他克莫司对转化生长因子-β诱导的肾小管上皮细胞转分化的抑制作用及对核因子-κB活性的影响

Effects of FK506 on transdifferentiation and NF-κB activity of human tubular cells induced by TGF-β in vitro

目的 :探讨他克莫司 (FK5 0 6 )对转化生长因子 β(TGF β)诱导的肾小管上皮细胞转分化的作用 ,及其与核因子 κB(NF κB)活性变化的关系。 方法 :以人类肾脏近曲小管上皮细胞株 (Humankidneycell,HKC)为研究对象 ,分为以下 4组 :①阴性对照组 ;②TGF β(8ng/ml)组 :③FK5 0 6 (0 1、1、10、5 0ng/ml)组 :④TGF β(8ng/ml) +FK5 0 6(0 1、1、10、5 0ng/ml)组。应用形态学、间接免疫荧光法 ,免疫组化技术和酶联免疫吸附法观察FK5 0 6对TGF β诱导的HKC细胞转分化的作用、细胞培养上清液纤连蛋白、I型胶原的变化及FK5 0 6对HKC细胞NF κB活性的影响。 结果 :FK5 0 6 (1,10 ,5 0ng/ml) +TGF β组比TGF β组诱导的HKC细胞E 钙粘蛋白和细胞角蛋白表达有所增强 ,波形蛋白和α 平滑肌肌动蛋白 (α smoothmuscleactin ,α SMA)的表达比TGF β组减弱 (P <0 0 5 ) ,同时HKC细胞NF κB的活性比TGF β组减弱 (P <0 0 5 ) ,培养上清液纤连蛋白及I型胶原的浓度比TGF β组降低 (P <0 0 5 ) ;FK5 0 6 (1、10、5 0ng/ml)使基础状态下HKC细胞NF κB的活性明显下降 (P <0 0 5 )和上清液中纤连蛋白及I型胶原的水平明显降低 (P <0 0 5 )。 结论 :①FK5 0 6剂量依赖性地抑制TGF

Objective:Tubular epithelial-myofibroblast transdifferentiation may be one of the important mechanisms of interstitial fibrosis in progressive renal diseases. The aim of this study is to examine effect of FK506 on transdifferentiation of human tubular epithelial cells induced by TGF-β and its relationship with NF-κB activity changes. Methodology: The human kidney cells (HKC) were cultured for 48 hours in different conditions:① Serum free as control;② Treated with TGF-β (8ng/ml); ③ Treated with FK506 at different concentration (0.1, 1, 10, 50ng/ml); and ④ Treated with FK506 (0.1, 1, 10, 50ng/ml) plus TGF-β (8ng/ml). The expression of vimentin, cytokeratin, E-cadherin, α-SMA and NF-κ B of HKC was assessed with indirect immunofluorescence, and immunohistochemical staining. The concentrations of fibronectin, and type Ⅰ collagen in culture medium supernatant were detected by ELISA. Results:The expression of cytokeratin and E-cadherin was markedly decreased in HKC treated with TGF-β, but not changed in HKC treated with FK506 alone. The expression of α-SMA, vimentin and NF-κB of HKC cultured with TGF-β was much notable than the control, and significantly decreased in HKC cultured with TGF-β plus FK506. Conclusions:FK506 at given concentrations may inhibit tubular epithelial-myofibroblast transdifferentiation, reduce synthesis and secretion of fibronectin and Ⅰ type collagen of HKC induced by TGF-β as well as NF-κB expression of HKC cells under basic or TGF-β induction condition in a dose-dependent manner. The inhibition of FK506 on transdifferentiation of HKC induced by TGF-β may be related to the down-regulation of NF-κB expression, which needs to be studied further.