ILK siRNA对高糖刺激的人肾小管上皮细胞GSK-3β及β-catenin表达的影响  

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

作　　者：闫喆[1,2] 姚芳[2] 张丽萍[1] 郝军[2] 吴海江[2] 段惠军[2] 

机构地区：[1]河北医科大学第二医院肾内科,河北石家庄050000 [2]河北医科大学病理教研室,河北石家庄050000

出　　处：《中国病理生理杂志》2014年第3期503-508,共6页Chinese Journal of Pathophysiology

基　　金：河北省卫生厅课题(No.08282)

摘　　要：目的:探讨高糖诱导肾小管上皮细胞转分化中整合素连接激酶小干扰RNA(ILK siRNA)对糖原合成酶激酶3β(GSK-3β)磷酸化和β-连环蛋白(β-catenin)核内表达的影响及意义。方法:体外培养人近端肾小管上皮细胞系HKC,分为正常对照组(NG)、高糖组(HG)、高糖+阴性转染对照组(HG+HK)和高糖+ILK siRNA组(HG+ILK siRNA)。倒置荧光显微镜下观察绿色荧光蛋白表达。RT-PCR及Western blotting检测ILK mRNA及蛋白表达水平;免疫细胞化学检测磷酸化GSK-3β(p-GSK-3β)和β-catenin表达。Western blotting检测总GSK-3β、pGSK-3β、核β-catenin、总β-catenin、E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)的表达水平。结果:(1)倒置荧光显微镜下可见绿色荧光蛋白表达,证实构建的siRNA重组质粒成功转染HKC细胞;(2)与HG和HG+HK组相比,HG+ILK siRNA组ILK mRNA及蛋白水平下降,但较NG组表达仍高;(3)HG+ILK siRNA组ILK基因沉默后,p-GSK-3β与核β-catenin蛋白表达较HG及HG+HK组均下降,但较NG组表达仍高。而总GSK-3β与总β-catenin在各组表达无明显差异。结论:ILK、GSK-3β和β-catenin可能参与了高糖介导的肾小管上皮细胞转分化过程。ILK可能通过调节Wnt/β-catenin途径下游效应蛋白GSK-3β和β-catenin的表达而促使肾小管上皮细胞转分化。To investigate the effects of siRNA targeting integrin-linked kinase （ILK） on the expression of glycogen synthase kinase 3β （GSK-3β） and β-catenin during epithelial-mesenchymal transition （EMT） in human kid ney proximal tubular epithelial cell line HKC induced by high glucose. METHODS： HKC cells were divided into 4 groups： normal glucose （NG） group, high glucose （HG） group, HG ＋ HK （a vector containing the non-specific siRNA designed as negative control） group and HG ＋ ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein （GFP）. The expression of ILK at mRNA and protein levels was detected by RT PCR and Western blotting. The expression of p-GSK-3 β and β-eatenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and a-smooth muscle actin （α-SMA） was measured by Western blotting. RESULTS： GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG ＋ ILK siRNA group compared with HG group and HG ＋ HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CON CLUSION： These data suoDort a functional role of ILK may promote tubular EMT＇by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.

关 键 词：糖尿病肾病 上皮-间充质转化 整合素连接激酶 小干扰RNA 糖原合成酶激酶313 β-连环蛋白 

分 类 号：R363[医药卫生—病理学]