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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 investigate the effect of cyclin-dependent protein kinase 5 (Cdk5) on the structure and function of podocytes by transfecting p25 gene in glomerulus podocytes. Methods In 2014, the mature mouse podocytes were cultured in RPMI 1640 medium containing 10% fetal bovine serum. The podocytes were divided into control group, empty-transfected group and p25-transfected group. The p25 gene was cloned by pAdTrans-CMV and transfected into podocytes for 48 h. The expression of Cdk5, p25, p35 and Cleaved caspase3 were detected by Western blotting. The expression of Cdk5 and p35 in podocyte and the arrangement of podocyte Actin were observed by immunofluorescence staining. Results The expression of Cdk5 and p35 were negative in HEK293 cells. The expressions of Cdk5 and p35 in renal cortex, podocytes and glomerulus were all different. The control group and empty transfected group p25 negative expression, p25 transfected group p25 positive expression. The expression level of Cleaved caspase 3 and Cdk5 in each group were significantly different (P <0.05). The expression level of Cleaved caspase 3 and Cdk5 in p25 transfected group were higher than those in control group and empty Transfection group (P <0.05). Actin in p25 transfected group was disorderly arranged and the morphology of cells changed abnormally. Actin in control group and empty transfected group were normal, and the morphology of cells did not change. Conclusion p25 induced Cdk5 over-activity can lead to glomerular podocyte morphological changes, Actin disorganized, induced apoptosis. Therefore, Cdk5 activity plays an important role in maintaining the normal structure and function of podocytes.