發(fā)布時間：2018-02-01 06:15

  本文關鍵詞： EML4-ALK alectinib 肝細胞生長因子 表皮生長因子受體 耐藥　出處：《廣西醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:間變性淋巴瘤激酶酪氨酸激酶抑制劑(anaplastic lymphoma kinase tyrosine kinase inhibitor,ALK-TKI)的耐藥已成為其臨床應用最普遍也是最大的障礙,旁路信號通路的激活是非小細胞肺癌(non-small cell lung cancer,NSCLC)靶向治療的耐藥機制之一。本研究旨在探討肝細胞生長因子(hepatocyte growth factor,HGF)、表皮生長因子(epidermal growth factor,EGF)及轉化生長因子-α(transforming growth factor-α,TGF-α)以旁路信號激活的方式誘導棘皮動物微管結合蛋白樣蛋白4-間變性淋巴瘤激酶(echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase,EML4-ALK)融合基因陽性肺癌細胞株H3122對alectinib的耐藥,并進一步探討旁路信號激活在alectinib耐藥中的作用。方法:1.ALK陽性肺癌細胞H3122對alectinib的敏感性檢測檢測ALK陽性肺癌細胞H3122對alectinib的敏感性,H3122細胞含EML4-ALK融合基因變體1。根據(jù)CCK-8使用說明書,檢測H3122細胞在不同濃度的alectinib處理下,細胞的增殖情況。HGF、EGF、TGF-α誘導后ALK陽性肺癌細胞H3122對alectinib的敏感性的檢測,應用HGF(50ng/m L)、EGF(100 ng/m L)、TGF-α(100 ng/m L)聯(lián)合不同濃度的alectinib處理H3122細胞,繼續(xù)培養(yǎng)72h后,檢測在有HGF、EGF、TGF-α存在的情況下ALK陽性肺癌細胞H3122細胞對alectinib敏感性變化。2.采用流式細胞術檢測H3122細胞在0.05μmol/L alectinib作用48h的凋亡率,檢測HGF(50 ng/m L)、EGF(100 ng/m L)、TGF-α(100 ng/m L)誘導后alectinib對H3122細胞的凋亡率。3.應用免疫印跡(Western blot)技術檢測alectinib單獨或聯(lián)合HGF(50ng/m L)、EGF(100 ng/m L)、TGF-α(100 ng/m L)處理后H3122細胞中ALK、MET、EGFR及磷酸化蛋白的表達,并觀察其下游信號通路關鍵蛋白AKT、ERK、p-AKT、p-ERK水平,探討分子機制。結果:1.ALK陽性肺癌細胞H3122對alectinib高度敏感Alectinib作用72h后,H3122的活性隨著alectinib藥物濃度的增加而逐漸下降,呈濃度依賴性,alectinib對H3122的IC50為0.042μmol/L。2.在外源性HGF、EGF、TGF-α的暴露下,H3122細胞對alectinib的敏感性顯著下降,HGF、EGF、TGF-α誘導后alectinib抑制H3122細胞的生長曲線往右移,HGF、EGF、TGF-α處理能夠緩解alectinib對H3122細胞活性的抑制作用。3.Alectinib對H3122細胞有促凋亡作用(P0.05),0.05μmol/L的alectinib作用H3122細胞株48h后的凋亡率為(20.12±1.36)%,而alectinib聯(lián)合HGF、EGF、TGF-α后的凋亡率分別為(7.85±1.03)%、(5.6±0.79)%、(4.58±1.00)%,顯著低于alectinib單藥處理(P㩳0.05)。4.蛋白檢測提示H3122細胞可測得ALK和下游信號通路蛋白AKT、ERK及其磷酸化蛋白,0.05μmol/L alectinib單藥作用2h后成功抑制ALK及其下游信號蛋白AKT、ERK的磷酸化。聯(lián)合50 ng/m L HGF、100 ng/m L EGF、100 ng/m L TGF-α后H3122細胞中的p-ALK被抑制,但仍然表達p-AKT、p-ERK,50 ng/m L HGF處理后明顯增加細胞中p-MET的表達,100 ng/m L EGF、100 ng/m L TGF-α處理后明顯增加細胞中p-EGFR的表達。結論:HGF、EGF、TGF-α可通過旁路激活的方式誘導EML4-ALK陽性肺癌細胞H3122對alectinib耐藥,其機制可能與HGF激活MET磷酸化,EGF、TGF-α激活EGFR磷酸化有關。
[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?yōu)娲绘�х殑鎶戝埗浣滅敤.3.Alectinib瀵笻3122緇嗚優(yōu)鏈変績鍑嬩骸浣滅敤(P0.05),0.05渭mol/L鐨刟lectinib浣滅敤H3122緇嗚優(yōu)鏍，

本文編號：1481155