旁路信号通路激活介导的EML4-ALK融合基因阳性肺癌细胞株H3122对alectinib继发耐药研究
发布时间：2018-02-01 06:15
本文关键词： EML4-ALK alectinib 肝细胞生长因子 表皮生长因子受体 耐药 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:间变性淋巴瘤激酶酪氨酸激酶抑制剂(anaplastic lymphoma kinase tyrosine kinase inhibitor,ALK-TKI)的耐药已成为其临床应用最普遍也是最大的障碍,旁路信号通路的激活是非小细胞肺癌(non-small cell lung cancer,NSCLC)靶向治疗的耐药机制之一。本研究旨在探讨肝细胞生长因子(hepatocyte growth factor,HGF)、表皮生长因子(epidermal growth factor,EGF)及转化生长因子-α(transforming growth factor-α,TGF-α)以旁路信号激活的方式诱导棘皮动物微管结合蛋白样蛋白4-间变性淋巴瘤激酶(echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase,EML4-ALK)融合基因阳性肺癌细胞株H3122对alectinib的耐药,并进一步探讨旁路信号激活在alectinib耐药中的作用。方法:1.ALK阳性肺癌细胞H3122对alectinib的敏感性检测检测ALK阳性肺癌细胞H3122对alectinib的敏感性,H3122细胞含EML4-ALK融合基因变体1。根据CCK-8使用说明书,检测H3122细胞在不同浓度的alectinib处理下,细胞的增殖情况。HGF、EGF、TGF-α诱导后ALK阳性肺癌细胞H3122对alectinib的敏感性的检测,应用HGF(50ng/m L)、EGF(100 ng/m L)、TGF-α(100 ng/m L)联合不同浓度的alectinib处理H3122细胞,继续培养72h后,检测在有HGF、EGF、TGF-α存在的情况下ALK阳性肺癌细胞H3122细胞对alectinib敏感性变化。2.采用流式细胞术检测H3122细胞在0.05μmol/L alectinib作用48h的凋亡率,检测HGF(50 ng/m L)、EGF(100 ng/m L)、TGF-α(100 ng/m L)诱导后alectinib对H3122细胞的凋亡率。3.应用免疫印迹(Western blot)技术检测alectinib单独或联合HGF(50ng/m L)、EGF(100 ng/m L)、TGF-α(100 ng/m L)处理后H3122细胞中ALK、MET、EGFR及磷酸化蛋白的表达,并观察其下游信号通路关键蛋白AKT、ERK、p-AKT、p-ERK水平,探讨分子机制。结果:1.ALK阳性肺癌细胞H3122对alectinib高度敏感Alectinib作用72h后,H3122的活性随着alectinib药物浓度的增加而逐渐下降,呈浓度依赖性,alectinib对H3122的IC50为0.042μmol/L。2.在外源性HGF、EGF、TGF-α的暴露下,H3122细胞对alectinib的敏感性显著下降,HGF、EGF、TGF-α诱导后alectinib抑制H3122细胞的生长曲线往右移,HGF、EGF、TGF-α处理能够缓解alectinib对H3122细胞活性的抑制作用。3.Alectinib对H3122细胞有促凋亡作用(P0.05),0.05μmol/L的alectinib作用H3122细胞株48h后的凋亡率为(20.12±1.36)%,而alectinib联合HGF、EGF、TGF-α后的凋亡率分别为(7.85±1.03)%、(5.6±0.79)%、(4.58±1.00)%,显著低于alectinib单药处理(P㩳0.05)。4.蛋白检测提示H3122细胞可测得ALK和下游信号通路蛋白AKT、ERK及其磷酸化蛋白,0.05μmol/L alectinib单药作用2h后成功抑制ALK及其下游信号蛋白AKT、ERK的磷酸化。联合50 ng/m L HGF、100 ng/m L EGF、100 ng/m L TGF-α后H3122细胞中的p-ALK被抑制,但仍然表达p-AKT、p-ERK,50 ng/m L HGF处理后明显增加细胞中p-MET的表达,100 ng/m L EGF、100 ng/m L TGF-α处理后明显增加细胞中p-EGFR的表达。结论:HGF、EGF、TGF-α可通过旁路激活的方式诱导EML4-ALK阳性肺癌细胞H3122对alectinib耐药,其机制可能与HGF激活MET磷酸化,EGF、TGF-α激活EGFR磷酸化有关。
[Abstract]:Objective: anaplastic lymphoma kinase tyrosine kinase inhibitors (anaplastic lymphoma kinase tyrosine kinase inhibitor, ALK-TKI) resistance has become the most common clinical application is the biggest obstacle, the bypass signal pathway is activated in non-small cell lung cancer (non-small cell lung cancer, NSCLC) targeting one of the resistance mechanisms of treatment. This study aimed to investigate the liver cells growth factor (hepatocyte growth, factor, HGF), epidermal growth factor (epidermal growth, factor, EGF) and transforming growth factor alpha (transforming growth factor- TGF- alpha, alpha) binding protein like protein 4- of anaplastic lymphoma kinase activation induced by bypass signal echinoderm microtubule (echinoderm microtubule-associated protein-like 4-anaplastic animal lymphoma kinase, EML4-ALK) the fusion gene positive lung cancer cell line H3122 resistant to alectinib, and to further explore the next Signal activation in alectinib MDR. Methods: the sensitivity of H3122 1.ALK positive lung cancer cells on the sensitivity of alectinib detection of ALK positive lung cancer cell H3122 on alectinib, H3122 cells containing EML4-ALK fusion gene variant 1. according to CCK-8 manual detection of H3122 cells in different concentrations of alectinib treatment, the cell proliferation of.HGF, EGF the detection sensitivity of H3122, ALK positive lung cancer cells on the alectinib induced by TGF- after the application of HGF (50ng/m L), EGF (100 ng/m L), TGF- alpha (100 ng/m L) combined with different concentration of alectinib treated H3122 cells, cultured 72h, EGF, HGF detection, ALK positive lung cancer cells H3122 the presence of alpha TGF- cells by H3122 cells was detected by flow cytometry in 0.05 mol/L alectinib 48h apoptosis rate of alectinib.2. detection sensitivity, HGF (50 ng/m L), EGF (100 ng/m L), TGF- Alpha (100 ng/m L) alectinib on H3122 cell apoptosis rate of.3. by Western blot after induction (Western blot) detection of alectinib alone or in combination with HGF (50ng/m L), EGF (100 ng/m L), TGF- alpha (100 ng/m L) after the treatment of H3122 cells in ALK, MET, EGFR expression and phosphorylation of eggs white, and observe its downstream signaling pathway protein AKT, ERK, p-AKT, p-ERK level, and to explore the molecular mechanism of H3122. Results: 1.ALK positive lung cancer cells are highly sensitive to alectinib Alectinib 72h, the activity of H3122 decreased with the increase of alectinib concentration, in a concentration dependent manner, alectinib of H3122 IC50 0.042 mol/L.2. in exogenous HGF, EGF, TGF- exposure under the sensitivity of H3122 cells to alectinib decreased significantly, HGF, EGF, alectinib inhibited H3122 cell growth curve induced by TGF- after HGF, EGF, to the right, TGF- treatment can alleviate the alectinib of H3122 鑳炴椿鎬х殑鎶戝埗浣滅敤.3.Alectinib瀵笻3122缁嗚優鏈変績鍑嬩骸浣滅敤(P0.05),0.05渭mol/L鐨刟lectinib浣滅敤H3122缁嗚優鏍，
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