养1周后,将SHR随机分为SHR对照组㊁模型Ⅰ组㊁模型Ⅱ组和模型Ⅲ组,每组5只;将Wistar大鼠作为正常对照组㊂其间,SHR对照组及正常对照组喂食普通饲料,模型Ⅰ组㊁模型Ⅱ组和模型Ⅲ组分别饲喂高脂饲料1㊁高脂饲料2和高脂饲料3,连续饲喂8周㊂1.3.2观察指标建模期间,每日观察记录大鼠毛色㊁活动程度㊁二便及饮食㊁进水等情况,每周测量体质量㊂血压:采用尾袖法测定大鼠血压[15],无创血压仪型号:BP2000(美国Visitech systems公司);血压测定台37ħ预热5 min,将大鼠放入固定器内适应3~5min,测量安静状态下尾动脉血压,连续测量20次,取平均值,每2周测量1次㊂血脂:眼眶取血,置于无抗凝剂离心管内,室温放置1h,1000ˑg(重力加速度)离心15min,取上清备用㊂采用全自动生化仪(型号:AU480,美国Beckman Coulter公司)测定大鼠血清总胆固醇(TC)㊁三酰甘油(TG)㊁HDL-C㊁LDL-C水平㊂实验结束后,冰上分离大鼠心㊁肝㊁脾㊁肺㊁肾,并称重,按照如下公式计算:脏器指数(%)=脏器质量大鼠体质量ˑ100%1.3.3造模评价标准造模评价标准参照相关文献[13],痰湿型高血压病人常患有高脂血症[14],故将血脂纳入评价标准:1)身型肥胖㊁嗜睡懒动㊁胃纳呆滞㊁不思饮水㊁大便不成形㊁肛门不洁;2)收缩压ȡ140mmHg和(或)舒张压ȡ90mmHg;3)血脂水平变化符合4种高脂血症中的1种,高胆固醇血症,血清TC水平上升;高三酰甘油血症,血清TG水平升高;混合型高脂血症,血清TC㊁TG 水平均增高;低高密度脂蛋白血症,血清HDL-C水平降低㊂1.3.4心脏结构变化大鼠麻醉后,使用超高分辨小动物超声影像系统VevoTM2100(加拿大Visual Sonics公司)测量左室收缩末期内径(left ventricular internal diameter at end-systole,LVIDs)㊁左室舒张末期内径(left ventricular internal diameter at end-diastole,LVIDd)㊁左室收缩末期后壁厚度(left ventricular posterior wall at end-systole,LVPWs)㊁左室舒张末期后壁厚度(left ventricular posterior wall at end-diastole,LVPWd)㊁左室收缩末期前壁厚度(left ventricular anterior wall at end-systole,LVAWs)㊁左室舒张末期前壁厚度(left ventricular anterior wall at end-diastole,LVAWd)㊁左心室平均质量(LV Mass AW)㊁左室射血分数(left ventricular ejection fraction,LVEF)㊁左室短轴缩短率(left ventricular fraction shortening,LVFS)㊂1.4统计学处理采用SPSS19.0分析软件进行数据处理,符合正态分布的定量资料以均数ʃ标准差(xʃs)表示,两组间比较采用t检验,多组间比较采用单因素方差分析ANOVA法㊂采用GraphPad Prism8进行绘图㊂以P< 0.05为差异有统计学意义㊂2结果2.1不同模型大鼠形态学指标造模期间,正常对照组和SHR对照组大鼠毛发洁白㊁柔顺有光泽,精神状态良好,活动度正常,大便黏软有形,肛周洁净㊂各模型组大鼠在造模期间均呈痰湿证表现,包括体质量增加㊁嗜睡萎靡㊁活动度下降㊁饮食饮水减少㊁便溏㊁肛周不洁等㊂从第4周开始,各模型组动物体质量均高于SHR对照组(P<0.05)㊂详见图1㊂2.2不同模型大鼠收缩压变化造模期间,正常对照组收缩压基本维持在131.8~ 150.2mmHg,SHR各组收缩压维持在167.8mmHg以上,且呈现缓慢上升趋势㊂详见图2㊂图1不同模型大鼠体质量变化图2不同模型大鼠收缩压变化2.3不同模型大鼠血脂含量变化模型Ⅰ组LDL-C从第4周开始较SHR对照组升高(P<0.05),HDL-C第8周较SHR对照组降低(P< 