AngⅡ对人肾小管上皮细胞TSP1、TGF-β1表达的影响

The study of the effection of angiotensin-Ⅱ on expression of thrombospondin-1 and transforming growth factorβ1 in renal tubular cells

目的探讨血管紧张素Ⅱ（angiotensin-Ⅱ，AngⅡ）对人肾小管上皮细胞（HK-2）血小板反应蛋白1（TSP1）、转化生长因子β1（TGF-β1）表达的影响及其机制。方法①以AngⅡ零浓度作为空白对照，观察低、中、高浓度（终浓度分别为10^-8、10^-7、10^-6mol／L）的AngⅡ对HK-2 TSP1、TGF-β1 mRNA和蛋白表达的影响；②以0h为空白对照，观察AngⅡ（10^-7mol／L）在0、3、6、12、24h对HK-2 TSP1、TGF-β1 mRNA及蛋白表达的影响；③观察TSP1抑制剂G肽对AngⅡ刺激的HK-2总TGF-β1和活性TGF-β1表达的影响。采用Real Time PCR法检测TSP1和TGF-β1的mRNA表达；采用Western blot检测TSP1和TGF-β1的蛋白表达；采用ELISA法检测各组总TGF-β1、活性TGF-β1表达。结果与对照组比，低、中、高浓度AngⅡ均增加了HK-2细胞TSP1 mRNA表达和TGF-β1 mRNA表达，同时伴有TSP1蛋白和TGF-β1蛋白表达的增加。终浓度为10^-7mol／L的AngⅡ以时间依赖的方式促进HK-2细胞TSP1、TGF-β1 mRNA表达，同时伴TSP1和TGF-β1的蛋白表达增加，其中TSP1的表达峰值在6h，TGF-β1的表达峰值在12h。与对照组比较，10^-7mol／L的AngⅡ上调总TGF-β1、活性TGF-β1表达，10μmol／L的G肽仅下调活性TGF-β1表达，对总TGF-β1表达无影响。结论AngⅡ能促进人肾小管上皮细胞TSP1、TGF-β1表达。TSP1依赖的TGF-β1活化可能是AngⅡ诱导的肾小管上皮细胞TGF-β1活化的重要途径。

Objective To investigate the effect of angiotensin-Ⅱ on the expression of thrombospondin-1 and transforming growth factorβ1 in renal tubular cells. Methods HK-2 cells were treated by different concent ration of angiotensin- Ⅱ （0 、 10^- 8 、10^- 7 、 10^- 6 mol/L）. The expression of mRNA of TSP1 and TGF-β1 were examined by Real Time PCR,and the protein levels were examined by Western blot. Real Time PCR and Western blot analysis were also performed to detect TSP1 and TGF-β1 expression in HK-2 cells stimulated by angiotensin-Ⅱ（10^-7mol/L ） for 0.3.6.12、24 h,0 h was as normal control group. ELISA were used to detect the expression of total and active TGF-β1 under angiotensin-Ⅱ（10^-7 mol/L ） and G peptid（G peptid 10umol/L ＋ angiotensin-Ⅱ 10^-7 mol/L）, un stimulated HK-2 cells was as normal control group. Results The expression of mRNA of TSP1and TGF-β1 were up-regulated significantly when stimulated by angiotensin-Ⅱ（10^-8、10^-7、 10^-6mol/L ） . And the protein level of TSP1 and TGF-β1 were also up-regulated. Angiotensin-Ⅱ enhanced the expression of TSP1 and TGF-β1 in mRNA and protein level in a time-dose dependent manner. And Angiotensin-Ⅱ could up-regulated the expression of total and active TGF-β1 （P〈0. 051. G peptid only down-regulated expression active TGF-β1 （ P〈0. 05）,it had no effect on total TGF-β1. Conclusions Angiotensin-Ⅱ can up-regulated the expression of TSP1 and TGF-β1. The path of TSP1- TGF-β1 acting TGF-β1 is most important in renal tubular cells.