【作者】 邢星；

【导师】 胡义珍；

【作者基本信息】 华中科技大学 ， 眼科学， 2010， 博士

【摘要】 第一部分tTG抑制剂对人晶状体上皮细胞表达FN、Col-Ⅳ的影响研究目的观察组织型转谷氨酰胺酶(tissue transglutaminase, tTG)抑制剂单丹磺酰尸胺monodansyl-cadaverine (MDC)对转化生长因子β2 (transforming growth factor-β2, TGF-β2)诱导的人晶状体上皮细胞(Human lens epithelial cells, HLECs)表达纤维连接蛋白(Fibrone-ctin, FN)和Ⅳ型胶原(collagenⅣ, Col-Ⅳ)的影响。方法将体外培养的人晶状体上皮细胞株HLE-B3分为5组：无血清培养基中培养的HLE-B3为正常对照组；加入10μg/L TGF-β2处理36h的HLE-B3为TGF-p2组;10μg／LTGF-p2与100、200和400μmol/L MDC共同处理36h的HLE-B3为不同浓度MDC组。用半定量RT-PCR检测tTG、FN、Col-Ⅳ在HLE-B3细胞中表达的变化。结果正常体外培养的HLE-B3细胞中可表达一定量的tTG, FN及Col-Ⅳ。与正常对照组相比,TGF-β2组中tTG, FN和Col-Ⅳ的表达明显增强,差异有统计学意义(P<0.01)。与TGF-β2组相比,100、200和400μmol/L MDC组中FN和Col-Ⅳ的表达含量明显减少(P<0.01)。100、200和400μmol/L MDC组各组间FN和Col-Ⅳ的表达差异亦具有显著性意义(P<0.01)。结论MDC可剂量依赖性的抑制TGF-p2诱导的LECs中FN和Col-Ⅳ表达的增加。tTG可能通过促进HLECs表达FN和Col-Ⅳ等细胞外基质成分参与白内障术后后囊膜混浊(posterior capsule opacification, PCO)的形成。第二部分RNA干扰对人晶状体上皮细胞tTG表达的影响研究目的研究RNA干扰抑制组织型转谷氨酰胺酶(tissue transglutaminase, tTG)对人晶状体上皮细胞(Human lens epithelial cells, HLECs)中tTG表达的影响。方法设计并合成特异性针对tTG的3对小干扰RNA(short interfering RNA, siRNA): siRNA-1、siRNA-2、siRNA-3,用Real-time RT-PCR和Western blot法检测转染了siRNA后人晶状体上皮细胞株(HLE-B3)中tTG基因在mRNA水平和蛋白水平的变化。结果转染siRNA-1、siRNA-2、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%,与空白对照组相比差异具有统计学意义(P<0.05)。转染siRNA-1、siRNA-2、siRNA-360h后,tTG蛋白的表达含量分别下降为空白对照组的60.49%,27.87%,55.91%(P<0.01)。Real-time RT-PCR与Western blot检测结果一致显示了siRNA-2对tTG基因的抑制效果最佳。结论我们设计并合成的靶向tTG的siRNA可以显著降低tTG的表达。第三部分tTG-siRNA对人晶状体上皮细胞转分化和细胞外基质沉积的作用研究目的探讨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 factorβ2, TGF-β2)诱导的人晶状体上皮细胞(Human lens epithelial cells, HLECs)转分化和细胞外基质沉积的抑制作用。方法将体外培养的人晶状体上皮细胞株HLE-B3分为正常对照组,TGF-β2组和TGF-β2+siRNA组。Western blot法检测各组细胞tTG、α平滑肌肌动蛋白(a-Smooth muscle actin, a-SMA)、纤维连接蛋白(Fibronectin, FN)和Ⅳ型胶原(collagen IV, Col-IV)的表达。结果与正常对照组相比,TGF-β2组中tTG、α-SMA、FN、Col-Ⅳ的表达量均明显增加(P<0.01)；与TGF-β2组相比,TGF-β2+siRNA组中tTG、α-SMA、FN、Col-Ⅳ的表达量均明显减少(P<0.01)。结论靶向tTG的siRNA可以明显抑制TGF-β2诱导的HLE-B3细胞合成a-SMA、FN、Col-Ⅳ。提示tTG是介导TGF-β2诱导人晶状体上皮细胞转分化和细胞外基质沉积的重要分子,tTG蛋白有望成为预防和治疗后囊膜混浊(posterior capsule opacification, PCO)的新靶点。更多还原

