本文選題：肺腫瘤　切入點：EML4-ALK融合基因　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:觀察mTOR信號通路在17-DMAG克服旁路信號通路激活誘導(dǎo)棘皮動物微管相關(guān)蛋白樣4與間變性淋巴瘤激酶(echinoderm microtubule-associated protein like4-anaplastic lymphoma kinase,EML4-ALK)融合基因陽性肺癌細胞株H3122(簡稱“H3122細胞”)對alectinib耐藥中的變化,并探討其內(nèi)在的調(diào)控機制。方法:加入100 ng/ml轉(zhuǎn)化生長因子α(transforming growth factor-α,TGF-α)和100 ng/ml表皮生長因子(epidermal growth factor,EGF)誘導(dǎo)H3122細胞對alectinib耐藥,觀察加入17-DMAG后上述耐藥能否被克服。采用CCK-8法檢測不同處理方式下H3122細胞的增殖,流式細胞術(shù)檢測細胞凋亡,蛋白質(zhì)免疫印跡(Western blot)技術(shù)檢測不同處理方式細胞中ALK、EGFR及磷酸化蛋白的表達,觀察其下游mTOR信號通路中關(guān)鍵蛋白及活化水平的表達。結(jié)果:Alectinib作用72 h后H3122細胞增殖率隨藥物濃度升高而下降,IC50為0.042μmol/L;聯(lián)合TGF-α或EGF后H3122細胞對alectinib不敏感,IC50遠大于10μmol/L;單藥17-DMAG處理后H3122細胞增殖率隨藥物濃度升高而下降,IC50為0.245μmol/L;聯(lián)合TGF-α或EGF后H3122細胞增殖率亦被17-DMAG抑制,且呈劑量依賴性,IC50分別為0.251μmol/L和0.301μmol/L。經(jīng)0.05μmol/L的alectinib作用72 h后H3122細胞凋亡率為(30.01±0.92)%,alectinib聯(lián)合TGF-α或EGF后的細胞凋亡率分別為(6.36±0.14)%和(6.13±0.21)%,顯著低于alectinib單藥處理組(P0.001);經(jīng)0.3μmol/L的17-DMAG單藥或聯(lián)合TGF-α、EGF作用72 h后H3122細胞凋亡率分別為(28.37±1.75)%、(26.69±1.2)%和(26.62±0.72)%,三組間差異無統(tǒng)計學(xué)意義(P0.05)。Alectinib可抑制H3122細胞中p-ALK、p-mTOR的表達,也可抑制mTOR上、下游關(guān)鍵蛋白的活化狀態(tài);EGF可明顯增加細胞中p-EGFR、p-mTOR及mTOR上、下游關(guān)鍵蛋白的活化水平表達;alectinib可抑制p-ALK表達,但聯(lián)合EGF后不能抑制mTOR及mTOR上、下游關(guān)鍵蛋白活化水平的表達;即使聯(lián)合EGF后,17-DMAG亦能抑制H3122細胞中ALK、EGFR、mTOR信號通路蛋白及其活化狀態(tài)蛋白的表達。結(jié)論:旁路信號通路激活介導(dǎo)EML4-ALK融合基因陽性肺癌細胞株H3122對alectinib耐藥的方式具有可行性,mTOR信號通路在17-DMAG克服旁路信號通路激活引起alectinib的獲得性耐藥過程中有一定作用。
[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- 偽 (TGF- 偽) and 100 ng/ml epidermal growth factor- (EGF- 偽). 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- 偽 or EGF, and the IC50 of H3122 cells was much more than 10 渭 mol 路L ~ (-1) after treatment with TGF- 偽 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- 偽 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 鹵0.92% in combination with TGF- 偽 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- 偽 (EGF 偽) combined with TGF- 偽 EGF. After 72 h treatment, the apoptosis rates of H3122 cells were 28.37 鹵1.75 and 26.69 鹵1.2% and 26.62 鹵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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2

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本文編號：1636637