Oxygen-Glucose Deprivation Activates 5-Lipoxygenase Mediated by Oxidant Stress through p38 MAPK

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基于无酶葡萄糖传感器的修饰电极及电化学性能

基于无酶葡萄糖传感器的修饰电极及电化学性能崔英磊;钱锋;张彰【摘要】The content of glucose detection has important significance in many fields and sensor technology has been applied in our daily life. Therefore glucose sensor becomes a hot topic in the study of biological sensors. In this article,four transition metal oxides of nano-material modified carbon paste electrodes were applied to detect glucose. The electrolyte concentration and the influence of NiO content in the modified materials were also stud-ied. High electric potential was used in processing the electrode to prove electrochemical performance of non-enzyme glucose sensor. This study lays a foundation for applications of non-enzyme electrochemical glucose sensors.%葡萄糖的含量检测在诸多领域都有着不可取代的作用,传感器技术也在日常生活中普遍使用,因此葡萄糖传感器成为了生物传感器研究中的热点.使用4种纳米过渡金属氧化物材料修饰的碳糊电极进行葡萄糖检测,并研究电解液浓度和修饰材料中NiO含量的影响.研究表明,高电位处理提高了无酶电化学葡萄糖传感器的性能,为无酶电化学葡萄糖传感器的进一步应用奠定了基础.【期刊名称】《上海大学学报（自然科学版）》【年(卷),期】2017(023)006【总页数】6页(P905-910)【关键词】传感器;无酶;葡萄糖;纳米材料电极【作者】崔英磊;钱锋;张彰【作者单位】上海大学环境与化学工程学院,上海200444;上海赛孚燃料检测股份有限公司,上海201206;上海大学环境与化学工程学院,上海200444【正文语种】中文【中图分类】TP212含酶葡萄糖电化学传感器有着很长的研究历史,并取得了不少成果.但在实际应用中,酶修饰电极的“短板”逐渐凸显,如电极稳定性较差、成本高、葡萄糖氧化酶固定过程复杂等.这些因素使得酶电极葡萄糖生物传感器的灵敏度、稳定性及重现性受到了一定的影响,应用受限.目前尚无一种可靠的方法,能够使葡萄糖电极中的酶既稳定又高效,且不易脱落、失活.基于此,科研人员着手于无酶葡萄糖传感器的研究.无酶葡萄糖传感器以其耐用、工艺简单、成本低、稳定性及重现性好的特点[1],深受青睐.目前研制的无酶葡萄糖传感器在使用中仍旧存在缺陷,例如:选择性较含酶传感器差;当使用镍电极检测时,样品中有抗坏血酸或尿酸大量存在,响应电流就会受到干扰[2];容易出现氯离子中毒等现象[3].因此,研究制备一种低成本、高选择性、能迅速精准检测葡萄糖的无酶葡萄糖传感器,具有良好的学术与应用价值.由于葡萄糖在某些材料表面通过电催化氧化能够转化为葡萄糖酸内酯,因此无酶葡萄糖传感器的研究热点聚焦在电极材料方面.纳米材料具有较高的比表面积及特殊的物理性质,以其修饰的电极在检测葡萄糖时表现出了线性范围广、检测限低、灵敏度高、电催化活性良好等诸多优点[4].Wang等[5]制备的纳米多孔和中孔Pt电极在葡萄糖的测定中得到了较好的结果.Rong等[6]用Al模板方法制备了Pt纳米管阵列修饰电极.郭合帅等[7]以超薄氧化铝(ultra-thin alumina mask,UTAM)为模板,采用真空镀膜制备了大面积高度有序的银纳米点阵活性基底,并经过表面预处理后用于检测葡萄糖灵敏度,效果良好.Yeo等[8]探索了过渡金属Ni,Mn,Cu,Fe合金电极在0.1mol·L−1NaOH介质中葡萄糖含量的测定,结果表明这些电极具有较好的灵敏度.过渡金属氧化物具有空穴的阴离子和混合价态的阳离子,通过改变阴离子和阳离子,可以调节过渡金属氧化物的电学、光学、磁学以及催化等物理化学性质.另外,它们具有较低的氧化还原电势,可以充当氧化还原电子媒介,且造价低廉、不易中毒.Zhang 等[9]发现将CuO用于电化学传感器构置对葡萄糖表现出了较高的电催化活性. 本工作将纳米级过渡金属氧化物Co3O4,NiO,CuO,Rh2O用于修饰碳糊电极来构造葡萄糖传感器,通过对葡萄糖进行检测筛选出合适的纳米材料,再进一步采用高电位法处理电极,提高无酶电化学葡萄糖传感器的检测性能.1 实验部分1.1 材料与仪器纳米级过渡金属氧化物Co3O4,NiO,CuO和Rh2O,由东莞市鸿博纳米材料有限公司提供;石蜡;石墨粉;葡萄糖,由南京古田化工有限公司提供.实验用水为二次超纯水,由美国Milli-pore公司Milli-Q超纯水制备系统生产,其他试剂均为光谱纯.标准熔点毛细管(直径1 mm),由苏州市东吴玻璃仪器有限公司提供.RST5200电化学工作站,由北京恒奥德仪器仪表有限公司提供.1.2 电极的制备首先以质量比1∶4混合石蜡和石墨粉,研磨形成碳糊,再将其装填在毛细管中,插入铜丝并固定,制得碳糊电极;然后分别把纳米级过渡金属氧化物Co3O4,NiO,CuO,Rh2O与碳糊按质量比1∶8混合研磨,再与石蜡按质量比1∶4混合,加热溶化后搅拌均匀,之后取约2 mL涂敷于碳糊电极顶端,冷却,以备葡萄糖检测使用.1.3 实验步骤选用三电极体系进行葡萄糖的检测,其中工作电极为碳糊电极,参比电极为甘汞电极,对电极使用铂丝.首先配制0.20 mol·L−1的NaCl溶液,再加入适量的固体KOH,使溶液中KOH浓度为0.60 mol·L−1,并将其用作电解液.全部实验都在20°C下完成. 本工作采用循环伏安法对不同修饰电极进行测试表征,葡萄糖的加入量为2.00 mmol·L−1.在考查OH−浓度对纳米NiO的碳糊电极检测葡萄糖的影响实验中,KOH的浓度分别选择为0.06,0.20和0.60 mol·L−1.考查修饰电极中NiO的含量对检测葡萄糖的影响实验中,碳糊和NiO的质量比分别为4∶1和8∶1.高电位处理时,使用循环伏安法将NiO修饰的碳糊电极在0.60 mol·L−1KOH电解液中以0∼1.1 V扫描.图1 三电极体系示意图Fig.1 Schematic diagram of three-electrode system 2 结果与讨论2.1 修饰电极图2是不同纳米级过渡金属氧化物修饰碳糊电极的情况下电解液的循环伏安曲线.葡萄糖加入量为2.00 mmol·L−1,扫描设定:含有 NaCl的KOH 溶液,其中 NaCl为0.20 mol·L−1,KOH为0.60 mol·L−1,扫描速度0.1 V/s.图2 不同纳米级过渡金属氧化物修饰碳糊电极的循环伏安曲线Fig.2 Cyclic voltammogram of electrolyte solutions based on diあerent transition metal oxides of nanomaterial modif i ed carbon paste electrodes由图2可以明显看出,在这4种过渡金属氧化物中,只有纳米NiO修饰的碳糊电极在加入葡萄糖时电流改变显著,即响应电流明显,而其他3种过渡金属纳米氧化物的修饰电极对葡萄糖的加入响应较弱.因此,后续实验主要围绕纳米NiO修饰的碳糊电极进行研究.2.2 电化学反应原理按照理论推测,使用NiO修饰的碳糊电极检测葡萄糖时涉及的反应过程可分为两个阶段.(1)第一阶段.NiO与水发生物理吸附,形成的水合氧化镍(NiO·H2O)牢固吸附在电极表层,而暴露在空气中的NiO·H2O与空气中的氧发生氧化还原反应生成Ni(OH)2,其中Ni(OH)2与NiOOH可以相互转化[10-11].NiO+物理吸附的水→NiO·H2O(牢固吸附在电极表层)+O2→Ni(OH)2,(2)第二阶段.葡萄糖在电极表面被NiOOH催化氧化为葡萄糖酸内酯,同时电极表面的NiOOH被还原为Ni(OH)2,且电位发生变化,所产生的电信号通过循环伏安法的响应电流而得以表征[12].NiOOH+葡萄糖−=⇒Ni(OH)2+葡萄糖酸内酯.2.3 KOH浓度根据上述反应机理,NiO与碳水化合物发生的电催化氧化反应需要在碱性溶液中进行.为此实验考察纳米级NiO材料修饰电极时不同浓度的KOH溶液中葡萄糖电化学反应响应电流的情况.KOH浓度不同时NiO修饰碳糊电极的循环伏安曲线如图3所示.由图可见,在KOH溶液浓度较高时,有两个显著的氧化还原峰出现.这可解释为,OH−的含量较高,促进了可逆反应的平衡右移,所对应的氧化还原峰亦趋于显著.由此可知,NiO检测葡萄糖的响应信号与溶液中的OH−浓度有关,且随着OH−浓度的增大而增强.因此,后续实验选择KOH溶液的浓度为0.60 mol·L−1.图3 KOH浓度不同时NiO修饰碳糊电极的循环伏安曲线Fig.3 Cyclic voltammogram of KOH solutions with diあerent concentrations based on NiO modif i ed carbon paste electrodes2.4 NiO含量为考察NiO含量对电化学反应的影响,实验自制了2种不同的NiO修饰碳糊电极并进行了测试比较,其结果如图4所示.从图中可以看出,当使用NiO含量较高的修饰电极时,氧化还原的两个峰值较显著;而当使用NiO含量较低的修饰电极时,虽有响应电流出现,但并无明显的氧化还原峰出现.分析原因可知:一方面,这是由于纳米材料的比表面积大,NiO的表面极易产生物理吸附,从而把水分子由空气中吸附到电极表面,导致电极表面羟基化,使表层NiO变成Ni(OH)2,从而进行检测时电信号不明显;另一方面,由于羟基化层的厚度较小,随着NiO含量的增大,电极表面的Ni(OH)2及NiOOH的浓度也逐步增大,致使Ni(OH)2/NiOOH可逆转化反应所产生的氧化还原峰逐步趋于显著.上述结果进一步验证了NiO催化氧化葡萄糖的机理.表面羟基化经常发生在诸多其他金属或其氧化物的表层,如Pt,Ni,MnO2等,而且碱性环境下会使羟基化加速[10,13].图4 NiO含量不同的修饰碳糊电极的循环伏安曲线Fig.4 Cyclic voltammogram of electrolyte solutions based on NiO modif i ed carbon paste electrodes with diあerent contens2.5 高电位处理基于上述实验的结果,对纳米NiO修饰电极进行高电位处理,推断高电位的处理会使NiO转化量大大增加,使NiOOH/Ni(OH)2氧化还原电对数量激增,从而有效提高葡萄糖电化学反应的响应信号.于是,在0∼1.2 V的范围内,对NiO修饰的碳糊电极进行循环伏安高电位处理后检测对不同浓度葡萄糖溶液的响应电流,并与处理前的电极作比较.图5为NiO修饰电极高电位处理前后葡萄糖溶液与响应电流的关系.通过比较拟合曲线的斜率可以看出:高电位处理后的拟合曲线斜率明显大于处理前,表明响应电流明显增大,电极灵敏度提高;线性相关系数与处理前相比更大,意味着经高电位处理过的电极能够更准确地检测出葡萄糖的含量.图5 NiO修饰电极高电位处理前后葡萄糖溶液浓度与响应电流的关系Fig.5Relations between glucose concentrations and currents based on NiO modif i ed carbon paste electrode before and after high electric potential 3 结束语本工作探索了4种纳米级过渡金属氧化物修饰的碳糊电极在葡萄糖检测中的应用.实验结果显示,采用NiO材料修饰的碳糊电极对葡萄糖有良好的催化氧化性能.同时实验还研究了电解液中KOH浓度、碳糊电极中NiO含量的影响,并由此得到了适宜的电化学反应条件:KOH的浓度为0.60 mol·L−1,电极中碳糊和NiO的质量比为4∶1.另外,高电位处理后的电极的灵敏度显著提升,与处理前相比,检测限更低,反应更为迅速.参考文献:[1]DI RESTA E,PRUDDEN J,SALEMME J,et al.Continuous glucose monitoring on-body sensor:U.S.2014/022637[P].2014-10-02.[2]刘忠银,史天录,张雪,等.利用纳米材料制备葡萄糖传感器的研究进展[J].功能材料与器件学报,2015,21(4):24-30.[3]姚伟.葡萄糖传感电极的制备与性能研究[D].济南:山东师范大学,2015.[4]KANYONG P,RAWLINSON S,DAVIS J.A non-enzymatic sensor based on the redox of ferrocene carboxylic acid on ionic liquid f i lm-modif i ed screen-printed graphite electrode for the analysis of hydrogen peroxide residues in milk[J].Journal of Electroanalytical Chemistry,2016,766:147-151.[5]WANG J,THOMAS D F,CHEN A.Nonenzymatic electrochemical glucose sensor based on nanoporous Pt-Pb networks[J].AnalyticalChemistry,2012,80(4):997-1004.[6]RONG L Q,YANG C,QIAN Q Y,et al.Study of the nonenzymatic glucose sensor based on highly dispersed Pt nanoparticles supported on carbonnanotubes[J].Talanta,2013,72(2):819-824.[7]郭合帅,付群,林伟,等.模板法制备银纳米点阵活性基底及其用于葡萄糖的高灵敏检测[J].上海大学学报(自然科学版),2015,21(1):54-63.[8]YEO I H,JOHNSON D C.Anodic response of glucose at copper-based alloy electrodes[J].Journal of Electroanalytical Chemistry,2012,484(2):157-163.[9]ZHANG X J,WANG G F,LIU X W,et al.Diあerent CuO nanostructures:synthesis,characterization,and applications for glucose sensors[J].The Journal of Physical Chemistry C,2011,112(43):16845-16849.[10]KURODA Y,HAMANO H,MORI T,et al.Specif i c adsorption behavior of water on a Y2O3surface[J].Langmuir,2000,16(17):6937-6947.[11]MEDWAY S L,LUCAS C A,KOWAL A,et al.In situ studies of the oxidation of nickel electrodes in alkaline solution[J].Journal of Electroanalytical Chemistry,2006,587(1):172-181.[12]LU L M,ZHANG L,QU F L,et al.A nano-Ni based ultrasensitive nonenzymatic electrochemical sensor for glucose:enhancing sensitivity through a nanowire array strategy[J].Biosensors andBioelectronics,2009,25(1):218-223.[13]张昱,豆小敏,杨敏,等.砷在金属氧化物/水界面上的吸附机制Ⅰ.金属表面羟基的表征和作用[J].2006,26(10):1586-1591.。

氧自由基与氧自由基清除剂依达拉奉

氧自由基与氧自由基清除剂依达拉奉山东大学齐鲁医院麻醉科（250012）于金贵一、氧自由基（一）自由基的概念自由基（freeradical，FR）是指外层轨道上有未配对电子的原子、原子团、分子或离子的总称。

因其含有未配对的电子，故化学性质非常活泼，极易与其生成部位的其他物质发生反应，而这种反应的最大特点是以连锁反应的形式进行。

氧原子上有未配对电子的自由基称为氧自由基。

人体吸入的分子氧，在正常状态下绝大多数（98%）都连接4个电子，它们最终与H+结合，代谢还原为H2O。

但有极少数氧（1~2%）在代谢过程中被夺去或接受一个电子而形成活性氧，即氧自由基。

（二）氧自由基的生理作用氧自由基在生理上是必需的物质，如合成ATP 和前列腺素、中性粒细胞杀灭细菌、酸性粒细胞杀灭寄生虫等过程都必须有氧自由基参与。

氧自由基在体内的生成与清除保持动态平衡，且在体内存在时间甚短。

由于其化学性极强，反应剧烈，过量产生会对机体造成极大危害。

（三）氧自由基的种类及其作用1. 超氧化物阴离子：氧自由基连锁反应的启动者，使生物膜、激素和脂肪酸过氧化。

2. 羟自由基（OH∙）：作用最强的自由基，可破坏氨基酸、蛋白质、核酸和糖类。

3. 过氧化氢（H2O2）：过渡型氧化剂，主要使巯基氧化，可氧化不饱和脂肪酸。

4. 单线态分子氧（1O2）：氧分子的激发状态，亲电子性强，在光作用下可由O2直接产生，对细胞有杀伤作用。

5.其他含氧的自由基如脂质过氧化物（ROOH）：易于分解再产生自由基，腐化脂肪，破坏DNA，可与蛋白质交联使之形成变性交聚物。

（四）机体抗氧化机制机制一：直接提供电子，以确保氧自由基还原；机制二：增强抗氧化酶的活性，以有效地消除或抵御氧自由基的破坏作用如酶类抗氧化剂超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-PX）；非酶类抗氧化剂如维生素E、维生素C、辅酶Q、还原型谷胱甘肽（GSH）、葡萄糖、含硫氨基酸和不饱和脂肪酸等。

