【摘要】：目的:構建肺腺癌細胞放射抗拒株模型;慢病毒介導sh RNA靶向沉默抗拒株細胞中HIF-1α的表達,探討其對β-catenin的調控及對肺腺癌細胞放射敏感性的影響。方法:采用亞致死劑量法誘導人肺腺癌H1299及A549放射抗拒細胞H1299R及A549R,克隆形成實驗驗證放射抗性,CCK-8實驗檢測增細胞殖能力變化,q RT-PCR及Western Blot檢測HIF-1α、β-catenin、Cyclin D1、Survivin的基因及蛋白表達差異;通過RNAi技術,設計靶向沉默HIF-1α的sh RNA,構建慢病毒表達載體;慢病毒轉染H1299R、A549R細胞,通過克隆形成實驗驗證放射抗拒性的變化,Western Blot檢測下調組及對照組細胞HIF-1α、β-catenin、Cyclin D1、Survivin的蛋白表達變化。結果:克隆形成實驗結果顯示:抗拒株細胞比親本株細胞具有更高的克隆形成率(P0.05),H1299R細胞SF2值是H1299細胞的1.56倍,A549R細胞SF2值是A549細胞的1.55倍,抗拒株細胞放療抗性增強;CCK-8結果提示:在接受4Gy射線干預后親本株細胞增殖能力顯著抑制(P0.05);q RT-PCR及Western Blot結果顯示:抗拒株細胞比親本株細胞中HIF-1α、β-catenin、Cyclin D1、Survivin的基因及蛋白表達水平明顯升高(P0.05);通過慢病毒介導sh RNA下調抗拒株細胞HIF-1α后,下調組細胞β-catenin、Cyclin D1、Survivin蛋白表達明顯下降(P0.05);在不同劑量射線干預后,隨照射劑量增加,下調組細胞HIF-1α、β-catenin、Cyclin D1、Survivin的蛋白表達明顯低于對照組(P0.05);克隆形成實驗結果顯示:下調組比對照組細胞克隆形成率明顯降低(P0.05),通過放射敏感性參數分析H1299R對照組細胞SF2值是下調組細胞的3.56倍,A549R對照組細胞SF2值是下調組細胞的2.45倍,下調組細胞放射敏感性顯著提高;CCK-8結果顯示:在細胞增殖能力方面,下調組比對照組細胞明顯降低(P0.05);在接受6Gy射線干預后,下調組細胞增殖能力顯著抑制(P0.05)。結論:HIF-1α可能調控β-catenin影響肺腺癌細胞放射敏感性,其機制可能與影響下游Cyclin D1、Survivin等蛋白的表達有關。
[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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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