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目的构建稳定的XRCC2基因沉默大肠癌细胞系及阐明沉默XRCC2基因表达的大肠癌细胞对电离辐射损伤的反应。方法采用Western blot法检测不同肿瘤细胞系人大肠癌细胞系(T84、HT29、Lovo)、食管癌EC9706细胞、乳腺癌MCF-7细胞和人正常胚肾HEK293细胞中XRCC2蛋白的表达水平。大肠癌T84细胞经0、2、4、8、12 Gy X射线照射后和8 Gy X线照射后0、6、24、48 h,用Western blot法和实时定量PCR法测定XRCC2蛋白和mRNA的表达水平。将XRCC2 shRNA质粒转染T84细胞,用嘌呤霉素进行细胞筛选,采用Western blot法和实时定量PCR法,检测沉默XRCC2蛋白和mRNA表达的效率。将XRCC2 shRNA质粒转染T84细胞,经X线照射后,采用单细胞凝胶电泳测定沉默XRCC2基因表达的T84细胞的DNA损伤修复。结果与人正常胚肾HEK293细胞相比,在不同肿瘤细胞系中XRCC2蛋白均呈不同程度的高表达,其中在大肠癌T84细胞中XRCC2蛋白表达最高。随着X线照射剂量的增加,T84细胞中XRCC2蛋白的表达水平逐渐升高,8 Gy照射后XRCC2蛋白表达水平在48 h内随着时间的延长而逐渐升高;XRCC2 mRNA表达也表现出随剂量增加和时间延长而逐渐升高。与未处理的细胞相比,转染XRCC2 shRNA质粒的细胞中XRCC2蛋白和mRNA表达明显下降,约分别减少了70%和60%。照射后的T84细胞DNA损伤修复中shRNA-XRCC2组TDNA%、TM和OTM均显著高于未处理组和shRNA-SC组,差异有统计学意义(t=15.12、11.36、13.15,P<0.01)。结论成功建立了稳定表达XRCC2基因沉默的大肠癌T84细胞系,XRCC2基因沉默导致辐射诱导的DNA损伤修复能力下降。
Objective To construct a stable XRCC2 gene silencing colorectal cancer cell line and to elucidate the response of XRCC2 gene silencing colorectal cancer cells to ionizing radiation damage. Methods The expression of XRCC2 protein in human colorectal cancer cell lines (T84, HT29, Lovo), esophageal cancer EC9706 cells, breast cancer MCF-7 cells and human normal embryonic kidney HEK293 cells was detected by Western blot. XRCC2 protein and mRNA were detected by Western blot and real-time quantitative PCR after 0, 2, 4, 8, 12 Gy X-irradiation in colorectal cancer T84 cells and 0, 6, 24, 48 h after 8 Gy X-ray irradiation. The expression level. The XRCC2 shRNA plasmid was transfected into T84 cells and screened by puromycin. Western blot and real-time quantitative PCR were used to detect the efficiency of silencing XRCC2 protein and mRNA expression. The XRCC2 shRNA plasmid was transfected into T84 cells and irradiated with X-rays. DNA damage repair of T84 cells silenced by XRCC2 gene expression was detected by single cell gel electrophoresis. Results Compared with normal human embryonic kidney HEK293 cells, XRCC2 protein was highly expressed in different degrees in different tumor cell lines, among which XRCC2 protein expression was highest in colorectal cancer T84 cells. With the increase of X-ray irradiation dose, the expression level of XRCC2 protein in T84 cells increased gradually. After 8 Gy irradiation, the expression of XRCC2 protein gradually increased with the prolongation of time within 48 hours; XRCC2 mRNA expression also showed Increased doses and prolonged periods gradually increased. The expression of XRCC2 protein and mRNA in the cells transfected with the XRCC2 shRNA plasmid was significantly decreased by approximately 70% and 60%, respectively, compared to untreated cells. The TDNA%, TM, and OTM in the shRNA-XRCC2 group after irradiation were significantly higher than those in the untreated group and shRNA-SC group (t=15.12, 11.36, 13.15, P<0.01). . Conclusion The T84 cell line that stably expresses XRCC2 gene silencing is successfully established. The silencing of XRCC2 gene leads to the decrease of radiation-induced DNA damage repair capacity.