211134068_盐酸氨基葡萄糖片中5-HMF的含量测定及形成动力学研究

盐酸氨基葡萄糖片中5-HMF 的含量测定及形成动力学研究Δ倪东宇 1， 2＊，王琼芬 2，张梦奇 2，李彬 2，石婧 2，徐虹 2，张帅 1 #（1.浙江海洋大学食品与药学学院，浙江舟山　316022；2.舟山市食品药品检验检测研究院，浙江舟山　316021）中图分类号 R 917 文献标志码 A 文章编号 1001-0408（2023）08-0950-05DOI 10.6039/j.issn.1001-0408.2023.08.11摘要目的建立测定盐酸氨基葡萄糖片中5-羟甲基糠醛（5-HMF ）含量的方法，分析其含量变化规律及影响因素。

方法采用高效液相色谱法对5-HMF 进行定量测定，以Shim-pack GIST C 18-AQ 为色谱柱，0.1%磷酸溶液-甲醇（90∶10，V /V ）为流动相，柱温为30 ℃，检测波长为284 nm ，流速为1.0 mL/min ，进样量为20 μL 。

通过不同温度反应动力学实验分析5-HMF 含量与反应温度、反应时间的相关性，建立其形成动力学模型。

结果 5-HMF 检测质量浓度的线性范围为0.057～5.698 μg/mL （r ＝0.999 9）；检测限为5.70 ng/mL ，定量限为17.09 ng/mL ；精密度、重复性和稳定性（24 h ）试验的RSD 均小于1.0%（n ＝6）；加样回收率为99.38%～99.73%（RSD ＝0.53%，n ＝9）。

8批样品含量为4.10～35.13 μg/g 。

反应动力学实验数据拟合结果显示，随着反应温度升高、反应时间延长，样品中5-HMF 含量越高。

50、60、70、80 ℃下，5-HMF 含量与反应时间均呈线性关系，符合零级动力学模型，反应速率常数分别为6.789、7.715、8.815、11.430。

结论所建含量测定方法专属性强、灵敏度高、准确度好；反应温度和反应时间是影响盐酸氨基葡萄糖片中5-HMF 形成的重要因素，其含量变化规律符合零级动力学模型。

银杏二萜内酯葡胺注射液对缺糖缺氧损伤的SH-SY5 Y细胞保护作用

银杏二萜内酯葡胺注射液对缺糖缺氧损伤的SH-SY5 Y细胞保护作用刘秋;许治良;周军;李娜;毕宇安;王振中;萧伟【期刊名称】《中国药理学通报》【年(卷),期】2015(000)007【摘要】Aim To investigate the protective effects of YXETNZ injection on SH-SY5 Y cells damaged by oxygen-glucose deprivation ( OGD ) , and explore its functional mechanisms. Methods After 4 h of OGD, the cells were treated with 25 mg·L-1 drugs for 1 h. Subsequently, cell viabilities were measured by cell counting kit-8 ( CCK-8 kit ) and cell apoptosis was measured by caspase-3/7 assay kit according to manu-facturer’ s instructions. Furthermore, cell death was also detected by ELISA. The levels of phospho-Akt, phospho-PKA,phospho-Bad were evaluated by western blot. Results Oxygen-glucose deprivation significant-ly decreased the cell viabilities of SH-SY5Y cells, while YXETNZ injection significantly increased cell vi-abilities, phospho-Akt, phospho-PKA and phospho-Bad. Furthermore, YXETNZ injection also reduced the activities of caspase-3/7 and cytoplasmic histone-asso-ciated-DNA-fragments contents. Conclusion Our re-searches demonstrat that YXETNZ injection shows good neuroprotective effects on SH-SY5 Y cells after oxygen-glucose deprivation. The underlying mechanisms may be associated with activation of PI3K/Akt/Bad/caspase-3/7 , cAMP/PKA/Bad/caspase-3/7 signaling pathway.%目的：探讨银杏二萜内酯葡胺注射液( YXETNZ)对缺糖缺氧( oxygen-glucose deprivation,OGD)损伤的人神经母细胞瘤细胞(SH-SY5Y)的保护作用及可能的机制。

平衡磷酸烯醇式丙酮酸节点通量强化筑波链霉菌合成他克莫司

2018年第37卷第6期 CHEMICAL INDUSTRY AND ENGINEERING PROGRESS·2347·化工进展平衡磷酸烯醇式丙酮酸节点通量强化筑波链霉菌合成他克莫司吕蒙蒙1，2，刘蛟1，2，刘欢欢1，2，陈红1，2，王成1，2，闻建平1，2（1天津大学化工学院系统生物工程教育部重点实验室，天津 300072；2天津大学天津化学化工协同创新中心，天津 300072）摘要：他克莫司（FK506）是最重要的免疫抑制剂之一，然而前体代谢供应不足制约着工业化生产。

通过优化平衡磷酸烯醇式丙酮酸（PEP ）节点支路通量可提高FK506产量。

本文首先在S. tsukubaensis D852中过表达基因fkb O （编码分支酸水合还原酶）和 fkb L （编码赖氨酸环化酶）得到S. tsukubaensis -OL1，FK506的产量仅从158.7mg/L 提高到163.9mg/L 。

随后调节PEP 节点支路回补途径和莽草酸途径通量强化FK506的合成：先分别将不同菌株中编码磷酸烯醇式丙酮酸羧化酶（PPC ）和3-脱氧-D-阿拉伯糖基-heptulosonate-7-磷酸合酶（DAHPS ）的基因在S. tsukubaensis -OL1中过表达，FK506的产量分别提高40%（ppc ，S. tsukubaensis ）和47%（dah P ，S. roseosporus ）；然后采用4个不同强度的组成型启动子（P ermE *，P sco 4503，P sco 3410 and P sco 5768）平衡ppc 和dah P 的表达水平获得9株工程菌，最终使FK506的产量由163.9mg/L 显著提高到350.3mg/L 。

这个结果说明优化平衡PEP 节点竞争支路通量是提高FK506产量的有效策略。

关键词：他克莫司；回补途径；莽草酸途径；组成型启动子；筑波链霉菌中图分类号：Q591 文献标志码：A 文章编号：1000–6613（2018）06–2347–07 DOI ：10.16085/j.issn.1000-6613.2017-1482Balancing carbon flux rebalancing around phosphoenolpyruvate node forenhancement of FK506 production in Streptomyces tsukubaensisLÜ Mengmeng 1，2，LIU Jiao 1，2，LIU Huanhuan 1，2，CHEN Hong 1，2，WANG Cheng 1，2，WEN Jianping 1，2（1Key Laboratory of System Bioengineering （Tianjin University ），Ministry of Education ，Tianjin 300072，China ；2SynBio Research Platform ，Collaborative Innovation Center of Chemical Science and Engineering (Tianjin)，School ofChemical Engineering and Technology ，Tianjin University ，Tianjin 300072，China ）Abstract ：Tacrolimus （FK506），as one of the widely used immunosuppressants produced by Streptomyces species ，has drawn much attention on clinic application. However ，the low FK506 fermentation titer restricts its industrial production ，which is mainly due to the insufficient precursor metabolism of the producing strain. In this work ，balancing carbon flux rebalancing around phosphoenolpyruvate （PEP ）node for enhancement of FK506 production were carried on. Firstly ，the genes fkb O and fkb L were overexpressed in S. tsukubaensis D852，achieving S. tsukubaensis -OL1，of which FK506 production changed only slightly from 158.7mg/L to 163.9mg/L. Then ，two precursor metabolic pathways ，the anaplerotic and shikimate pathways emanating from PEP node ，were fine-tuned for eliminating the inefficient supply of precursors of DHCHC and pipecolate. The genes encoding PPC and DAHPS were cloned from various species and expressed in S. tsukubaensis -OL1，自然科学基金项目（21376171）。

酶在贮藏保鲜中的应用

解决氧化问题的根本办法是脱氧。葡萄糖氧化酶是一种理想的除氧保鲜剂，可有效防止食品因氧化而引起的质量下降和变质。罐藏食品可以使用含葡萄糖氧化酶的吸氧保鲜袋防止氧化，罐装果汁、酒和水果罐头等可以直接加入葡萄糖氧化酶以保持品质，另外葡萄糖氧化酶也可以有效地防止罐装容器的氧化作用。
3、防止微生物繁殖
用葡萄糖氧化酶—过氧化氢酶体系将还原糖分子上的醛基转变成羧基，这样就消除了美拉德反应中的底物之一----还原糖。反应中需要不断地供给氧气，因此使用葡萄糖氧化酶脱糖时应分次加入适量的H2O2溶液，这样同葡萄糖氧化酶一起使用的过氧化氢酶就能分解H2O2，以补充反应所需要的氧。
2、脱氧保鲜
葡萄糖氧化酶本身并不具有抗微生物的作用，但由于葡萄糖氧化酶能去除氧，所以能防止好气菌的生长繁殖；形成的葡萄糖酸引起pH下降，也有抑制作用；同时由于产生过氧化氢具有细胞毒性，也可起到杀菌的作用。因此葡萄糖氧化酶可用于在特殊情况下防止微生物的繁殖。
二、溶菌酶在食品保鲜方面的应用
溶菌酶（ N- 乙酰胞壁质聚糖水解酶， EC 3.2.1.17）又称为胞壁质酶，细胞壁溶解酶。是一种专门作用于微生物细胞壁的水解酶。溶菌酶是由129个氨基酸构成的单纯碱性球蛋白，化学性质非常稳定。
1、脱糖保鲜
用葡萄糖氧化酶去除食品中残留的葡萄糖，目前应用最多的是脱水制品如蛋白粉、蛋白片的生产。由于蛋白中含有0.5%～0.6%的葡萄糖，因此在蛋类制品加工和贮藏过程中，极易发生葡萄糖分子中的羰基与蛋白质分子的氨基结合生成黑蛋白的
美拉德反应。
美拉德反应不但导致食品中葡萄糖和游离氨基消失，还会使食品褐变、营养损失，风味也会发生变化，甚至产生有毒物质。因此，在蛋制品加工过程中往往要先进行蛋白的脱糖处理，以防止食品因氧化而引起的品质下降和变质。早期在蛋制品工艺中多是采用干或湿酵母发酵的方法除去葡萄糖，该方法的缺点是周期长，卫生条件差，产品质量也不理想。近几年来，在干制蛋品加工中已普遍采用葡萄糖氧化酶进行脱糖处理。

cx3cl1 cx3cr1-mediated microglia activation plays a detrimental role in ischemic mice brain via

