医药学论文：还原型谷胱甘肽对培养乳鼠心肌细胞缺氧复氧损伤的影响

【摘要】 目的 研究还原型谷胱甘肽对乳鼠心肌细胞缺氧/复氧(A/R)损伤的影响。方法 建立心肌细胞A/R损伤模型，随机分为５组：A组，正常对照组；B组，单纯缺氧/复氧(A/R低氧2h，复氧1h)组；C、D、E组为还原型谷胱甘肽处理组，加入还原型谷胱甘肽(GSH)分别使其终浓度为40、80、160 mg/L，后A/R。于复氧后测定各组培养液中乳酸脱氢酶(LDH)变化及细胞内丙二醛(MDA)含量和细胞存活率。结果 与正常组相比，单纯低氧/复氧组LDH漏出量、MDA水平显著升高(P< 0.01)，细胞存活率显著降低(P< 0.01)。与缺氧/复氧组比较，各谷胱甘肽处理组上述变化明显减轻(P< 0.05)。结论 还原型谷胱甘肽对乳鼠心肌细胞A/R损伤具有保护作用，并具有浓度依赖性。

【关键词】 还原型谷胱甘肽 心肌细胞 缺氧 复氧

The effects of reduced glutathione sodium on anoxia/reoxygenation injuries of neonatal rat cardiomyocytes

【Abstract】 Objective To study the effects of reduced glutathione sodium(GSH) on anoxia /reoxygenation injuries of neonatal rat cardiomyocytes.Methods A/R model of myocardial cells of neonatal rats was established. The cells of neonatal rats were randomly divided into five groups: control group(A): without any treatment; A/R group(B):2-hour anoxia followed by 1-hour reoxygenation; GSH conditioned group(C):adding GSH into culture medium till the final concentration of 40mg/L then A/R; GSH conditioned group (D):adding GSH into culture medium till the final concentration of 80mg/L then A/R; GSH conditioned group (E):adding GSH into culture medium till the final concentration of 160mg/L then A/R. The activity of lactate dehydrogenase(LDH)，the contents of cellular melondadehyde(MDA) and the rate of cell viability of each group were evaluated after reoxyenation.Results Compared with group A，the A/R group showed a great increase in levels of LDH，MDA and a decrease in the rate of cell viability(P< 0.01). Compared with group B，the GSH conditioned groups significantly attenuated these changes(P< 0.05).Conclusion GSH could be a protective effect on A/R injury of neonatal rat cardiomyocytes in a concentration dependent manner.

【Key words】 reduced glutathione sodium cardiomyocytes anoxia reoxygenation

1 材料与方法

1.1 材料 实验用1～3天的Wistar大鼠乳鼠，山东大学实验动物中心提供，雌雄不拘；DMEM培养基由爱博公司出品；胎牛血清为美国　Gibco公司出品；胰酶为Sigma公司出品；丙二醛（MDA），乳酸脱氢酶（LDH）试剂盒均购自南京建成生物研究所；还原型谷胱甘肽（古拉定）由 Laboratorio Farmaceutico C.T.S.R.1生产(批号：4027)。

1.2 乳鼠心肌细胞培养 无菌取出生1～3天大鼠乳鼠心脏，剪碎后用0.25％胰酶消化4～5次，取除第1次以外的上清，用含15％胎牛血清的高糖DMEM 中止消化，离心取心肌细胞沉淀，加入培养液，集中混匀制成悬液，应用差速贴壁法分离纯化心肌细胞，以5×105/ml的培养密度接种于２４孔培养板中， 在CO２培养箱中培养，48h后换液。

1.3 实验分组及操作程序 试验分为５组，每组重复８孔，A组为正常对照组（培养板置培养箱中持续孵育3h结束实验）；B组为单纯缺氧／复氧组（A/R 缺氧2h，复氧1h）：将培养板置于充有99.５％氮气的缺氧孵育器中37℃密闭培养2h，再以９5％氧气＋５％二氧化碳进行复氧1h；C组，D组，E组为古拉定处理组，加入古拉定使其终浓度分别为40、80、160mg/L。各组缺氧前、复氧前均给药。

1.4 实验检测指标 (1)计数细胞搏动：在倒置显微镜观察并计数心肌细胞搏动的频率及节律。(2)细胞存活率计数：采用台盼蓝排斥法检测，各组取少许细胞混悬液，0.4%台盼蓝混匀2min，按白细胞计数法在光镜下计数蓝染细胞。台盼蓝摄取率＝蓝染细胞／（蓝染细胞＋未蓝染细胞）×100%。(3)LDH及MDA活性及含量测定：实验结束后按试剂盒说明测定。