【摘要】：目的:構(gòu)建肺腺癌細(xì)胞放射抗拒株模型;慢病毒介導(dǎo)sh RNA靶向沉默抗拒株細(xì)胞中HIF-1α的表達(dá),探討其對β-catenin的調(diào)控及對肺腺癌細(xì)胞放射敏感性的影響。方法:采用亞致死劑量法誘導(dǎo)人肺腺癌H1299及A549放射抗拒細(xì)胞H1299R及A549R,克隆形成實(shí)驗(yàn)驗(yàn)證放射抗性,CCK-8實(shí)驗(yàn)檢測增細(xì)胞殖能力變化,q RT-PCR及Western Blot檢測HIF-1α、β-catenin、Cyclin D1、Survivin的基因及蛋白表達(dá)差異;通過RNAi技術(shù),設(shè)計(jì)靶向沉默HIF-1α的sh RNA,構(gòu)建慢病毒表達(dá)載體;慢病毒轉(zhuǎn)染H1299R、A549R細(xì)胞,通過克隆形成實(shí)驗(yàn)驗(yàn)證放射抗拒性的變化,Western Blot檢測下調(diào)組及對照組細(xì)胞HIF-1α、β-catenin、Cyclin D1、Survivin的蛋白表達(dá)變化。結(jié)果:克隆形成實(shí)驗(yàn)結(jié)果顯示:抗拒株細(xì)胞比親本株細(xì)胞具有更高的克隆形成率(P0.05),H1299R細(xì)胞SF2值是H1299細(xì)胞的1.56倍,A549R細(xì)胞SF2值是A549細(xì)胞的1.55倍,抗拒株細(xì)胞放療抗性增強(qiáng);CCK-8結(jié)果提示:在接受4Gy射線干預(yù)后親本株細(xì)胞增殖能力顯著抑制(P0.05);q RT-PCR及Western Blot結(jié)果顯示:抗拒株細(xì)胞比親本株細(xì)胞中HIF-1α、β-catenin、Cyclin D1、Survivin的基因及蛋白表達(dá)水平明顯升高(P0.05);通過慢病毒介導(dǎo)sh RNA下調(diào)抗拒株細(xì)胞HIF-1α后,下調(diào)組細(xì)胞β-catenin、Cyclin D1、Survivin蛋白表達(dá)明顯下降(P0.05);在不同劑量射線干預(yù)后,隨照射劑量增加,下調(diào)組細(xì)胞HIF-1α、β-catenin、Cyclin D1、Survivin的蛋白表達(dá)明顯低于對照組(P0.05);克隆形成實(shí)驗(yàn)結(jié)果顯示:下調(diào)組比對照組細(xì)胞克隆形成率明顯降低(P0.05),通過放射敏感性參數(shù)分析H1299R對照組細(xì)胞SF2值是下調(diào)組細(xì)胞的3.56倍,A549R對照組細(xì)胞SF2值是下調(diào)組細(xì)胞的2.45倍,下調(diào)組細(xì)胞放射敏感性顯著提高;CCK-8結(jié)果顯示:在細(xì)胞增殖能力方面,下調(diào)組比對照組細(xì)胞明顯降低(P0.05);在接受6Gy射線干預(yù)后,下調(diào)組細(xì)胞增殖能力顯著抑制(P0.05)。結(jié)論:HIF-1α可能調(diào)控β-catenin影響肺腺癌細(xì)胞放射敏感性,其機(jī)制可能與影響下游Cyclin D1、Survivin等蛋白的表達(dá)有關(guān)。
[Abstract]:Aim: to establish a lung adenocarcinoma cell line model of radioresistance and to investigate the effect of lentivirus-mediated HIF-1 偽 expression on 尾-catenin and radiosensitivity of lung adenocarcinoma cells. Methods: human lung adenocarcinoma cell lines H1299R and A549R were induced by sublethal dose method. The radioresistance of H1299R and A549R was verified by clone formation assay. The colonization ability was detected by CCK-8 assay. HIF-1 偽 and 尾-catenin, were detected by Q RT-PCR and Western Blot. The expression of survivin gene and protein in Cyclin D1 was different. The expression vector of lentivirus was constructed by designing sh RNA, targeting silencing HIF-1 偽 by RNAi technique. Lentivirus was transfected into H1299RtA549R cells. The change of radioresistance was verified by clone formation assay. The expression of HIF-1 偽, 尾-catenin,Cyclin D1 survivin was detected by, Western Blot in down-regulation group and control group. Results: the clone forming rate of resistant cell was higher than that of parent cell (P0.05). The SF2 value of H1299R cell was 1.56 times of that of H1299 cell, and the SF2 value of A549R cell was 1.55 times of that of A549 cell. The cell resistance to radiotherapy of the resistant strain was enhanced. The results of CCK-8 showed that the cell proliferation ability of parental lines was significantly inhibited after 4Gy irradiation (P0.05). The results of Q RT-PCR and Western Blot showed that the expression of HIF-1 偽, 尾-catenin,Cyclin D1 survivin gene and protein in resistant cell were significantly higher than those in parent cell line (P0.05). After down-regulation of HIF-1 偽 by sh RNA mediated by lentivirus, the expression of 尾-catenin,Cyclin D1 survivin protein decreased significantly (P0.05). The protein expression of HIF-1 偽, 尾-catenin,Cyclin D 1 and survivin was significantly lower than that in control group with the increase of irradiation dose after different doses of radiation (P0.05). The results of clone formation test showed that the colony formation rate of down-regulation group was significantly lower than that of control group (P0.05). The SF2 value of H1299R control group was 3.56 times of that of down-regulation group by radiosensitivity parameter analysis. The SF2 value of A549R control group was 2.45 times of that of down-regulated cells, and the radiosensitivity of down-regulated cells was significantly increased. CCK-8 results showed that the cell proliferation ability of down-regulation group was significantly lower than that of control group (P0.05), and that of down-regulation group was significantly inhibited after 6Gy irradiation (P0.05). Conclusion: HIF-1 偽 may regulate 尾-catenin to affect radiosensitivity of lung adenocarcinoma cells, and its mechanism may be related to the expression of downstream Cyclin D1 survivin and other proteins.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2

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