血管紧张素Ⅱ激活肾间质成纤维细胞的实验研究

Experimental study of angiotensin Ⅱ activating renal interstitial fibroblast

目的观察血管紧张素Ⅱ(AngⅡ)对正常大鼠肾间质成纤维细胞株NRK-49F功能变化的影响,探讨其参与肾小管间质纤维化的作用机制。方法用不同浓度AngⅡ(10-6,10-7,10-8,10-9mol/L)刺激NRK-49F(6 h,12 h,24 h和48 h)。蛋白免疫印迹法检测α平滑肌动蛋白(α-SMA)、纤维连接蛋白(FN)和TGF-β受体(TβR I)的表达。ELISA方法检测细胞上清液中TGF-β1的浓度。明胶酶谱法检测细胞上清液中MMP2/9的表达量。结果①10-7mol/L AngⅡ能刺激NRK-49F细胞上调FN和α-SMA表达,随着AngⅡ浓度的增加,该细胞表达FN和α-SMA的量亦随之增加,呈明显的剂量依赖性;②10-9mol/L AngⅡ能够上调NRK-49F细胞TβR I的表达,10-7mol/L AngⅡ刺激该细胞6 h后,TGF-β1的表达开始增加,12 h后达到峰值,24 h和48 h仍能维持较高的水平;③10-7mol/L AngⅡ能够刺激NRK-49F细胞表达MMP2和MMP9,其中MMP2的表达量显著多于MMP9的表达量。结论AngⅡ能够激活间质成纤维细胞,并可能以此参与肾小管间质纤维化的发生发展。

Objective It is to approach the mechanism of angiotensin Ⅱ（AngⅡ） on renal tubule interstitial fibrosis through observing the influence of AngⅡ on the function change of NRK-49F in renal interstitial fibroblast of normal rat.Methods Different concentrations of AngⅡ（10-6 mol/L,10-7 mol/L,10-8 mol/L and 10-9 mol/L） were used to stimulate NRK-49F for 6 h,12 h,24 h and 48 h.The expressions of α-smooth muscle actin（α-SMA）,Fibronectin（FN） and TGF-β receptor（TβRI） were detected with Western Blot method.The concentration of TGF-β1 in the supernatant of cell was detected with ELISA method.The expression quantity of MMP2/9 was detected with gelatin zymography.Results ①10-7 mol/L AngⅡ could stimulate NRK-49F to up-regulate the expressions of FN and α-SMA.The quantity of FN and α-SMA expressed by NRK-49F were increased along with the increase of AngⅡ concentration,which was showed as obvious dose-dependent.②10-9 mol/L AngⅡ could up-regulate NRK-49F to express TβRI.After 10-7 mol/L AngⅡ stimulating NRK-49F for 6 h,the expression of TGF-β1 began increase,12 h to peak,and after 24 h and 48 h still kept the higher level.③10-7 mol/L AngⅡ could stimulate NRK-49F to express MMP2 and MMP9.Among those,the quantity of MMP2 expressed was significant more than that of MMP9.Conclusion AngⅡ can activate interstitial fibroblast,with which it maybe participate the occurrence and development of renal tubule interstitial fibrosis.