野生型DNA聚合酶β转染对细胞应对交链孢霉酚损伤作用的影响

发布时间：2018-01-26 01:25

本文关键词： DNA聚合酶β 转染 NIH3T3细胞 p53 交链孢霉酚　出处：《郑州大学》2006年硕士论文　论文类型：学位论文

【摘要】：背景及目的： DNA聚合酶β(DNA polymerase β，DNA po1β)是参与体内碱基切除修复(base excision repair，BER)过程的重要的损伤修复酶，其功能的正常发挥对维持细胞的正常进程和遗传的稳定性具有重要作用。DNA po1β主要修复自由基氧化或胞嘧啶和甲基胞嘧啶脱氨基等过程产生的DNA损伤，也能解决外源性因素如亚硝胺类产生的烷基化碱基，在DNA和RNA水平上进行损伤修复。正常细胞中，DNApo1β在整个细胞周期中都是低表达的，由于DNA po1β缺少3'-5'校读功能，是所有聚合酶中保真度最低的酶。高表达的DNA po1β可能扰乱细胞内的各种聚合酶类功能，参与其它DNA合成过程，并可导致NER过程的易错合成，因此会引起基因组的不稳定性，，导致肿瘤的发生。研究发现，在多种肿瘤细胞和癌旁组织中发现DNA po1β基因突变及DNA po1β蛋白高表达。p53基因是最具代表性的抑癌基因，野生型p53蛋白在维持细胞正常生长、抑制恶性增殖中起着重要作用。交链孢霉酚(alternariol，AOH)是河南林州市食管癌高发区比较常见的一种霉菌毒素，能通过多种机制引起细胞癌变。本研究建立了高表达野生型DNA po1β的细胞系，并用AOH损伤细胞后，间接研究DNA po1β的损伤修复作用及与抑癌基因p53的关系，并为食管癌的病因学提供一定的资料。方法：将已构建的含野生型DNA po1β的绿色荧光真核表达载体pEGFP-C3-po1β用脂质体介导转染NIH3T3细胞，经G418筛选获得稳定转染的细胞系，通过荧光倒置显微镜观察转染基因在细胞中的表达。经交链孢霉酚作用48小时后，流
[Abstract]:Background and purpose: DNA polymerase 尾 -DNA polymerase 尾 -DNA po1 尾 is involved in base excision repair in vivo. Excision repairBERs are important damage repair enzymes in the process. Its normal function plays an important role in maintaining the normal progress of cells and genetic stability. Po1 尾 mainly repairs DNA damage caused by free radical oxidation or cytosine and methyl cytosine deaminination. It can also solve exogenous factors such as alkylation bases produced by nitrosamines and repair the damage at the level of DNA and RNA. The expression of po1 尾 in normal cells is low in the whole cell cycle. DNA po1 尾 is the least fidelity enzyme of all polymerase due to its lack of 3- 5'reading function. The high expression of DNA po1 尾 may disturb the functions of various polymerase types in cells. Participate in other DNA synthesis processes, and can lead to the error-prone synthesis of NER process, thus causing genomic instability, leading to tumorigenesis. The mutation of DNA po1 尾 gene and the overexpression of DNA po1 尾 protein. P53 gene are the most representative tumor suppressor genes in various tumor cells and adjacent tissues. Wild type p53 protein plays an important role in maintaining normal cell growth and inhibiting malignant proliferation. AOH) is a common mycotoxin in the high incidence area of esophageal cancer in Linzhou City, Henan Province, which can cause cell carcinogenesis through many mechanisms. In this study, a cell line with high expression of wild-type DNA po1 尾 was established. The damage and repair of DNA po1 尾 and the relationship between DNA po1 尾 and tumor suppressor gene p53 were studied indirectly, and some data were provided for the etiology of esophageal cancer. Methods: The constructed green fluorescent eukaryotic expression vector pEGFP-C3-po1 尾 containing wild-type DNA po1 尾 was transfected into NIH3T3 cells by liposome mediated transfection. The stable transfected cell lines were obtained by G418 screening, and the expression of transfected genes in the cells was observed by fluorescence inverted microscope. After 48 hours of treatment with Alternazol, the expression of transfected cells was observed.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363

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