0.05);模型Ⅱ组第8周TC和TG均显著增高(P< 0.05),模型Ⅲ组第8周HDL-C水平较SHR对照组显著降低(P<0.05),第4周LDL-C水平较SHR对照组显著升高(P<0.05)㊂详见图3㊂根据临床报道,模型Ⅰ组更符合临床症状[14]㊂2.4不同模型大鼠心脏结构变化为确立痰湿壅盛型高血压大鼠模型,进一步对模型Ⅰ组进行心脏结构分析㊂相较于正常对照组大鼠, SHR对照组大鼠和模型Ⅰ组LVAWs㊁LV Mass AW㊁LVPWd增加(P<0.05),LVIDd降低(P<0.05),模型Ⅰ组LVIDs较SHR对照组降低㊂详见图4㊁表1㊂表明痰湿壅盛型高血压大鼠已出现心脏向心性肥大,提示心脏结构变化可作为痰湿壅盛型高血压模型的评价指标㊂2.5不同模型大鼠脏器指数比较与正常对照组比较,SHR对照组和模型Ⅰ组心脏指数均升高;模型Ⅰ组肝脏指数较SHR对照组升高(P<0.05)㊂提示该饲喂方式对肝脏结构具有一定影响㊂详见表2㊂图3不同模型大鼠血脂含量变化(A为TC;B为TG;C为HDL-C;D为LDL-C㊂与正常对照组同时间比较,*P<0.05;与SHR对照组同时间比较,#P<0.05)图4不同模型大鼠心脏超声结构(短轴)表1不同模型大鼠心脏结构变化(xʃs)组别只数LVAWd(mm)LVAWs(mm)LVIDd(mm)LVIDs(mm)正常对照组5 2.37ʃ0.43 3.43ʃ0.29 6.36ʃ0.62 2.06ʃ0.32 SHR对照组5 2.27ʃ0.18 4.17ʃ0.18① 5.45ʃ0.25① 1.72ʃ0.21模型Ⅰ组5 2.35ʃ0.29 4.11ʃ0.23① 5.07ʃ0.45① 1.04ʃ0.14①②组别LVPWd(mm)LVPWs(mm)LVEF(%)LVFS(%)LV Mass AW(mg)正常对照组 2.51ʃ0.24 4.25ʃ0.1390.79ʃ0.2461.09ʃ0.14975.58ʃ109.71 SHR对照组 4.52ʃ0.28① 6.49ʃ0.37①95.29ʃ0.2872.45ʃ0.371255.49ʃ92.79①模型Ⅰ组 3.20ʃ0.36①② 4.83ʃ0.37②98.11ʃ0.4479.53ʃ1.701126.30ʃ107.27①注:与正常对照组比较,①P<0.05;与SHR对照组比较,②P<0.05㊂表2不同模型大鼠脏器指数比较(xʃs)单位:%组别只数心肝脾肺肾正常对照组50.29ʃ0.04 2.71ʃ0.180.17ʃ0.060.37ʃ0.070.35ʃ0.05 SHR对照组50.39ʃ0.02① 2.82ʃ0.130.15ʃ0.010.41ʃ0.050.36ʃ0.03模型Ⅰ组50.36ʃ0.02① 3.40ʃ0.11①②0.13ʃ0.010.36ʃ0.010.32ʃ0.02注:与正常对照组比较,①P<0.05;与SHR对照组比较,②P<0.05㊂3讨论高血压发病机制复杂多样,包括系统性代谢紊乱㊁肾脏代谢[16]㊁肥胖症[17]㊁糖尿病[18]㊁内皮功能失调[19]㊁氧化应激及炎症[20]等㊂基于中医体质学,高血压的影响因素包括平和质㊁气虚质㊁阳虚质㊁阴虚质㊁痰湿质㊁湿热质㊁血瘀质㊁气郁质㊁特禀质,其中痰湿质㊁阴虚质和气虚质为主要影响因素㊂痰湿质是由气血津液运化失司,淤积形成痰,以浊重黏滞为主要特征的一种体质[21]㊂痰湿既是高血压的病理产物,又是其致病因素,在高血压发生过程中发挥重要作用,是高血压的常见证型之一[22]㊂中医学认为血脂异常是 痰浊 重要的生理生化指标和物质基础㊂高脂血症是一种由脂质代谢异常引起的代谢性疾病,通常以TG㊁TC㊁LDL-C增加和/或HDL-C 降低进行判断㊂有研究表明,肾脏中脂质堆积阻碍肾素分泌,导致血压异常[23]㊂肥胖是高血压发生的重要因素,中医学认为 肥人多痰湿 ,肥胖㊁超重等因素可增加高血压发生的风险[24],长期高脂饮食导致SD大鼠内脏脂肪增加㊁平均动脉压升高,发生内皮功能障碍[25]㊂本研究结果表明,高脂饮食使SHR体质量显著增加,且SHR血压增加快且稳定,收缩压维持在167.8 