X射线照射鼻咽癌CNE1细胞后MRE11蛋白表达的初步研究

发布时间：2018-10-24 09:38

【摘要】： 肿瘤DNA损伤修复是放疗失败的主要原因。电离辐射造成细胞DNA损伤类型包括脱氧核糖变化、碱基变化、DNA单链断裂、DNA双链断裂、形成DNA-DNA交联和DNA-蛋白质交联等,其中DNA双链损伤(double-stranded breaks, DSBs)是最严重的损伤,导致细胞失去分裂增生能力而死亡,是电离辐射致肿瘤细胞死亡主要机制。DNA双链损伤修复在正常细胞中,可增强放疗靶区中正常组织的耐辐射能力,但在肿瘤细胞中,可能导致肿瘤局部复发和转移。修复机制包括同源重组修复(homologous recombination repair, HRR)和非同源末端连(nonhomologous end joining, NHEJ)。研究表明MRE11-RAD50-NBS1复合物在DSBs修复中发挥重要作用。其中MRE11的N端具有4个保守的磷酸酯酶区域,C端有2个DNA结合区,并具有单链／双链核酸内切酶的活性和3′→5′核酸外切酶活性,能识别并结合到DSBs处,与RAD50、NBS1相互作用共同完成修复。研究X射线照射后肿瘤细胞中MRE11基因及其蛋白表达的规律,并探讨其与肿瘤放疗失败的相关性,为提高肿瘤放射治疗疗效开辟新途径,提供新的靶点。目的：鼻咽癌细胞株CNE1经2Gy、4Gy、6Gy、8Gy不同剂量X线照射后4小时时间点各组MRE11mRNA表达与未照射组比较,观察射线照射对MRE11mRNA的表达变化。CNE1细胞株经4GyX线照射1.5h、4h、6h、8h、12h、24h后MRE11蛋白的表达变化。从基因和蛋白水平探讨了鼻咽癌细胞株CNE 1经照射后MRE 11基因及其蛋白的表达改变。方法：选取人鼻咽癌CNE 1细胞株进行体外培养及X线照射。按单次照射0Gy、2Gy、4Gy、6Gy、8Gy剂量分组,照射后4小时收集,利用RT-PCR技术检测MRE11mRNA的表达;CNE 1细胞株单次照射4Gy于第0h、1.5h、4h、6h、8h、12h、24h时间点收集固定,利用免疫荧光技术检测MRE11蛋白的表达,并采用Image-Pro Plus 5.0软件分析测定各组荧光的平均光密度值(mOD)。结果：RT-PCR检测CNE 1细胞株分别经0Gy、2Gy、4Gy、6Gy、8GyX线照射后4小时MRE11mRNA半定量灰度值分别为1.1±0.43,0.87±0.42,0.81±0.32,0.87±0.48,0.83±0.21,经单因素方差分析(Kruskal-Wallis检验),P=0.9797(P0.05),各组之间MRE11mRNA表达无统计学差异。免疫荧光技术检测CNE1细胞株经4GyX线照射后0h、1.5h、4h、6h、8h、12h、24h时间点MRE11蛋白的表达,由荧光强度表示和平均光密度(mOD)表示。发现荧光强度先增强后减弱,4小时荧光强度最强。各组mOD分别138.7±24.91、238.1±19.57、721.5±14.60、326.4±22.74、262.0±27.68、228.6±20.32、132.2±24.73,经单因素方差分析(Kruskal-Wallis检验),1.5h、4h、6h、8h、12h组与0h组比较,P0.05, MRE11蛋白表达差异有统计学意义;24h组与0h组比较,P0.05,差异无统计学意义。结论：CNE1细胞株MRE11mRNA的表达与X线照射剂量递增无相关性。但CNE1细胞株经X线照射后MRE11蛋白的表达随时间的延长先增强后减弱,且在照射4小时表达最强,24小时后恢复到未照射水平。
[Abstract]:DNA damage repair is the main reason for the failure of radiotherapy. The types of DNA damage induced by ionizing radiation include deoxyribose changes, base changes, DNA single strand breaks, DNA double strand breaks, DNA-DNA crosslinking and DNA- protein cross-linking, among which DNA double strand damage (double-stranded breaks, DSBs) is the most serious. The main mechanism of tumor cell death caused by ionizing radiation is that the cells lose the ability to divide and proliferate. The repair of DNA double strand damage in normal cells can enhance the radioresistance of normal tissues in the target region of radiotherapy, but in tumor cells, It may lead to local recurrence and metastasis of tumor. Repair mechanisms include homologous recombination repair (homologous recombination repair, HRR) and non-homologous terminal linked (nonhomologous end joining, NHEJ). It has been shown that MRE11-RAD50-NBS1 complex plays an important role in DSBs repair. The N-terminal of MRE11 has four conserved phosphatase regions, and the C-terminal has two DNA binding regions. It has the activity of single-stranded / double-stranded nucleic acid endonuclease and the exonuclease activity of 3'5 'nucleic acid, which can recognize and bind to DSBs. Work with RAD50,NBS1 to complete the repair. To study the expression of MRE11 gene and its protein in tumor