c—Abl通过p53信号通路促进高糖诱导的足细胞凋亡

c-Abl mediates glucose-induced apoptosis via p53 signaling pathway in podocytes

目的研究非受体酪氨酸激酶蛋白c-Abl在高糖诱导足细胞损伤中的作用及可能的信号转导通路。方法体外培养条件永生性小鼠足细胞，并用不同浓度葡萄糖不同时间刺激已分化足细胞，免疫荧光染色和Western印迹检测足细胞中c—Abl蛋白的表达和分布改变。采用c-Abl siRNA转染沉默足细胞表达c—Abl，细胞分成正常对照组（5mmol／L葡萄糖）、高渗对照（25mmol／L甘露醇）组、高糖组、高糖＋c—Abl siRNA组。应用流式细胞术及Hoechst33258染色评价足细胞凋亡水平，Western印迹法检测c—Abl及p53蛋白的表达水平，免疫荧光染色观察足细胞内c—Abl分布的改变，免疫共沉淀法检测e—Abl与p53蛋白的结合水平。结果与对照组比较，高糖刺激导致足细胞凋亡率增加（P〈0．05）；c—Abl表达水平增加（P〈0．05），并以时间和浓度依赖方式增加且存在核转移现象；同时p53蛋白表达水平上调，c-Abl与p53的结合水平增加（均P〈0．05）。与高糖组比较，c—Abl siRNA转染后c—Abl蛋白表达水平降低（P〈0．05）且核转移减弱，高糖诱导足细胞凋亡率下降（P〈0．05），且c—Abl与p53结合水平降低（P〈0．05）。上述各项高渗组与对照组差异均无统计学意义（均P〉0．05）。结论c—Abl蛋白是高糖诱导足细胞损伤的促凋亡因子，其作用机制与p53信号通路有关。

Objective To investigate the role of the non- receptor tyrosine kinase c- Abl in high glucose- induced podocyte injury and its possible signal transduction pathway. Methods Differentiated mouse podocytes were exposed to different glucose concentration conditions in different time slots. Western blotting and immunofluorescence assay were used to measure c-Abl expression and distribution, c-Abl siRNA was transfected to block c-Abl expression. Then podocytes were divided into normal control group （5 mmol/L glucose）, high permeability （25 mmol/L mannitol） group, high glucose group and high glucose ＋ c-Abl siRNA group. The podocytes apoptosis was assessed by flow cytometry and Hoechst 33258 staining. The protein expression of c-Abl and p53 was detected by Western blotting. The distribution of c- Abl was observed by immunofluorescence. Co- immunoprecipitation assay was used to evaluate the interaction between c-Abl and p53. Results Compared with normal group, high glucose-induced podocytes had increased apoptosis （P 〈 0.05）, and up-regulated c- Abl expression （P 〈 0.05） in time and concentration dependent manner. More c-Abl was distributed in the nuclei. Moreover, high glucose promoted the expression of p53 and the interaction of c-Abl and p53 （all P 〈 0.05）. Compared with high glucose group, transfection of c-Abl siRNA restored high glucose- induced podocytes apoptosis （P〈 0.05）. The reduction of c- Abl and p53 expression, and the combination of c-Abl and p53 were also diminished in high glucose＋c-Abl siRNA group （P 〈 0.05）. There was no significant difference between normal control group and high permeability group （all P 〉 0.05）. Conclusion During the process of high glucose-induced podocytes damage, c- Abl is an apoptosis factor, associated with p53 signaling pathway.