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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。
OBJECTIVE: To evaluate the protective effect of glucose-regulated protein-75 on cells under hypoglycemic and glucose-reperfusion conditions after 12-hour sugar-free culture and then with complete medium. Methods: The experiment was performed in the Laboratory of Medical and Cytogenetics, School of Medicine, Fudan University from September 2002 to February 2004. The Chinese hamster lung cell line was used as materials to culture in sugar-free condition for 12 h, then the sugar-containing medium was replaced to continue the culture to establish the model of ischemia-reperfusion in vitro and the liposome-mediated method was used to adjust the glucose The protein 75 expression vector (pcDNA3 / glucose-regulated protein 75) was transfected into Chinese hamster lung cells to obtain cell clones overexpressing glucose-regulated protein 75. Methyl thiazolyl tetrazolium (MTT) micro-enzyme reaction colorimetric assay, lactate dehydrogenase Release rate determination and other methods to assess the degree of cell damage. Results: (1) Western blotting analysis of cloned cells with glucose-regulated protein 75 showed that the expression of glucose-regulated protein 75 in cloned cells was significantly higher than that of the control group. (2) MTT colorimetric assay showed that the proliferation ability of untransfected cells was significantly lower than that of 20 h after culture (P <0.05) <0.05). After reperfusion, the proliferation of cells overexpressing glucose-regulated protein 75 was significantly higher than that of the control group (P <0.01). (3) The results of lactate dehydrogenase release showed that the percentage of lactate dehydrogenase released from the transfected cells was significantly higher than that of the control group (P <0.01) The percentage of cell lactate dehydrogenase released by regulatory protein 75 over-expression was significantly lower than that of the control group (P <0.01). CONCLUSIONS: Cell clones overexpressing glucose-regulated protein 75 were used to simulate energy metabolism stress in glucose-free culture to observe the protective effect of glucose-regulated protein 75 on cells in energy-metabolic stress and the recovery of cells after stress. It was found that cells expressing glucose-regulated protein 75 had higher survival rates than non-transfected cells, with less cell membrane damage and DNA damage, indicating that glucose-regulated protein 75 has some protective effect on cell-induced glucose-deficient reperfusion injury.