摘要
目的研究足细胞内热休克蛋白27(HSP27)是否与转录激活因子5(ATF5)形成复合物及其在足细胞凋亡中的意义。方法高糖刺激体外培养的小鼠肾小球足细胞系建立细胞凋亡模型,应用Hochest染色、流式细胞技术测定细胞凋亡。应用Western印迹分析技术检测丝裂原活化蛋白激酶(MAPK)信号途径的活化,应用免疫共沉淀技术检测HSP27和ATF5形成复合物的变化,阻断实验观察MAPK信号途径对高糖调节足细胞HSP27与ATF5形成复合物以及足细胞凋亡的影响。结果成功建立了高糖刺激足细胞凋亡的模型,30mmol/L高糖刺激48h凋亡率(27.2%±8.9%)显著高于5.5mmol/L正常糖(对照)48h组(10.6%±2.7%,P<0.05)。正常糖(对照)组细胞HSP27与ATF5即可形成复合物,而在高糖刺激12hHSP27与ATF5形成的复合物明显增加为正常糖组的195%±36%(P<0.05)。高糖刺激30min即可使细胞外信号调节激酶(ERK1/2)和p38活化。进一步阻断实验显示ERK1/2活化抑制剂可以减弱高糖刺激引起HSP27和ATF5形成复合物的效应(PD98059+高糖组为109%±19%,高糖组211%±46%,P<0.05),同时加重高糖诱导细胞凋亡(PD98059+高糖组凋亡率为51%±4%,高糖组凋亡率为27%±9%)(P<0.05)。p38活化抑制剂不影响高糖刺激引起的HSP27和ATF5形成复合物(SB20358+高糖组为290%±43%,高糖组为231%±20%,P>0.05),但可减轻高糖诱导细胞凋亡(SB20358+高糖组凋亡率为16%±6%,高糖组凋亡率为27%±9%)(P<0.05)。结论高糖依赖ERK1/2信号通路而不是p38信号通路刺激足细胞HSP27与ATF5形成复合物的增加,蛋白复合物可能在高糖诱导的足细胞凋亡中起保护作用。
Objective To investigate the regulation of heat shock protein （HSP）27/activating transcription factor （ATF）-5 complex in podocytes induced by high glucose and relevant mechanisms. Metlmds Mice kidney podocytes were cultured in culture fluid with D-glucose at normal concentration （5.5 tool/L） （Group NG） or with D-glucose at high concentration （30 mmol/L） （Group HG） cells of these 2 groups were collected at different time points after glucose stimulation to detect the cell apoptosis by Hoechst 33342 staining and fluorescence microscopy and flow cytometry. Western blotting was used to analyze the activation of extracellular signal-regnlated kinase （ ERK = MAPK） and p38 signaling pathway. The HSP27/ATF5 complex was assessed by co-immunoprecipation. ERK pathway blocker PD98059 and p38 signal pathway blocker SB203580 were added into the culture fluid of Group HG and Group NG respectively, and then the podocytes were collected at different time points to detect the high glucose-induced HSP27/ ATF5 complex and cell apoptusis. Results The apoptotic rate of the podocytes of Group HG 24 hours after high glucose incubation was 14.3% ±6.2%, and that 48 h after was 27.2% ±8.9%, significantly higher than that of Group NG （10.6% ±2.7%, P〈0.05）. HSP27/ATF5 complex could detected in the cells of Group NG too, however, the level of HSP27/ATF5 complex in Group HG 12 hours after incubation was 195% ±36% that of Group NG （ P 〈0.05）. Both the ERK signal pathway and p38 signal pathway of Group HG began to be activated 10 min after incubation, peaked 30 min after, remained at the highest level the 1 hour after, and returned almost to the baseline level 2 hours after. No activation of these 2 pathways was observed in Group NG. The HSP27/ATF5 complex level of the PD98059 ＋ high glucose group was 109% ± 19% that of Group NG, significantly lower than that of Group HG （211% ±46% that of Group NG, P 〈 0.05）. The apoptotic rate of the PD98059 ＋ high glucose group was 51% ±4%, significantl
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第6期394-398,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目(30270612
30570851)
北京大学"211"工程人类功能基因与疾病基因研究项目
