周期素依赖性蛋白激酶5过度活性与肾小球足细胞凋亡的关系研究

Relationship Between Overactivation of Cdk5 and the Glomerulus Podocyte Apoptosis

目的通过在肾小球足细胞中转染p25基因,探讨周期素依赖性蛋白激酶5(Cdk5)的活性对足细胞结构及功能的影响。方法 2014年度,于含10%胎牛血清的RPMI 1640培养液中培养分化成熟的小鼠离体足细胞,将足细胞分为对照组、空转染组和p25转染组。应用p Ad Track-CMV病毒载体克隆p25基因,并转染足细胞,48 h收集细胞;采用Western blotting法测定Cdk5、p25、p35及细胞凋亡相关蛋白Cleaved caspase3的表达水平;应用免疫沉淀和同位素γ-32P标记法测定Cdk5活性;足细胞免疫荧光化学染色,观察Cdk5、p35在足细胞中的表达,以及足细胞Actin的排列情况。结果 HEK293细胞Cdk5表达阳性,p35表达阴性,肾脏皮质、足细胞和肾小球中均有不同程度的Cdk5和p35的表达。对照组和空转染组p25表达阴性,p25转染组p25表达阳性。各组细胞凋亡相关蛋白Cleaved caspase3表达水平和Cdk5活性比较,差异有统计学意义(P<0.05);其中p25转染组细胞凋亡相关蛋白Cleaved caspase3表达水平和Cdk5活性高于对照组和空转染组(P<0.05)。p25转染组足细胞内Actin排列紊乱,细胞形态发生异常改变,对照组和空转染组细胞内Actin排列正常,细胞形态没有发生改变。结论 p25引起Cdk5过度活性可致肾小球足细胞形态改变,Actin排列紊乱,诱发细胞凋亡。因此,Cdk5的活性在维持足细胞正常结构和功能方面发挥重要作用。

Objective To study the influence of Cdk5 activity on the structure and function of glomerulus podocyte by transfection of p25 gene. Methods In 2014,differentiated and mature isolated mouse podocytes were cultured in RPMI 1640 medium with 10% fetal bovine serum and divided into three groups including control group,empty group and p25 group. p25 gene was cloned through the p Ad Track-CMV viral vector,and transfected into podocytes. After 48 hours,cells were collected for further analysis. Cdk5,p25,p35,Cleaved caspase3 expressions were detected by Western blotting method; Cdk5 activity were tested through immunoprecipitation and isotope γ-32 P marking methods. In order to observe the expression of Cdk5,p35 and structure of Actin in podocyte, immunofluorescent staining were conducted. Results Cak5 in HEK293 cells had positive expression while p35 had negative expression; there were different levels of Cdk5 and p35 in renal cortex, podocyte and glomerulus. p25 had negative expression in control group and empty group while positive expression in p25 group. As for the expression levels of Cleaved caspase3 and Cdk5 activity in different groups,differences were of statistical significance（ P〈0. 05）; the expression levels of Cleaved caspase3 and Cdk5 activity in p25 group was higher than those in the other two groups（ P〈0. 05）. In p25 group,Actin within podocyte were arranged in disorder and forms of cells were abnormal,while those conditions were on the contrary in the other two groups. Conclusion p25 causes overactivation of Cdk5 and induces podocyte morphological change, Actin arrangement disorder and cell apoptosis. Therefore, Cdk5 activity plays an important role in maintaining normal structure and function of podocyte.