ORIGINAL ARTICLECX3CL1/CX3CR1-mediated microglia activation plays a detrimental role in ischemic mice brain via p38MAPK/PKC pathwayYong Liu 1,5,Xiao-Mei Wu 2,3,5,Qian-Qian Luo 2,4,Suna Huang 4,Qing-Wu Qian Yang 1,Fa-Xiang Wang 1,Ya Ke 3and Zhong-Ming Qian 2,4INTRODUCTIONMicroglia is key modulators of the immune response in the brain.1Under normal physiologic conditions,these cells constantly survey their microenvironment for noxious agents and injurious processes,2respond to extracellular signals and are responsible for clearing debris and toxic substances by phagocytosis,thereby maintaining normal cellular homeostasis in the central nervous system.3Under the pathologic circumstance,these cells could be activated at very early stage.4In animal models of cerebral ischemia,the processes of microglial activation have been studied extensively,however,the exact function of these activated cells is not fully understood 5and the ﬁndings reported are controversial.6–8The answers to the question of whether activated microglial responses are destructive or bene ﬁcial after ischemic injury remains equivocal.4,8Microglial activation is usually regulated by the chemokine fractalkine (CX3CL1)CX3CL1and its receptor,CX3CR1.4CX3CL1is a relatively new member of the chemokine (chemotactic cytokine)family and the sole member of the CX3C chemokine class,9–10which exists in both membrane bound and soluble forms.9In contrast to many other chemokines,CX3CL1binds only one receptor CX3CR1.11In the brain,CX3CL1is a unique chemokine,being constitutively expressed by neurons where it is tethered to the extracellular membrane by a mucin stalk.9,12The CX3CL1receptor CX3CR1is a G-protein-coupled receptor and exclusively expressed by microglia.13The CX3CL1/CX3CR1signaling pathway has been shown to play an important role in the maintenance of neural –immune communication and the bidirectional interaction between neurons and microglia in health and disease.14,15A number of studies have been conducted to investigate the role of CX3CL1/CX3CR1signaling in brain ischemic injury,however,the relevant ﬁndings are also controversial.The destructive or bene ﬁcial roles of CX3CL1/CX3CR1in brain ischemic injury have also been reported.Mice de ﬁcient in CX3CL1were found to be less susceptible to cerebral ischemia –reperfusion injury when compared with wild-type littermates.16And lack or de ﬁciency of CX3CR1was shown to reduce signi ﬁcantly ischemic damage and in ﬂammation in mice with focal cerebral ischemia 171Department of Neurology,Xinqiao Hospital,The Third Military Medical University,Chongqing,China;2Department of Neurobiology,Institute for Nautical Medicine,Nantong University,Nantong,China;3School of Biomedical Sciences,Faculty of Medicine,The Chinese University of Hong Kong,New Territories,Hong Kong,China and 4Laboratory of Neuropharmacology,Fudan University School of Pharmacy,Pu Dong,Shanghai,China.Correspondence:Dr Y Ke,School of Biomedical Sciences,Faculty of Medicine,The Chinese University of Hong Kong,New Territories,Hong Kong,China or Dr ZM Qian,Laboratory of Neuropharmacology,Fudan University School of Pharmacy,Pu Dong,Shanghai 201203,China.E-mail:yake@.hk or qianzhongming@The studies in our laboratories were supported by the Competitive Earmarked Grants of The Hong Kong Research Grants Council (GRF 466713),National 973Programs (2011CB510004and 2014CB541604),General Grant of National Natural Science Foundation of China (NSFC;81070930,81471108,31271132and 31371092),and Key Project Grant of NSFC (31330035-2013).5These authors shared ﬁrst authorship.Received 23February 2014;revised 8April 2015;accepted 13April 2015Journal of Cerebral Blood Flow &Metabolism (2015),1–9©2015ISCBFM All rights reserved 0271-678X/15$and to suppress activation and neurotoxicity of microglia/macro-phage in experimental ischemic stroke.18In addition,CX3CL1-and CX3CR1-knockout mice revealed to have less severe brain injury on permanent19and transient20middle cerebral artery occlusion. These studies suggested that CX3CL1–CX3CR1expression is detrimental to recovery after ischemic injury.15,16However,it has also been reported that CX3CR1deﬁciency worsens the behavioral impairment induced by transient global cerebral ischemic injury and silencing CX3CR1expression exacerbates the learning and memory deﬁcits.4Moreover,administration of exogenous CX3CL1 was found to reduce ischemia-induced cerebral infarct size and neurologic deﬁcits in in vivo murine models of permanent middle cerebral artery occlusion.19These conﬂicting data to date do not provide a coherent conclusion on the role of CX3CL1/CX3CR1in brain injury and disease.4,20To further explore the roles of this signaling pathway andmicroglial activation in brain ischemic injury,we investigated theeffects of CX3CR1siRNA(silencing CX3CR1expression)onexpression of CX3CR1,p38mitogen-activated protein kinase(p38MAPK),protein kinase C(PKC),tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6,and also microglia activation,white matter lesions,and cognitive function in the brain in mice modelof bilateral common carotid artery stenosis(BCAS)in vivo.We alsoexamined the effects of the addition of exogenous CX3CL1withCX3CR1siRNA and SB2035080(SB,a p38MAPK inhibitor)on theexpression of TNF-αand IL-1βin OGD(oxygen–glucose depriva-tion)treated BV2microglial cells in vitro.The p38MAPK waschosen to be examined because this signaling molecule has beenwell documented to be associated with the expression ofinﬂammatory and cytotoxic mediators.21Ourﬁndings indicatedthat CX3CL1,acting on the CX3CR1of microglia,activates p38MAPK/PKC and promotes the generation of TNF-αand IL-1βinducing a detrimental effect in the brain of ischemic mice under our experimental conditions.MATERIALS AND METHODSMaterialsUnless otherwise stated,all chemicals were obtained from Sigma Chemical Company,St.Louis,MO,USA.Rabbit polyclonal TNF-αwas purchased from Afﬁniti Research,Devon,UK,and rabbit monoclonal IL-1βand rabbit polyclonal IL-6from Lab Vision Corporation,Fremont,CA,USA. Rabbit polyclonal anti-CX3CR1was obtained from Merck Millipore,Billerica, MA,USA,antibodies against p38MAPK and PKC were purchased from CST,Cell Signaling Technology,Danvers,MA,USA and TNF-αand IL-1βELISA(enzyme-linked immunosorbent assay)kits from R&D Systems China,Shanghai,PRC.Sheep antirat biotinylated IgG,antirabbit secondary antibody–conjugated horseradish peroxidase(HRP),and HRP–streptavidin were obtained from Zhongshan Biotech,Beijing,China;rat monoclonal anti-CD11b antibody from AbD Serotec,Kidlington,UK;and Bradford assay kit from Bio-Rad,Hercules,CA,USA.CX3CR1siRNA(against CX3CR1 retrovirus)and negative siRNA(negative control)were obtained from GeneChem,Shanghai,PRC.Animals,BV2Microglia,and Primary Cultured NeuronsMale C57BL/6J mice(8to10weeks)weighing20to24g were supplied by the Animal Center of the Third Military Medical University(Chongqing, China)and housed in individual cages under a standard12-hour light–dark cycle with water and food supplied ad libitum.All animal handling and surgical procedures were approved by the Animal Research Ethics Committee of the Third Military Medical University in accordance with the guidelines of the Chongqing City Health Department on Animal Care. The experiments were performed in accordance with the Animal Research: Reporting In Vivo Experiments guidelines.BV2microglial cells were cultured in Dulbecco's modiﬁed Eagle's medium(DMEM;Gibco,Carlsbad,CA,USA)supplemented with10%fetal bovine serum(Gibco)and100μg/mL penicillin–streptomycin(Gibco)at 37°C in humidiﬁed atmosphere containing5%CO2.22The primary cortical neuronal culture was prepared from embryonic day14to15C57BL/6J mice using a method as described previously.23Experimental designIn vivo study.Two-vessel occlusion caused tissue injury in C57BL/6mice, which is highly variable because of the anatomic variations of the posterior communicating artery.24–26We therefore premeasured cerebral bloodﬂowbefore and2hours after BCAS with laser-Dopplerﬂowmetry as describedpreviously27to exclude animals that are less susceptible(cerebral blood ﬂow2hours after BCAS/cerebral bloodﬂow before⩾65%)to ischemic insult.To determine the role of CX3CL1/CX3CR1-mediated activation ofmicroglia and the relevant mechanisms under the conditions of ischemia,a total of27susceptible C57BL/6J mice were randomly assigned into three groups.The mice in BCAS or BCAS+siRNA group were pretreated with3μL of saline(BCAS group,n=9)or CX3CR1siRNA(BCAS+siRNA group,n=11) by intracerebroventricular injection,andﬁve days later,underwent with BCAS.The animals in the sham-operated group(n=7)underwent the same surgical procedure as the above two groups without BCAS.At day14after BCAS,the expression of CX3CR1,p38MAPK,and PKC,and the contents of TNF-α,IL-1β,and IL-6were determined by Western blot assay,and also microglia activation and white matter lesions were evaluated by histochemical method,cognitive function assessed by water maze test, and the expression of CX3CR1was also assessed by immunostain analysis.In vitro study.To further explore the relevant mechanisms involved in the role of fractalkine/CX3CR1-mediated activation of microglia under ischemia,the BV2microglial cells were treated with Oxygen–glucose deprivation(OGD)in the presence of CX3CL1(0,12.5,25,and50ng/mL),or infected with10Multiplicity of Infection(MOI)CX3CR1siRNA or negative siRNA for72hours or pretreated with20μmol/L of SB2035080only for 1hour,before subjected to OGD for4hours in the presence of0or50ng/ mL of CX3CL1.To investigate whether neurons prepared from C57BL/6J mice could secrete and express CX3CL1and whether OGD affect the release and expression of CX3CL1in neurons,neurons were treated with OGD for4hours and then reperfusion(R)for24hours.The contents of CX3CL1in neuronal medium was detected by ELISA and the expression of CX3CL1in neurons were observed by immunostaining.OGD was performed by exposing the cells to serum-free DMEM without glucose in a hypoxic incubator(INVIVO2,RUSKINN,Sanford,Maine,USA)with1%O2, 94%N2,and5%CO2at37°C for4hours as previously described.28After the treatments,the measurements of TNF-α,IL-1β,and CX3CL1were conducted using ELISA.Intracerebroventricular InjectionInjections(intracerebroventricular)were accomplished via an indwelling guide cannula stereotaxically implanted into the right lateral cerebral ventricle(bregma−0.58mm,lateral1.20mm,and depth2.25mm).28Bilateral Common Carotid Artery StenosisMale C57BL/6J mice were subjected to BCAS,which was performed by applying the microcoils(Sawane Spring,Osaka,Japan)with an inner diameter of0.18mm to common carotid arteries(CCAs).26Brieﬂy,both CCAs were exposed and freed from their sheaths,through a midline cervical incision,after the mice were intraperitoneally anesthetized with sodium pentobarbital(50mg/kg).Two4to0silk sutures were placed around the distal and proximal parts of the right CCA.Then,the artery was gently lifted by these sutures and placed between the loops of the microcoil just below the carotid bifurcation.The microcoil was twined by rotating it around the CCA.After30minutes,another microcoil of the same size was twined around the left CCA.The rectal temperature was maintained between36.5°C and37.5°C.Sham-operated mice underwent the same surgical procedure without using coils.Western Blot AssayThe expression of CX3CR1,p38MAPK,PKC,TNF-α,IL-1β,and IL-6in brain tissue of the mice were determined as described previously.29Total protein content was determined using the Bradford assay kit(Bio-Rad).The blots were probed with primary antibodies:rabbit polyclonal anti-CX3CR1 (1:1,000),p38MAPK(1:1,000),PKC(1:1,000),rabbit polyclonal TNF-α(1:500), rabbit monoclonal IL-1β(1:1,000),and rabbit polyclonal IL-6(1:1,000),and then antirabbit secondary antibody–conjugated horseradish peroxidase (1:2,500).The intensity of the speciﬁc bands was detected and analyzed by Odyssey infrared imaging system(Li-Cor Biosciences,Lincoln NE,USA).To ensure even loading of the samples,the same membrane was probed with rabbit antiratβ-actin polyclonal antibody at a1:2,000dilution.CX3CL1/CX3CR1and microglia activationY Liu et al 2Enzyme-Linked Immunosorbent AssayTNF-αand IL-1βconcentrations in the culture medium of BV2microglia and CX3CL1in the culture medium of neurons were determined using commercially available ELISA kits according to the manufacturer ’s instruction (R&D Systems,China).The optical density at 450nm was read by using an ELX-800microplate assay reader (Elx800,Bio-tek,Winooski,VT,USA).The average absorbance values for each set of standards and samples were calculated from the standard curve.30ImmunohistochemistryCoronal sections (30μm)were treated with 3%H 2O 2in 0.01mol/L phosphate-buffered saline and preincubated in 5%normal goat serum.Sections were then incubated with the primary antibody,rat monoclonal anti-CD11b antibody (1:100)at 4°C overnight,then with sheep antirat biotinylated IgG (1:200)for 1hour at room temperature and ﬁnally incubated in HRP –streptavidin (1:200)for 1hour at room temperature.The color reaction was conventionally developed with 3,3′-diaminobenzidine and H 2O 2.White Matter Lesion EvaluationAt day 14after BCAS,the brains were removed and post ﬁxed in 4%paraformaldehyde in 0.1mol/L phosphate buffer (PB)(pH 7.4)for 12hour,and then stored in 30%sucrose in 0.1mol/L PB (pH 7.4).Serial coronal sections were cut on a cryostat,spanning from the anterior aspect oftheFigure 1.CX3CR1siRNA signi ﬁcantly inhibits the increased expression of CX3CR1,p38MAPK,and PKC,and microglia activation induced by bilateral common carotid artery stenosis (BCAS)in mice brain in vivo .The mice in BCAS (n =9)or BCAS+siRNA (n =11)group were pretreated with saline or CX3CR1siRNA,and then underwent with BCAS.The animals in the sham-operated group (n =7)underwent the same surgical procedure as the above two groups without BCAS.At day 14after BCAS,the expression of CX3CR1,p38MAPK,and PKC (A and B )was determined by Western blot assay.The expression of CX3CR1was also assessed by immunostain analysis (C )and microglia activation was evaluated by histochemical method using rat monoclonal anti-CD11b antibody (D ),and the data were quanti ﬁed respectively (E ,CX3CR1;F ,anti-CD11b).Scale bar,50μm in C and D .Data were represented as mean ±s.e.m.*P o 0.05versus sham;#P o 0.05versus BCAS.IOD,integrated optical density;OD,optical density.CX3CL1/CX3CR1and microglia activation Y Liu et al3corpus callosum(bregma0.26mm)to the anterior aspect of the hippocampus(bregma−0.58mm).Everyﬁfth section(180μm)was cut at10μm and processed for Klüver-Barrera staining.The severity of the lesions in the corpus callosum was graded as normal(grade0), disarrangement of the nerveﬁbers(grade1),formation of marked vacuoles(grade2),or disappearance of myelinatedﬁbers(grade3).26 Water Maze TestThe cognitive function was assessed using a Morris water maze paradigm at day14after BCAS.31The testing was conducted inﬁve consecutive days. Each mouse was subjected to eight trials per day with a5-minute break between trials.A white and circular pool(1.5-m diameter and45-cm deep) wasﬁlled with water to a25-cm depth.Water temperature was maintained at~22°C.A clear Plexiglas platform(11×12cm)was positioned1cm below the surface of water.Mice were placed in the tank facing the wall, randomizing to one of four starting locations(north,south,east,or west), and given90seconds toﬁnd the platform,mount the platform,and remain on it for5seconds.The mice were then placed under a heat lamp to dry before their next trial.The time until the mouse mounted the platform (escape latency)was measured and recorded.The data of Morris water maze test were analyzed by the repeated measures analysis of variance. Statistical AnalysisStatistical analyses were performed using SPSS software for Windows (version10.0;SPSS,Chicago,IL,USA).Data were presented as mean±s.e.m. The difference between or among the means was determined by Kruskal–Wallis test followed by Mann–Whitney test for multiple comparisons or one-way or two-way analysis of variance in appropriate experiments followed by Newman–Keuls post hoc test.A probability value of P o0.05 was considered to be statistically signiﬁcant.RESULTSCX3CR1siRNA Signiﬁcantly Inhibits the Increased Expression of CX3CR1as well as p38MAPK and PKC Induced by Bilateral Common Carotid Artery Stenosis in Mice Brain In VivoTo determine the role of CX3CL1/CX3CR1-mediated activation ofmicroglia and the relevant mechanisms under ischemia,we initially investigated the effects of BCAS on the expression ofCX3CR1and also p38MAPK and PKC in mice brain.Western blot analysis showed that the contents of CX3CR1as well as p38MAPKand PKC in the brain of mice treated with BCAS all were signiﬁcantly higher than those of mice in sham-operated group(Figures1A and1B).Immunostain analysis also showed that BCAS could signiﬁcantly increase CX3CR1expression(Figure1C).Theseevidenced that ischemia induced by BCAS could induce a signiﬁcant increase in the expression of the receptor and molecules involved in the signal transduction pathway.We thenobserved the effects of CX3CR1siRNA on the expression of CX3CR1,p38MAPK,and PKC in the brain of the ischemic mice to ﬁnd out whether the increased expression of p38MAPK and PKC is associated with the expression of CX3CR1.We showed that thelevels of CX3CR1,p38MAPK,and PKC in the mice treated with CX3CR1siRNA and then BCAS were markedly lower than thecorresponding values in the mice treated with BCAS only(Figures 1A and1C).Theﬁnding indicated that the reduced expression of CX3CR1induced by CX3CR1siRNA could lead to a remarkable reduction in the expression of p38MAPK and PKC in the brain of BCAS mice and evidenced that the signiﬁcant increase in the expression of p38MAPK and PKC induced by BCAS is CX3CR1 dependent in mice brain.CX3CR1siRNA Signiﬁcantly Inhibits Microglia Activation Induced by Bilateral Common Carotid Artery Stenosis in Mice Brain In Vivo Second,we investigated whether the ischemia induced by BCAS could activate microglia by immunohistochemical analysis using CD11b,which was used to identify ischemia-induced activation of microglia.32Immunohistochemical data(Figure1D)revealed that microglia have swollen and larger cell bodies with thicker and shorter processes that form thick bundles around an enlarged cell body or a large cell body with almost absent processes in the brain of mice treated with BCAS,indicating that BCAS could lead to activation of microglia.We then evaluated the effects of CX3CR1siRNA(inhibiting CX3CR1expression)on microglia activation induced by BCAS to understand whether the activation is related to the expression of CX3CR1.We showed that the morphologic changes found in microglia in the mice treated with BCAS disappeared mostly in the brain of the mice treated with CX3CR1siRNA and then BCAS.Theﬁndings evidenced that treatment with CX3CR1siRNA could signiﬁcantly attenuate the BCAS-induced microglia activation and also implied that CX3CR1 played a key and essential role in BCAS-induced microglia activation in mice brain.CX3CR1siRNA Signiﬁcantly Inhibits the Increased Expression of Tumor Necrosis Factor-α,Interleukin-1β,and Interleukin-6in Mice Brain In VivoThird,we examined the effects of ischemia induced by BCAS on the expression of microglia cytokines,including TNF-α,IL-1β,and IL-6,in mice brain.Western blot analysis showed that the contents of TNF-αas well as IL-1βand IL-6proteins in the brain of mice treated with BCAS were signiﬁcantly higher than those of mice in the sham-operated group(Figures2A and2B).This showed that ischemia could induce a remarkable increase in the expression of these microglia cytokines.We then explored the effects ofCX3CR1Figure2.CX3CR1siRNA signiﬁcantly reduces the increased expres-sion of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6in mice brain in vivo.The mice in bilateral common carotid artery stenosis(BCAS,n=9)or BCAS+siRNA(n=11)group were pretreated with saline or CX3CR1siRNA by intracerebroventricular injection, and then treated with BCAS.The animals in the sham-operated group(n=7)underwent the same surgical procedure as the above two groups without BCAS.At day14after BCAS,the expression of TNF-α,IL-1β,and IL-6was determined by Western blot assay.(A)A representative experiment of Western blot.(B)Quantiﬁcation of expression of proteins.Data were represented as mean±s.e.m. *P o0.05versus sham;#P o0.05versus BCAS.OD,optical density.CX3CL1/CX3CR1and microglia activationY Liu et al 4siRNA on the expression of TNF-α,IL-1β,and IL-6in the brain of the ischemic mice toﬁnd out whether the increased expression of TNF-α,IL-1β,and IL-6is associated with the expression of CX3CR1. We showed that the levels of TNF-α,IL-1β,and IL-6in the mice treated with CX3CR1siRNA and then BCAS were signiﬁcantly lower than those in the mice treated with BCAS only.Theﬁnding suggested that the reduced expression of CX3CR1induced by CX3CR1siRNA could lead to a remarkable reduction in the expression of TNF-α,IL-1β,and IL-6in the brain of BCAS mice and evidenced that the signiﬁcant increase in the expression oftheseFigure3.CX3CR1siRNA signiﬁcantly attenuates white matter lesions and cognitive deﬁcits induced by bilateral common carotid artery stenosis(BCAS)in mice.The mice in BCAS(n=9)or BCAS+siRNA(n=11)group were pretreated with saline or CX3CR1siRNA by intracerebroventricular injection,and then underwent BCAS.The animals in the sham-operated group(n=7)underwent the same surgical procedure as the above two groups without BCAS.At day14after BCAS,grading scores=white matter rarefaction grading scores(A)were evaluated by histochemical method,and cognitive function,Mean Escape latency(seconds)=mean escape latency(seconds;B)assessed by water maze test.(C)A representative histologic analysis of white matter lesion.Data were represented as mean±s.e.m.*P o0.05versus sham;#P o0.05versusBCAS.Figure4.Treatment with oxygen–glucose deprivation(OGD)and CX3CL1signiﬁcantly increase the expression of tumor necrosis factor-α(TNF-α)and interleukin(IL)-1βin BV2microglia in vitro.BV2microglial cells were treated with OGD for4hours in the presence of CX3CL1(0,12.5,25,and50ng/mL),and then the measurements of TNF-αand IL-1βin the medium were conducted using enzyme-linked immunosorbent assay(ELISA)as described in the Materials and Methods section.(A and B)The levels of TNF-αand IL-1βin the medium of BV2microglia treated with OGD for4hours.(C and D)The contents of TNF-αand IL-1βin the medium of BV2microglia treated with OGD plus different concentrations of CX3CL1.Data were represented as mean±s.e.m.(n=5).*P o0.05versus the control(A and B)or0ng/mL CX3CL1(C and D).CX3CL1/CX3CR1and microglia activationY Liu et al5microglia cytokines induced by BCAS is also CX3CR1dependent in mice brain.CX3CR1siRNA Signiﬁcantly Attenuates White Matter Lesions and Cognitive Deﬁcits Induced by Bilateral Common Carotid Artery Stenosis in MiceFinally,we examined the effects of BCAS and CX3CR1siRNA on white matter lesion and cognitive function by histochemical method and water maze test.Histochemical analysis(Figures3A and3C)showed that the white matter rarefaction grading scores in mice of the BCAS group were signiﬁcantly higher than those in mice of the sham-operated group.However,the scores in mice in the BCAS group were signiﬁcantly lower than those in mice in the BCAS+siRNA group.Theﬁndings implied that BCAS could lead to white matter lesion,while CX3CR1siRNA could dramatically suppress the lesion in mice.The results obtained from water maze test(Figure3B)showed that the mean escape latency in mice of the BCAS group were signiﬁcantly longer than those in mice of the sham-operated group.However,the mean escape latency in mice in the BCAS group were signiﬁcantly shorter than those in mice in the BCAS+siRNA group.The data indicated that BCAS could lead to cognitive deﬁcits,while CX3CR1siRNA could signiﬁcantly attenuate the deﬁcits in mice.Theﬁndings also showed that CX3CR1-associated microglia activation plays a detrimental role in ischemic mice under our experimental conditions.CX3CL1Induced a Further Enhancement in Tumor Necrosis Factor-αand Interleukin-1βExpression,Which Could be Signiﬁcantly Attenuated by CX3CR1siRNA or the p38MAPK Inhibitor in Oxygen–Glucose Deprivation–Treated BV2Microglial Cells In Vitro To further investigate the relevant mechanisms involved in the detrimental role of CX3CR1-associated activation of microglia under ischemia,we then investigated the effects of exogenous CX3CL1with CX3CR1siRNA and SB2035080on the expression of microglia cytokines TNF-αand IL-1βin OGD-treated BV2micro-glial cells in vitro.CX3CL1was used in this study because its role in the CX3CR1-associated activation of microglia was well demonstrated.11Theﬁndings(Figures4A and4B)showed that the levels of TNF-αand IL-1βin the culture medium were signiﬁcantly higher in OGD-treated BV2microglia than in control cells, indicating that OGD could induce a signiﬁcant increase in TNF-αand IL-1βrelease from BV2microglia.Also,the addition of different concentrations(0,12.5,25,or50ng/mL)of CX3CL1led to a progressive increase in the contents of TNF-αand IL-1βin the BV2microglial culture medium(Figures4C and4D),implying that CX3CL1has a role to increase TNF-αand IL-1βexpression in BV2 microglia treated without OGD.In addition,the treatment with OGD plus50ng/mL of CX3CL1induced a more signiﬁcantincreaseFigure5.CX3CR1shRNA or SB2035080(SB,a p38MAPK inhibitor)signiﬁcantly reduce the increased expression of tumor necrosis factor-α(TNF-α)and interleukin(IL)-1βinduced by oxygen–glucose deprivation(OGD)and CX3CL1in BV2microglia in vitro.BV2microglial cells were infected with CX3CR1siRNA or negative siRNA for72hours(A and B)or pretreated with20μmol/L of SB2035080only for1hour(C and D), then subjected to OGD for4hours in the presence of50ng/mL of CX3CL1.After the treatments,the measurements of TNF-αand IL-1βwere conducted using enzyme-linked immunosorbent assay(ELISA)as described in the Materials and Methods section.(A and C)TNF-α;(B and D) IL-1β.Data were represented as mean±s.e.m.(n=5).*P o0.05versus the control;#P o0.05versus OGD;and@P o0.05versus CX3CL1+OGD.CX3CL1/CX3CR1and microglia activationY Liu et al6in the expression of TNF-αand IL-1βin BV2microglia as compared with the cells treated with OGD only(Figure5).This increase in the expression of TNF-αand IL-1βinduced by OGD and exogenous CX3CL1could be dramatically suppressed by pretreatment of the cells with CX3CR1siRNA(Figures5A and5B)or the p38MAPK inhibitor(20μmol/L of SB;Figures5C and5D).Theseﬁndings showed that the increased expression of TNF-αand IL-1βinduced by the addition of CX3CL1in OGD-treated BV2microglia is CX3CR1and also p38MAPK dependent.Bilateral Common Carotid Artery Stenosis and Oxygen–Glucose Deprivation Induced a Signiﬁcant Increase in CX3CL1Expression In Vivo and In VitroIn vivo experiments showed that there was a basal level of CX3CL1 expression in mice brain cortex(Figure6A—sham),while BCAS could induce a signiﬁcant increase in CX3CL1expression in this brain region(Figure6A).Immunostaining analysis showed thatthere also was a basal level of CX3CL1expression in normalcultured neurons(Figure6B).Meanwhile,OGD/R treatmentinduced a signiﬁcant increase in CX3CL1level in the neurons(Figure6B)and also in the culture medium(Figure6C).These ﬁndings evidenced that CX3CL1was not only expressed in neurons but also released from neurons in the brain under theconditions of ischemia.DISCUSSIONIn the present study,we showed that ischemia induced by BCAScould lead to white matter lesion,as reﬂected by the signiﬁcantlyincreased white matter rarefaction grading scores and inducecognitive deﬁcits as evidenced by the much longer mean escapelatency in ischemic mice.By using CD11b identiﬁcation,weFigure6.Effects of bilateral common carotid artery stenosis(BCAS)and oxygen–glucose deprivation(OGD)on CX3CL1expression in the brain cortex in vivo and primary cultured neurons in vitro.Mice were treated with BCAS as described in the Materials and Methods section and thenCX3CL1expression was examined by immunostaining(A).Primary cultured neurons were treated with OGD for4hours followed by reoxygenation(R)for24hours.The CX3CL1expression in neurons was then detected by immunostaining(B)and CX3CL1contents in the neuronal culture medium measured by enzyme-linked immunosorbent assay(ELISA;C).Data were represented as mean±s.e.m.(n=3).*P o0.05versus the control.CX3CL1/CX3CR1and microglia activationY Liu et al7。

