Rad51基因沉默对铅诱导TK6细胞DNA双链断裂损伤修复的影响

Effect of Rad51 gene silencing on repair of lead-induced DNA double-strand breaks in TK6 cells

目的研究Rad51基因沉默后对醋酸铅染毒致人淋巴母细胞(TK6细胞)DNA双链断裂损伤的修复作用的影响。方法构建Rad51沉默慢病毒载体及阴性对照,感染对数期TK6细胞,荧光定量PCR和Western blot验证感染效果。运用480μmol/L的醋酸铅染毒TK6细胞24 h(Control组、shRNA-NC组和shRNA-Rad51组),采用免疫荧光法检测TK6细胞的磷酸化组蛋白H2AX(γ-H2AX)的表达,Western Blot检测TK6细胞的Rad51、BRCA1、53BP1蛋白的表达。结果 shRNA-Rad51组的Rad51 mRNA表达水平和Rad51蛋白表达水平均低于Control组及shRNA-NC组(P <0.01);shRNA-Rad51组的γ-H2AX阳性率为(27.48±1.66)%,与Control组的(14.77±1.21)%及shRNA-NC组的(14.04±1.31)%比较,差异均有统计学意义(P <0.01);shRNA-Rad51组的BRCA1蛋白表达水平为(0.25±0.03),与Control组的(0.55±0.04)及shRNA-NC组的(0.51±0.04)比较,差异均有统计学意义(P <0.01);shRNARad51组的53BP1蛋白表达水平为(3.24±0.27),与Control组的(2.01±0.19)及shRNA-NC组的(2.11±0.17)比较,差异均有统计学意义(P <0.01)。结论 Rad51基因沉默后,TK6细胞HR修复通路受抑制和NHEJ修复通路激活,TK6细胞对铅遗传毒性的敏感性增强。

Objective To study the effect of Rad51 gene silencing on the repair of DNA double-strand breaks in human lymphoblastic cells(TK6 cells) induced by lead acetate.Methods The TK6 cells were infected with Rad51 silent lentiviral vector to construct Rad51 gene silencing cell,which were confirmed by fluorescence quantitative PCR and Western blot.TK6 cells(control,shRNA-NC and shRNA-Rad51)were exposed to 480 μmol/L lead acetate for 24 hours.The expression of phosphorylated histone H2 AX (γ-H2 AX)and the expression of Rad51,BRCA1 and 53 BP1 proteins in TK6 cells were detected by Western Blot.Results The expression of Rad51 mRNA and Rad51 protein in the shRNA-Rad51 group were significantly lower than those in the Control group and the shRNA-NC group(P < 0.01).The positive rate ofγ-H2 AX in the shRNA-Rad51 group was(27.48 ± 1.66)%,which was higher than that in the Control group(14.77 ±1.21)% and the shRNA-NC group(14.04 ± 1.31)%(P < 0.01).The expression level of BRCA1 protein in the shRNA-Rad51 group was(0.25 ± 0.03),which was lower than that in the Control group(0.55 ± 0.04) and the shRNA-NC group(0.51 ± 0.04)(P < 0.01).The expression level of 53 BP1 was(3.24 ± 0.27)in the shRNA-Rad51 group,which was higher than that in the Control group(2.01 ± 0.19)and the shRNA-NC group(2.11 ± 0.17)(P <0.01).Conclusions After Rad51 gene silencing,HR repair pathway was inhibited and NHEJ repair pathway was activated in TK6 cells,and the sensitivity of TK6 cells to lead genotoxicity was enhanced.