本文關(guān)鍵詞：抑癌基因PTEN在同源重組修復(fù)及其敲除對(duì)Rad51基因表達(dá)的影響及機(jī)制研究　出處：《重慶醫(yī)科大學(xué)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)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(fù)與其敲除對(duì)Rad51基因表達(dá)的作用及可能的機(jī)制。 方法:半定量RT-PCR比較對(duì)照小鼠胚胎成纖維細(xì)胞PTEN+/+MEFs與PTEN基因敲除的小鼠胚胎成纖維細(xì)胞PTEN-/-MEFs兩種細(xì)胞內(nèi)同源重組修復(fù)相關(guān)的部分基因包括Rad51, Rad51相關(guān)蛋白1 (Rad51 associated protein 1,Rad51ap1), Rad52及Rad52b mRNA表達(dá)水平的差異;免疫熒光原位雜交技術(shù)比較兩種細(xì)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(xì)胞技術(shù)比較兩種細(xì)胞穩(wěn)定轉(zhuǎn)染Rad52基因后同源重組修復(fù)水平的差異;克隆形成實(shí)驗(yàn)比較PTEN+/+MEFs與PTEN-/-MEFs細(xì)胞輻射敏感性的差異;半定量RT—PCR檢測(cè)輻射后兩種細(xì)胞Rad51基因表達(dá)的差異以及不同濃度LY—294002作用PTEN-/-MEFs細(xì)胞Rad51表達(dá)水平的變化。 結(jié)果:同對(duì)照細(xì)胞相比,PTEN-/-MEFs細(xì)胞部分參與同源重組修復(fù)的基因Rad51, Rad51ap1,Rad52及Rad52b mRNA表達(dá)水平降低,γ射線誘導(dǎo)的DNA DSBs損傷水平增高,而穩(wěn)定轉(zhuǎn)染Rad52基因后同源重組修復(fù)缺陷部分得到矯正。同對(duì)照細(xì)胞相比,PTEN-/-MEFs細(xì)胞輻射敏感性降低,輻射后Rad51表達(dá)增強(qiáng),不同濃度PI3K/AKT信號(hào)通路抑制劑LY—294002作用PTEN-/-MEFs細(xì)胞后,Rad51表達(dá)水平增高。 結(jié)論:PTEN可能通過(guò)影響DNA雙鏈斷裂損傷同源重組修復(fù)相關(guān)基因Rad52等的轉(zhuǎn)錄,促進(jìn)DNA雙鏈斷裂損傷的修復(fù),并由此維持基因組穩(wěn)定性。PTEN缺失后細(xì)胞輻射敏感性降低,Rad51表達(dá)異常,PTEN可能通過(guò)拮抗抑制PI3K/AKT信號(hào)通路對(duì)Rad51基因的轉(zhuǎn)錄進(jìn)行調(diào)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 陳漢春;Rad51同系物與DNA重組修復(fù)[J];國(guó)外醫(yī)學(xué).遺傳學(xué)分冊(cè);2003年04期

2 金問(wèn)森;金一尊;;Rad51的異常表達(dá)與腫瘤治療[J];國(guó)際腫瘤學(xué)雜志;2006年10期

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本文編號(hào)：1375147