抑制细胞周期素依赖性蛋白激酶5对小鼠足细胞凋亡的影响

摘要

目的：探讨细胞周期素依赖性蛋白激酶5（Cdk5）在高糖刺激体外培养小鼠足细胞中的表达及抑制 Cdk5表达对高糖诱导足细胞凋亡的影响。方法（1）体外培养条件性小鼠足细胞，分为正常糖组、甘露醇组、高糖组，高糖组按不同刺激时间分为0、6、12、24、48 h 5组，应用蛋白质印迹（Western blot）法检测足细胞中 Cdk5蛋白表达水平。（2）应用 Cdk5 miRNA 干扰质粒抑制足细胞中 Cdk5基因的表达水平。分别设立：NG 组（正常糖）；HG 组（高糖）；HG ＋ S 组（高糖＋ Scrambled 阴性质粒）；HG ＋ C 组（高糖＋ Cdk5 miRNA 质粒）。以上各组细胞培养48 h 后，应用流式细胞术和末端脱氧核苷酰基转移酶介导性 dUTP 切口末端标记（TUNEL）法检测足细胞凋亡水平，Western blot 法检测凋亡相关蛋白 B 细胞淋巴瘤/白血病-2基因（Bcl-2）、Bcl-2相关 X 蛋白（Bax）和分裂型半胱氨酸天冬氨酸蛋白酶-3（cleaved caspase-3）的蛋白表达水平。结果与正常糖组相比，高糖刺激后，足细胞中Cdk5的蛋白表达水平明显增加（P＜0．05）。 Cdk5 miRNA 干扰质粒能显著抑制足细胞中 Cdk5的表达水平。与 HG 组和 HG ＋ S组相比，HG ＋ C 组足细胞凋亡率显著降低（P＜0．05），凋亡相关蛋白 cleaved caspase-3蛋白表达水平和 Bax/Bcl-2蛋白表达比率也有明显降低（P＜0．05）。结论高糖刺激可以增加足细胞中 Cdk5蛋白的表达水平；抑制 Cdk5表达可以降低高糖刺激诱导的足细胞凋亡。%Objective To investigate the expression of Cdk5 in high glucose-induced mouse podocytes and the effects of inhibi-ting Cdk5 expression on podocyte apoptosis .Methods (1)Mouse podocytes cultured in vitro were divided into normal glucose group ,mannitol group ,high glucose group and the high glucose group was further divided in to 0 ,6 ,12 ,24 ,48 h gorup depend on the induce time .Changes of Cdk5 expression in each group were detected by Western blotting .(2)The expression of Cdk5 in podo-cyte was inhibited by Cdk5 miRNA plasmid .The podocytes were divided into 4 groups :NG group(normal glucose) ,HG group(high glucose) ,HG + S group(high glucose + scrambled plasmid) and HG + C group(high glucose + Cdk5 miRNA plasmid) .The podo-cyte apoptosis rates were detected by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) , and the expressions of Bcl-2 ,Bax and cleaved caspased-3 were detected by Western blotting respectively after 48 h .Results Com-pared with normal glucose group ,the expression of Cdk5 was significantly increased in podocytes of high glucose group (P< 0 .05) . The expression of Cdk5 in podocytes was inhibited by Cdk5 miRNA plasmid .Compared with HG and HG + S group ,the podocyte apoptosis rate of HG + C group was significantly decreased(P< 0 .05) .The expression of cleaved caspase-3 protein and Bax /Bcl-2 ratio were also decreased (P< 0 .05) .Conclusion High glucose could increased the expression of Cdk5 in podocytes .The downregu-lation of Cdk5 expression could decrease the podocyte apoptosis induced by high glucose .