tTG小干扰RNA对人晶状体上皮细胞转分化和细胞外基质沉积的抑制作用  

Effect of tissue tTG-siRNA on transdifferentiation and extracellular matrix deposition of human lens epithelial cells

作　　者：邢星[1] 胡义珍[2] 李世洋[1] 马红利[1] 

机构地区：[1]中国人民解放军第150中心医院眼科,河南省洛阳市471031 [2]华中科技大学同济医学院附属协和医院眼科,湖北省武汉市430022

出　　处：《眼科新进展》2012年第1期11-14,19,共5页Recent Advances in Ophthalmology

基　　金：湖北省卫生厅科研基金资助(编号:JX3B09)~~

摘　　要：目的探讨RNA干扰抑制组织型转谷氨酰胺酶(tissue transglutaminase,tTG)的表达对人转化生长因子β2(transforming growth factor-beta2,TGF-β2)诱导的晶状体上皮细胞(lens epithelial cells,LEC)转分化和细胞外基质沉积的抑制作用。方法设计并合成针对tTG的3对小干扰RNA(siRNA):tTG-siRNA-1、tTG-siRNA-2、tTG-siRNA-3,用Real-time PCR和Western blot检测转染了siR-NA后HLE-B3细胞表达tTG mRNA和tTG蛋白的变化。然后将体外培养的HLE-B3细胞分为正常对照组、TGF-β2组和TGF-β2+siRNA组。48h后,用Western blot检测各组tTG、平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤维连接蛋白(fibronec-tin,FN)和Ⅳ型胶原(collagenⅣ,Col-Ⅳ)的表达。结果转染tTG-siRNA-1、tTG-siRNA-2、tTG-siRNA-348h后,tTG mRNA的表达分别下降为空白对照组的46.60%、12.84%、66.75%(均为P<0.05);tTG蛋白的表达量分别下降为空白对照组的60.49%、27.87%、55.91%(均为P<0.01)。Real-time PCR与Western blot检测结果一致显示了tTG-siRNA-2对tTG基因的抑制效果最佳。与正常对照组相比,TGF-β2组中tTG、α-SMA、FN、Col-Ⅳ的表达量均明显增加(均为P<0.01);与TGF-β2组相比,TGF-β2+tTG-siRNA-2组中tTG、α-SMA、FN、Col-Ⅳ的表达量均明显减少(均为P<0.01)。结论靶向tTG的siRNA可以显著降低tTG的表达水平,同时可以明显抑制TGF-β2诱导的HLE-B3细胞合成α-SMA、FN、Col-Ⅳ。提示tTG是介导TGF-β2诱导人LEC转分化和细胞外基质沉积的重要分子。Objective To observe the effect of tissue transglutaminase（tTG） siRNA on transdifferentiation and extracellular matrix deposition of human lens epithelial cells（HLEC） induced by TGF-β2.Methods Three specific siRNAs of tTG（tTG-siRNA-1,tTG-siRNA-2 and tTG-siRNA-3） were designed and synthesized,and were transiently transfected into human lens epithelial cell line（HLE-B3）.Real-time PCR and Western blot were used to detect the tTG expression at mRNA and protein levels.Then cultured HLE-B3 cells in vitro were divided into normal control group,TGF-β2-group and TGF-β2＋siRNA group.Western blot was used to assay the expression of tTG,α-smooth muscle actin（α-SMA）,fibronectin（FN）,and collagen Ⅳ（Col-Ⅳ） in HLE-B3 after 48 hours.Results After the transfection with tTG-siRNA-1,tTG-siRNA-2 and tTG-siRNA-3 for 48 hours,the tTG mRNA expression were decreased to 46.60%,12.84% and 66.75% of that in blank control group,respectively.The tTG protein expression were decreased to 60.49%,27.87 % and 55.91% of that in blank control group,respectively.All differences had statistical significant（all P0.05 or 0.01）.The same result was showed in Real-time RT-PCR and Western blot that tTG-siRNA-2 had the best inhibitive effect on tTG.The expression of tTG,α-SMA,FN and Col-Ⅳ were markedly increased in TGF-β2 group as compared with that in normal control group（all P0.01）.And the expression of tTG,α-SMA,FN and Col-Ⅳ were significantly decreased in TGF-β2＋siRNA group as compared with that in TGF-β2 group（all P0.01）.Conclusion siRNA targeting tTG gene can effectively inhibit the expression of tTG gene,and the synthesis of α-SMA,FN,Col-Ⅳ in HLE-B3 cells induced by TGF-β2.It suggests that tTG play an important role in transdifferentiation and extracellular matrix deposition of HLECs induced by TGF-β2.

关 键 词：RNA干扰 组织型转谷氨酰胺酶 细胞外基质 平滑肌肌动蛋白 晶状体上皮细胞 后囊膜混浊 

分 类 号：R344.1[医药卫生—基础医学]