乙酸铅染毒对人淋巴母细胞DNA双链断裂损伤及修复的研究

Study on damage and repair of DNA double-strand breaks induced by lead acetate in TK6 cells

目的 研究乙酸铅染毒致人淋巴母细胞(TK6细胞)DNA双链断裂损伤及细胞的DNA损伤修复作用与机制。方法 将TK6细胞与不同浓度(0μmol/L、120μmol/L、240μmol/L、480μmol/L)的Pb(Ac)2孵育24 h,检测γ-H2AX蛋白、细胞增殖、细胞周期和细胞凋亡变化情况,以及DNA双链断裂损伤修复蛋白RAD51、BRCA1、53BP1的表达情况。结果 与阴性对照组比较,随着乙酸铅染毒浓度的升高,TK6细胞γ-H2AX阳性率和γ-H2AX蛋白表达水平均显著升高(F值分别为138.34、289.89,P均<0.01);TK6细胞的增殖率降低(F=43.04,P<0.01);G1期的细胞比例出现升高趋势(F=4.19,P<0.05),S期的细胞比例出现降低趋势(F=81.94,P<0.01),G2期的细胞比例则无明显变化(F=1.73,P>0.05);TK6细胞的凋亡率升高(F=77.44,P<0.01),TK6细胞RAD51蛋白先升高后降低,BRCA1蛋白明显降低,53BP1蛋白明显升高(F值分别为263.16、69.76、86.17,P均<0.01)。结论 乙酸铅染毒可诱导TK6细胞的DNA双链断裂,抑制细胞增殖,阻滞细胞周期停滞于G1期,促进细胞凋亡。乙酸铅染毒抑制TK6细胞的HR修复通路,激活NHEJ修复通路,修复过程倾向于NHEJ修复。

Objective To study the DNA double-strand breaks and DNA damage repair and mechanism of TK6 cells induced by lead acetate.Methods TK6 cells were incubated with Pb(Ac)2of different concentrations (0μmol/L,120μmol/L,240μmol/L,480μmol/L) for 24 h.The changes of γ-H2AX protein,cell proliferation,cell cycle and apoptosis,the protein expression of RAD51,BRCA1 and 53BP1 were detected.Results Through cmoparing with the negative control group,with the increase of lead acetate concentration,the positive rate of γ-H2AX and the expression level of γ-H2AX protein in TK6 cells were significantly increased (F=138.34,289.89,P<0.01).The cell proliferation rate was significantly decreased(F=43.04,P<0.01).The proportion of G1 phase cells was significantly increased (F=4.19,P<0.05).The percentage of cells in S phase was significantly decreased (F=81.94,P<0.01).The percentage of cells in G2 phase did not change significantly (F=1.73,P>0.05).The percentage of cells apoptosis increased significantly(F=77.44,P<0.01).The results of western blot showed that the RAD51 protein,BRCA1 protein and 53BP1 protein in TK6 cells were significantly increased(F=263.16,69.76,86.17,P<0.01).Conclusion Lead acetate cas induce DNA double-strand breaks in TK6 cells,which inhibit cell proliferation,arrest cell cycle and promote cell apoptosis.Lead acetate inhibits HR repair pathway,activates NHEJ repair pathway in TK6 cells.The repair process of DNA double-strand breaks tends to be NHEJ repair.