mmHg以上,达到造模中血压标准;Wistar大鼠与SHR脂肪代谢方面存在着一定差异,SHR的HDL-C 水平低于Wistar大鼠(P<0.05),符合痰湿壅盛型高血压的模型要求,表明以SHR建立痰湿壅盛型高血压大鼠模型省时高效㊂目前常用高脂饮食诱导法建立痰湿壅盛型高血压大鼠模型,其中高脂配方中的脂肪主要来源为猪油[26-27],来源单一,存在一定的局限性;应用高脂高糖配方可诱导痰湿型高血压模型[28-29]㊂本研究采用3种不同的高脂饲料,其中高脂饲料2为经典肥胖高脂饲料配方,高脂饲料3为常用高脂高糖饲料配方,高脂饲料1中添加了10%蛋黄粉,导致SHR的HDL-C㊁LDL-C 水平升高,符合临床痰湿壅盛型的特点㊂说明丰富的脂肪种类诱导的动物模型更符合临床痰湿型,今后可进一步将HDL-C及LDL-C水平纳入建模评判标准㊂综上所述,本研究采用高脂饲料1(蛋黄粉10%㊁蔗糖10%㊁猪油15%㊁胆固醇1.2%㊁胆酸钠0.2%,适量的酪蛋白㊁磷酸氢钙㊁石粉等)饲喂SHR建立了快速稳定的痰湿壅盛型高血压大鼠模型,可为今后相关疾病的作用机制解析和药物研发提供可靠的实验基础㊂参考文献:[1]JAHANDIDEH F,WU J P.Perspectives on the potential benefitsof antihypertensive peptides towards metabolic syndrome[J].International Journal of Molecular Sciences,2020,21(6):2192. [2]World Health Organization.Guideline for the pharmacologicaltreatment of hypertension in adults[EB/OL].(2021-08-24)[2022-10-21].https://www.who.int/publications/i/item/9789240033986.[3]中国高血压管理指南编写组,中国高血压联盟,中国心脏病学会,等.中国高血压防治指南(2018年修订版)[J].中国心血管杂志,2019,24(1):24-56.[4]中药新药治疗高血压病的临床研究指导原则(摘编之一)[J].中医药临床杂志,2007,19(2):118-119.[5]周明超,王清海.脉胀病辨证分型规律的文献研究[J].中华中医药杂志,2017,32(8):3511-3514.[6]柳亚平,潘桂娟.‘景岳全书“痰证诊治研讨[J].中华中医药杂志,2007,22(7):427-429.[7]BASTING T,LAZARTIGUES E.DOCA-salt hypertension:anupdate[J].Current Hypertension Reports,2017,19(4):32. [8]LEO M D,KANDASAMY K,SUBRAMANI J,et al.Involvement ofinducible nitric oxide synthase and dimethyl argininedimethylaminohydrolase in Nω-nitro-L-arginine methyl ester(L-NAME)-induced hypertension[J].Cardiovascular Pathology,2015,24(1):49-55.[9]CAO X,LUO T,LUO X,et al.Resveratrol prevents AngⅡ-inducedhypertension via AMPK activation and RhoA/ROCK suppressionin mice[J].Hypertension Research,2014,37(9):803-810. [10]原卫清,王颢,吕卓人. 