【作者】 张丽；

【导师】 姚克；

【作者基本信息】 浙江大学 ， 眼科学， 2009， 博士

【摘要】 研究目的:后发障是现代白内障术后最常见的并发症,后发障的发病机制至今尚未完全明确。上皮间质转分化是后发障发生过程中主的要病理改变,转化生长因子β（TGF-β）作为间质转分化最有力的诱导因子,在后发障发病过程中发挥重要作用。阻断TGF-β诱导的间质转分化的生物学过程为后发障的防治提供了新的思路。结缔组织生长因子（CTGF）是一种在纤维化过程中起重要作用的间质细胞生长因子,近年来CTGF在TGF-β诱导的一系列生物学过程,特别是间质转分化过程中的作用受到重视。本课题旨在总结以往研究TGF-β与后发障相关研究的基础上,采用siRNA干扰这一高效、特异的基因沉默手段抑制体外培养的人晶状体上皮细胞CTGF的表达,观察基因干扰对TGF-β₂诱导的上皮间质转分化的影响,分析TGF-β₂和CTGF在晶状体上皮细胞间质转分化过程中的作用及信号转导机制,进一步明确后发障的发病机制,为基因治疗后发障提供靶标。研究方法:采用体外培养的人晶状体上皮细胞株HLE B-3,用不同浓度的TGF-β₂处理细胞,应用免疫印迹法及RT-PCR观察细胞CTGF表达及间质转分化相关蛋白connexin43、a-SMA表达的影响。建立后发障离体研究模型,采用脂质体介导的siRNA干扰技术转染HLE B-3细胞,特异性沉默CTGF的表达。应用免疫印迹、免疫荧光等方法观察CTGF基因干扰对TGF-β₂诱导的晶状体上皮细胞间质转分化及粘附功能的影响。研究结果:TGF-β₂能够诱导体外培养人晶状体上皮细胞表达CTGF蛋白,CTGF在晶状体上皮细胞内的表达主要位于细胞浆。外源性TGF-β₂及CTGF均能诱导人晶状体上皮细胞发生间质转分化。siRNA干扰能够成功的从基因和蛋白水平抑制TGF-β₂诱导的人晶状体上皮细胞CTGF的表达,并且能够抑制TGF-β₂诱导的人晶状上皮细胞间质转分化和粘附作用及相关蛋白的表达。研究结论:CTGF作为TGF-β₂的下游分子,在TGF-β₂诱导的晶状体上皮细胞的间质转分化过程中发挥关键作用。基因干扰CTGF的表达能够抑制TGF-β₂诱导的晶状体上皮细胞间质转分化,为后发障的防治提供理论依据。更多还原

【Abstract】 Objective:Posterior capsular opacification（PCO） is the main complication of modern cataract surgery and cause of poor vision post-operation.Residual lens epithelial cells （LECs） undergo epithelial mesenchymal transition（EMT） in the progress of PCO. Previous work has revealed that transforming growth factor beta（TGF-β）play an important role in PCO.Though it is extensively studied,the mechanism of TGF-βon lens is complex and not completely understood yet.Connective tissue growth factor （CTGF） is a matricellular protein that has been found to participate in EMT.So it is critical to understand the mechanism of CTGF,a potent growth factor in ocular media, in process of EMT in LECs.On the basis of previous studies,we established a research model for PCO in vitro.We also took the siRNA technique to selectively silence the expression of CTGF in LECs to investigate the role of CTGF in TGF-β₂ induced EMT in human lens epithelial cells.Methods:HLE B-3 cells were treated with different concentrations of TGF-β₂ to investigate effect of TGF-β₂ on expression of CTGF,connexin43 and a-SMA with western blot and RT-PCR.siRNA technique was operated to selectively silence the expression of CTGF gene.Then we analyzed the effect of siRNA interfere on TGF-β₂ induced EMT and adhesion of LECs with western blot and immunofluorescence.Results:HLE B-3 cells underwent morphological alteration and EMT under TGF-β₂ stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels.The expression of Connexin43 was decreased after treatment of TGF-β₂,while the expression of a-SMA was increased.CTGF silencing by CTGF siRNA approaches showed that TGF-β₂-induced EMT and adhesion were CTGF dependent.Conclusion:CTGF is an essential downstream mediator of TGF-β₂-induced EMT in lens epithelial ceils.Selectively silence the expression of CTGF could be a potent therapeutic target of PCO.更多还原