【摘要】：背景與目的:目前放射治療仍為鼻咽癌首選的治療方式。放射線抗拒是鼻咽癌治療失敗的主要原因之一，研究其抗拒機制、尋求一些輔助手段或藥物降低腫瘤細胞的放射抵抗、提高放射敏感性、提高療效。CCDC7蛋白家族均含有一個CCDC7保守結構域，在進化上高度保守，在高等哺乳動物中高度同源，但其功能知之甚少。目前關于該基因與鼻咽癌的關系國內外尚未見報道。目的：研究CCDC7基因增強鼻咽癌細胞株（CNE2）放射敏感性的功能及其分子機制。 方法:1.Western blot檢測CNE-1、HNE-1、CNE-2三種細胞株CCDC7基因蛋白的表達。2.分組:(1)瞬時轉染CCDC7基因：①對照組（空白對照：NS；陰性對照:Lip-Control），②CCDC7組(Lip-CCDC7)，③照射(R)組，④聯(lián)合組(Lip-CCDC7+R)；(2)瞬時轉染siCCDC7干擾序列：①對照組（空白對照：NS；陰性對照:Lip-Control），②siCCDC7組(Lip-siCCDC7)，③照射(R)組，④聯(lián)合組(Lip-siCCDC7+R)。3.檢測CNE-2細胞的CCDC7/siCCDC7轉染水平：瞬時轉染CCDC7基因24h后，采用倒置熒光顯微鏡，觀察轉染效率；瞬時轉染siCCDC7基因24h后QPCR檢測其干擾效率；4.克隆形成實驗測定人鼻咽癌細胞株（CNE-2）在60Coγ射線照射后的存活分數(shù)，用多靶單擊模型擬合劑量-存活曲線；5.MTT法檢測腫瘤細胞增殖活力、生長速度的改變；6.流式細胞術檢測鼻咽癌細胞株CNE-2處理后的凋亡率；7.Western Blot檢測相關蛋白的表達變化。 結果:1.Western blot檢測CNE-1、HNE-1、CNE-2三種細胞株CCDC7基因蛋白的表達，CNE-2細胞株的CCDC7蛋白的表達較CNE-1、HNE-1兩細胞株的CCDC7蛋白表達要高，本實驗選取CNE-2細胞株來研究CCDC7基因對鼻咽癌放射敏感性的影響。2.瞬時轉染CCDC7基因，實驗組轉染效率約50%，對照組轉染效率約60%。3.瞬時轉染siCCDC724h后，采用QPCR檢測其干擾效率，結果顯示CNE-2細胞CCDC7的表達量明顯下調，實驗組與對照組比較（0.546±0.092vs0.992±0.234，，P=0.011）,有顯著差異，僅為對照組的55%。4.細胞克隆實驗函數(shù)模型參數(shù)顯示Lip-siCCDC7+R組的Dq、D0及SF2均明顯低于R組，Lip-siCCDC7+R組較R組具有更高的輻射敏感性；Lip-CCDC7+R的Dq,D0，SF2與單純照射組比較無明顯差異。5.MTT法顯示Lip-siCCDC7+R組細胞存活率明顯低于對照組(6Gy)（42.98%±2.711%vs57.11%±2.39%, P0.05）。Lip-CCDC7+R組細胞存活率與對照組（6Gy）（55.47%±2.58%vs58.47%±2.89%，P0.05）比較無明顯差異。6.流式實驗結果顯示干擾人鼻咽癌細胞株CNE-2的CCDC7的表達聯(lián)合輻射誘導細胞凋亡率明顯高于單純照射(4Gy)（45.8±2.63vs24.56±3.012，P=0.017）。7.Western blotting結果顯示Lip-siCCDC7聯(lián)合照射PI3K的表達與對照組比較明顯降低。 結論：干擾人鼻咽癌細胞株CNE-2的CCDC7基因表達可增強該細胞株的放射敏感性，可能通過下調PI3K的表達水平來誘導細胞株（CNE-2）凋亡有關。
[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 緯 -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 鹵0.092vs0.992 鹵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% 鹵2.711% vs 57.11% 鹵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% 鹵2.71 11% 鹵2.39%). There was no significant difference in cell survival rate between Lip-CCDC7 R group and control group (6Gy) (55.47% 鹵2.58 vs 58.47% 鹵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 鹵2.63vs24.56 鹵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.
【學位授予單位】：廣西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.63

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相關期刊論文 前10條

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本文編號：2410052