C末端Src激酶在高糖诱导足细胞骨架重构中的作用

Role of C-terminal Src Kinase in Glucose-induced Cytoskeletal Rearrangement in Podocytes

目的:研究糖尿病小鼠肾小球及条件永生性人足细胞C末端Src激酶(Csk)的表达变化,探讨Csk在高糖诱导足细胞骨架重构中的作用。方法:(1)体内实验:健康雄性C57BL/6小鼠12只,按随机数字表法分为对照组和糖尿病组,采用尾静脉注射链脲佐菌素(STZ)制备1型糖尿病模型,于实验第12周留取两组小鼠肾组织,采用免疫荧光法观察Csk在肾小球的表达及分布,实时荧光定量PCR及Western印迹法检测肾小球Csk mRNA及蛋白表达变化。(2)体外实验:取条件永生性人足细胞培养,分为正常葡萄糖组(NG组:5mmol/L葡萄糖)、高渗组(MA组:5mmol/L葡萄糖,25mmol/L甘露醇)、高糖组(HG组:30mmol/L葡萄糖)、高糖+Csk siRNA转染组(HG+Csk siRNA组),同体内实验方法观察足细胞Csk mRNA及蛋白表达;同时采用异硫氰酸荧光素(FITC)-鬼笔环肽(Phalloidin)染色观察足细胞骨架改变。结果:(1)体内实验结果显示,肾小球Csk主要沿毛细血管袢分布;与对照组相比,糖尿病组小鼠肾小球Csk蛋白和mRNA表达增加(P<0.01)。(2)体外实验,HG组足细胞Csk mRNA及蛋白表达较NG组增加(P<0.05),足细胞出现骨架重构;Csk siRNA转染足细胞可下调Csk表达,减轻高糖诱导的足细胞骨架重构,降低F-actin评分(P<0.05)。结论:高糖可通过诱导Csk表达上调参与足细胞骨架重构。

Objective:To investigate the effect of the high glucose on the expression of C-terminal Src kinase(Csk)in STZ-induced diabetic mice model and high glucose-treated cultured human podocytes,and to explore the role of Csk in high-glucose induced cytoskeletal rearrangement of podocytes.Method:Twelve male C57BL/6 mice were randomly allocated to the group with STZ treatment or the control group.The mice were sacrificed at 12 weeks post STZ injection.The expression of glomerular Csk was analyzed by immune-fluorescence,Western blotting and real-time fluorescent quantitative PCR.In vitro,conditionally immortalized human podocytes were divided into normal control group(5 mmol/L glucose),high permeability(5 mmol/L glucose,25 mmol/L mannitol)group,high glucose group(30mmol/L glucose)and high glucose+Csk siRNA group.The mRNA and protein expression of Csk were detected by real-time fluorescent quantitative PCR and Western blotting.After transfection with Csk siRNA,FITC-conjugated Phalloidin was used to stain F-actin in podocytes,to investigate the role of Csk in high glucose-induced cytoskeletal rearrangement.Results:Compared with control group,the protein and mRNA expression of glomerular Csk in STZ treated mice were increased(P<0.05).In vitro,high glucose induced the mRNA and protein expression of Csk in podocytes compared with normal group(P<0.01).Furthermore,high glocose-induced podocytes cytoskeletal rearrangement.Knockdown Csk with siRNA stabilized the actin filaments in cultured podocytes(P<0.05).There was no significant difference between normal control group and high permeability group(all P>0.05).Conclusion:The enhanced expression of Csk may be involved in high glucose-induced podocytes cytoskeletal rearrangement.