摘要
目的:以无糖培养12h后再以完全培养基继续培养的方法模拟细胞缺血后再灌注的损伤情况,观察葡萄糖调节蛋白75在缺糖及缺糖再灌注条件下对细胞的保护作用。方法:实验于2002-09/2004-02在复旦大学医学院医学与细胞遗传系实验室完成。以中国仓鼠肺细胞株为材料,置于无糖条件下培养12h后,重换含糖培养基继续培养以建立缺血再灌注体外培养模型,并采用脂质体介导的方法,以葡萄糖调节蛋白75表达载体(pcDNA3/葡萄糖调节蛋白75)转染中国仓鼠肺细胞细胞,获得过表达葡萄糖调节蛋白75的细胞克隆,运用四甲基偶氮唑盐微量酶反应比色法,乳酸脱氢酶释放率测定等方法评估细胞损伤程度。结果:①克隆细胞用葡萄糖调节蛋白75抗体对其蛋白质产物进行印迹分析,克隆细胞葡萄糖调节蛋白75蛋白表达量明显高于对照组。②四甲基偶氮唑盐微量酶反应比色法显示,未转染细胞缺糖再灌注的增殖能力比完全培养20h增殖能力明显降低(P<0.05),且低于无糖培养20h(P<0.05);重新灌注后,葡萄糖调节蛋白75过表达的细胞的增殖能力明显高于对照组(P<0.01)。③乳酸脱氢酶释放测定结果显示,转染细胞缺糖再灌注乳酸脱氢酶释放百分比显著高于完全培养20h(P<0.01),与无糖培养20h无明显差别(P>0.05),葡萄糖调节蛋白75过表达的细胞乳酸脱氢酶释放百分比显著低于对照组(P<0.01)。结论:通过建立过表达葡萄糖调节蛋白75的细胞克隆以无糖培养模拟能量代谢应激,观察葡萄糖调节蛋白75对处于能量代谢性应激中细胞的保护作用,以及应激后细胞的恢复情况。发现表达葡萄糖调节蛋白75的细胞较未转染的细胞具有更高的存活率,细胞膜损伤和DNA损伤较轻,说明葡萄糖调节蛋白75在细胞缺糖后再灌注损伤中具有一定的保护作用。
AIM： To simulate the ischemia-reperfusion injury by means of culture with glucose deprivation for 12 hours, and continue to culture in complete medium, and observe the protective effect of glucose-regulated protein 75 on cells under ischemia and ischemia-reperfusion. METHODS：The experiment was carried out in the laboratory of Department of Medical Science and Cytogenetics, Fudan University between .September 2002 and February. The pulmonary cell strains of Chinese hamsters were cultured under the condition of glucose deprivation for 12 hours, and continued to culture in the changed culture medium containing glucose to establish in vitro cultured models of ischemiareperfusion. The pulmonary cells of Chinese hamsters were transfected with the expression vector of glucose-regulated protein 75 （pcDNA3/glucoseregulated protein 75） by means of liposomes mediation to obtain the cell clone of the overexpressed glucose-regulated protein 75. The severity of cell injury was evaluated with methyl-thiazol-tetrazolium colorimetry and detection of the lactate dehydrogenase leakage. RESULTS： ① The results of the blotting analysis of the protein production of the cloning cells with the antibody of glucose-regulated protein 75 showed that the amount of the protein expression of glucoseregulated protein 75 was obviously higher in the cloning cells than in the control group. ② The results of methyl-thiazol-tctrazolium colorimetry showed that tile proliferative ability of the untransfected cells of ischemia reperfusion was obviously decreased as compared with those completely cultured for 20 hours （P 〈 0.05）, and lower than those cultured under glucose deprivation for 20 hours （P 〈 0.05）; After reperfusion, the proliferative ability of cells with overexpressed glucose-regulated protein 75 was obviously higher than that in the control group （P 〈 0.01）. ③ The results of detection of the lactate dehydrogenase leakage indicated that the percentage of lactate dehydrogentase leakage was significantly highe
出处
《中国临床康复》
CSCD
北大核心
2005年第27期67-69,共3页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助(39670239)
美国中华医学基金会资助项目(96-635)~~
