CCDC7基因增强人鼻咽癌细胞放射敏感性的研究

发布时间：2019-01-17 12:18

【摘要】：背景与目的:目前放射治疗仍为鼻咽癌首选的治疗方式。放射线抗拒是鼻咽癌治疗失败的主要原因之一，研究其抗拒机制、寻求一些辅助手段或药物降低肿瘤细胞的放射抵抗、提高放射敏感性、提高疗效。CCDC7蛋白家族均含有一个CCDC7保守结构域，在进化上高度保守，在高等哺乳动物中高度同源，但其功能知之甚少。目前关于该基因与鼻咽癌的关系国内外尚未见报道。目的：研究CCDC7基因增强鼻咽癌细胞株（CNE2）放射敏感性的功能及其分子机制。方法:1.Western blot检测CNE-1、HNE-1、CNE-2三种细胞株CCDC7基因蛋白的表达。2.分组:(1)瞬时转染CCDC7基因：①对照组（空白对照：NS；阴性对照:Lip-Control），②CCDC7组(Lip-CCDC7)，③照射(R)组，④联合组(Lip-CCDC7+R)；(2)瞬时转染siCCDC7干扰序列：①对照组（空白对照：NS；阴性对照:Lip-Control），②siCCDC7组(Lip-siCCDC7)，③照射(R)组，④联合组(Lip-siCCDC7+R)。3.检测CNE-2细胞的CCDC7/siCCDC7转染水平：瞬时转染CCDC7基因24h后，采用倒置荧光显微镜，观察转染效率；瞬时转染siCCDC7基因24h后QPCR检测其干扰效率；4.克隆形成实验测定人鼻咽癌细胞株（CNE-2）在60Coγ射线照射后的存活分数，用多靶单击模型拟合剂量-存活曲线；5.MTT法检测肿瘤细胞增殖活力、生长速度的改变；6.流式细胞术检测鼻咽癌细胞株CNE-2处理后的凋亡率；7.Western Blot检测相关蛋白的表达变化。结果:1.Western blot检测CNE-1、HNE-1、CNE-2三种细胞株CCDC7基因蛋白的表达，CNE-2细胞株的CCDC7蛋白的表达较CNE-1、HNE-1两细胞株的CCDC7蛋白表达要高，本实验选取CNE-2细胞株来研究CCDC7基因对鼻咽癌放射敏感性的影响。2.瞬时转染CCDC7基因，实验组转染效率约50%，对照组转染效率约60%。3.瞬时转染siCCDC724h后，采用QPCR检测其干扰效率，结果显示CNE-2细胞CCDC7的表达量明显下调，实验组与对照组比较（0.546±0.092vs0.992±0.234，，P=0.011）,有显著差异，仅为对照组的55%。4.细胞克隆实验函数模型参数显示Lip-siCCDC7+R组的Dq、D0及SF2均明显低于R组，Lip-siCCDC7+R组较R组具有更高的辐射敏感性；Lip-CCDC7+R的Dq,D0，SF2与单纯照射组比较无明显差异。5.MTT法显示Lip-siCCDC7+R组细胞存活率明显低于对照组(6Gy)（42.98%±2.711%vs57.11%±2.39%, P0.05）。Lip-CCDC7+R组细胞存活率与对照组（6Gy）（55.47%±2.58%vs58.47%±2.89%，P0.05）比较无明显差异。6.流式实验结果显示干扰人鼻咽癌细胞株CNE-2的CCDC7的表达联合辐射诱导细胞凋亡率明显高于单纯照射(4Gy)（45.8±2.63vs24.56±3.012，P=0.017）。7.Western blotting结果显示Lip-siCCDC7联合照射PI3K的表达与对照组比较明显降低。结论：干扰人鼻咽癌细胞株CNE-2的CCDC7基因表达可增强该细胞株的放射敏感性，可能通过下调PI3K的表达水平来诱导细胞株（CNE-2）凋亡有关。
[Abstract]:Background & objective: radiotherapy is still the first choice for nasopharyngeal carcinoma. Radiation resistance is one of the main reasons for the failure of nasopharyngeal carcinoma (NPC) treatment. The CCDC7 family contains a conserved domain of CCDC7, highly conserved in evolution and highly homologous in higher mammals, but its function is poorly understood. The relationship between the gene and nasopharyngeal carcinoma has not been reported at home and abroad. Aim: to study the function of CCDC7 gene in enhancing radiosensitivity of nasopharyngeal carcinoma cell line (CNE2) and its molecular mechanism. Methods: 1.Western blot was used to detect the expression of CCDC7 gene protein in three CNE-1,HNE-1,CNE-2 cell lines. 2. Groups: (1) transient transfection of CCDC7 gene: 1 control group (blank control: NS; negative control: Lip-Control), 2CCDC7 group (Lip-CCDC7), 3 irradiated (R) group, 4 combined group (Lip-CCDC7 R);) (2) transient transfection of siCCDC7 interference sequence: 1 control group (blank control: NS; negative control: Lip-Control), 2siCCDC7 group (Lip-siCCDC7), 3 irradiated (R) group, 4 combined group (Lip-siCCDC7 R). 