葡萄糖饥饿对HeLa细胞形态与结构的影响

葡萄糖饥饿对HeLa细胞形态与结构的影响曾杰;陈佳;邵邻相;苏佳佳【摘要】为了探究葡萄糖饥饿对HeLa细胞形态与结构的影响,采用噻唑蓝法检测了细胞活力;用倒置显微镜、荧光显微镜和电子显微镜观察了细胞形态与结构的变化;采用激光共聚焦免疫荧光技术观察了细胞微丝与微管的分布.结果表明:葡萄糖饥饿能够抑制HeLa细胞增殖,破坏细胞骨架,改变细胞形态;使细胞皱缩、染色质凝集,出现凋亡小体,微丝微管解聚,表现出典型的凋亡特征,且细胞凋亡程度呈葡萄糖浓度依赖性和处理时间依赖性.总之,葡萄糖饥饿能抑制HeLa细胞活性,改变细胞形态,诱导细胞凋亡.%It was investigated the effects of glucose deprivation on morphology and structure of HeLa cells. The cell viability was analyzed by MTT assay,and the cell morphology and structure were observed by inverted mi-croscopy,fluorescence and electron microscope. Additionally, immunofluorescence analysis and confocal mi-croscopy were performed to observe the distribution of microtubules and microfilaments. The results showed that glucose deprivation could obviously inhibit the growth of HeLa cells and induce changes of morphology by destroying their skeletons. The cells exhibited typical apoptotic phenotype,including cell shrinkage,chroma-tin condensation,apoptotic bodies appeared,the microtubules and microfilaments depolymerized. With loss of the glucose concentration and increase of processing time, the degree of apoptosis increased. Glucose starva-tion could inhibit HeLa cell viability,modify cell shape,induce cell apoptosis.【期刊名称】《浙江师范大学学报（自然科学版）》【年(卷),期】2018(041)001【总页数】5页(P92-96)【关键词】葡萄糖饥饿;HeLa细胞;细胞形态;细胞结构;凋亡【作者】曾杰;陈佳;邵邻相;苏佳佳【作者单位】浙江师范大学化学与生命科学学院,浙江金华 321004;浙江师范大学化学与生命科学学院,浙江金华 321004;浙江师范大学化学与生命科学学院,浙江金华 321004;浙江师范大学化学与生命科学学院,浙江金华 321004【正文语种】中文【中图分类】Q291葡萄糖作为真核生物的主要能源物质和新陈代谢中间产物，对生物体的生长发育具有重要作用，因此，体内葡萄糖水平对生物体的健康具有重要影响.近几年的研究表明:体内高浓度的葡萄糖可增加大多数癌症的发病机率[1]，并可促进癌细胞的增殖、转移和粘附[2];低浓度葡萄糖有益于人体对抗由高糖分解癌症和转移性肿瘤(HGCM)引发的疾病[3]，并可降低癌细胞的增殖[4]．肿瘤细胞表现出异常的新陈代谢，其葡萄糖的摄取和利用速度增加，糖酵解代谢增强，胞外乳酸堆积增多，肿瘤细胞从细胞外环境摄取的葡萄糖的量大约是正常细胞的10多倍[5]．葡萄糖为肿瘤细胞的生长提供了充分的能量，因此，通过剥夺葡萄糖的癌症治疗方法受到人们的广泛关注．文献[6]首次解析了人源葡萄糖转运蛋白GLUT1的晶体结构，有可能实现葡萄糖转运的人工干预，通过特异性阻断对癌细胞的葡萄糖供应，抑制癌细胞生长．文献[7]用碳酸氢钠去除肿瘤内的氢离子，可破坏乳酸根和氢离子的协同作用，从而快速有效地杀死处于葡萄糖饥饿或缺乏的肿瘤细胞．葡萄糖饥饿是指通过降低培养基中的葡萄糖浓度而对细胞进行饥饿处理．本实验通过对HeLa细胞进行葡萄糖饥饿处理，观察细胞形态与结构的变化，为肿瘤的限食疗法提供理论依据．1 材料与方法1.1 主要试剂与仪器高糖DMEM培养基为Gibco公司产品；胎牛血清为兰州百灵生物技术有限公司产品；无糖DMEM培养基、葡萄糖、噻唑蓝(MTT)、二甲基亚砜(DMSO)、吖啶橙(AO)、溴化乙锭(EB)、牛血清蛋白(BSA)、Anti-α-Tubulin鼠多克隆抗体和鬼笔环肽(FITC-Phallodin)为Sigma公司产品；Alexa Fluor® 488驴抗鼠IgG为Invitrogen公司产品；3111型CO2细胞培养箱为热电公司产品；iMark Microplate Reader为Bio-Rad公司产品；TS-100型倒置显微镜为尼康公司产品；S-4800日立扫描电子显微镜(SEM)为Hitachi公司产品；TCS SP5Ⅱ激光共聚焦扫描显微镜为Leica公司产品．1.2 细胞培养0.0,1.0,1.5,2.0和25.0 mmol/L葡萄糖培养基由无糖培养基配置；对照组培养基内葡萄糖浓度为25.0 mmol/L．培养方法参照文献[8]．1.3 葡萄糖饥饿最适葡萄糖浓度的筛选根据人体内的葡萄糖浓度(5.5 mmol/L)及参考文献[9]，设置葡萄糖浓度梯度为0.0，1.0，1.5和2.0 mmol/L(以上浓度未计算细胞培养时加入的原胎牛血清中的5.5 mmol/L糖浓度)的培养基，分别处理HeLa细胞24 h后，每孔加入5mg/mL MTT溶液10 μL，培养3 h，弃去培养基，每孔加入75 μL DMSO，震荡15 min，酶标仪570 nm下测定吸光度值，并计算抑制率．抑制率1.4 倒置显微镜观察细胞形态HeLa细胞经葡萄糖饥饿分别处理24，48及72 h后，倒置显微镜下进行观察，拍照．1.5 荧光染色观察细胞核形态HeLa细胞经葡萄糖饥饿分别处理24，48及72 h后，取出细胞爬片，磷酸盐缓冲液清洗3次．临用前将AO(5 μg/mL)和EB(5 μg/mL)溶液等体积混合．在爬片上滴加混合液进行染色，荧光显微镜观察，拍照．1.6 扫描电镜观察细胞表面超微结构HeLa细胞经葡萄糖饥饿处理48 h后，使用扫描电子显微镜观察细胞表面结构，实验方法参照文献[10]．1.7 激光共聚焦免疫荧光观察细胞骨架HeLa细胞经葡萄糖饥饿处理48 h后，3.7%甲醛溶液4 ℃固定过夜；磷酸盐缓冲液清洗3次；0.1%TritonX-100透膜20 min，磷酸盐缓冲液清洗3次；5%BSA室温封闭30 min；α-Tubulin鼠多克隆抗体(1%BSA稀释液1∶1 000)4 ℃过夜；磷酸盐缓冲液清洗3次；Alexa Fluor®488驴抗鼠IgG(1∶1 000)室温避光孵育30 min；磷酸盐缓冲液清洗3次；4′,6-二脒基-2-苯基吲哚(DAPI)染色液染色10 min，磷酸盐缓冲液清洗2次，双蒸水漂洗2次；加防荧光淬灭剂，4 ℃晾干，封片；在激发光为496 nm，发射光范围为515～525 nm条件下观察，拍照．微丝(F-Actin)染色方法：3.7%甲醛溶液4 ℃固定过夜；磷酸盐缓冲液清洗3次；FITC-Phalloidin染色(1%BSA稀释液1∶40)室温避光孵育30 min；以下操作步骤同上．1.8 统计分析应用SPSS 19.0软件进行单因素方差分析，数据统计结果用表示，以P<0.05为差异有显著性意义，P<0.01 为差异有极显著性意义．2 结果2.1 葡萄糖饥饿最适葡萄糖浓度的筛选结果如表1所示：HeLa细胞经葡萄糖饥饿处理24 h后，葡萄糖浓度为0.0，1.0，1.5和2.0 mmol/L组细胞增殖抑制率分别为56.16%，36.30%，25.00%和16.44%．细胞增殖抑制率随着葡萄糖浓度的降低而逐渐升高．为了实验的方便性与准确性，选择1.0 mmol/L葡萄糖浓度作为葡萄糖饥饿的最适处理浓度．表1 葡萄糖饥饿24 h最适葡萄糖浓度的筛选葡萄糖浓度/(mmol\5L-1)OD值抑制率/%对照組0.292±0.0480.00.128±0.049**56.161.00.186±0.036**36.301.50.219±0.038** 25.002.00.244±0.033*16.44注：与对照组比较，*表示P<0.05，**表示P<0.01．2.2 葡萄糖饥饿对细胞形态的影响如图1所示：HeLa细胞经葡萄糖饥饿处理24 h后，对照组和25.0 mmol/L组的细胞生长良好，细胞形态无明显变化，其分布均匀，贴壁性好，形态饱满；1.0 mmol/L组的细胞密度减小，少数细胞收缩变圆；0.0 mmol/L组的细胞大多数都收缩变圆．48 h后，对照组和25.0 mmol/L组的细胞密度变大，细胞形态正常；1.0 mmol/L组的细胞大多数变圆；0.0 mmol/L组的细胞几乎全部变圆．72 h后，对照组和25.0 mmol/L组的细胞生长状态较好，增殖速度快，细胞间出现相互挤压的现象，但细胞贴壁性好，形态饱满；1.0 mmol/L与0.0 mmol/L组的细胞几乎全部变圆．A：对照组；B：c(葡萄糖)=25.0 mmol/L组；C：c(葡萄糖)=1.0 mmol/L组；D：c(葡萄糖)=0.0 mmol/L组.标尺长度为50 μm图1 倒置显微镜观察葡萄糖饥饿对HeLa细胞形态的影响2.3 葡萄糖饥饿对HeLa细胞核的影响如图2所示：HeLa细胞经葡萄糖饥饿分别处理24，48及72 h后，AO/EB双重荧光染色观察细胞核染色质的分布，吖啶橙(AO)能透过完整的细胞，嵌入细胞核DNA，使之发出明亮的绿色荧光，溴化乙锭(EB)仅能透过胞膜受损的细胞，嵌入核DNA，发橘红色荧光．葡萄糖饥饿24 h后，对照组、25.0 mmol/L组的细胞结构完整，呈分布均匀的绿色荧光，细胞核完整，边界清晰；1.0 mmol/L组的少部分细胞发生膜皱缩，胞浆稀少，细胞收缩变圆，体积减小，核染色体固缩、边聚，出现凋亡小体等细胞凋亡特征；0.0 mmol/L组多数细胞的细胞核被染成黄色，处于中期凋亡状态．48 h后，对照组、25.0 mmol/L组的细胞结构完整，呈分布均匀的绿色荧光；1.0 mmol/L组多数细胞的细胞核被染成黄色，处于中期凋亡状态；0.0 mmol/L组细胞几乎全部被染成橙红色，处于晚期凋亡的状态．72 h后，对照组、25.0 mmol/L组的细胞结构完整，呈分布均匀的绿色荧光；1.0 mmol/L组与0.0 mmol/L组细胞全部被染成橙红色，处于晚期凋亡的状态．2.4 葡萄糖饥饿对HeLa细胞表面超微结构的影响由图3可见：HeLa细胞经葡萄糖饥饿处理48 h后，对照组和25.0 mmol/L组细胞形态饱满，多呈规则的梭形或多角形，贴壁性好，细胞表面具有丰富的微绒毛，伪足发达且延展性好，相邻细胞的伪足相互紧密连接．1.0 mmol/L组的细胞形状变圆，体积缩小，贴壁性降低，细胞表面的微绒毛和伪足等结构几乎全部消失，出现凋亡小体结构．0.0 mmol/L组的细胞收缩变圆，体积进一步缩小，细胞表面的微绒毛和伪足等结构近乎完全消失，细胞膜出现褶皱．A：对照组；B：c(葡萄糖)=25.0 mmol/L组；C：c(葡萄糖)=1.0 mmol/L组；D：c(葡萄糖)=0.0 mmol/L组.标尺长度为25 μm图2 AO/EB双重荧光染色观察葡萄糖饥饿对HeLa细胞核染色质分布的影响A：对照组；B：c(葡萄糖)=25.0 mmol/L组；C：c(葡萄糖)=1.0 mmol/L组；D：c(葡萄糖)=0.0 mmol/L组图3 扫描电镜观察葡萄糖饥饿对HeLa细胞表面超微结构的影响2.5 葡萄糖饥饿对HeLa细胞微管分布的影响由图4可见：HeLa细胞经葡萄糖饥饿处理48 h后，对照组和25.0 mmol/L组细胞轮廓清晰，立体感强；微管从微管组织中心散发呈辐射状，围绕在细胞核周围，均匀地分布在整个细胞中．微管着色均一，连贯性好，呈清晰的丝状纤维结构．1.0 mmol/L组细胞整体轮廓清晰度下降，呈圆形或三角形，可见凋亡小体；微管蛋白解聚，聚集于细胞边缘，丝状纤维结构消失．0.0 mmol/L组细胞收缩变小，呈圆形；微管蛋白完全解聚，聚集于细胞边缘两端，丝状纤维结构消失．A：对照组；B：c(葡萄糖)=25.0 mmol/L组；C：c(葡萄糖)=1.0 mmol/L组；D：c(葡萄糖)=0.0 mmol/L组图4 激光共聚焦免疫荧光观察葡萄糖饥饿对HeLa细胞微管分布的影响2.6 葡萄糖饥饿对HeLa细胞微丝分布的影响由图5可见：HeLa细胞经葡萄糖饥饿处理48 h后，对照组和25.0 mmol/L组细胞形态规则，立体感强，肌动蛋白丝聚合形成束状结构并分布于细胞边缘，细胞间连接紧密．1.0 mmol/L组细胞的细胞核收缩变圆，轮廓模糊．细胞收缩变小，呈圆形或三角形．肌动蛋白丝消失，表面微绒毛减少，细胞间的连接减少或中断．0.0 mmol/L组细胞进一步收缩变圆，微丝蛋白解聚，着色变浅，细胞表面微绒毛完全消失．A：对照组；B：c(葡萄糖)=25.0 mmol/L组；C：c(葡萄糖)=1.0 mmol/L组；D：c(葡萄糖)=0.0 mmol/L组图5 激光共聚焦免疫荧光观察葡萄糖饥饿对HeLa细胞微丝分布的影响3 讨论在葡萄糖饥饿条件下，降低癌细胞的异常新陈代谢，诱导癌细胞的凋亡[11]．Foster等[9]用0.0，0.5和25.0 mmol/L葡萄糖浓度的条件处理多种肿瘤细胞120 h.结果表明，0.0 mmol/L葡萄糖浓度处理组的所有肿瘤细胞的生长均受到抑制作用；0.5 mmol/L葡萄糖浓度处理可以抑制多种肿瘤细胞的生长．闫珊[12]研究发现，缺糖能降低HeLa细胞的生存率，并诱导细胞发生凋亡．本实验通过对HeLa细胞进行葡萄糖饥饿处理，倒置显微镜下观察细胞形态，结果发现，细胞形态发生改变，细胞由规则的梭形或多角形逐渐转变成三角形或圆形，并出现凋亡小体等凋亡特征，这与闫珊等[12]的研究结果相一致；AO/EB染色结果显示，细胞膜皱缩，胞浆稀少，细胞收缩变圆，体积减小，核染色体固缩，边聚，出现凋亡小体等细胞凋亡特征，这与蒋时红等[13]的研究结果相一致；扫描电镜观察发现：细胞表面的微绒毛和伪足等结构近乎完全消失，细胞膜出现褶皱；免疫荧光检测结果显示：微丝蛋白减少，细胞形态发生改变，微管结构被破坏，微管蛋白解聚并聚集于细胞边缘，并且随着葡萄糖饥饿葡萄糖浓度的降低，细胞骨架被破坏的程度加深．综上所述，葡萄糖饥饿条件下，HeLa细胞表面的微绒毛和伪足结构消失，微丝蛋白减少，排列混乱，微管蛋白解聚并聚集于细胞边缘，丝状纤维结构消失，染色质凝集，进而诱导HeLa细胞形态变化和凋亡．葡萄糖饥饿能够抑制HeLa细胞活性，改变细胞形态，诱导细胞凋亡，这为临床上饥饿疗法治疗肿瘤提供理论依据．参考文献：[1]Giovannucci E,Harlan D M,Archer M C,et al.Diabetes and cancer:A consensus report[J].Diabetes Care,2010,33(7):1674-1685.[2]Graham N A,Tahmasian M,Kohli B,et al.Glucose deprivation activates a metabolic and signaling amplification loop leading to celldeath[J].Molecular Systems Biology,2012,8(1):589.[3]Mathews E H,Liebenberg L.Can successful cancer therapies build onwhat we learn from complex disorders[J].MedicalHypotheses,2012,78(5):687-689.[4]Bost F,Sahra I B,Marchand-Brustel Y L,et al.Metformin and cancer therapy[J].Current Opinion in Oncology,2012,24(1):103-108.[5]Kallinowski F,Vaupel P,Runkel S,et al.Glucose uptake,lactate release,ketone body turnover,metabolic micromilieu,and pH distributions in human breast cancer xenografts in nude rats[J].Cancerresearch,1988,48(24):7264-7272.[6]Deng D,Xu C,Sun P C,et al.Crystal structure of the human glucose transporter GLUT1[J].Nature,2014,510(7503):121-125.[7]Chao M,Wu H,Jin K,et al.A nonrandomized cohort and a randomized study of local control of large hepatocarcinoma by targeting intratumoral lactic acidosis[J].Elife,2016,2(5):e15691.[8]成文召,麻艳芳,邵邻相，等.佛手叶挥发油对HeLa细胞形态与结构的影响[J].浙江师范大学学报:自然科学版,2013,36(3):331-336.[9]Foster R,Griffin S,Grooby S,et al.Multiple metabolic alterations exist in mutant PI3K cancers,but only glucose is essential as a nutrientsource[J].Plos One,2012,7(9):1-8.[10]麻艳芳,邵邻相,张均平，等.佛手挥发油对MDA-MB-435人乳腺癌细胞体外增殖的影响[J].中国药学杂志,2010，45,(22):1737-1741.[11]Knouzy B,Dubourg L,Baverel G,et al.Ifosfamide metabolite chloroacetaldehyde inhibits cell proliferation and glucose metabolism without decreasing cellular ATP content in human breast cancer cells MCF-7[J].Journal of Applied Toxicology,2010,30(3):204-211.[12]闫珊.缺糖增加Bcl-2抑制剂S1诱导人宫颈癌细胞凋亡敏感性的实验研究[D].长春:吉林大学,2014.[13]蒋时红,孙超龙,刘燕,等.麦门冬汤诱导人肺腺癌A549细胞凋亡作用及其机制[J].中华中医药杂志,2015(4):1236-1238.。