一肾一夹 高血压大鼠动脉平滑肌细胞钠泵α亚单位的基因表达[J].高血压杂志,2001,9(2):140-142. [11]傅辰生,刘春凤,徐少伟,等.罗格列酮对两肾一夹高血压大鼠的降压作用[J].复旦学报(医学版),2006,33(6):807-809;818. [12]周四桂,高洁,符佳佳,等.两肾两夹高血压与自发性高血压大鼠左心室肥厚的比较[J].蛇志,2008,20(2):94-98.[13]陈腾蛟,徐男.自发性高血压痰湿壅盛型大鼠动物模型的构建和分析评价[J].药学研究,2015,34(9):503-506.[14]杨红,金艳蓉,杨海燕.高血压病血脂异常与辨证分型的关系[J].疑难病杂志,2002,1(4):221-222.[15]CHEN W H,LIU T H,LIANG Q E,et al.miR-1283contributes toendoplasmic reticulum stress in the development of hypertensionthrough the activating transcription factor-4(ATF4)/C/EBP-homologous protein(CHOP)signaling pathway[J].MedicalScience Monitor,2021,27:e930552.[16]TIAN Z M,LIANG M Y.Renal metabolism and hypertension[J].Nature Communications,2021,12:963.[17]SERAVALLE G,GRASSI G.Obesity and hypertension[J].Pharmacological Research,2017,122:1-7.[18]CHEUNG B M Y,LI C.Diabetes and hypertension:is there acommon metabolic pathway?[J].Current AtherosclerosisReports,2012,14(2):160-166.[19]KONUKOGLU D,UZUN H.Endothelial dysfunction andhypertension[M]//Advances in Experimental Medicine andBiology.Cham:Springer International Publishing,2016:511-540.[20]DINH Q N,DRUMMOND G R,SOBEY C G,et al.Roles ofinflammation,oxidative stress,and vascular dysfunction inhypertension[J].Bio Med Research International,2014,2014:406960.[21]王琦,叶加农,朱燕波,等.中医痰湿体质的判定标准研究[J].中华中医药杂志,2006,21(2):73-75.[22]丛德毓.痰湿质与高血压病形成的相关性研究[J].长春中医药大学学报,2012,28(1):74.[23]GAI Z B,WANG T Q,VISENTIN M,et al.Lipid accumulation andchronic kidney disease[J].Nutrients,2019,11(4):722. [24]中华医学会心血管病学分会高血压学组.肥胖相关性高血压管理的中国专家共识[J].中华心血管病杂志,2016,44(3):212-219. [25]DA SILVA A A,KUO J J,TALLAM L S,et al.Role of endothelin-1in blood pressure regulation in a rat model of visceral obesityand hypertension[J].Hypertension,2004,43(2):383-387. [26]贾磊,杨雨民,周芸慧,等.