DJ-1癌基因在肾间质纤维化中表达的实验研究

Study of oncogene DJ-1 expression in renal interstitial fibrosis

目的观察DJ-1癌基因在肾纤维化过程中表达水平、细胞内定位及高表达DJ-1基因对人肾小管上皮细胞E钙黏蛋白（E—cadherin）、波形蛋白（vimentin）蛋白表达和β连环素（β-catenin）酪氨酸磷酸化水平的影响。方法体外实验以人肾小管上皮细胞为研究对象，10μg／LTGF-β1刺激72h诱导人肾小管上皮细胞转分化；Western印迹法检测正常组和TGF—β1干预组细胞内E—cadherin、vimentin和DJ-1蛋白表达；RT—PCR法检测两组细胞内DJ-1 mRNA表达水平；应用激光共聚焦显微镜观察DJ-1在细胞内的定位。体内实验以SD大鼠为研究对象，5／6肾切除法制作慢性肾纤维化模型；常规检测BUN和Scr水平；Masson染色检测肾组织纤维化水平；免疫组化法检测肾组织内DJ-1蛋白的表达和分布。脂质体法介导含野生型DJ-1基因的重组真核表达质粒pEGFP—N1-DJ-1或空载体转染人肾小管上皮细胞，倒置荧光显微镜和Western印迹法鉴定转染效率后，Western印迹法检测正常组、pEGFP—N1-DJ-1转染组和空载体转染组细胞内E—cadherin、vimentin蛋白的表达及各组β—catenin酪氨酸磷酸化水平。结果正常组细胞表达E—cadherin和DJ-1，几乎不表达vimentin；TGF—β1干预组细胞表达vimentin，表达较少E—cadherin，DJ-1 mRNA和蛋白表达均较正常组细胞显著增多（P〈0．05）。DJ-1在正常细胞内大多分布在细胞质，部分分布在细胞核；在转分化细胞内胞质和胞核内表达均有增加，且胞核内增加更显著。假手术组大鼠肾功能正常，肾组织内未见纤维组织，DJ-1主要表达于肾小管，肾小球内几乎没有表达。模型组大鼠肾功能不全，肾组织内可见明显纤维化结构，肾小管内DJ-1表达明显增加。pEGFP—N1-DJ-1转染组较正常组和空载体转染组细胞内DJ-1和vimentin蛋白表达明显增加且β-catenin酪氨酸磷酸化水平升高，而E—cadherin蛋白表达减少。结论DJ-1基因高表达可能促进了肾间质纤维化。

Objective To observe the expression and localization of DJ-1 in renal fibrosis, and to investigate the expressions of E-cadherin, vimentin and the level of β-catenin tyrosine phosphorylation in human tubular epithelial cells. Methods In vitro, the human tubular epithelial cells （HKC cell line） were cultured with 10 μg/L TGF-β1 for 72 h. The protein expressions of E-cadherin, vimentin and DJ-1 were measured by Western blot. RT-PCR was used to detect the expression of DJ-1 mRNA. The intracellular distribution of D J-1 was observed by confocal microscope. In vivo, Masson stain was used to evaluate the level of renal fibrosis. The expression and disposition of DJ-1 in renal tissue were detected by immunohistochenfistry. HKC cells were transfected with pEGFP-N1-DJ-1 via lipofectamine 2000. The efficiency of transfection was detected by fluorescence microscope. The expressions of DJ-1, E-cadherin, vimentin and β-catenin tyrosine phosphorylation level in the transfected cells were detected by Western blot. Results The expressions of DJ-1 protein and DJ-1 mRNA were up-regulated in renal tubular EMT cells. Most of DJ-1 protein localized in cytoplasm, and some was in nucleus. After stimulation by TGF-β1, the expressions of DJ-1 protein both in cytoplasm and nucleus was greatly increased, especially in nucleus. In vivo, renal tissue expressed DJ-1 in tubular epithelia, but little expression in glomeruli. In renal tissue from 5/6-nephrectomized rats, DJ-1 expression was greatly increased. In the DJ-1 transfectants, the expressions of DJ-1, vimentin and β-catenin tyrosine phosphorylation level were up-regulated, but E-cadherin expression was suppressed. Conclusion The increased expression of DJ-1 may promote renal fibrosis.