摘要
目的观察肾小管上皮细胞转分化过程中PTEN的表达和分布，并研究上调DJ-1对PTEN表达和分布及P13K—Akt通路活化的影响。方法以人。肾小管上皮细胞为研究对象，10μg／L TGF—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PTEN、E—cadherin和α-SMA蛋白表达；RT—PCR法检测两组细胞内PTEN mRNA表达水平。脂质体法介导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PTEN蛋白表达；RT—PCR法检测各组细胞内PTEN mRNA表达。pEGFP-N1-DJ-1转染前1h用P13K抑制剂LY294002预处理，Western印迹检测正常组、pEGFP．N1一DJ-1转染组和空载体转染组及LY294002预处理1h后pEGFP-N1-DJ-1转染组的p-Akt和Akt蛋白的表达。激光共聚焦显微镜下观察正常组、TGF-β1干预组和pEGFP—N1-DJ．1转染组细胞内PTEN蛋白的分布。结果正常组细胞表达E—cadherin和PTEN，几乎不表达α-SMA；TGF—β1干预组α—SMA表达显著高于正常组（P〈0．05），而E—cadherin表达显著低于正常组（P〈0.05），PTEN mRNA和蛋白表达均显著低于正常组（P〈0．05）。pEGFP-N1-DJ-1和空载体转染后，细胞绿色荧光表达均在80％以上；pEGFP—N1-DJ-1转染组细胞内DJ-1表达显著高于正常组（P〈0．05），而PTEN mRNA和蛋白表达均显著低于正常组（P〈0．05）；pEGFP—N1-DJ-1转染组p-Akt表达显著高于正常组（P〈0．05），但经LY294002干预后与正常组表达基本一致。正常组细胞内PTEN分布于细胞质和细胞核；TGF—β1干预组细胞质内PTEN几乎完全消失，而细胞核PTEN略有增加；pEGFP—N1-DJ-1转染组细胞核表达PTEN，但细胞质几乎无PTEN表达，这与TGF—β1干预组相似。结论肾间质纤维化中高表达DJ-1可抑制PTEN表达，并促进P13K—Akt通路活化。
Objective To observe the expression and distribution of PTEN in renal epithelial-mesenchymal transition （EMT）, and to investigate the effect of DJ-1 up-regulation on the expression and distribution of PTEN and the activation of PI3K-Akt signal pathway. Methods Human tubular epithelial cells （HKC cell line） were cultured with 10 μg/L TGF-131 for 72 h. Protein expressions of PTEN, E-eadherin and α-SMA were measured by Western blot. RT-PCR was used to detect the expression of PTEN mRNA. To over express DJ-1, HKC cells were transfected with pEGFP-N1-DJ-1 via Iipofectamine 2000 to induce up-regulation of DJ-1. The efficiency of transfection was examined by fluorescence microscope and Western blot. The expressions of D J-1, PTEN, p-Akt and Akt in the transfected cells were detected by Western blot, and PTEN mRNA was detected by RT-PCR. The intracellular distribution of PTEN in normal HKC cells, ceils stimulated by TGFq31 and cells transfected with pEGFP-N1-DJ-1 was observed by confocal microscope. Results Normal HKC cells expressed PTEN, E-cadherin, but almost did not express α-SMA. The expressions of PTEN protein, PTEN mRNA and E-cadherin in cells stimulated by TGF-β1 were less as compared to normal cells （P〈0.05）, while α-SMA expression was increased （P〈0.05）. The effenciency of green fluorescence was more than 80% in transfected cells. In the DJ-1 transfectants, the expressions of PTEN and PTEN mRNA were suppressed, but p-Akt expression was up-regulated as compared to normal cells. In normal HKC cells, PTEN distributed both in cytoplasm and nucleus. In cells stimulated by TGF-β1, cytoplasmic PTEN was completely lost while nuclear PTEN was increased slightly. In the DJ-1 transfectants, only the nuclear PTEN was observed which was similar to the cells stimulated by TGF-β1. Conclusion In renal interstitial fibrosis, the over-expression of DJ-1 can suppress PTEN expression and activate PI3K- Akt signal pathway.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2009年第10期782-787,共6页
Chinese Journal of Nephrology
基金
国家自然科学基金（30370657,30871172）
青年科学基金（30800525）
教育部新世纪优秀人才支持计划（NCET-04-0712）