cells after X-ray irradiation, and to explore the relationship between the expression of MRE11 gene and the failure of radiotherapy, and to provide a new target for improving the therapeutic effect of radiotherapy. Objective: to observe the changes of MRE11mRNA expression in nasopharyngeal carcinoma (NPC) cell line CNE1 at 4 hours after irradiation with different doses of 2 Gy, 4 Gy, 6 Gy or 8 Gy, and to observe the changes of MRE11mRNA expression in CNE1 cell line irradiated with 4GyX for 1. 5 h, 4 h, 6 h, 8 h, 12 h and 24 h after 4GyX irradiation. The expression of MRE 11 gene and its protein in nasopharyngeal carcinoma cell line CNE 1 after irradiation were studied at gene and protein levels. Methods: human nasopharyngeal carcinoma (NPC) CNE 1 cell line was cultured in vitro and irradiated by X-ray. The expression of MRE11mRNA was detected by RT-PCR technique, and the expression of MRE11 protein was detected by immunofluorescence technique, and the expression of MRE11 protein was detected by immunofluorescence technique, and the expression of MRE11 protein was detected by immunofluorescence technique, and the expression of MRE11 protein was detected by immunofluorescence technique at the time point of 12 h and 12 h after single irradiation of 0 Gy ~ 2 Gy ~ (2) Gy ~ (4) Gy ~ (6) Gy. The average optical density (mOD).) of fluorescence in each group was determined by Image-Pro Plus 5.0 software. Results: the semi-quantitative gray value of CNE 1 cell line was 1.1 卤0.43 卤0.82 卤0.82 卤0.82 卤0.87 卤0.88 卤0.83 卤0.21 at 4 hours after 0 Gy ~ 2 Gy ~ 4 Gy ~ 6 Gy ~ (8) Gy X-ray irradiation, respectively. There was no significant difference in the expression of MRE11mRNA between the three groups by Kruskal-Wallis test (P 0.05). Immunofluorescence technique was used to detect the expression of MRE11 protein in CNE1 cell line at 24 h after 4GyX irradiation. The expression of MRE11 protein was expressed by fluorescence intensity and average optical density (mOD). It was found that the fluorescence intensity first increased and then decreased, and the fluorescence intensity was the strongest in 4 hours. MOD in each group was 138.7 卤24.91238.1 卤19.57721.5 卤14.60326.4 卤22.74262.0 卤27.68228.6 卤20.32132.2 卤24.73.The expression of P0.05 and MRE11 protein was not significantly different between 24 h group and 0 h group by single factor variance analysis (Kruskal-Wallis test). Conclusion: there is no correlation between the expression of MRE11mRNA in CNE1 cell line and the increasing dose of X-ray irradiation. However, the expression of MRE11 protein in CNE1 cells increased first and then decreased with the prolongation of time, and the expression of MRE11 protein was strongest at 4 hours of irradiation, and recovered to the level of no irradiation after 24 hours of irradiation.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.63

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