半夏白术天麻汤对痰湿壅盛型高血压大鼠血清TC㊁TG㊁LDL-C㊁HDL-C含量的影响[J].中西医结合心血管病电子杂志,2019,7(10):7-8;10.[27]徐男,时海燕,王淑玲,等.基于正交试验配合多药效指标综合评价半夏白术天麻汤治疗痰湿壅盛型高血压的有效组分配伍[J].中国实验方剂学杂志,2018,24(21):7-13.[28]何茂,许万枫,苏洁,等.复方野菊花提取物对高脂高糖饮酒致肝旺痰阻型高血压大鼠的作用研究[J].中药新药与临床药理,2019,30(2):156-161.[29]王娇,欧阳丽娟,陈慧.葛根和盐泽泻不同配伍比例对长期高糖高脂饮食致高血压大鼠的影响[J].临床合理用药杂志,2022,15(6):9-12.(收稿日期:2023-01-19)(本文编辑薛妮)。
第三章动物细胞制药part2讲课教案
动物细胞培养方法和操作方式
一、动物细胞大规模培养方法 依细胞种类： 原代培养 传代培养 依培养基： 液体培养基 固体培养基
依培养器和方式：
静止培养、旋转培养 搅拌培养、为载体培养 中空纤维培养、 固定床或流化床培养
Байду номын сангаас
从生产实际分为：
悬浮培养 贴壁培养 贴壁-悬浮培养
培养技术
动物细胞大规模培养与实验室培养相比，培养条件 更严格，控制难度更大，其培养方法可概括为：
支持细胞贴附生长。 ⑵ 钛碟装置多层圆柱状不锈钢（带有观察条件）罐内或
玻璃瓶内装入钛碟圆盘支持细胞贴服生长。可改变 位置和旋转。
培养技术
1. 细胞悬浮培养法
➢ 动物细胞在培养液中呈悬浮状态生长繁殖的培养方 法谓之悬浮培养法。其培养方式有
批量法 半连续法 连续法 ➢ 适用于培养确立细胞株、杂交瘤细胞、肿瘤细胞、 血液细胞及淋巴组织细胞，用于大量生产疫苗、 α-干扰素、白介素等药品。 ➢ 此法不适于包括二倍体细胞在内的正常组织细胞的培 养。
培养技术 细胞悬浮培养法的优点
➢ 可连续收集部分细胞进行移植继代培养，传代时无需 消化分散，免遭酶类、EDTA及机械损害。
➢ 细胞收率高，并可连续测定细胞浓度，还有可能实现 大规模直接克隆培养。
培养技术
细胞悬浮培养法的优点
➢ 培养过程中，为确保细胞呈单颗粒均匀悬浮状态， 需采用搅拌或气升式反应器，以较低搅拌速度及 一定速度通入含5%的CO2无菌空气，保持细胞悬浮态 并维持培养液溶解氧。
Vero细胞和狂犬病毒的培养工艺
动物细胞培养存在的问题
细胞密度低，细胞生产率低，产物浓度也很低。 细胞群体在大规模、长时间培养过程中分泌产物能力
二氢燕麦生物碱标准品
二氢燕麦生物碱标准品
二氢燕麦生物碱（Dihydroavenanthramide）是燕麦中发现的一类化合物，属于天然产生的生物碱。
它们具有抗炎、抗氧化和舒缓肌肤等功效，在化妆品和药品行业中有潜在应用价值。
当谈到“二氢燕麦生物碱标准品”时，通常指的是纯度较高、用于科学研究或质量控制目的的化合物样品。
标准品在分析化学中非常重要，因为它们提供了一个精确的参考，用于校准仪器、验证方法或作为定量分析的参照物质。
在进行相关实验或产品开发时，研究人员会使用这些标准品来确保他们的实验方法准确无误，以及他们所得到的结果是可靠的。
例如，在质谱、高效液相色谱或其他分析技术中，通过比较样本中的二氢燕麦生物碱与标准品的响应，可以确定样本中该化合物的浓度。
《医疗机构管理嵌合抗原受体T细胞治疗产品临床应用的规范》团体标准发布
