抑癌基因PTEN在同源重组修复及其敲除对Rad51基因表达的影响及机制研究

发布时间：2018-01-03 18:45

本文关键词：抑癌基因PTEN在同源重组修复及其敲除对Rad51基因表达的影响及机制研究　出处：《重庆医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:研究PTEN缺失与否在DNA双链断裂损伤(DSBs)同源重组修复与其敲除对Rad51基因表达的作用及可能的机制。方法:半定量RT-PCR比较对照小鼠胚胎成纤维细胞PTEN+/+MEFs与PTEN基因敲除的小鼠胚胎成纤维细胞PTEN-/-MEFs两种细胞内同源重组修复相关的部分基因包括Rad51, Rad51相关蛋白1 (Rad51 associated protein 1,Rad51ap1), Rad52及Rad52b mRNA表达水平的差异;免疫荧光原位杂交技术比较两种细胞γ射线照射后DNA双链断裂(DSBs)损伤程度;免疫荧光及流式细胞技术比较两种细胞稳定转染Rad52基因后同源重组修复水平的差异;克隆形成实验比较PTEN+/+MEFs与PTEN-/-MEFs细胞辐射敏感性的差异;半定量RT—PCR检测辐射后两种细胞Rad51基因表达的差异以及不同浓度LY—294002作用PTEN-/-MEFs细胞Rad51表达水平的变化。结果:同对照细胞相比,PTEN-/-MEFs细胞部分参与同源重组修复的基因Rad51, Rad51ap1,Rad52及Rad52b mRNA表达水平降低,γ射线诱导的DNA DSBs损伤水平增高,而稳定转染Rad52基因后同源重组修复缺陷部分得到矫正。同对照细胞相比,PTEN-/-MEFs细胞辐射敏感性降低,辐射后Rad51表达增强,不同浓度PI3K/AKT信号通路抑制剂LY—294002作用PTEN-/-MEFs细胞后,Rad51表达水平增高。结论:PTEN可能通过影响DNA双链断裂损伤同源重组修复相关基因Rad52等的转录,促进DNA双链断裂损伤的修复,并由此维持基因组稳定性。PTEN缺失后细胞辐射敏感性降低,Rad51表达异常,PTEN可能通过拮抗抑制PI3K/AKT信号通路对Rad51基因的转录进行调控。
[Abstract]:Aim: to investigate the effect of homologous recombination repair and knockout of PTEN deletion on Rad51 gene expression and its possible mechanism. Methods: Semi-quantitative RT-PCR was used to compare the PTEN / FB of mouse embryonic fibroblasts. Some genes related to homologous recombination repair of MEFs and PTEN knockout mouse embryonic fibroblasts (PTEN-/-MEFs) include Rad51. Rad51 associated protein 1 / Rad51 associated protein 1 / Rad51ap1). The expression level of Rad52 and Rad52b mRNA was different. Immunofluorescence in situ hybridization technique was used to compare the damage degree of DNA double strand breaks (DSBs) after 纬-ray irradiation. Immunofluorescence and flow cytometry were used to compare the level of homologous recombination repair after stable transfection of Rad52 gene between the two cells. The difference of radiosensitivity between PTEN / MEFs and PTEN-/-MEFs cells was compared by clone forming assay. The difference of Rad51 gene expression between two kinds of cells after irradiation by semi-quantitative RT-PCR and the expression level of Rad51 in PTEN-/-MEFs cells exposed to different concentrations of LY-294002. Change. Results: compared with the control cells, PTEN-R / -MEFs cells partially participated in the homologous recombination repair gene Rad51, Rad51ap1. The expression of Rad52 and Rad52b mRNA decreased, and the level of DNA DSBs damage induced by 纬 -ray increased. After stable transfection of Rad52 gene, the homologous recombination repair defects were corrected. Compared with the control cells, the radiosensitivity of PTEN-R / -MEFs cells was decreased, and the expression of Rad51 was enhanced after irradiation. The expression of Rad51 in PTEN-/-MEFs cells was increased after the treatment of LY-294002 with different concentrations of PI3K/AKT signaling pathway inhibitor. ConclusionPTEN may promote the repair of DNA double strand break injury by affecting the transcription of Rad52 and other genes associated with homologous recombination repair of DNA double strand break damage. The radiosensitivity of the cells decreased after maintaining genomic stability. PTEN deletion. PTEN may regulate the transcription of Rad51 gene by antagonistic inhibition of PI3K/AKT signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346

【参考文献】