發(fā)布時間：2018-04-05 09:32

  本文選題：肺癌細胞　切入點：ERCC1　出處：《江蘇大學》2017年碩士論文

【摘要】：目的探討分別單沉默和共沉默ERCC1和BRCA2基因后順鉑(DDP)對耐藥肺腺癌細胞(A549/DDP)的細胞毒性變化及其可能的機制?方法運用細胞脂質(zhì)體轉(zhuǎn)染技術將靶向于ERCC1和BRCA2基因的siRNAs(ERCC1-siRNA和BRCA2-siRNA)分別和同時轉(zhuǎn)染于體外培養(yǎng)的A549/DDP細胞。Western Blot檢測該細胞轉(zhuǎn)染前后ERCC1和BRCA2蛋白表達水平以確認轉(zhuǎn)染成功。用不同濃度DDP處理A549/DDP細胞,測定ERCC1基因沉默前后FANCD2單泛素化水平(以FANCD2-L/S比值表示),以及BRCA2基因沉默前后BRCA1和RAD51蛋白的表達水平。同時測定DDP敏感肺腺癌細胞(A549細胞)經(jīng)不同濃度的DDP誘導后FANCD2單泛素化、BRCA1和RAD51蛋白的表達水平。應用CCK-8法和Annexin V/PI流式細胞術分別測定ERCC1和BRCA2基因單沉默及共沉默前后經(jīng)不同濃度DDP處理的A549/DDP細胞增殖率與凋亡率變化。免疫熒光染色法分別測定ERCC1基因沉默前后的FANCD2核聚小體表達及BRCA2基因沉默前后RAD51小體表達。結果(1)Western Blot結果顯示,應用ERCC1-siRNA和BRCA2-siRNA分別轉(zhuǎn)染A549/DDP細胞后ERCC1和BRCA2蛋白的表達水平均明顯低于轉(zhuǎn)染前(P均0.05),表明ERCC1-siRNA和BRCA2-siRNA轉(zhuǎn)染有效,ERCC1和BRCA2基因被沉默。(2)Western Blot和免疫熒光法結果顯示,A549/DDP細胞的FANCD2單泛素化水平、BRCA1和RAD51蛋白的表達水平隨著DDP濃度的增加總體上呈上升趨勢,而A549細胞并未顯示出這一趨勢,提示范可尼貧血(FA)通路和同源重組修復(HRR)通路參與了A549/DDP細胞對DDP的耐藥機制。ERCC1基因沉默后經(jīng)DDP處理的FANCD2蛋白單泛素化水平和核聚小體表達較沉默前均顯著下降(P均0.05);BRCA2基因沉默后經(jīng)DDP處理的BRCA1和RAD51蛋白表達及RAD51核聚小體表達水平亦較沉默前顯著下降(P均0.05),表明FA通路和HRR通路DNA修復功能受到抑制。(3)CCK-8法和流式細胞術測定結果示,分別沉默ERCC1基因和BRCA2基因后均能增強DDP對A549/DDP細胞的增殖抑制作用和促凋亡作用(P均0.05),這兩個基因共沉默較單基因沉默可進一步增強DDP對A549/DDP的細胞毒性作用(P均0.05)?這一結果表明同時沉默ERCC1和BRCA2基因在增強DDP對耐藥肺癌細胞的殺瘤效應方面具有協(xié)同作用。結論沉默ERCC1與BRCA2基因均可逆轉(zhuǎn)耐藥肺癌細胞A549/DDP對DDP的耐藥性,且兩個基因共沉默這種逆轉(zhuǎn)DDP耐藥的作用更明顯。這種逆轉(zhuǎn)耐藥作用主要是通過抑制FA通路和HRR通路的DNA損傷修復功能功能來實現(xiàn)的。
[Abstract]:Objective to investigate the cytotoxicity of cisplatin (DDP) after single silencing and co-silencing of ERCC1 and BRCA2 genes to drug-resistant lung adenocarcinoma cell line A549 / DDP and its possible mechanism.Methods siRNAs(ERCC1-siRNA and BRCA2-siRNAs targeting ERCC1 and BRCA2 genes were detected by cell liposome transfection technique, and the expression levels of ERCC1 and BRCA2 protein were detected by Western Blot before and after transfection.A549/DDP cells were treated with different concentrations of DDP. The level of FANCD2 monoubiquitin (expressed as FANCD2-L/S ratio) before and after ERCC1 gene silencing and the expression of BRCA1 and RAD51 protein before and after BRCA2 gene silencing were measured.At the same time, the expression levels of FANCD2 monoubiquitin B BRCA1 and RAD51 protein were detected in DDP sensitive lung adenocarcinoma cell line (A549) induced by different concentrations of DDP.CCK-8 assay and Annexin V/PI flow cytometry were used to detect the proliferation and apoptosis of A549/DDP cells treated with different concentrations of DDP before and after ERCC1 and BRCA2 gene single silencing and co-silencing.The expression of FANCD2 nucleopolymer before and after ERCC1 gene silencing and the expression of RAD51 corpuscle before and after BRCA2 gene silencing were detected by immunofluorescence staining.Results Western Blot showed that,搴旂敤ERCC1-siRNA鍜孊RCA2-siRNA鍒嗗埆杞煋A549/DDP緇嗚優(yōu)鍚嶦RCC1鍜孊RCA2铔嬬櫧鐨勮〃杈炬按騫沖潎鏄庢樉浣庝簬杞煋鍓，

本文編號：1714189