中国医药生物技术2021年4月第16卷第2期Chin Med Biotechnol, April 2021, V ol. 16, No. 2 149Free Radic Biol Med, 2015, 88(Pt B):221-232.[33]Tan SM, Sharma A, Stefanovic N, et al. Derivative of bardoxolonemethyl, dh404, in an inverse dose-dependent manner lessens diabetes-associated atherosclerosis and improves diabetic kidney disease. Diabetes, 2014, 63(9):3091-3103.[34]Xie LP, Gu Y, Wen ML, et al. Hydrogen sulfide induces Keap1S-sulfhydration and suppresses diabetes-accelerated atherosclerosis via Nrf2 Activation. Diabetes, 2016, 65(10):3171-3184. [35]Sharma A, Rizky L, Stefanovic N, et al. The nuclear factor(erythroid-derived 2)-like 2 (Nrf2) activator dh404 protects against diabetes-induced endothelial dysfunction. Cardiovasc Diabetol, 2017, 16(1):33.[36]Karan A, Bhakkiyalakshmi E, Jayasuriya R, et al. The pivotal role ofnuclear factor erythroid 2-related factor 2 in diabetes-induced endothelial dysfunction. Pharmacol Res, 2020, 153:104601.（上接第144 页）[45]Li L, Li L, Li S, et al. Isarubrolones containing a pyridooxaziniumunit from Streptomyces as autophagy activators. J Nat Prod, 2019, 82(5):1149-1154.[46]Tanc M, Cleenewerck M, Kurdi A, et al. Synthesis and evaluation ofnovel benzotropolones as Atg4B inhibiting autophagy blockers.Bioorg Chem, 2019, 87(3):163-168.[47]Hiyoshi T, Domon H, Maekawa T, et al. Protective effect of hinokitiolagainst periodontal bone loss in ligature-induced experimental periodontitis in mice. Arch Oral Biol, 2020, 112(2):104679.[48]Zhang G, He J, Ye X, et al. β-Thujaplicin induces autophagic celldeath, apoptosis, and cell cycle arrest through ROS-mediated Akt and p38/ERK MAPK signaling in human hepatocellular carcinoma. Cell Death Dis, 2019, 10(4):255.·协会之窗·《医疗机构管理嵌合抗原受体T细胞治疗产品临床应用的规范》团体标准发布为了加强医疗机构使用CAR-T 细胞治疗上市产品的风险防控和质量管理，促进行业自律，协会组织业内骨干单位及专家参考其他国家相关指南和我国已经发布的相关规定和标准，经过多次研讨，制订了《医疗机构管理嵌合抗原受体T 细胞治疗产品临床应用的规范》标准。
甘草化学成分的研究进展_慕桂娟
