整合素连接激酶在草酸钙晶体诱导肾小管细胞上皮间质转换中的作用

Role of Integrin Linked Kinase in Epithelial Mesenchymal Transition of Renal Tubular Cells Induced by Calcium Oxalate Monohydrate

目的探讨人肾小管上皮细胞(HK-2)暴露于一水草酸钙(COM)6、24和48 h后,整合素连接激酶(ILK)在HK-2细胞中表达水平的变化以及与上皮间质转换(EMT)的关系。方法本实验以人HK-2细胞为研究对象,分为空白对照(NC)组和COM组(6、24和48 h)共4组,用CCK-8实验检测HK-2细胞存活率;倒置显微镜观察HK-2细胞形态学变化;实时荧光定量PCR(qRT-PCR)检测基质金属蛋白酶(MMP-2、MMP-9)、组织金属蛋白酶抑制剂-1(TIMP-1)和ILK基因表达水平;免疫印迹法(Western blot)检测HK-2细胞EMT相关蛋白,E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)和细胞外基质(ECM)重塑相关蛋白,MMP-2、MMP-9、TIMP-1、Ⅰ型胶原蛋白α1(COL1A1)、纤维连接蛋白(Fibronectin)以及ILK蛋白表达水平。结果与NC组相比,COM组COM对HK-2细胞具有损伤作用,HK-2表现为长梭形,形成纤维细胞样外观,细胞之间排列较为松散,细胞的间隙增大。COM抑制HK-2细胞的增殖活性,随着HK-2细胞暴露于COM时间的延长,HK-2细胞存活率下降(P均<0.01)。qRT-PCR结果显示,24、48 h时COM组MMP-2和MMP-9基因表达水平均降低(P均<0.01),TIMP-1和ILK基因表达水平均升高(P均<0.01)。Western blot结果显示,COM组E-cadherin蛋白表达水平均降低(P均<0.01);α-SMA和Vimentin蛋白表达水平升高(P均<0.01);ECM重塑相关蛋白MMP-2和MMP-9表达水平降低(P均<0.01);TIMP-1、COL1A1和Fibronectin蛋白表达水平增高(P均<0.01);ILK蛋白表达水平增高(P均<0.01)。结论ILK可能参与COM诱导的HK-2细胞EMT改变。

Objective To investigate the expression and relationship with epithelial mesenchymal transition(EMT)of integrin linked kinase(ILK)in human renal tubular epithelial cells(HK-2)exposed to calcium oxalate monohydrate(COM)for 6,24 and 48 h.Methods Human HK-2 cells were divided into blank control group(NC)and COM(6,24 and 48 h).The survival rate of HK-2 cells was detected by CCK-8 assay.The morphological changes of HK-2 cells were observed by inverted microscope.The expression levels of Matrix metalloproteinase-2(MMP-2),Matrix metalloproteinase-9(MMP-9),tissue inhibitor of metalloproteinase-1(TIMP-1)and ILK genes were detected by quantitative Real-time PCR(qRT-PCR).The EMT related proteins E-cadherin,αsmooth muscle actin(α-SMA),vimentin and extracellular matrix(ECM)remodeling related proteins MMP-2、MMP-9、TIMP-1,recombinant collagen typeIα1(COL1A1)and fibronectin in HK-2 cells treated with COM were detected by Western blot.Results Compared with NC group,in COM group,COM was damage to HK-2 cells,HK-2 was long spindle shaped,forming a fibroblast like appearance,the arrangement between cells was loose,and the gap between cells was increased.With the extension of exposure time of HK-2 cells to COM,the cell viability of HK-2 cells decreased gradually(P all<0.01).qRT-PCR results showed that in COM group,the expression levels of MMP-2 and MMP-9 genes decreased(P all<0.01),and the expression levels of TIMP-1 and ILK increased(P all<0.01).Western blot showed that the expression level of E-cadherin protein decreased significantly(P all<0.01).The expression levels ofα-SMA and vimentin protein increased(P all<0.01).The expression levels of ECM remodeling related proteins MMP-2 and MMP-9 decreased and TIMP-1,COL1A1 and fibronectin increased(P all<0.01).The expression level of ILK protein increased(P all<0.01),ILK expression increased significantly with the extension of exposure time of HK-2 cells to COM.Conclusion ILK may be involved in the EMT changes of HK-2 cells induced by COM.