缺氧复氧对大鼠离体肾小管上皮细胞凋亡及缝隙连接的影响

Effects of hypoxia/reoxygenation on apoptosis and gap junction in isolated renal tubular epithelial cells of rats

目的 评价缺氧复氧对大鼠离体肾小管上皮细胞凋亡及缝隙连接的影响.方法 肾小管上皮细胞以1＆#215;105个/ml密度接种于6孔培养板（2 ml/孔）,采用随机数字表法分为2组（n=18）：正常对照组（C组）和缺氧复氧组（H/R组）.C组细胞置于常氧恒温CO2细胞培养箱28 h,H/R组细胞置于厌氧培养罐中缺氧培养24 h后,取出置于常氧恒温CO2细胞培养箱进行复氧培养4h.使用Hoechst33258染色法及流式细胞术检测细胞凋亡率；采用特异的“细胞接种荧光示踪法”检测细胞缝隙连接功能,Western blot法检测缝隙连接蛋白Connexin 43（Cx43）表达水平.结果 与C组比较,H/R组细胞凋亡率升高,细胞缝隙连接功能及Cx43蛋白表达降低（P＜0.05）.结论 缺氧复氧通过破坏细胞缝隙连接促进大鼠肾小管上皮细胞凋亡.

Objective To evaluate the effects of hypoxia/reoxygenation （H/R） on apoptosis and gap junction in isolated renal tubular epithelial cells of rats.Methods The renal tubular epithelial cells were seeded in 6-well plates at a density of 1 ＆#215; 105 cells/ml and randomly divided into 2 groups （n =18 wells each） using a random number table：control group （group C） and H/R group.In group C,the cells were cultured in a regular incubator with 21% oxygen at 37 ℃.In group H/R,the cells were incubated in an anaerobic chamber for 24 h and then returned to a regular incubator and incubated for 4 h.Hoechst33258 staining method and flow cytometry were used to detect the cell apoptosis.Gap junction function was determined by specific parachute assay.Gap function protein connexin 43（Cx43） expression was measured by Western blot.Results Compared with group C,the apoptosis rate was significantly increased and gap junction function and Cx43 expression were decreased in group H/R （P 〈 0.05）.Conclusion H/R promotes apoptosis in isolated renal tubular epithelial cells through destroying intercellular gap junction in rats.