3). The CCDC7/siCCDC7 transfection level of CNE-2 cells was measured: 24 hours after transient transfection of CCDC7 gene, transfection efficiency was observed by inverted fluorescence microscope, QPCR interference efficiency was detected after 24 h transient transfection of siCCDC7 gene. 4. The survival fraction of human nasopharyngeal carcinoma cell line (CNE-2) after 60Co 纬 -irradiation was determined by clone formation assay, and the dose-survival curve was fitted by multi-target click model. The proliferation activity and growth rate of tumor cells were detected by 5.MTT assay. 6. Apoptosis rate of nasopharyngeal carcinoma cell line treated with CNE-2 was detected by flow cytometry and expression of related protein was detected by 7.Western Blot. Results: the expression of CCDC7 gene protein in three CNE-1,HNE-1,CNE-2 cell lines was detected by 1.Western blot. The expression of CCDC7 protein in CNE-2 cell line was higher than that in CNE-1,HNE-1 cell line. In this study, CNE-2 cell lines were selected to study the effect of CCDC7 gene on radiosensitivity of nasopharyngeal carcinoma. 2. The transfection efficiency of CCDC7 gene was about 50% in the experimental group and 60. 3% in the control group. After transient transfection of siCCDC724h, QPCR was used to detect its interference efficiency. The results showed that the expression of CCDC7 in CNE-2 cells was significantly down-regulated, and there was a significant difference between the experimental group and the control group (0.546 卤0.092vs0.992 卤0.234). Only for the control group 55. 4. The Dq,D0 and SF2 of Lip-siCCDC7 R group were significantly lower than that of R group, and the radiosensitivity of Lip-siCCDC7 R group was higher than that of R group. The Dq,D0,SF2 of Lip-CCDC7 R was significantly lower than that of the control group (6Gy, 42.98% 卤2.711% vs 57.11% 卤2.39%, P < 0.05), and the cell survival rate of Lip-siCCDC7 R group was significantly lower than that of the control group (42.98% 卤2.71 11% 卤2.39%). There was no significant difference in cell survival rate between Lip-CCDC7 R group and control group (6Gy) (55.47% 卤2.58 vs 58.47% 卤2.89 P 0.05). The results of flow cytometry showed that the rate of apoptosis induced by interference of CCDC7 expression in human nasopharyngeal carcinoma cell line CNE-2 combined with radiation was significantly higher than that of 4Gy (45.8 卤2.63vs24.56 卤3.012). 7.Western blotting results showed that the expression of PI3K in combined Lip-siCCDC7 irradiation was significantly lower than that in the control group. Conclusion: interfering with the expression of CCDC7 gene in human nasopharyngeal carcinoma cell line CNE-2 can enhance the radiosensitivity of the cell line, which may be related to the apoptosis of the cell line (CNE-2) by down-regulating the expression level of PI3K.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.63

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