【Abstract】 Part I Effect of tissue transglutaminase inhibitor on the expression of FN and Col-Ⅳin human lens epithelial cellsObjective To observe the effect of the tissue transglutaminase(tTG) inhibitor monodansyl cadaverineon(MDC) on the expression of fibronectin(FN) and collagenⅣ(Col-Ⅳ) induced by transforming growth factor-β2 (TGF-β2) in human lens epithelial cells (HLECs).Methods Cultured human lens epithelial cell line(HLE-B3) in vitro were divided into five groups, including:(1)group1, normal control group, (2)group2, 10μg/L TGF-β2-treated group, (3)group3,10μg/L TGF-β2+100μmol/L MDC-treated group, (4)group4,10μg/L TGF-β2+200μmol/L MDC-treated group and (5)group5,10μg/L TGF-β2+400μmol/L MDC-treated group. Semiquantitative RT-PCR was used to assay the expression of tTG, FN and Col-Ⅳin HLE-B3 cells.Results HLE-B3 cells cultured in vitro expressed certain quantity of tTG, FN and Col-Ⅳ. And the expression of them were markedly increased in group2 as compared with that in group 1 (P<0.01). The expression of FN and Col-Ⅳwere significantly inhibited in group3, 4 and 5 by MDC in a concentration-dependent manner as compared with that in group2(P<0.01). Conclusion MDC can inhibit the enhanced expression of FN and Col-IV induced by TGF-β2 in HLECs. tTG may be involved in posterior capsule opacification(PCO) through up-regulating the expression of FN and Col-IV in HLECs.Part II Inhibition of tissue transglutaminase expression in human lens epithelial cells by RNA interferenceObjective To investigate the inhibition of tissue transglutaminase (tTG) expression in human lens epithelial cells(HLECs) by RNA interference.Methods Three specific short interfering RNA (siRNA) of tTG (siRNA-1, siRNA-2, siRNA-3) were designed and synthesized, and transiently transfected into HLE-B3 cells. Real-time RT-PCR and Western blot were used to measure the expression of tTG mRNA and protein respectively.Results after the transfection with siRNA-1, siRNA-2, 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, after the transfection with siRNA-1, siRNA-2, siRNA-3 for 60hours, 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 significance (P<0.05～P<0.01). The same result was showed in Real-time RT-PCR and Western blot that siRNA-2 had the best inhibition effect on tTG.Conclusion siRNA targeting tTG gene is effective in suppressing the tTG expression. PartⅢThe role of tTG-siRNA in transdifferentiation and extracellular matrix deposition of human lens epithelial cellsObjective To investigate the effect of tissue transglutamiase (tTG) siRNA on transdifferentiation and extracellular matrix deposition of human lens epithelial cells(HLECs) induced by TGF-P2.Methods Cultured HLE-B3 cells in vitro were divided into normal control group, TGF-P2-group and TGF-β2+siRNA-group. Western blot was used to assay the expression of tTG, a-smooth muscle actin(α-SMA), fibronectin(FN), and collagenⅣ(Col-Ⅳ) in HLE-B3 cells.Results The expression of tTG,α-SMA, FN and Col-Ⅳwere markedly increased in TGF-P2-group as compared with that in normal control group (P<0.01). And the expression of tTG,α-SMA, FN and Col-Ⅳwere significantly decreased in TGF-P2+siRNA-group as compared with that in TGF-β2-group (P<0.01).Conclusion siRNA targeting tTG gene can obviously inhibit the synthesis of a-SMA, FN and Col-IV in HLE-B3 cells induced by TGF-β2. It suggests that tTG may play an important role in transdifferentiation and extracellular matrix deposition of HLECs induced by TGF-β2. tTG is hopeful to be a new target for the prevention and treatment of posterior capsule opacification(PCO).更多还原