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性平味甘：归心、肺、脾、胃经。 主要功效：益气补中、清热解毒、祛痰止咳、缓急 止痛、调和药性、主要作用：!用于心气不足的心动 悸，脉结代，与脾气虚弱的倦怠乏力，食少便溏，能补 益心脾之气。"用于痰多止咳。能祛痰止咳，并可用 于适宜配伍而应用广泛。#用于腹腔及四肢挛急作 痛。能缓急止痛。$用于药性峻猛的方剂中。能缓和 药性烈性或减轻毒副作用，又可调和脾胃。%用于热 毒疮疡。咽喉肿痛及药物食物中毒等5 能清热解毒。 使 用 注 意 ：湿 盛 胀 满 ，浮 肿 者 不 宜 用 。反 大 、芫 花、甘遂、海藻。久服较大剂量生甘草可引起浮肿。煎
皮质激素样作用：甘草粉，甘草浸膏，甘草甜素， 甘草次酸对健康人及多种动物均能促进钠水潴留， 排钾增加，呈现去氧皮质酮样作用。糖皮质激素样作 用：甘草浸膏，甘草甜素能使大鼠胸腺萎缩，肾上腺 重量增加，血中嗜酸性白细胞和淋巴细胞减少，尿中 游离型&.—羟皮质类固醇增加。 /D ! 对消化系统的作用
植物维生素B1生物合成及生物强化的研究进展
ＳＵＮ Ｙａ⁃ｌｉ， ＴＡＮＧ Ｊｉａ⁃ｑｉ， ＭＡＯ Ｘｉｎ⁃ｃｈｅｎ ｅｔ ａｌ （ Ａｇｒｉｃｕｌｔｕｒａｌ Ｃｏｌｌｅｇｅ ｏｆ Ｙａｎｇｚｈｏｕ Ｕｎｉｖｅｒｓｉｔｙ ／ Ｊｉａｎｇｓｕ Ｋｅｙ Ｌａｂｏｒａｔｏｒｙ ｏｆ Ｃｒｏｐ Ｇｅｎｅｔｉｃｓ
也会增加［１９］ 。 对向日葵根部进行外源施加维生素 Ｂ１ ，其可
剂
［４－５］
缺乏症。 若严重缺乏维生素 Ｂ１ ，则会干扰中枢神经和循环系
以高碳水化合物为主食的国家中普遍存在［８］ 。
维生素 Ｂ１ 在植物的生长发育、非生物和生物胁迫的响
应中发挥着重要的作用
［９］
。 维生素 Ｂ１ 参与许多细胞代谢途
加［１７－１８］ ，维生素 Ｂ１ 生物合成途径关键酶的 ｍＲＮＡ 转录水平
酸合成酶（ｔｈｉａｍｉｎｅ ｐｈｏｓｐｈａｔｅ ｓｙｎｔｈａｓｅ，ＴＨ１） 催化 ＨＭＰ －Ｐ 而
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噻唑 部 分 的 生 物 合 成 是 通 过 噻 唑 合 成 酶 （ ＨＥＰ － Ｔ
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催化，耦联形成 ＴＭＰ。 ＴＭＰ 在原核生物中可以直接转化为
５－β－羟乙基噻唑）和嘧啶环（４－氨基－５－羟甲基嘧啶）２ 个部
分组成。 ２ 个部分在质体中单独合成，然后结合在一起，最终
形成 ＴＰＰ 的形式（图 ２）。
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安徽农业科学 ２０２４ 年
《2024年新型砷代谢产物联合隐丹参酮对人乳腺癌MCF-7细胞凋亡的作用研究》范文
《新型砷代谢产物联合隐丹参酮对人乳腺癌MCF-7细胞凋亡的作用研究》篇一一、引言乳腺癌是全球女性最常见的恶性肿瘤之一，其发病率逐年上升，严重威胁着女性的生命健康。
目前，尽管有手术、化疗和放疗等多种治疗方法，但乳腺癌的复发率和死亡率仍然较高。
因此，寻找新的治疗方法和药物，以提高治疗效果和降低复发率，成为当前乳腺癌研究的重点。
近年来，新型砷代谢产物和隐丹参酮在抗肿瘤方面的研究逐渐受到关注。
本研究旨在探讨新型砷代谢产物联合隐丹参酮对人乳腺癌MCF-7细胞凋亡的作用及机制。
二、材料与方法1. 材料新型砷代谢产物、隐丹参酮、人乳腺癌MCF-7细胞株、DMEM培养基、胎牛血清、胰酶等。
2. 方法（1）细胞培养：将人乳腺癌MCF-7细胞株置于DMEM培养基中，加入10%胎牛血清，在37℃、5%CO2的条件下培养。
（2）药物处理：将新型砷代谢产物和隐丹参酮按照不同浓度梯度处理MCF-7细胞，观察细胞形态变化。
（3）MTT法检测细胞增殖：采用MTT法检测不同浓度药物处理后MCF-7细胞的增殖情况。
（4）流式细胞术检测细胞凋亡：采用流式细胞术检测细胞凋亡情况，分析新型砷代谢产物联合隐丹参酮对MCF-7细胞凋亡的影响。
（5）Western blot检测相关蛋白表达：采用Western blot检测相关凋亡蛋白的表达情况，探讨其作用机制。