川芎对神经细胞保护作用活性成分的研究

【Ｋｅｙｗｏｒｄｓ】ＬｉｇｕｓｔｉｃｕｍＣｈｕａｎｘｉｏｎｇ；ＳｅｎｋｙｕｎｏｌｉｄｅＩ；Ｌｉｇｕｓｔｉｌｉｄｅ；ＦｅｒｕｌｉｃＡｃｉｄ；ＯｘｙｇｅｎＧｌｕｃｏｓｅＤｅｐｒｉｖａｔｉｏｎ；ＳＨ－ＳＹ５Ｙ
脑缺血（ＣｅｒｅｂｒａｌＩｓｃｈｅｍｉａ）又称为缺血性脑中
ＤＯＩ：１０．１３９３５／ｊ．ｃｎｋｉ．ｓｊｚｘ．２１０５０２基金项目：国家自然科学基金资助项目（８１８７３０４１，８２０３０１２４）作者单位：１．中国中医科学院，西苑医院基础医学研究所，北京１０００９１；２．北京中医药大学，北京１０００９１通信作者：刘建勋，Ｅｍａｉｌ：ｌｉｕｊｘ０３２４＠ｓｉｎａ．ｃｏｍ
风（ＩｓｃｈｅｍｉｃＳｔｒｏｋｅ）。近年来缺血后神经保护的研究成为热点，脑缺血后可引起偏瘫、学习记忆障碍等神经损伤症状，目前尚无公认的针对缺血后神经损伤治疗的制剂上市，因此急需寻找并开发有效的治疗缺血后神经损伤的药物。中医药在治疗脑缺血后神经损伤具有一定的优势和潜力，许多学者已经
实验室前期已研究并报道了川芎提取物在缺血性脑损伤方面的神经保护作用并检测了提取物中的主要成分［７］，本研究在此基础上，为进一步阐释川芎发挥神经保护的物质基础，拟以ＳＨ－ＳＹ５Ｙ（人神经母细胞瘤细胞系）氧糖剥夺／复氧复糖（ＯｘｙｇｅｎａｎｄＧｌｕｃｏｓｅＤｅｐｒｉｖａｔｉｏｎ－Ｒｅｏｘｙｇｅｎａｔｉｏｎ，ＯＧＤ－Ｒ）为模型［８］，筛选了川芎中几种主要化合物（图１），洋川芎内酯Ｉ（ＳｅｎｋｙｕｎｏｌｉｄｅＩ，ＳＥＩ）、洋川芎内酯Ｈ（ＳｅｎｋｙｕｎｏｌｉｄｅＨ，ＳＥＨ）、洋川芎内酯Ａ（ＳｅｎｋｙｕｎｏｌｉｄｅＡ，ＳＥＡ）、阿魏酸（ＦｅｒｕｌｉｃＡｃｉｄ，ＦＡ）、藁本内酯（Ｌｉｇｕｓｔｉｌｉｄｅ，ＬＩＧ）的神经保护活性，为阐释川芎神经保护的物质基础及以中药成分为核心的神经保护制剂的开发提供参考和依据。

不同活性氧产生途径对高糖诱导内皮细胞活性氧生成及高糖对细胞凋亡的影响

2．方法：（ 1）原代人脐静脉内皮细胞分离：取长约 20cm 的无菌新鲜脐带，以生理盐水冲洗脐静脉 3 次，0． 1% 的Ⅱ型胶原酶（ Promega 公司）灌注脐静脉 15min，收集灌注液，800r / min 离心 6min，收集内皮细胞，20% 胎牛血清（ HYCLONE 公司）的 M199 混合培养基（ GIBCO 公司）培养。（ 2 ）细胞内ＲOS 的检测：用 2，7 －二氯荧光黄双乙酸盐（ DCFH － DA）荧光染色检测细胞内ＲOS，严格按照碧云天公司试剂盒说明书操作。（ 3） Hochest 染色： ①胰酶消化、离心收集内皮细胞； ② 加 PBS 洗 1 遍； ③Hochest 染液染色 10min； ④加入 PBS 洗两遍： ⑤取 50μl 滴于玻片上； ⑥倒置显微镜观察。（ 4） PI 染色： ①收集细胞，离心 5min，弃去培养液； ②PBS 洗涤 1 次； ③离心去 PBS，加入冰预冷的 70% 的乙醇固定； ④离心弃去固定液， PBS 重悬 5min； ⑤用 PI 染液染色，4℃ 避光 30min； ⑥流式细胞仪检测。（ 5 ） Western blot 法检测：细胞裂解蛋白经 SDS － PAGE 电泳后转移到 PVDF 膜上，封闭过夜后，与相应一抗（ Santa Cruz 产品）孵育后，再与辣根过氧化物酶标记的二抗（ Promega 产品）孵育，洗膜后，应用 ECL 试剂盒进行化学发光反应，最后曝光后显影，定影。
中图分类号Ｒ363． 1 + 3
文献标识码 A
DOI 10． 3969 / j． issn． 1673-548X． 2015． 02． 022
Effect of Different Pathways of ＲOS on ＲOS and Apoptosis Induced by High Glucose in HUVECs． Wang Lei，Su Huabin，Lu Qiong． Department of Basical Medicine，Hunan University of Medicine，Hunan 418000，China

右美托咪定对器官功能的影响及机制

右美托咪定对器官功能的影响及机制李晓燕;解雅英【摘要】右美托咪定（Dexmedetomidine，DEX）激动α2受体后通过抗交感、抗凋亡、抑制细胞炎症和氧化应激反应、活化细胞保护信号通路等多种途径重要脏器发挥保护作用。

通过查阅近年 PubMed、知网等数据库中有关 DEX 对脑、心、肺、肾、肝、肠道等器官功能的影响及机制，并进行总结。

DEX 具有多器官保护作用，但尚需更多、更细致的研究。

【期刊名称】《河北医药》【年(卷),期】2016(000)004【总页数】3页(P601-603)【关键词】右美托咪定;α2 肾上腺素能受体;器官功能的影响【作者】李晓燕;解雅英【作者单位】010010 呼和浩特市，内蒙古医科大学第一附属医院麻醉科;010010 呼和浩特市，内蒙古医科大学第一附属医院麻醉科【正文语种】中文【中图分类】R97E-mail:**************DEX是一种高选择性的α2-肾上腺素能受体(α2-AR)激动药(α2∶α1=1 620∶1)，通过激动α2-AR发挥镇痛、抗焦虑、抑制交感神经活动性、产生类似于人体自然睡眠的镇静等作用。

DEX被广泛应用于神经外科、泌尿外科、以及产科分娩镇痛等麻醉中。

除此之外，DEX对大脑、心脏、肾脏、肠道、肺等器官功能的影响也被广泛的关注。

本文主要针对DEX对器官功能的影响及机制作一综述。

1.1 脑功能的影响DEX对脑功能的保护作用是1991年由Hoffman WE等在大鼠短暂全脑缺血模型中首先发现的。

他们在实验中发现，DEX预处理可以使血浆中肾上腺素和去甲肾上腺素的水平显著下降，并且这种效应可以被阿替美唑拮抗;进一步分析表明血浆中肾上腺素和去甲肾上腺素的浓度与神经系统功能的受损程度呈正相关。

