外分泌GRP78通过激活EGFR-Src-STAT3信号通路使肝癌细胞发生索拉非尼耐药

发布时间：2018-08-13 09:01

【摘要】：目的探讨外分泌的GRP78和细胞膜表面EGFR相互作用的情况,确定外分泌GRP78和EGFR在肝癌细胞产生索拉非尼耐药过程中的作用及其机制,根据外分泌GRP78含量的不同以及其产生的耐药机制的原理,将其作为临床诊断的标志和治疗的靶点。方法1.用蛋白质印迹分析各肝癌细胞外分泌GRP78的含量和细胞表面EGFR的表达,并选择合适的细胞株。2.收集肝癌患者的血清和正常人的血清,利用ELISA分析肝癌患者血清中GRP78含量和正常人血清中GRP78含量的差异。3.在细胞水平检测rh GRP78是否影响索拉非尼对肝癌细胞的敏感性及其作用机制。利用MTT,Western blot,GST pull down和集落形成实验分析rh GRP78存在情况下肝癌细胞对索拉非尼敏感性的影响和rh GRP78与EGFR相互作用产生耐药性的信号传导通路。4.在动物水平建立肿瘤模型,并将动物分为三组,rh GRP78组,索拉非尼组和rh GRP78+索拉非尼组,采用灌胃方式进行治疗,一段时间后取出小鼠肿瘤组织进行瘤体称重和免疫组化检测,比较三组结果的差异。结果1.GRP78可以由HCC细胞分泌2.外分泌的GRP78促进细胞增殖并抑制索拉非尼诱导的细胞凋亡3.分泌的GRP78与EGFR相互作用并诱导其活化4.EGFR介导HCC细胞对分泌的GRP78的反应结论由肿瘤细胞分泌的GRP78可以以自分泌或旁分泌的方式激活EGFR-Src-STAT3信号通路导致肝癌细胞对索拉非尼的耐药。并且EGFR决定HCC细胞对外分泌GRP78的反应性
[Abstract]:Objective to investigate the interaction between exocrine GRP78 and EGFR on cell membrane, and to determine the role and mechanism of exocrine GRP78 and EGFR in the production of solafenib resistance in hepatoma cells. According to the different levels of exocrine GRP78 and the mechanism of drug resistance, it is regarded as a marker of clinical diagnosis and a target of treatment. Method 1. The content of exocrine GRP78 and the expression of EGFR on the surface of hepatoma cells were analyzed by Western blot, and the appropriate cell line. 2. The serum levels of GRP78 in HCC patients and those in normal controls were analyzed by ELISA. 3. 3. To determine whether rh GRP78 affects the sensitivity of Solafenib to hepatoma cells and its mechanism at cell level. The effects of rh GRP78 on the sensitivity of hepatoma cells to Solafenil and the signal transduction pathway of drug resistance between rh GRP78 and EGFR were analyzed by using MTTX Western blottle pull down and colony formation assay. The tumor model was established at the animal level, and the animals were divided into three groups: GRP78 group, Solafenil group and rh GRP78 sorafenil group. The tumor tissues of mice were taken out for weighing and immunohistochemical examination after a period of time. The differences among the three groups were compared. Results 1.GRP78 could be secreted by HCC cells. Exocrine GRP78 promotes cell proliferation and inhibits the apoptosis induced by Solafenib. The secreted GRP78 interacts with EGFR and induces its activation 4.EGFR mediates the response of HCC cells to secreted GRP78. Conclusion GRP78 secreted by tumor cells can activate the EGFR-Src-STAT3 signaling pathway in an autocrine or paracrine manner leading to the drug resistance of HCC cells to Solafenib. And EGFR determines the response of HCC cells to exocrine GRP78
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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