错配修复蛋白hMLH1在硒化合物诱导DNA损伤应答途径中的作用

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	错配修复蛋白hMLH1在硒化合物诱导DNA损伤应答途径中的作用
Alternative Title	The Role of Mismatch Repair Protein hMLH1 in Selenium-induced DNA Damage Response
	漆永梅
Thesis Advisor	张迎梅
	2011-05-08
Degree Grantor	兰州大学
Place of Conferral	兰州
Degree Name	博士
Keyword	硒共济失调毛细血管扩张突变蛋白人MutL同源体-1 DNA损伤应答 HCT116结肠直肠癌细胞活性氧自由基 G2/M细胞周期检验点应答
Abstract	动物模型和流行病学研究表明，硒化合物能有效降低结肠直肠癌的发生。然而，其发挥抗癌作用的分子机制尚不清楚。大多数结肠直肠癌的典型特征是DNA错配修复功能缺失。人MutL同源体-1蛋白（hMLH1）作为碱基错配修复的关键蛋白，虽然其功能在错配修复途径中得到普遍认可，但在DNA损伤应答途径即抗癌的分子途径中是否发挥作用并不清楚。因此，本文采用错配修复功能缺失（hMLH1缺失）的结肠直肠癌HCT116细胞和功能获得（载体导入hMLH1蛋白）的HCT116+hMLH1细胞，研究hMLH1在硒诱导的DNA损伤应答途径中的作用。本研究通过细胞克隆形成和MTT（四甲基偶氮唑盐）比色实验探讨了硒化合物对结肠直肠癌HCT116细胞的毒性效应和作用机制；通过免疫荧光实验检测了硒化合物致HCT116细胞DNA损伤效应；借助流式细胞术检测了硒化合物对HCT116细胞周期的影响；通过免疫沉淀和免疫印迹实验探究了硒化合物对错配修复途径中重要的蛋白二聚体hMLH1-hPMS2的影响；并进一步探索了活性氧自由基（Reactive oxygen species，ROS）和DNA损伤应答途径关键调控蛋白ATM（共济失调毛细血管扩张突变蛋白）对细胞周期和hMLH1-hPMS2蛋白相互作用的影响。最终，试图明确硒化合物是否通过错配修复蛋白hMLH1调节ATM依赖的DNA损伤应答途径。
Other Abstract	Animal and epidemiological studies strongly implicate selenium as an effective chemoprevention agent against colon cancer; however, the molecular mechanism by which Se mitigates colorectal tumorigenesis is largely unknown. The majority of colorectal cancers is characterized by microsatellite instability due to a defective mismatch repair system. It’s known that hMLH1 (human MutL homologue-1) plays an important role in reparing DNA base mismatches. Although hMLH1 is acknowledged as a DNA repair protein, whether it functions in DNA damage response pathway is unknown. Here, the MMR-deficient HCT116 colorectal cancer cells and the MMR-proficient HCT116 cells with hMLH1 complementation are employed to investigate the role of hMLH1 in selenium induced DNA damage response, a tumorigenesis barrier. The effects of selenium compounds on HCT116 cell viability were detected by colony formation and MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. DNA damages were checked by immunofluorescence analysis. Cell cycle profiles were analyzed by flow cytometry. By using immunoprecipitation and immunoblotting, the association between hMLH1and hPMS2 was studied. Futhermore, the roles of reactive oxygen species (ROS) and DNA damage response protein ATM (Ataxia telangiectasia mutated) in regulating cell cycle progression and mediating hMLH1 and hPMS2 (Post-meiotic segregation protein-2) association were explored. This study was trying to figure out if selenium compounds could activate ATM-dependent DNA damage response via the mismatch repair protein hMLH1 in colorectal cancer cells.
URL	查看原文
Language	中文
Document Type	学位论文
Identifier	https://ir.lzu.edu.cn/handle/262010/221614
Collection	生命科学学院
Recommended Citation GB/T 7714	漆永梅. 错配修复蛋白hMLH1在硒化合物诱导DNA损伤应答途径中的作用[D]. 兰州. 兰州大学,2011.

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