Sanders等［1］研究发现，DEX能够剂量依赖性的提高离体皮质神经元的生存率，特别是0.1～100 μmol/L的DEX能够剂量依赖性的减少渥曼青霉素所导致的细胞凋亡，产生神经保护作用。

细胞内氧化应激活性氧ROS初级荧光测定试剂盒产品说明书中文版主要用途

细胞内氧化应激活性氧（RoS）初级荧光测定试剂盒产品说明书（中文版）主要用途细胞内氧化应激活性氧（ROS）初级荧光测定试剂是一种旨在通过透膜荧光染色剂二氯荧光乙酰乙酸盐，在细胞氧化条件下，产生荧光，来定量检测细胞内活性氧族的生成和增加的权威而经典的技术方法。

该技术由大师级科学家精心研制、成功实验证明的。

可以被用于细胞凋亡、信号传递、衰老、代谢和营养学等的研究。

产品严格无菌，即到即用，操作简易，活体检测，性能稳定。

技术背景超氧自由基阴离子（superoxideradica1：O2）＞过氧化氢（hydrogenperoxide；H2O2）、羟自由基或氢氧基（hydroxy1radica1：0H）、过氧化基（peroxy1radica1；R00'氢过氧自由基Chydroperoxy1;H00烷氧自由基（a1coxy!radica1）.氮氧基（niiricOxide；NO）、过氧亚硝基阴离子（PeroXyniIri1eanion；ONOO）次氯酸（hyρoch1orousacid；HoC1）、半酸自由基（semiquinoneradica1单线态氧气（Sing1etoxygen）等细胞内活性氧族（ReaciiveOxygenSpecies;ROS）的产生和增多，将导致细胞哀老或凋亡。

2,,7-二氯荧光乙酰乙酸盐（2'.7idich1orof1uOreSCeindiaCe1ate,DCFH-DA）一种完全自由通过细胞膜的染色剂。

一旦被过氧化氢、过氧化基、过氧亚硝基阴离子等氧化，便产生荧光。

据此测定细胞内活性氧族的浓度。

产品内容清理液（Reagen1A）亳升染色液（Reagen1B）微升稀释液（Reagen1e）亳升保存液（Reagen1D）受升产品说明书1份保存方式保存染色液（Reagen1B）在一20七冰箱里，严格避免光照；其余的保存在4C冰箱里，有效保证12月用户自备胰蛋白酶乙二胺四乙酸混合液（GMS12024）：用于细胞脱离完全细胞培养液（GMSI2052）：用于细胞操作所需的培养基15亳升锥形离心管：用于细胞操作的容器15亮升离心管：用于细胞染色的容器血细胞计数仪：用于细胞计数台式离心机：用于沉淀细胞细胞流式仪：用于细胞荧光分析（共聚焦）荧光显微镜：用于观察荧光细胞荧光分光光度仪或衡标仪：用于定量检测荧光细胞实验步骤一、间接法实验开始前，将一20℃冰箱里的试剂盒中的染色液（ReagentB）置入冰槽里融化，稀释液（ReagentC）放进37°C恒温水槽里预热。

靶向厌氧环境的蛋白

靶向厌氧环境的蛋白
靶向厌氧环境的蛋白通常指的是在缺氧或低氧环境中发挥特定功能的蛋白质。

这些蛋白质可能有助于细胞适应厌氧条件，并在细胞内执行各种生物学过程。

以下是一些与厌氧环境相关的蛋白质及其功能：
HIF-1α (Hypoxia-Inducible Factor 1 Alpha)：HIF-1α是一个重要的转录因子，在缺氧条件下被激活。

它参与调控一系列基因的表达，包括调节细胞的代谢、血管新生等，帮助细胞适应缺氧环境。

VEGF (Vascular Endothelial Growth Factor)：VEGF是一种血管内皮细胞生长因子，在低氧环境中被HIF-1α调控。

它促进血管新生，有助于维持组织的氧供应。

Glut1 (Glucose transporter 1)：Glut1是一种葡萄糖转运蛋白，在低氧环境中的细胞中表达增加，有助于提高细胞对葡萄糖的摄取，以应对能量代谢需求的变化。

CA IX (Carbonic Anhydrase IX)：CA IX是一种碳酸酐酶，在低氧环境中被HIF-1α调控。

它参与调节细胞内pH值，帮助维持细胞内部的酸碱平衡。

BNIP3 (BCL2/adenovirus E1B 19kDa protein-interacting protein 3)：BNIP3是一种与线粒体相关的蛋白质，在缺氧条件下表达增加。

它参与调节线粒体的功能和凋亡过程，有助于维持细胞的生存和适应性。

这些蛋白质在厌氧环境中的表达和功能调控对于细胞的生存和适应至关重要，它们的研究有助于理解细胞对于氧气水平变化的应对机制，并可能为治疗一些与低氧环境相关的疾病提供新的治疗靶点。

SDS-PAGE凝胶配方

SDS-PAGE凝胶配制试剂盒产品简介：碧云天生产的SDS-PAGE凝胶配制试剂盒提供了配制SDS-PAGE凝胶所需的各种试剂，用户只需自备制胶器具和蒸馏水，即可配制PAGE胶(即聚丙烯酰胺凝胶)。

SDS-PAGE凝胶配制试剂盒不仅可用于配制SDS-PAGE凝胶，也可用于配制非变性(native)PAGE凝胶。

本试剂盒约可配制30-50块常规大小的PAGE胶。

保存条件：1M Tris-HCl, pH8.8、10% SDS、Ammonnium persulfate (过硫酸铵)和1M Tris-HCl, pH6.8室温保存。

30% Acr-Bis (29:1)和TEMED 4℃避光保存。

过硫酸铵配制成10%溶液后，分装成小管-20℃保存，通常半年内有效。

注意事项：过硫酸铵配制成10%溶液后，应当-20℃保存。

同时应尽量减少室温存放时间，以防失效。

TEMED易挥发，使用后请盖紧瓶盖。

另外凝胶凝聚的速度和温度及光照关系密切，可通过适当调节TEMED的用量，控制在不同的室内环境下凝胶凝聚的速度。

为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用说明：1. 根据目的蛋白的分子量大小选择合适的凝胶浓度，再按照下面的表格配制SDS-PAGE的分离胶(即下层胶)：使用本产品的文献：1. Deng XQ, Chen LL, Li NX.The expression of SIRT1 in nonalcoholic fatty liver disease induced by high-fat diet in rats.Liver Int. 2007 Jun;27(5):708-15.2. Wang PH, Gu ZH, Huang XD, Liu BD, Deng XX, Ai HS, Wang J, Yin ZX, Weng SP, Yu XQ, He JG.An immune deficiency homolog from the white shrimp, Litopenaeus vannamei, activates antimicrobial peptide genes. Mol Immunol. 2009 May;46(8-9):1897-904.3. Liang QL, Wang BR, Li GH.DcR3 and survivin are highly expressed in colorectal carcinoma and closely correlated to its clinicopathologic parameters. J Zhejiang Univ Sci B. 2009;10(9):675-82.4. Deng XQ, Cheng JL, Zhang YP, Li NX, Chen LL.Effects of caloric restriction on SIRT1 expression and apoptosis of islet beta cells in type 2 diabetic rats.Springer Verlag. 2009.5. Cao X, Zhang Y, Zou L, Xiao H, Chu Y, Chu X.Persistent oxygen-glucose deprivation induces astrocytic death through two different pathways and calpain-mediated proteolysis of cytoskeletal proteins during astrocytic oncosis.Neurosci Lett. 2010;479(2):118-22. Epub 2010 May 21.6. Cao X, Xiao H, Zhang Y, Zou L, Chu Y, Chu X.1, 5-Dicaffeoylquinic acid-mediated glutathione synthesis through activation of Nrf2 protects against OGD/reperfusion-induced oxidative stress in astrocytes. Brain Res. 2010;1347:142-8. Epub 2010 Jun 1.7. Huang L, Bi HC, Liu YH, Wang YT, Xue XP, Huang MCAR-mediated up-regulation of CYP3A4 expression in LS174T cells by Chinese herbal compounds.Drug Metab Pharmacokinet. 2011;26(4):331-40.8. Li Q, Lei RX, Zhou XD, Kolosov VP, Perelman JM.Regulation of PMA-induced MUC5AC expression by heparin in human bronchial epithelial cells.Mol Cell Biochem. 2012 Jan;360(1-2):383-91.9. Luan HF, Zhao ZB, Zhao QH, Zhu P, Xiu MY.Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusioninjury mediated by the JAK2/STAT3 survival pathway.Braz J Med Biol Res. 2012 May 31.10.Chen Z, Qing J, Qin G, Hu LConstruction and characterization of bifunctional TIM-3-EGFP fusion proteins.Protein Expr Purif. 2012 Nov;86(1):1-6. doi: 10.1016/j.pep.2012.08.004. Epub 2012 Aug 23.11.Huang L, Huang M, Li YH, Li RM, Zeng Y, Kuang SY, Zhang L, Wang YT, Bi HC.Up-regulatation of CYP3A expression through pregnent X receptor by praeruptorin D isolated from Peucedanumpraeruptorum Dunn.J Ethnopharmacol. 2013 Jul 9;148(2):596-602. doi: 10.1016/j.jep.2013.05.008. Epub 2013 May 20.12.Zhang H, Wang ZW, Wu HB, Li Z, Li LC, Hu XP, Ren ZL, Li BJ, Hu ZP.Transforming growth factor-β1 induces matrix metalloproteinase-9 expression in rat vascular smooth muscle cellsvia ROS-dependent ERK-NF-κB pathways. Mol Cell Biochem. 2013 Mar;375(1-2):11-21. doi: 10.1007/s11010-012-1512-7. Epub 2012 Dec 29.13.Liu R, Liu X, Zheng Y, Gu J, Xiong S, Jiang P, Jiang X, Huang E, Yang Y, Ge D, Chu Y.MicroRNA-7 sensitizes non-small cell lung cancer cells to paclitaxel.Oncol Lett. 2014 Nov;8(5):2193-2200. Epub 2014 Sep 4.14.Zhao W, Zhao J, Hou M, Wang Y, Zhang Y, Zhao X, Zhang C, Guo D.HuR and TIA1/TIAL1 are involved in regulation of alternative splicing of SIRT1 pre-mRNA.Int J Mol Sci. 2014 Feb 20;15(2):2946-58. doi: 10.3390/ijms15022946.。

血、尿、脑脊液葡萄糖Glucose测定-检验科生化室作业书

血、尿、脑脊液葡萄糖Glucose测定实验原理VitrosGLU试剂是一种干燥，多涂层的，在透明的聚合物支撑基片上涂有分析成份的化学干片。

将一滴10ul的患者样品滴于干片上，分布层会使之均匀地分布并使溶解的分子渗透到下面的试剂层。

于是葡萄糖氧化酶氧化样品中的葡萄糖，生成过氧化氢和葡萄糖酸，随后过氧化酶催化染料前体生成呈色剂。

呈色剂的强度可用反射光谱法测定。

测定方式：比色法波长：540nm测试时间和温度：37℃约５分钟反应过程：β-D-葡萄糖+氧气+水——葡萄糖氧化酶→D-葡萄糖酸+过氧化氢过氧化氢+4-氨基氨替比林+二羟萘——过氧化酶→红色呈色剂2.标本：2.1病人准备：12小时禁食。