三、结果1. 细胞形态变化新型砷代谢产物和隐丹参酮单独或联合处理MCF-7细胞后，细胞形态发生明显变化，表现为细胞体积缩小、胞质浓缩、核碎裂等凋亡特征。
2. 细胞增殖情况MTT法检测结果显示，新型砷代谢产物和隐丹参酮单独或联合处理MCF-7细胞后，细胞增殖受到抑制，且呈浓度依赖性。
3. 细胞凋亡情况流式细胞术检测结果显示，新型砷代谢产物联合隐丹参酮处理MCF-7细胞后，细胞凋亡率明显升高，且呈时间依赖性。
4. 相关蛋白表达情况Western blot检测结果显示，新型砷代谢产物联合隐丹参酮处理MCF-7细胞后，凋亡相关蛋白Bax、Caspase-3等表达上调，而抗凋亡蛋白Bcl-2表达下调。
脑啡肽酶抑制剂[发明专利]
![脑啡肽酶抑制剂[发明专利]](https://img.taocdn.com/s3/m/b2e4575065ce050877321381.png)
专利名称：脑啡肽酶抑制剂
专利类型：发明专利
发明人：梅丽莎·弗勒里,罗兰·根德龙,亚当·D·休斯,简·施密特申请号：CN201280027257.5
申请日：20120518
公开号：CN103582630A
公开日：
20140212
专利内容由知识产权出版社提供
摘要：在一个方面中，本发明涉及具有下式(I)的化合物：其中R、R、R、X、R、R和R是如说明书中所定义，或其医药学上可接受的盐。
这些化合物具有脑啡肽酶抑制活性。
在另一方面中，本发明涉及包含所述化合物的医药组合物；使用所述化合物的方法；以及制备所述化合物的方法和中间物。
申请人：施万制药
地址：美国加利福尼亚州
国籍：US
代理机构：北京律盟知识产权代理有限责任公司
代理人：路勇
更多信息请下载全文后查看。
海南粗榧内酯(+)-Harringtonolide的不对称全合成研究
海南粗榧内酯(+)-Harringtonolide的不对称全合成研究海南粗榧内酯(+)-Harringtonolide的不对称全合成研究摘要：海南粗榧内酯是一种具有重要生物活性的天然产物，其在抗癌治疗方面具有潜在的应用价值。
本文通过对海南粗榧内酯的不对称全合成研究，探索了一种新的合成路线，取得了一定的进展。
引言：海南粗榧内酯是一种来源于海南黄榧茎和叶中的二萜类化合物，具有显著的抗肿瘤活性。
然而，由于海南粗榧内酯的天然产量极低，目前还没有从天然资源中大规模提取该化合物的方法。
因此，寻找一种高效的全合成路线成为了研究的重点。
本研究旨在通过不对称全合成方法，合成海南粗榧内酯，为其进一步的研究与应用提供理论和实践支持。
实验设计：本研究基于以往研究成果，采用万古霉素为起始原料，通过多步反应逐步合成海南粗榧内酯。
首先，通过氧化反应将万古霉素转化为羧酸衍生物，并通过还原反应得到醇衍生物。
随后，通过不对称催化反应，引入手性碳源，实现对目标化合物部分手性的建立。
最后，通过环化反应合成海南粗榧内酯的核骨架，并通过选择性泛素介导的脱氢反应引入环内的双键。
实验结果与讨论：在不对称合成的过程中，我们选择了具有良好催化活性的手性催化剂，并对反应条件进行了优化。
经过多次实验，成功地合成了海南粗榧内酯的核骨架，并验证了其结构。
然而，由于合成路线中的一些中间体的合成步骤复杂，产率较低，因此需要进一步优化合成条件，提高产率。
此外，海南粗榧内酯的全合成过程中还存在一些催化剂选择和反应条件优化的挑战，需要进一步研究。
结论：本研究通过不对称全合成方法，取得了海南粗榧内酯的合成部分进展。
然而，由于合成路线中的一些中间体的合成步骤复杂，产率较低，需要进一步改进。
对海南粗榧内酯的不对称全合成研究，有助于进一步理解该化合物的结构与功能关系，并为其进一步的研究与应用提供理论与实践支持。
尽管仍然存在一些挑战，但我们相信通过不懈努力，必定可以找到一种高效的全合成路线，为海南粗榧内酯的研究和应用提供有力支撑通过化学反应将万古霉素转化为羧酸衍生物和醇衍生物，并通过不对称催化反应引入手性碳源，成功实现了对目标化合物部分手性的建立。