本文選題：肺癌細(xì)胞　切入點(diǎn)：ERCC1　出處：《江蘇大學(xué)》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(duì)耐藥肺腺癌細(xì)胞(A549/DDP)的細(xì)胞毒性變化及其可能的機(jī)制?方法運(yùn)用細(xì)胞脂質(zhì)體轉(zhuǎn)染技術(shù)將靶向于ERCC1和BRCA2基因的siRNAs(ERCC1-siRNA和BRCA2-siRNA)分別和同時(shí)轉(zhuǎn)染于體外培養(yǎng)的A549/DDP細(xì)胞。Western Blot檢測(cè)該細(xì)胞轉(zhuǎn)染前后ERCC1和BRCA2蛋白表達(dá)水平以確認(rèn)轉(zhuǎn)染成功。用不同濃度DDP處理A549/DDP細(xì)胞,測(cè)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(dá)水平。同時(shí)測(cè)定DDP敏感肺腺癌細(xì)胞(A549細(xì)胞)經(jīng)不同濃度的DDP誘導(dǎo)后FANCD2單泛素化、BRCA1和RAD51蛋白的表達(dá)水平。應(yīng)用CCK-8法和Annexin V/PI流式細(xì)胞術(shù)分別測(cè)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(xì)胞增殖率與凋亡率變化。免疫熒光染色法分別測(cè)定ERCC1基因沉默前后的FANCD2核聚小體表達(dá)及BRCA2基因沉默前后RAD51小體表達(dá)。結(jié)果(1)Western Blot結(jié)果顯示,應(yīng)用ERCC1-siRNA和BRCA2-siRNA分別轉(zhuǎn)染A549/DDP細(xì)胞后ERCC1和BRCA2蛋白的表達(dá)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(jié)果顯示,A549/DDP細(xì)胞的FANCD2單泛素化水平、BRCA1和RAD51蛋白的表達(dá)水平隨著DDP濃度的增加總體上呈上升趨勢(shì),而A549細(xì)胞并未顯示出這一趨勢(shì),提示范可尼貧血(FA)通路和同源重組修復(fù)(HRR)通路參與了A549/DDP細(xì)胞對(duì)DDP的耐藥機(jī)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(dá)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(dá)及RAD51核聚小體表達(dá)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(fù)功能受到抑制。(3)CCK-8法和流式細(xì)胞術(shù)測(cè)定結(jié)果示,分別沉默ERCC1基因和BRCA2基因后均能增強(qiáng)DDP對(duì)A549/DDP細(xì)胞的增殖抑制作用和促凋亡作用(P均0.05),這兩個(gè)基因共沉默較單基因沉默可進(jìn)一步增強(qiáng)DDP對(duì)A549/DDP的細(xì)胞毒性作用(P均0.05)?這一結(jié)果表明同時(shí)沉默ERCC1和BRCA2基因在增強(qiáng)DDP對(duì)耐藥肺癌細(xì)胞的殺瘤效應(yīng)方面具有協(xié)同作用。結(jié)論沉默ERCC1與BRCA2基因均可逆轉(zhuǎn)耐藥肺癌細(xì)胞A549/DDP對(duì)DDP的耐藥性,且兩個(gè)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(guò)抑制FA通路和HRR通路的DNA損傷修復(fù)功能功能來(lái)實(shí)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(hào)：1714189