2.2样品类型：血清、肝素，氟化钠/草酸钾和EDTA抗凝血浆；尿液；脑脊液。

2.3标本处理：血清和血浆标本采集后30分钟内以1000X离心10分钟，然后吸出血清或血浆，从而防止细胞糖代谢（室温下1小时约7%）。

尿液标本不需加防腐剂，防腐剂可能会产生干扰。

脑脊液（CSF）。

根据实验室的标准程序定时收集的样品；样品采集后1小时内离心，吸出上清液。

一定量的特殊物质（如纤维蛋白）会覆盖分布层而限制氧扩散，从而产生负面干扰。

为避免此类情况发生，必须等样品完全凝固以后再离心。

3.标本的存放：血清、血浆15～25℃室温下最多1天；4～8℃冷藏最多7天；-20℃冷冻保存可长达1年。

尿液样品在测试前要冷藏。

脑脊液2-8℃冷藏最多可保存7天。

4.标本的运输：样品要放在带盖的容器内以防污染和蒸发。

5.标本拒收的标准：溶血，抗凝剂不符合要求，标本放置时间过长。

6.试验材料：6.1测试GLU所需的物品包括：Vitros化学定标物Kit1；Vitros特制质控液；Vitros7%BSA稀释液。

强生厂家提供原装进口，试剂盒外包装上标明了测试项目名称，试剂标签代码，有效期限和储藏温度。

6.1.1试剂组成：干片成分：反应成份有葡萄糖氧化酶，过氧化酶，二羟萘和4-氨基氨替比林（染料前体）。

英文：硫辛酸改善PC12细胞中GSH的水平：帕金森治疗的新启示

Pre-treatment with R -Lipoic Acid Alleviates the Effects of GSH Depletion in PC12Cells:Implications for Parkinson’sDisease TherapyS.Bharath 1,*,B.C.Cochran 1,M.Hsu 1,J.Liu 2,3,B.N.Ames 2,3,J.K.Andersen 11Buck Institute for Age Research,8001Redwood Blvd.,Novato,CA 94945,USA2Division of Biochemistry and Molecular Biology,University of California,Berkeley,CA 94720,USA 3Children’s Hospital Oakland Research Institute,Oakland,CA 94609,USAReceived 22August 2001;accepted 12April 2002AbstractOxidative stress is believed to play a key role in the degeneration of dopaminergic neurons in the substantia nigra (SN)of Parkinson’s disease (PD)patients.An important biochemical feature of PD is a signiﬁcant early depletion in levels of the thiol antioxidant compound glutathione (GSH)which may lead to the generation of reactive oxygen species (ROS),mitochondrial dysfunction,and ultimately to subsequent neuronal cell death.In earlier work from our laboratory,we demonstrated that depletion of GSH in dopaminergic PC12cells affects mitochondrial integrity and speciﬁcally impairs the activity of mitochondrial complex I.Here we report that pre-treatment of PC12cells with R-lipoic acid acts to prevent depletion of GSH content and preserves the mitochondrial complex I activity which normally is impaired as a consequence of GSH loss.#2002Elsevier Science Inc.All rights reserved.Keywords:Parkinson’s disease;Glutathione (GSH);R -lipoic acid;Mitochondrial complex IINTRODUCTIONParkinson’s disease (PD)is a progressive,neurode-generative disorder which involves the loss of dopa-minergic neurons of the substantia nigra (SN)pars compacta (Beal,1992)The disease condition occurs mostly in late midlife with clinical features including motor impairment,gradual loss of cognition,and depression.Oxidative stress appears to play an impor-tant role in neuronal degeneration associated with PD (Beal,1992;Burke,1998;Adams et al.,2001;Sayre et al.,2001).Dopaminergic neurons are particularly prone to oxidative stress due to oxidation of dopamine leading to the generation of reactive oxygen species(ROS).ROS act to oxidize various biological macro-molecules thereby disturbing homeostasis within the cell ultimately leading to cell death (Beal,1992;Burke,1998;Adams et al.,2001;Sayre et al.,2001).It has been observed that the SN of early PD patients has signiﬁcantly decreased levels of glutathione (GSH)(Perry and Yong,1988).GSH is a tripeptide localized both within the cytoplasm and mitochondria of neurons and other cell types.It acts as the major non-proteinac-eous antioxidant and redox modulator within the brain.The depletion of GSH in the SN is the earliest known indicator of oxidative stress in presymptomatic PD,preceding both decreases in mitochondrial complex I activity and dopamine levels (Jenner,1993).GSH is synthesized by a two-step reaction involving the enzymes g -glutamyl cysteine ligase (g -GCL)and glutathione synthetase.g -GCL is the rate-limiting enzyme in this process and brain GSH appearstoNeuroToxicology 23(2002)479–486*Corresponding author.Tel.:þ1-415-209-2000;fax:þ1-415-209-2231.E-mail address :bsrinivas@ (S.Bharath).0161-813X/02/$–see front matter #2002Elsevier Science Inc.All rights reserved.PII:S 0161-813X (02)00035-9primarily arise through synthesis from its constituent amino acids via this enzyme(Meister,1988).g-GCL is a dimer composed of a heavy catalytic subunit and a light regulatory subunit(Huang et al.,1993).GSH is synthesized in the cytosol and transported into the mitochondria via an energy-dependent transporter (Meister,1988).In earlier studies from our laboratory, we reported on the effects of partial depletion of GSH levels via g-GCL inhibition on mitochondrial physio-logy in PC12cells(Jha et al.,2000).When GSH levels in PC12were down regulated by simultaneous dox-ycycline(Dox)-induced expression of antisense g-GCL light and heavy subunit mRNAs and protein levels, it resulted in decreased mitochondrial GSH levels, increased oxidative stress,and decreased mitochon-drial function.Decreased mitochondrial activity in these cells appeared to be due to a selective inhibition of complex I activity as a result of thiol oxidation of the enzyme complex.These results suggested that the early observed GSH losses in the SN of PD patients could be linked to decreases in complex I activity(via increased ROS)and subsequent mitochondrial dysfunction which ultimately leads to dopaminergic cell death associated with the disease(Jha et al.,2000;Beal,1992).R-lipoic acid plays a fundamental role in mitochon-drial metabolism as a coenzyme for pyruvate dehy-drogenase and alpha-ketoglutarate dehydrogenase and as a substrate for the NADPH-dependent enzyme glutathione reductase.In recent years,R-lipoic acid has received considerable attention as an antioxidant (Fuchs et al.,1997;Packer et al.,1995;Sen et al., 1999).It has been demonstrated to have neuroprotec-tive effects on neuronal cells(Wolz and Krieglstein, 1996,1997;Tirosh et al.,1999;Sen et al.,1998). Studies using both in vitro and in vivo models have suggested that pretreatment with R-lipoic acid increases cellular levels of GSH,probably by prevent-ing its depletion thereby protecting mitochondrial integrity(Suzuki et al.,1991;Scott et al.,1994;Han et al.,1997;Xu and Wells,1996;Lykkesfeldt et al., 1998;Kagen et al.,1992).To address the question of the possible mechanism of R-lipoic acid-mediated protection,we have analyzed the effect of pretreatment with R-lipoic acid on GSH-depleted PC12cells.We have used buthionine sulfox-imine(BSO),an irreversible inhibitor of the enzyme g-GCL,to decrease GSH levels within PC12.BSO is a widely used pharmacological agent for decreasing GSH content in peripheral tissues as well as in cell culture models(Minchinton et al.,1984;Lee et al., 1988;Thanislass et al.,1995;Andersen et al.,1996). We demonstrate that pre-treatment of GSH-depleted cells with R-lipoic acid prevents depletion of the GSH pool within the cytoplasm and mitochondria.We also show that this pretreatment leads to preservation of mitochondrial complex I activity lost due to GSH depletion.MATERIALS AND METHODSPC12cells were obtained from American Type Culture Collection(ATCC),Rockville,MD.All tissue culture materials were procured from Life Technolo-gies or Cellgro.Chemicals were obtained from Sigma Chemical Company.The GSH assay kit was obtained from OXIS Research Inc.R-lipoic acid was a gift from ASTA Medica(Frankfurt/Main,Germany).Cell CulturePC12cells were cultured in DMEM medium con-taining10%horse serum,5%fetal bovine serum(FBS) and antibiotics(2units penicillin/ml and0.02mg streptomycin/ml)at378C and5%CO2.Cells were subcultured once a week by gentle cell scraping.Stock solutions of buthionine-S,R-sulfoximine(BSO)and R-lipoic acid were prepared in Hanks balanced salt solution(HBSS).Preparation of MitochondriaMitochondria were prepared by the method of Trounce et al.(1996).Brieﬂy,PC12cells were har-vested,washed in Buffer H(5mM HEPES,210mM mannitol,70mM sucrose,1mM EGTA and0.5% BSA)then resuspended in the same buffer.The cell suspension was then homogenized and centrifuged at 800Âg for5min at48C.The supernatant which was enriched in mitochondria was then centrifuged at 10,000Âg for20min at48C.The resultant mitochon-drial pellet was resuspended in buffer H and stored as aliquots atÀ208C.Estimation of Total Glutathione(GSHþGSSG) in Whole Cells and MitochondriaTotal cellular and mitochondrial glutathione estima-tion was carried out using a kit from OXIS Research Inc.following the instructions provided by the manu-facturer.Cells were plated at1Â106cells/ml in either 6-well dishes or90mm diameter plates.All estima-tions were conducted in triplicate.Cells were incubated with BSO and/or R-lipoic acid for the indicated time intervals at378C.Cells were then harvested,disrupted480S.Bharath et al./NeuroToxicology23(2002)479–486by sonication and cellular proteins precipitated by tricholoroacetic acid.The soluble fraction,which is enriched for total glutathione,was estimated using the OXIS kit.Brieﬂy,samples wereﬁrst buffered and then the reducing agent,tris(2-carboxyethyl)phosphine (TCEP)was added to reduce any oxidized glutathione (GSSG).The chromogen4-chloro-1-methyl-7-triﬂuor-omethylquinolinium methylsulfate was then added which forms thioethers with all thiols present in the sample.Upon addition of base to raise the pH to greater than13,a b-elimination speciﬁc to the GSH-thioether results in a chromophoric thione.The absorbance of this thione measured at420nm is directly proportional to total glutathione concentration.All values obtained were normalized per protein and tabulated. Mitochondrial Complex I Enzyme Assay Complex I enzyme assays were carried out as described by Trounce et al.(1996).Brieﬂy,the assay was initiated by addition of aliquots of sonicated cell samples to50mM potassium phosphate pH7.4, 500m M EDTA,1%BSA,200m M NADH and200m M decylubiquinoneþ2m M rotenone in the presence of KCN with0.002%dichloroindophenol(DCIP)as a secondary electron acceptor.The decrease in the absorbance at600nm was recorded as a measure of enzyme reaction rate at308C for10min and speciﬁc activity was calculated.The results were plotted as relative rotenone-sensitive speciﬁc activity.c-Glutamyl Cysteine Ligase Enzyme Assayg-GCL assays were carried out as described by Seelig and Meister(1985).Brieﬂy,cells obtained after various treatments were washed in1ÂPBS and resuspended in0.1M Tris–HCl,pH8.0.The cell suspension was sonicated and used as a source for g-GCL enzyme activity.The enzyme activity was determined at378C by adding cell lysates to a reaction mixture containing0.1M Tris–HCl buffer,pH8.0, 150mM KCl,5mM sodium ATP,2mM phosphoenol-pyruvate,10mM L-glutamate,10mM a-amino buty-rate,20mM MgCl2,2mM EDTA,0.2mM NADH, 17U pyruvate kinase and17U lactate dehydrogenase. The absorbance at340nm was monitored as a measure of enzyme activity.Assays run in the absence of a-aminobutyrate served as blank.All the g-GCL activ-ity values were normalized per protein.RESULTSPC12cells represent a good model system for the study of oxidative stress in dopamine-containing cells as they exhibit several physiological properties of dopaminergic neurons(Greene and Tischler,1976). PC12cells were treated with100m M of BSO for different time points and total glutathione content measured.Following BSO treatment,there was a gradual decrease in the levels of total cellular GSH content with time(Fig.1);the maximum decrease was observed at24h(data not shown).For subsequent experiments,addition of100m M of BSO for a time period of6h was employed to maintain approximately 50%depletion in total cellular GSH content,similar to the decreases observed in the PD SN.The unbound form of R-lipoic acid has two–SH groups and has been found to be an effective thiol anti-oxidant(Suzuki et al.,1991;Scott et al.,1994;Han et al.,1997;Xu and Wells,1996;Lykkesfeldt et al., 1998;Kagen et al.,1992).Therefore,R-lipoicacid Fig.1.The effect of100mM BSO on total cellular glutathione levels in PC12cells at various time points.Significance of difference fromthe control was determined by ANOV A single factor analysis.Data is expressed as meanÆS:E:M:(n¼3).ÃP<0:005vs.untreated cells;ÃÃP<0:001vs.untreated cells.S.Bharath et al./NeuroToxicology23(2002)479–486481pretreatmentwas testedtoseeifthiscould helpovercome the dramatic decreases in BSO-induced glutathione levels.PC12cells were incubated with 100m M of freshly prepared R -lipoic acid for 24h and then sub-jected to BSO treatment for 6h as previously described.Pretreatment with R -lipoic acid prevents depletion of glutathione levels within BSO-treated PC12cells (Fig.2a ).Mitochondrial glutathione content estimated from BSO-treated cells pre-treated with R -lipoic acid versus untreated cells demonstrate that mitochondrial levels of glutathione were decreased as signi ﬁcantly as cellular glutathione levels by BSO treatment and that they too are completely preserved upon pre-treatment with R -lipoic acid (Fig.2b ).These results emphasize the role of R -lipoic acid as a potential anti-oxidant with the ability to help cells maintain their total GSH pool.It has been well documented in PD that mitochon-drial dysfunction is a hallmark of disease pathogenesis (Beal,1992).This abnormality is manifested by a selective decrease in the enzymatic activity of mitochondrial complex I (Beal,1992;Jenner,1993;Hillered and Chan,1988;Haas et al.,1995;Jha et al.,2000).Since pretreatment with R -lipoic acid signi ﬁ-cantly prevented BSO-mediated depletion of cellular and mitochondrial total glutathione levels,it was logi-cal to analyze whether this type of pretreatment could lead to preservation of mitochondrial complex I activ-ity following BSO treatment.Hence,mitochondrial complex I enzyme assays were carried out on cells which had been pretreated with R -lipoic acid versus un-pretreated cells prior to BSO application.Fig.3shows data demonstrating that pre-incubation ofPC12Fig.2.The effect of pre-treatment with 100mM R -lipoic acid on the total glutathione (GSH þGSSG)levels in BSO treated and untreated (a)whole cell extracts and (b)mitochondria.Significance of difference from control was determined by ANOV A single factor analysis.Data is expressed as mean þS :E :M :(n ¼3).(a)ÃP <0:0001vs.untreated cells;ÃÃP <0:0001vs.untreated cells;ÃÃÃP <0:001vs.BSO alone treated cells.(b)ÃP <0:001vs.untreated cells;ÃÃP <0:0001vs.untreated cells;ÃÃÃP <0:005vs.BSO alone treatedcells.Fig.3.The effect of pre-treatment with 100m M R -lipoic acid on mitochondrial complex I activity in BSO-treated and untreated PC12cells.Relative rotenone-sensitive specific activities are plotted.Significance of difference from control was determined by ANOV A single factor analysis.Data is expressed as mean ÆS :E :M :(n ¼6).ÃP <0:005vs.control.482S.Bharath et al./NeuroToxicology 23(2002)479–486cells with R -lipoic acid preserves impaired complex I activity caused by BSO insult.The effect of R -lipoic acid demonstrated here might be due to either an increase in the activity of g -GCL,the rate-limiting enzyme in the glutathione production,or due to the direct anti-oxidant activity of R -lipoic acid which may prevent glutathione depletion.To distinguish between these two,we carried out enzyme assays for g -GCL from extracts obtained after various treatments.The result of these experiments (Fig.4)demonstrate that upon BSO treatment alone there is a decrease ($50%)in the g -GCL activity whereas cells treated with either R -lipoic acid alone or R -lipoic acid þBSO do not show any difference in the activity compared to control cells.DISCUSSIONOne of the earliest detectable events during the course of PD is a signi ﬁcant decrease in cellular levels of GSH in the SN (Pearce et al.,1997;Jenner,1993).This may contribute to oxidative stress and ensuing neuronal cell death of dopaminergic neurons in this brain region.Based on our earlier published ﬁndings,we proposed that early GSH depletion in the SN may be directly responsible for selective inhibition of mito-chondrial complex I activity and the concomitant loss of mitochondrial function which leads to neuronal cell death in this disorder (Jha et al.,2000).Selective reductions in GSH levels which precede losses in mitochondrial complex I activity have been reported to occur not only in PD but also in associated toxin models of the disease such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)administration (Perry and Yong,1988;Hallman et al.,1985;Sriram et al.,1998).Whether the inhibition of complex I activity and subsequent decreases in ATP levels fol-lowing MPTP administration can be totally accounted for by decreases in GSH levels is unclear as complex I also appears to be directly inhibited by interaction with 1-methyl-4-phenyl pyridium (MPP þ)formed during monoamine oxidase-B-mediated oxidation of MPTP (Jha et al.,1999).However,decreasing GSH levels by treatment with the pharmacological agent BSO has been shown to potentiate the neurodegenerative effects of MPTP in the SN and to alone cause sublethal damage to the nigrostriatal system (Wullner et al.,1996;Andersen et al.,1996).GSH is an anti-oxidant molecule well known to protect proteins during oxidative stress.It acts by conjugating oxidized thiol groups of target proteins forming mixed disul ﬁdes which are then processed by GSH reductase,thioredoxin or protein disulphide isomerase to reduced protein thiol residues and GSH (Ravindranath and Reed,1990;Jung and Thomas,1996).Thus,GSH serves as a major non-protein cellular defense against oxidative stress by maintaining the reduced state of cellular thiol proteins.Since GSH depletion in the SN is well documented in PD,it is of paramount importance to search for and test com-pounds which could prevent depletion of cellular levels of GSH in PD patients.To screen for such compounds,we have utilized the PC12cell culture model system in which activity of g -GCL has been partially impaired by BSO treatment.Although g -GCL activity levels do not appear to be speci ﬁcally impaired in sporadic cases of PD,the overall effect of BSO treatment in the dopaminergic PC12cells mimics that which is seen in the Parkinsonian brain,i.e.a decrease in GSHlevelsFig.4.The effect of pre-treatment with 100m M R -lipoic acid on g -glutamyl cysteine ligase activity in BSO-treated and untreated PC12cells.Relative %specific activity is plotted.Significance of difference from control was determined by ANOV A single factor analysis.Data is expressed as mean ÆS :E :M :(n ¼6).ÃP <0:0001vs.control.S.Bharath et al./NeuroToxicology 23(2002)479–486483leading to increased oxidative stress(Wullner et al., 1996;Andersen et al.,1996).Furthermore,though acute depletion of GSH appeared to have no effect on overall cell viability or growth even after48h(Jha et al.,2000),prolonged mitochondrial dysfunction would likely eventually result in decreased cell viability like that observed in dopaminergic SN neurons in PD. As shown in Figs.1and2,increased exposure to BSO results in a notable decrease in the GSH content both in whole cell preparations and in the mitochondria. Signiﬁcant changes occur in the mitochondrial phy-siology during oxidative stress and in diseases like PD (Beal,1992).In this current study,we found that BSO treatment eliciting an increase in oxidative stress via a 50%decrease in total cellular GSH content resulted a 40%decrease in the speciﬁc activity of mitochondrial complex I(Fig.3).This correlates well with our earlier ﬁndings in which we observed a similar decrease in mitochondrial complex I activity following50%GSH depletion,with no apparent effects on the activities of the other mitochondrial complexes(Jha et al.,2000). Mitochondrial complex I is considered to be the mitochondrial complex the most severely affected by oxidative stress(Lenaz et al.,1997).In synaptic mitochondria,complex I exerts a major control over oxidative phosphorylation such that at only25%inhi-bition,energy metabolism is disturbed resulting in decreased ATP synthesis(Davey et al.,1998).R-lipoic acid,a thiol containing compound,is a widely used antioxidant(Fuchs et al.,1997;Packer et al.,1995;Sen et al.,1999).R-lipoic acid functions as a redox regulator of proteins such as myoglobin, prolactin,thioredoxin,and NF-kappa B transcription factor(Bushtamante et al.,1998;Packer et al.,1995, 1997).It has been utilized to treat or prevent peripheral neuropathy and cardiac autonomic neuropathy(Ziegler and Gries,1997),insulin resistance in type II diabetes (Jacob et al.,1996),retinopathy and cataracts(Maitra et al.,1996),glaucoma(Head,2001),HIV/AIDS (Packer et al.,1995),cancer(Ames,1999),liver disease (Bushtamante et al.,1998),Wilson’s disease(Yamamoto et al.,2001),cardiovascular disease(Matalon et al., 1984)and lactic acidosis caused by inborn errors of metabolism(Yoshida et al.,1990).It has also been used for treatment of Alzheimer type dementia(Hager et al., 2001).R-lipoic acid administration has been reported to result in increased ambulatory activity and improved memory in aged animals and to partially restore age-associated mitochondrial decay in both the liver and heart(Hagen et al.,1999,2000;Suh et al.,2001). It therefore seems likely that thiol antioxidants such as R-lipoic acid which can enhance mitochondrial function,scavenge free radicals,and increase the levels of the antioxidants GSH and ascorbate might be useful neuroprotective agents via their reducing actions onvital protein thiol residues(Han et al.,1997;Xu and Wells, 1996).Our results together with previous studies suggest that R-lipoic acid may be an effective neuroprotective agent in age-associated neurodegeneration.Utilizing the PC12cell model system,we propose that R-lipoic acid administration could be an effective way of circumventing or delaying mitochondrial dys-function associated with PD.Treatment with R-lipoic acid alone seems to signiﬁcantly increase GSH levels only in whole cell preparations but not in mitochon-drial extracts(see Fig.2a and b).However,pretreat-ment of cells with R-lipoic acid appears to prevent BSO-mediated GSH depletion in both whole cells and mitochondria.These data suggest that only during BSO treatment is there a R-lipoic acid-mediated increase in mitochondrial GSH to counter BSO generated ROS production.We were curious to know whether the effect of R-lipoic acid on GSH levels was due to an induction of g-GCL activity.Han et al.(1997)have shown that R-lipoic acid increases de novo synthesis of glutathione by improving cystine utilization.It is also possible that reduction of R-lipoic acid to dihydrolipo-ate in vivo might cause oxidation of GSH to GSSG in turn inducing GCL activity.However,since total glu-tathione estimations in our experiments involved quan-titation of both GSHþGSSG,depletion of GSH cannot be due to the accumulation of GSSG thus ruling out the possibility that increased GSSG concentration might be affecting the g-GCL activity.Fig.4shows that BSO treated cells exhibit$50%decrease in g-GCL activity compared to the untreated cells.Interestingly, cells pretreated with R-lipoic acid upon incubation with BSO showed an increase in g-GCL activity compared to cells treated with BSO alone.These data suggest that R-lipoic acid might contribute to an increased GSH pool during BSO treatment due to the induction of g-GCL activity.However,when cells are treated with R-lipoic acid alone there is a signiﬁ-cant increase($1.6fold)in GSH levels without a concomitant increase in g-GCL activity suggesting that GSH levels are preserved by the direct anti-oxidant properties of R-lipoic acid and not due to increased g-GCL activity(Fig.2a).Decreases in mitochondrial NADH dehydrogenase(complex I)activity associated with GSH depletion also appear to be preserved via R-lipoic acid pretreatment(Fig.3).Taken together, these data suggest that GSH depletion,which may lead to oxidation of protein sulfhydryl residues in the mito-chondrial complex I important for normal functions484S.Bharath et al./NeuroToxicology23(2002)479–486resulting in profound effects on subsequent mitochon-drial performance,may be prevented by the direct action of R-lipoic acid.Our earlierﬁndings suggested that the activity of NADH dehydrogenase,the enzy-matic component of complex I,appears to be thiol-regulated(Jha et al.,2000).The preservation of mito-chondrial complex I activity via R-lipoic acid suggests that it protects against the oxidation of proteins by preventing the use of the cellular GSH pool or by increasing the de novo synthesis of cellular GSH in the presence of BSO-mediated oxidative stress.Whether such treatment would be effective in actually altering brain levels of GSH and thus providing beneﬁcial effects in vivo has not yet been demonstrated. However,our data suggest that maintaining thiol homeostasis may be critical for protecting dopami-nergic neurons of the SN against neurodegeneration and that antioxidants such as R-lipoic acid may be of therapeutic value in PD.It would be of interest to conduct additional experiments using intermediates of dopamine metabolism or MPTP to explore thera-peutic value of R-lipoic acid pretreatment in PD. Nevertheless,since GSH depletion is the earliest event in PD pathogenesis,our data clearly suggests that R-lipoic acid can prevent the deleterious effects that follow by preventing mitochondrial GSH depletion.ACKNOWLEDGEMENTSThis work was supported by a grant from the National Institute of Health(NIH,R01AG12141) to JKA and by a grant from the Ellison Foundation (SS-0422-99),the National Institute of Health (NIH,R01,AG17140),and the National Institute of Environmental Health Sciences Center(NIH,P30, ES01896)to BNA.We thank Dr.M.Jyothi Kumar for technical help and criticaldiscussionsandDr.Simon Melov,Buck Institute,for use of his double beamed spectrophotometer.REFERENCESAdams Jr,JD,Chang ML,Klaidman L.Parkinson’s disease-redox mechanisms.Curr Med Chem2001;8:809–14.Ames BN.Micronutrients prevent cancer and aging.Toxicol Lett 1999;103:5–18.Andersen JK,Mo JQ,Hom DG,Lee FY,Harnish P,Hamill RW, McNeill TH.Effect of buthionine sulfoximine,a synthesis inhibitor of the antioxidant glutathione,on murine nigrostriatal neurons.J Neurochem1996;67:2164–71.Beal MF.Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses.Ann Neurol1992;31:119–30.Burke RE.Parkinson’s disease.In:Koliatos,Ratan RR,editors. Cell death and disease of the nervous system.Totowa(NJ): Humana Press,1998.p.459–75.Bushtamante J,Lodge JK,Marcocci L,Tritschler HJ,Packer L, Rihn BH.Alpha-lipoic acid in liver metabolism and disease. Free Radic Biol Med1998;24:1023–39.Davey GP,Peuchen S,Clark JB.Energy thresholds in brain mitochondria:potential involvement in neurodegeneration.J Biol Chem1998;273:12753–7.Fuchs J,Packer L,Zimmer G.Lipoic acid in health and disease. New York:Marcel Dekker,1997.Greene L,Tischler A.Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.Proc Natl Acad Sci USA1976;73: 2424–8.Haas RH,Nasirian F,Nakano K,Ward D,Pay M,Hill R,Shults CW.Low platelet mitochondrial complex I and complex II/III activity in early untreated Parkinson’s disease.Ann Neurol 1995;37:714–22.Hagen TM,Ingersoll RT,Lykkesfeldt J,Liu J,Wehr CM,Vinarsky V,Barthlomew JC,Ames AB.(R)-alpha-lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage,and increased metabolic rate. FASEB J1999;13:411–8.Hagen TM,Vinarsky V,Wehr CM,Ames BN.(R)-alpha-lipoic acid reverses the age-associated increase in susceptibility of hepatocytes to tert-butylhydroperoxide both in vitro and in vivo. Antiox Redox Signal2000;2:473–83.Hager K,Marahrens A,Kenklies M,Riederer P,Munch G.Alpha-lipoic acid as a new treatment option for Alzheimer type dementia.Arch Gerontol Geriat2001;32:275–82.Hallman H,Lange J,Olson L,Stromberg I,Jonsson G. Neurochemical and histochemical characterization of neurotoxic effects of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on brain catecholamine neurons in the mouse.J Neurochem1985;44: 117–27.Han D,Handelman G,Marcocci L,Sen CK,Roy S,Kobuchi H, Tritschler HJ,Flohe L,Packer L.Lipoic acid increases de novo synthesis of cellular glutathione by improving cystine utilization. Biofactors1997;6:321–38.Head KA.Natural therapies for ocular disorders,part two: cataracts and glaucoma.Alternative Med Rev2001;6:141–66. Hillered L,Chan PH.Effects of arachidonic acid on respiratory activities in isolated brain mitochondria.J Neurosci Res 1988;19:94–100.Huang CS,Chang LS,Anderson ME,Meister A.Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase.J Biol Chem1993;268:19675–80. Jacob S,Henriksen EJ,Tritschler HJ,Augustin HJ,Dietz GJ. Improvement of insulin-stimulated glucose disposal in type2 diabetes after repeated parenteral administration of thioctic acid.Endocrinol Diabetes1996;104:284–8.Jenner P.Altered mitochondrial function,iron metabolism and glutathione levels in Parkinson’s disease.Acta Neurol Scand Suppl1993;146:6–13.Jha N,Andersen JK.Loss of glutathione(GSH)in Parkinson’s disease:how does GSH act to protect dopaminergic neurons of the substantia nigra?In:Pandalai SG,editor.Recent researchS.Bharath et al./NeuroToxicology23(2002)479–486485。

