鼻咽癌细胞株中EGFR的表达及吉非替尼对鼻咽癌细胞放射敏感性的影响

发布时间：2018-04-28 23:23

  本文选题：鼻咽癌 + 表皮生长因子受体　； 参考：《广西医科大学》2012年硕士论文

【摘要】：目的鼻咽癌的发生和发展是由多种病因通过多种途径长期共同影响及诱导的过程,病因学的研究表明与EB病毒感染、地域环境因素影响、饮食习惯以及家族遗传等因数相关。除此之外,某些特定蛋白和基因的过度表达及变异也在鼻咽癌发展中也发挥着重要作用。表皮生长因子受体(epidermal growth factor receptor, EGFR)在鼻咽癌(nasopharyngeal carcinoma, NPC)细胞中存在大量表达,并和鼻咽癌的发生和发展有着密切关系。本研究通过对比单纯使用吉非替尼、单纯照射及吉非替尼联合照射对鼻咽细胞杀伤的体外实验,旨在研究表皮生长因子受体的表达对放射照射的影响。探讨吉非替尼对鼻咽癌放射治疗作用的影响及可能机制,为临床治疗提供依据。方法取对数生长期人鼻咽癌CNE1和HONE1细胞接种于96孔板中并随机分组,分组分别设立对照组、吉非替尼组、单纯照射组及药物照射组。吉非替尼组投入吉非替尼浓度为0、0.1μmol/l、1μmol/l、5μmol/l、10μmol/l并继续孵育72小时后进行检测。用剂量率为81.78cGy/min的60Co机进行放射治疗,单纯照射组分别以0、2Gy、4Gy、6Gy、8Gy的单次剂量照射后继续在培养箱中孵育5到7天。药物照射组,在放射治疗30分钟前先投入1μmol/l浓度的吉非替尼,再给予不同剂量照射培养5-7天后检测以CCk-8测量细胞存活率；并用免疫荧光和免疫印迹分别检测EGFR蛋白的表达水平。细胞集落实验检测吉非替尼对不同鼻咽癌细胞株放射敏感性的影响,设0Gy、2Gy、4Gy、6Gy、8Gy共5个剂量点。单纯照射组和药物照射组均射剂量大小以1000个细胞的密度接进行照射后,在37℃恒温CO2培养箱中培养14天,在显微镜下观测并计数细胞的克隆数(含有≥50个细胞的集落)。用单靶多击数学模型拟合细胞存活曲线,分别计算各细胞株的放射增敏比(SER)。结果免疫荧光检测和免疫印迹均显示CNE1细胞和HONE1细胞内均有较高EGFR表达,细胞存活实验显示,药物照射组较单纯照射组在低放射量组时既有显著差异,(CNE1细胞株2Gy组P0.013,HONE1细胞株,2Gy组P0.008,4Gy组P0.02)。用单靶多击数学模型拟合细胞存活曲线表示,CNE1细胞株的单纯照射组细胞存活曲线的Do为2.069Gy,Dq为3.99Gy；药物照射组Do为1.67Gy, Dq为2.49Gy,放射增敏比(SER)为1.23。HONE1细胞株照射组Do为2.0Gy,Dq为3.59Gy；药物照射组Do为1.490Gy, Dq为2.71Gy,SER为1.35。结论EGFR在两种细胞中均有较高表达,吉非替尼联合放射照射较单纯放射照射能提高鼻咽癌的治疗效果。吉非替尼能增强鼻咽癌细胞株的放射敏感性。
[Abstract]:Objective the occurrence and development of nasopharyngeal carcinoma (NPC) are influenced and induced by a variety of etiological factors, including Epstein-Barr virus (EBV) infection, regional environmental factors, diet habits and family heredity. In addition, overexpression and variation of certain proteins and genes also play an important role in the development of nasopharyngeal carcinoma. Epidermal growth factor receptor (EGFR) is highly expressed in nasopharyngeal carcinoma (NPCs) cells, and is closely related to the occurrence and development of nasopharyngeal carcinoma (NPC). The purpose of this study was to study the effects of epidermal growth factor receptor (EGF) expression on the radiation of nasopharyngeal cells in vitro by comparing the killing effects of gifitinib alone, simple irradiation and combined irradiation with gefitinib on nasopharynx cells. To investigate the effect and possible mechanism of gefitinib on radiotherapy of nasopharyngeal carcinoma (NPC). Methods Human nasopharyngeal carcinoma (NPC) CNE1 and HONE1 cells in logarithmic phase were inoculated in 96-well plate and randomly divided into control group, gefitinib group, simple irradiation group and drug irradiation group. The concentration of gefitinib was 0. 1 渭 mol / L ~ (-1) 渭 mol / L ~ (-1) 渭 mol / L ~ (1) 5 渭 mol / L ~ (10) 渭 mol/l and incubated for 72 hours. Radiation therapy was performed with 60Co machine at dose rate of 81.78cGy/min. The irradiation group was incubated in incubator for 5 to 7 days after a single dose of 0 ~ 2 Gy ~ 4 Gy ~ 6 Gy ~ 8 Gy. In the drug irradiation group, 1 渭 mol/l concentration of gifitinib was injected 30 minutes before radiotherapy. After 5-7 days of different doses of irradiation, the survival rate of the cells was measured by CCk-8, and the expression of EGFR protein was detected by immunofluorescence and Western blotting. The effects of gefitinib on radiosensitivity of different nasopharyngeal carcinoma cell lines were detected by colony assay. After exposure to 1000 cells in the single irradiation group and drug irradiation group, the cells were cultured in a constant temperature CO2 incubator at 37 鈩，

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