TGF-β1对话Hedgehog-Gli1信号调控肾小管上皮细胞表型转化和胶原累积  

Dialogue between TGF-β1 and Hedgehog-Gli1 Signaling Regulates Phenotypic Changes of Renal Tubular Epithelial Cells and Collagen Deposition

作　　者：梁勇[1] 陆红[2] 林成成[1] 王斯璐[1] 夏鹏[3] 吴存造[3] 陈必成[1] 白永恒[1] 

机构地区：[1]温州医科大学附属第一医院外科实验室,温州325000 [2]温州医科大学附属第一医院医学检验中心,温州325000 [3]温州医科大学附属第一医院移植科,温州325000

出　　处：《中国细胞生物学学报》2015年第1期24-30,共7页Chinese Journal of Cell Biology

基　　金：浙江省自然科学基金(批准号:LQ12H05001;LY12H05004);温州市科技计划项目(批准号:Y20110028);浙江省临床医学"重中之重"一级学科开放基金资助的课题~~

摘　　要：该研究旨在探讨Hedgehog-Gli1（HH）信号在肾小管上皮细胞表型转化（epithelial-mesenchymal transition,EMT）和胶原累积中的作用及与TGF-β1信号的对话机制。该实验通过体外培养大鼠肾小管上皮细胞NRK-52E,以溶剂作为对照组,以1～50 ng/m L重组蛋白sonic hedgehog（Shh）或5 ng/m L TGF-β1作为诱导组,以加入或不加入HH信号特异性阻断剂环靶明（cyclopamine,Cyp）5μmol/L为干预组。细胞培养24 h,采用ELISA、q RT-PCR、免疫细胞荧光染色和Western blot等方法检测HH信号相关分子（Ptch1、Smo和Gli1）、TGF-β1、EMT相关分子（Rac1蛋白、肌成纤维细胞标志物α-SMA和上皮细胞标志物E-cadherin）、III型胶原m RNA或蛋白的表达。结果发现,外源性Shh上调Smo和Gli1表达,抑制Ptch1表达,继而激活HH信号;HH信号活化抑制肾小管上皮细胞E-cadherin的表达,上调α-SMA、III型胶原和TGF-β1的表达。环靶明干预后,Smo表达下调,进而抑制HH信号、EMT和胶原累积,并下调TGF-β1的表达。应用TGF-β1诱导小管上皮细胞EMT,同时也上调HH信号分子Smo和Gli1的表达,下调Ptch1的表达,提示TGF-β1可诱导HH信号活化。综上所述,HH信号和TGF-β1均参与了肾小管上皮细胞EMT和胶原累积过程。HH信号活化可促进TGF-β1的表达,同时TGF-β1能激活HH信号,推测TGF-β1与HH信号可能存在交叉对话以调控EMT和胶原累积。The aim of this study was to investigate effect of Hedgehog-Glil （HH） signaling in phenotypic changes of renal tubular epithelial cells and its association with TGF-β1. Rat renal tubular epithelial cells （NRK-52E） were treated with 1-50 ng/mL of exogenous recombinant sonic hedgehog （Shh）, or 5 ng/mL of TGF-β1, or HH signaling inhibitor cyclopamine （5 μmol/L） for 24 hours, qRT-PCR, immunoftuorescence staining and Western blot were performed to detect mRNA or protein expressions of HH signaling-related molecules （Ptchl, Smo and Glil）, TGF-β1, EMT markers （α-SMA, E-cadherin and Racl） and type III collagen. The levels of TGF-β1 were detected by ELISA assay. Our results showed that Shh increased the expression of Smo and Glil and decreased the expression of Ptchl, suggesting that HH signaling was activated. Activated HH signaling resulted in down-regulated expression of E-cadherin and up-regnlated expression of α-SMA and type Ⅲ collagen. In Shh-treated NRK-52E cells, the levels of TGF-β1 were also enhanced, but inhibited by cyclopamine. Cyclopamine also inhibited EMT and ECM deposition. In cultured NRK-52E cells, recombinant TGF-β1 not only induced EMT, but also increased the activity of HH signaling. Thus, these findings suggested that both HH signaling and TGF-β1 were involved in EMT and ECM accumulation in vitro. Activation of HH signaling can promote TGF-β1 expression, and TGF-β1 also can activate HH signaling, sug- gesting that cross-talk between HH signaling and TGF-β1 regulates renal EMT and ECM deposition.

关 键 词：TGF-Β1 Hedgehog-Gli1信号 肾小管上皮细胞 上皮-间质表型转化 胶原累积 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] Q756[医药卫生—基础医学]