聚二甲基硅氧烷芯片自由酶反应器检测葡萄糖

聚二甲基硅氧烷芯片自由酶反应器检测葡萄糖作者：仲海燕周洁余晓冬陈洪渊【摘要】应用电泳中介微分析（EMMA）技术，构建聚二甲基硅氧烷（PDMS）芯片自由酶反应器，在线检测葡萄糖（Glu），在十字形的芯片通道上，采用自制的碳纤维微电极检测葡萄糖氧化酶（GOD）催化氧化Glu生成的H2O2，并对检测电位、GOD浓度、GOD进样时间、分离电压等参数进行了优化，测定了该自由酶反应器的线性范围和检出限，考察了其重现性及稳定性。

结果表明，此自由酶反应器制作方便，操作简单，重现性好，Glu浓度在0.1～20 mmol/L之间有较好的线性关系（r=0.997），检出限为19.8 μmol/L（S/N=3）。

【关键词】自由酶反应器；葡萄糖；电泳中介微分析；聚二甲基硅氧烷芯片The Key Lab of Analytical Chemistry for Life Science, Ministry of Education, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093)Abstract Microfluidic enzyme based reactor could be used to detect the biomolecules with high sensitivity and selectivity. Based on the electrophoretic mediated microanalysis (EMMA) method, a new kind of free enzyme based poly(dimethylsiloxane) (PDMS) microchip was developed to detect glucose (Glu). On line catalysis reaction of Glu by glucose oxidase (GOD) was carriedout on the chip. The product H2O2 was detected using single carbon fibre cylindrical electrode. Factors influencing the separation and detection, such as detection potential, GOD concentration, GOD injection time and separation voltage, were investigated and optimized. Results showed that the peak current had a good linear relationship with Glu concentration in the range of 0.1－20 mmol/L (R=0.997). The detection limit of Glu was 19.8 μmol/L (S/N=3). In addition, the PDMS enzyme based reactor had long term stability and excellent reproducibility (RSD=2.02%, n=10). It was easy to fabricate and operate, which showed great potential application in bioanalysis.Keywords Free enzyme based microchip; Glucose; Electrophoretic mediated microanalysis; Poly(dimethylsiloxane)1 引言近年来，基于酶催化反应的微流控芯片备受关注。

泽泻醇在小胶质细胞中对基质金属蛋白酶3与一氧化氮的抑制作用

泽泻醇在小胶质细胞中对基质金属蛋白酶3与一氧化氮的抑制作用刘瑜【摘要】This paper investigates the inhibitory effect and mechanism of alismol on neuroinflamination in the activated BV2 microglial cells which are stimulated by lipopolysaccharides(LPS). NO was measured by using Griess reagent. RT-PCR and Western blot are used to analyse ERK, JNK, Akt, and MMP3 . Alismol can significantly inhibit LPS-induced NO production and the MMP3 expression. The mechanism is involved to its inhibition of PI3K/Akt pathway.%利用脂多糖(LPS)刺激小鼠小胶质细胞BV2,研究泽泻醇对炎症相关分子的抑制及机制.Griess法测定一氧化氮(NO)浓度,RT-PCR和Western blot法检测细胞外调节蛋白激酶(ERK)、p38、c-Jun氨基末端激酶(JNK)、蛋白激酶B(Akt)、基质金属蛋白酶3(MMP3)的变化.研究结果表明,泽泻醇不仅对LPS刺激小胶质细胞产生的NO有明显抑制作用,还能在mRNA与蛋白质水平抑制MMP3的表达,这种抑制与其对PI3K/Akt通路的干预相关.阐述了泽泻醇对小胶质细胞的抑制与PI3K/Akt通路的相关机制.【期刊名称】《实验技术与管理》【年(卷),期】2012(029)010【总页数】4页(P47-50)【关键词】小胶质细胞;泽泻醇;一氧化氮;基质金属蛋白酶3【作者】刘瑜【作者单位】南开大学医学院,天津 300071【正文语种】中文【中图分类】R914Abstract：This paper investigates the inhibitory effect and mechanism of alismol on neuroinflammation in the activated BV2microglial cells whichare stimulated by lipopolysaccharides（LPS）.NO was measured by using Griess reagent.RT-PCR and Western blot are used to analyse ERK，JNK，Akt，and MMP3 .Alismol can significantly inhibit LPS-induced NO production and the MMP3expression.The mechanism is involved to its inhibition of PI3K/Akt pathway.Key words：microglia；alismol；NO；MMP3小胶质细胞是中枢神经系统内的免疫细胞，长期激活而形成中枢神经系统的慢性炎症，其释放的大量氧自由基、炎症介质细胞因子以及基质金属蛋白酶（matrix metalloproteinases，MMP）是神经元损伤的重要原因之一，也是阿尔茨海默病（Alzheimer’s disease，AD）与帕金森病（Parkinson’s disease，PD）等许多中枢神经退行性疾病发生与发展的重要因素之一［1-2］。

脊髓缺血再灌注损伤的发病机制与治疗进展_艾琪勇

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实用医学杂志 2014 年第 30 卷第 16 期
脊髓缺血再灌注损伤的发病机制与治疗进展
艾琪勇罗越刘辉洪国峰蔡林
脊髓缺血再灌注损伤（spinal cord ischemia ／ reperfusion injury，SCII）常发生于脊髓长期缓慢受压迫如黄韧带、后纵韧带增厚骨化、椎间盘突出引起椎管狭窄或椎管内肿瘤逐渐增大压迫脊髓引起脊髓缺血性损伤的患者在通过手术减压后随即出现更严重的部分或原受压平面以下全脊髓损伤症状［1］。如不及时处理，脊髓功能可能长期或永久损伤，严重影响患者生活质量，造成个人、家庭甚至社会的负担。 1 脊髓缺血再灌注损伤的发病机制
doi：10．3969 ／ j．issn．1006-5725.2014.16．052 作者单位：430071 武汉大学中南医院骨科（艾琪勇，罗越，刘辉，蔡林）；430071 武汉大学口腔医院（洪国峰）通信作者：蔡林 E-mail：guke3559＠ali yun．com
实用医学杂志 2014 年第 30 卷第 16 期
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可以通过提高神经细胞对氧化的耐力，从而达到保护神经细胞的功能。［22－23］ 2．7 α-2a 肾上腺素激动剂治疗磷酸化环 AMP 反应元件蛋白（CREB）是神经元存活所必需的因子，α-2a 肾上腺素激动剂如右旋美托嘧啶可以通过激活 CREB 磷酸化上调抗凋亡因子及脑源性神经保护因子（BDNF）以达到脊髓神经元的保护。［24］ 2．8 地尔硫卓治疗地尔硫卓属于钙离子拮抗剂。在 SCII 中使用钙离子拮抗剂可有效减轻细胞钙超载，具有细胞保护的作用。也有研究表明，地尔硫卓可以在使用后 3 ～ 24 h 内有效控制 IL-6 的释放，减轻炎性损伤。［25］ 2．9 米诺环素治疗小胶质神经细胞是 SCII 后大量聚集并诱导炎症趋化因子聚集损伤神经元细胞［6］。米诺环素属于大环内酯类抗生素的一种，有研究表明米诺环素可以通过抑制小胶质神经细胞的活性以达到保护神经元功能。它是预防及治疗 SCII 的潜在药物。［26］ 2．10 亚低温技术应用亚低温指 28 ～ 35 ℃水平的温度。而 31 ～ 33 ℃为最佳温度段。亚低温抑制 IL-8 的释放，减少神经细胞凋亡、白质空泡及白质灰质神经胶质化以达到减轻缺血再灌注损伤造成的再次脊髓损伤。［27］且亚低温技术的应用相对安全，适于临床推广。 2．11 高压氧治疗动物实验证明高压氧预处理急性脊髓损伤的小鼠可减少继发性脊髓损伤细胞凋亡，同时促进脊髓损伤后修复。高压氧预处理可以促进氧自由基的生成。氧自由基可刺激缺氧诱导因子－1 （HIF-1 ）的生成从上调 EPO 的表达，从而减轻 SCII 。无论从动物实验还是临床观察均提示围手术期中重复高压氧治疗可减轻脊髓缺血－再灌注对神经细胞的损伤，保护神经功能进一步损伤。 2．12 电针治疗有研究表明 SCII 患者通过电针刺激足三里及曲池穴可能通过降低 TNF-α 及 IL-6 等炎症因子的释放以达到脊髓功能的保护。 2．13 脂肪干细胞移植治疗有动物实验表明向因 SCII 而导致脊髓功能受损的动物髓鞘内注射脂肪干细胞后 15 min 脊髓功能得到改善。脂肪干细胞的移植在 SCII 发生 72 h 后上调 BDNF 的表达及增加神经营养因子以防止运动神经元受胶质细胞增生损坏。