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

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。

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

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 P<0.05 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 P<0.01).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 P<0.01).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.