氧化损伤修复基因的表达及表观遗传学改变与年龄相关性白内障的关系

Age-related cataract (ARC) causes visual impairment in the elderly and its etiology remains largely unknown.The previous research was concerned with studying the relationships between human groups and the structure variation of oxidative damage repair genes. we did not detect sequence variation association of some oxidative damage repair genes such as HOGG1、CSB with ARC, but we did found the altered expression of those genes in ARC lens.Our studies also found there were changes of epigenetics in ARC. Hereby, we hypothesize that those altered expression oxidative damage repair genes in the ARC lens might due to epigenetical changes. It is known that the methylation of DNA CpG islands and histone regulates strongly gene expression and many oxidative damage repair genes contain abundant CpG islands in their regulatory regions. The current proposal is set to investigate the epigenetic changes in the lens along with its development of ARC. We will collect lens from various subtypes of ARC patients and normal lens as well, use microarray to determine the differentiated expression of oxidative damage repair genes in those lens, apply RT-PCR, Western Blot and immunochemistry to confirm the results from microarray and previous chareterized HOGG1, WRN and CSB, adopt Qiagen MethyLight and ChIP methods to compare the levels of DNA and histone methylation of above selected genes in the lens from various subtypes of ARC patients and normal lens. Finally we will use human lens epithelium cell line to establish demethylation model to link the functional changes of gene expression with the epigenetical changes. The results can unveil the roles of epigenetical change in the development of lens opacity associated with ARC, gain an insight view of ARC pathogenesis and provide molecular targets in ARC therapy and prevention.

年龄相关性白内障（ARC）的发病机制尚不清楚。我们之前的流行病学人群氧化损伤清除与修复ARC易感基因的研究发现：不少患者HOGG1和CSB等基因并无明显序列改变但其混浊晶状体内的基因产物却显著减少，同时我们的预试验还初步表明ARC存在表观遗传学改变，提示ARC患者氧化损伤修复基因可能存在表观遗传修饰。本课题拟用微点阵法从正常人透明晶状体和ARC患者混浊晶状体中筛选出表达差别显著的氧化损伤修复基因；RT-PCR和免疫组化及荧光、Western Blot检测其表达；彗星分析晶状体上皮细胞DNA损伤和基因表达的关系；MethyLight法和ChIP法分析比对透明/混浊晶状体上述基因的DNA甲基化和组蛋白修饰水平。并建立去甲基化和高乙酰化模型观察各种因素的相互作用，以探讨氧化损伤修复基因的表达及表观遗传学改变与ARC的关系，为临床转化设计ARC特异性防治措施提供靶点。

年龄相关性白内障（ARC）是国内外最常见的致盲性眼病，其病因及发病机制尚不明确。本项目通过收集南通大学附属医院眼科ARC住院患者为ARC组（662例）和本院眼科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（305例），使用微点阵分析DNA氧化损伤修复基因，qRT-PCR、Western Blot、免疫荧光的方法检测氧化损伤清除基因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与ARC的关系。结果发现：与对照组相比，ARC组中10个DNA修复基因的mRNA水平是下调，1个是上升的；MGMT、WRN、GSTM3、GSTP1、ERCC6启动子在ARC组中呈现高甲基化，其他基因两组比较无差异。与对照组相比，ARC组LECs中组蛋白H3乙酰化水平较低而H3K9三甲基化水平则较高，ARC组LECs和外周血淋巴细胞氧化损伤加剧。因此，MGMT、WRN、GSTM3、GSTP1、ERCC6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。已发表SCI论文12篇，总影响因子27.75，中文文章5篇，参加国内外会议14次。