mTOR信号通路在17-DMAG克服EML4-ALK阳性肺癌细胞株H3122对alectinib耐药中的作用机制研究

发布时间：2018-03-20 00:12

本文选题：肺肿瘤　切入点：EML4-ALK融合基因　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:观察mTOR信号通路在17-DMAG克服旁路信号通路激活诱导棘皮动物微管相关蛋白样4与间变性淋巴瘤激酶(echinoderm microtubule-associated protein like4-anaplastic lymphoma kinase,EML4-ALK)融合基因阳性肺癌细胞株H3122(简称“H3122细胞”)对alectinib耐药中的变化,并探讨其内在的调控机制。方法:加入100 ng/ml转化生长因子α(transforming growth factor-α,TGF-α)和100 ng/ml表皮生长因子(epidermal growth factor,EGF)诱导H3122细胞对alectinib耐药,观察加入17-DMAG后上述耐药能否被克服。采用CCK-8法检测不同处理方式下H3122细胞的增殖,流式细胞术检测细胞凋亡,蛋白质免疫印迹(Western blot)技术检测不同处理方式细胞中ALK、EGFR及磷酸化蛋白的表达,观察其下游mTOR信号通路中关键蛋白及活化水平的表达。结果:Alectinib作用72 h后H3122细胞增殖率随药物浓度升高而下降,IC50为0.042μmol/L;联合TGF-α或EGF后H3122细胞对alectinib不敏感,IC50远大于10μmol/L;单药17-DMAG处理后H3122细胞增殖率随药物浓度升高而下降,IC50为0.245μmol/L;联合TGF-α或EGF后H3122细胞增殖率亦被17-DMAG抑制,且呈剂量依赖性,IC50分别为0.251μmol/L和0.301μmol/L。经0.05μmol/L的alectinib作用72 h后H3122细胞凋亡率为(30.01±0.92)%,alectinib联合TGF-α或EGF后的细胞凋亡率分别为(6.36±0.14)%和(6.13±0.21)%,显著低于alectinib单药处理组(P0.001);经0.3μmol/L的17-DMAG单药或联合TGF-α、EGF作用72 h后H3122细胞凋亡率分别为(28.37±1.75)%、(26.69±1.2)%和(26.62±0.72)%,三组间差异无统计学意义(P0.05)。Alectinib可抑制H3122细胞中p-ALK、p-mTOR的表达,也可抑制mTOR上、下游关键蛋白的活化状态;EGF可明显增加细胞中p-EGFR、p-mTOR及mTOR上、下游关键蛋白的活化水平表达;alectinib可抑制p-ALK表达,但联合EGF后不能抑制mTOR及mTOR上、下游关键蛋白活化水平的表达;即使联合EGF后,17-DMAG亦能抑制H3122细胞中ALK、EGFR、mTOR信号通路蛋白及其活化状态蛋白的表达。结论:旁路信号通路激活介导EML4-ALK融合基因阳性肺癌细胞株H3122对alectinib耐药的方式具有可行性,mTOR信号通路在17-DMAG克服旁路信号通路激活引起alectinib的获得性耐药过程中有一定作用。
[Abstract]:Objective: to observe the effect of mTOR signaling pathway on the activation of acanthoderm microtubule-associated protein like4-anaplastic lymphoma kinaseEML4-ALK fusion gene positive lung cancer cell line H3122 ("H3122 cells") by activating the 17-DMAG overcome bypass signaling pathway and inducing acanthoderm microtubule-associated protein like4-anaplastic lymphoma kinase- EML4-ALK to induce acanthoderm microtubule-associated protein-like 4. Changes in alectinib resistance, Methods: the drug resistance of H3122 cells to alectinib was induced by adding 100 ng/ml transforming growth factor- 伪 (TGF- 伪) and 100 ng/ml epidermal growth factor- (EGF- 伪). CCK-8 assay was used to detect the proliferation of H3122 cells under different treatments, and flow cytometry was used to detect the apoptosis of H3122 cells. Western blotting technique was used to detect the expression of ALKG FR and phosphorylated protein in different treatments. Results the proliferation rate of H3122 cells decreased with the increase of drug concentration at 72 h, and the IC50 of H3122 cells was 0.042 渭 mol / L after treatment with TGF- 伪 or EGF, and the IC50 of H3122 cells was much more than 10 渭 mol 路L ~ (-1) after treatment with TGF- 伪 or EGF. The proliferation rate of H3122 cells treated with single drug 17-DMAG decreased with the increase of drug concentration, the IC50 was 0.245 渭 mol / L, and the proliferation rate of H3122 cells treated with TGF- 伪 or EGF was also inhibited by 17-DMAG. In a dose-dependent manner, IC50 was 0.251 渭 mol/L and 0.301 渭 mol / L, respectively. The apoptotic rate of H3122 cells was 30.01 卤0.92% in combination with TGF- 伪 or EGF after 72 h of alectinib treatment with 0. 05 渭 mol/L, respectively, which was significantly lower than that of alectinib single drug treatment group (P 0.001), 17 DMAG single drug (0.3 渭 mol/L) or TGF- 伪 (EGF 伪) combined with TGF- 伪 EGF. After 72 h treatment, the apoptosis rates of H3122 cells were 28.37 卤1.75 and 26.69 卤1.2% and 26.62 卤0.72%, respectively. There was no significant difference among the three groups. P0.05, Alectinib could inhibit the expression of p-ALKN p-mTOR in H3122 cells. EGF could also inhibit the expression of p-ALK in mTOR, and the activation state of downstream key proteins could significantly increase the expression of p-EGFRR p-mTOR and mTOR, and the activation level of downstream key proteins could inhibit the expression of p-ALK, but after combined with EGF, it could not inhibit the expression of p-ALK on mTOR and mTOR. Expression of downstream key protein activation level; The expression of ALK-EGFR mTOR signaling pathway protein and its activated state protein in H3122 cells could be inhibited even if combined with EGF. Conclusion: Bypass signal pathway activation mediates EML4-ALK fusion gene positive lung cancer cell line H3122 has the ability of drug resistance to alectinib. The transversal mTOR signaling pathway plays a role in the process of 17-DMAG overcoming the acquired drug resistance of alectinib due to the activation of the bypass signaling pathway.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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