年龄相关性白内障患者晶状体组织GST基因的表达及表观遗传学改变

Age-related cataract (ARC) is one of the most common eye diseases and its etiology remains largely unknown. We were granted "The Molecular Epidemiological Study of ARC and DNA repair" in year 2010 by National Natural Foundation China to explore the relationship between ARC and genes of DNA repair. We collected the DNA of ARC and the control in "Jiangsu Eye Study" and found the copy number polymorphisms of GSTT1, a gene functioning in removal of oxidative damaged molecules, is associated with ARC. It was reported that the red blood cell of ARC patients had lower GST level than that of controls. Hereby, we hypothesize that those altered expression of GST in the ARC might also occur in the lens of ARC patients, which might due to epigenetical changes. It is known that the methylation of DNA CpG islands regulates strongly gene expression and GST gene contains abundant CpG islands in their regulatory regions. The current proposal is to investigate the expression and epigenetic changes of GST in the lens along with its development of ARC. We will collect lens at different levels of opacity from various subtypes of ARC patients and normal lens. The collected lens will subject to qRT-PCR, Western Blot and immunochemitry to measure GST expression, comet assay to analyze DNA damage in lens epithelial cell to observe the relationship between GST and DNA damage, MethyLight method to compare the levels of DNA methylation of GST genes. Finally we will use human lens epithelium cell line to establish demethylation model to link the functional changes of GST expression with the epigenetical changes. The results can unveil the roles of GST epigenetical change in the development of lens opacity associated with ARC, gain an insight view of ARC pathogenesis and provide molecular target in ARC therapy and prevention.

影响年龄相关性白内障（ARC）的病因及确切的分子机制至今仍不清楚。我们曾经收集"江苏眼病研究"的ARC和对照人群DNA研究ARC与氧化损伤通路的易感/保护基因，发现氧化损伤清除基因GSTT1的拷贝数多态性与ARC发病有关。加上已有报道ARC患者外周血红细胞GST水平比正常人低且这种变化与GST多态性有关。因此我们推测ARC患者GST基因在晶状体中可能存在表达的变化和/或表观遗传修饰。本课题拟采用qRT-PCR、免疫组化及荧光、Western Blot、测DNA损伤的彗星分析和测DNA序列CpG岛甲基化的MethyLight法等多种技术，检测GST在人透明/混浊晶状体的表达，分析DNA损伤与晶状体上皮细胞GST表达的关系，并比对透明/混浊晶状体GST的DNA甲基化修饰水平，建立去甲基化模型，探讨GST的表达和表观遗传学改变与ARC晶状体混浊的关系。

年龄相关性白内障（ARC）是我国首位致盲性眼病，其确切的病因及分子机制至今仍不清楚。本项目通过收集南通大学附属医院眼科ARC住院患者为病例组（120例）和诊断为玻璃体视网膜疾病，因年龄或手术等因素需要在玻璃体切除手术中同时作透明晶状体摘除者为对照组（60例），使用实时定量PCR、Western Blot、免疫荧光的方法检测氧化损伤清除基因GST在混浊晶状体和透明晶状体中的表达，重亚硫酸盐测序PCR及焦磷酸测序法检测基因的甲基化状态，染色质免疫共沉淀测定样品中被修饰的组蛋白（甲基化或乙酰化组蛋白）水平，qRT-PCR、免疫荧光分析多种组蛋白甲基转移酶、乙酰基转移酶的表达，结合彗星试验，探讨基因表观遗传学改变和氧化损伤清除与ARC的关系。结果发现：与对照组相比，三种类型ARC患者晶状体LECs及皮质中GSTM3、GSTP1的表达量均下降，GSTM3、GSTP1启动子在ARC组中呈现高甲基化；与对照组相比，ARC组LECs中组蛋白H3乙酰化水平较低而H3K9甲基化水平则较高，ARC组LECs和外周血淋巴细胞氧化损伤加剧。因此，GSTM3、GSTP1基因表观遗传学改变可能与ARC的发生发展相关；这些基因甲基化调节自身蛋白质的表达，进而影响其DNA的修复功能，可作为ARC防治的新靶点。