本文關鍵詞：抑癌基因PTEN在同源重組修復及其敲除對Rad51基因表達的影響及機制研究　出處：《重慶醫(yī)科大學》2009年碩士論文　論文類型：學位論文

  更多相關文章： PTEN Rad52 Rad51 雙鏈斷裂損傷(DSBs) 同源重組

【摘要】： 目的:研究PTEN缺失與否在DNA雙鏈斷裂損傷(DSBs)同源重組修復與其敲除對Rad51基因表達的作用及可能的機制。 方法:半定量RT-PCR比較對照小鼠胚胎成纖維細胞PTEN+/+MEFs與PTEN基因敲除的小鼠胚胎成纖維細胞PTEN-/-MEFs兩種細胞內同源重組修復相關的部分基因包括Rad51, Rad51相關蛋白1 (Rad51 associated protein 1,Rad51ap1), Rad52及Rad52b mRNA表達水平的差異;免疫熒光原位雜交技術比較兩種細胞γ射線照射后DNA雙鏈斷裂(DSBs)損傷程度;免疫熒光及流式細胞技術比較兩種細胞穩(wěn)定轉染Rad52基因后同源重組修復水平的差異;克隆形成實驗比較PTEN+/+MEFs與PTEN-/-MEFs細胞輻射敏感性的差異;半定量RT—PCR檢測輻射后兩種細胞Rad51基因表達的差異以及不同濃度LY—294002作用PTEN-/-MEFs細胞Rad51表達水平的變化。 結果:同對照細胞相比,PTEN-/-MEFs細胞部分參與同源重組修復的基因Rad51, Rad51ap1,Rad52及Rad52b mRNA表達水平降低,γ射線誘導的DNA DSBs損傷水平增高,而穩(wěn)定轉染Rad52基因后同源重組修復缺陷部分得到矯正。同對照細胞相比,PTEN-/-MEFs細胞輻射敏感性降低,輻射后Rad51表達增強,不同濃度PI3K/AKT信號通路抑制劑LY—294002作用PTEN-/-MEFs細胞后,Rad51表達水平增高。 結論:PTEN可能通過影響DNA雙鏈斷裂損傷同源重組修復相關基因Rad52等的轉錄,促進DNA雙鏈斷裂損傷的修復,并由此維持基因組穩(wěn)定性。PTEN缺失后細胞輻射敏感性降低,Rad51表達異常,PTEN可能通過拮抗抑制PI3K/AKT信號通路對Rad51基因的轉錄進行調控。
[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 緯-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 緯 -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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R346

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本文編號：1375147