【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(jī)制、尋求一些輔助手段或藥物降低腫瘤細(xì)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(gè)CCDC7保守結(jié)構(gòu)域，在進(jìn)化上高度保守，在高等哺乳動(dòng)物中高度同源，但其功能知之甚少。目前關(guān)于該基因與鼻咽癌的關(guān)系國內(nèi)外尚未見報(bào)道。目的：研究CCDC7基因增強(qiáng)鼻咽癌細(xì)胞株（CNE2）放射敏感性的功能及其分子機(jī)制。 方法:1.Western blot檢測(cè)CNE-1、HNE-1、CNE-2三種細(xì)胞株CCDC7基因蛋白的表達(dá)。2.分組:(1)瞬時(shí)轉(zhuǎn)染CCDC7基因：①對(duì)照組（空白對(duì)照：NS；陰性對(duì)照:Lip-Control），②CCDC7組(Lip-CCDC7)，③照射(R)組，④聯(lián)合組(Lip-CCDC7+R)；(2)瞬時(shí)轉(zhuǎn)染siCCDC7干擾序列：①對(duì)照組（空白對(duì)照：NS；陰性對(duì)照:Lip-Control），②siCCDC7組(Lip-siCCDC7)，③照射(R)組，④聯(lián)合組(Lip-siCCDC7+R)。3.檢測(cè)CNE-2細(xì)胞的CCDC7/siCCDC7轉(zhuǎn)染水平：瞬時(shí)轉(zhuǎn)染CCDC7基因24h后，采用倒置熒光顯微鏡，觀察轉(zhuǎn)染效率；瞬時(shí)轉(zhuǎn)染siCCDC7基因24h后QPCR檢測(cè)其干擾效率；4.克隆形成實(shí)驗(yàn)測(cè)定人鼻咽癌細(xì)胞株（CNE-2）在60Coγ射線照射后的存活分?jǐn)?shù)，用多靶單擊模型擬合劑量-存活曲線；5.MTT法檢測(cè)腫瘤細(xì)胞增殖活力、生長速度的改變；6.流式細(xì)胞術(shù)檢測(cè)鼻咽癌細(xì)胞株CNE-2處理后的凋亡率；7.Western Blot檢測(cè)相關(guān)蛋白的表達(dá)變化。 結(jié)果:1.Western blot檢測(cè)CNE-1、HNE-1、CNE-2三種細(xì)胞株CCDC7基因蛋白的表達(dá)，CNE-2細(xì)胞株的CCDC7蛋白的表達(dá)較CNE-1、HNE-1兩細(xì)胞株的CCDC7蛋白表達(dá)要高，本實(shí)驗(yàn)選取CNE-2細(xì)胞株來研究CCDC7基因?qū)Ρ茄拾┓派涿舾行缘挠绊憽?.瞬時(shí)轉(zhuǎn)染CCDC7基因，實(shí)驗(yàn)組轉(zhuǎn)染效率約50%，對(duì)照組轉(zhuǎn)染效率約60%。3.瞬時(shí)轉(zhuǎn)染siCCDC724h后，采用QPCR檢測(cè)其干擾效率，結(jié)果顯示CNE-2細(xì)胞CCDC7的表達(dá)量明顯下調(diào)，實(shí)驗(yàn)組與對(duì)照組比較（0.546±0.092vs0.992±0.234，，P=0.011）,有顯著差異，僅為對(duì)照組的55%。4.細(xì)胞克隆實(shí)驗(yàn)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(xì)胞存活率明顯低于對(duì)照組(6Gy)（42.98%±2.711%vs57.11%±2.39%, P0.05）。Lip-CCDC7+R組細(xì)胞存活率與對(duì)照組（6Gy）（55.47%±2.58%vs58.47%±2.89%，P0.05）比較無明顯差異。6.流式實(shí)驗(yàn)結(jié)果顯示干擾人鼻咽癌細(xì)胞株CNE-2的CCDC7的表達(dá)聯(lián)合輻射誘導(dǎo)細(xì)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(jié)果顯示Lip-siCCDC7聯(lián)合照射PI3K的表達(dá)與對(duì)照組比較明顯降低。 結(jié)論：干擾人鼻咽癌細(xì)胞株CNE-2的CCDC7基因表達(dá)可增強(qiáng)該細(xì)胞株的放射敏感性，可能通過下調(diào)PI3K的表達(dá)水平來誘導(dǎo)細(xì)胞株（CNE-2）凋亡有關(guān)。
[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(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王宏梅;陳龍華;鄭小康;李啟生;伍新堯;夏云飛;;抑制ATM/PI3K功能區(qū)表達(dá)對(duì)鼻咽癌細(xì)胞CNE1輻射增敏的研究[J];癌癥;2006年09期

2 何本夫;孫愛民;黃碧燕;王雯s

本文編號(hào)：2410052