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目的深入了解PARG的功能及探讨PARG基因沉默在调节六价铬诱导细胞周期改变的作用。方法使用前期构建的PARG基因缺陷细胞(sh PARG细胞)和正常16HBE细胞作为研究对象,进行0、0.3、0.6、1.2、2.5和5.0μmol/L的六价铬[Cr(Ⅵ)]溶液染毒处理24 h,利用免疫荧光法检测PAR的表达、流式细胞仪观察细胞周期、RTPCR分析ATM和P53基因mRNA水平的表达。结果 Cr(Ⅵ)染毒处理后,正常16HBE细胞的S期明显延长,0、0.3、0.6、1.2、2.5和5.0μmol/L剂量组S期细胞比例分别为18.3%、21.6%、26.0%、30.9%、38.8%和43.2%,G2期明显缩短;sh PARG细胞的S期延长,但比例增幅远小于正常16HBE细胞,0、0.3、0.6、1.2、2.5和5.0μmol/L剂量组S期细胞比例分别为17.0%、19.0%、20.1%、21.2%、24.5%和31.3%。正常16HBE细胞内P53基因表达随Cr(Ⅵ)作用剂量的增加而逐渐升高,PARG缺陷细胞内P53基因表达改变不明显(与对照组相比,P>0.05);Cr(Ⅵ)染毒处理后,两种细胞内ATM基因的表达均增加,但正常16HBE细胞内增加更明显,如:5.0μmol/L的Cr(Ⅵ)作用时,正常16HBE细胞内ATM基因的表达升高可达6.67倍(与对照组相比,P<0.01),而sh PARG细胞内ATM基因表达仅升高2.27倍(与对照组相比,P<0.01)。结论 PARG基因沉默可对抗Cr(Ⅵ)诱导的细胞周期时相改变。
Objective To understand the function of PARG and to explore the role of PARG gene silencing in the regulation of hexavalent chromium-induced cell cycle changes. Methods The PARG gene-deficient cells (sh PARG cells) and normal 16HBE cells were used to study the effects of hexavalent chromium [Cr (Ⅵ)] solutions at 0, 0.3, 0.6, 1.2, 2.5 and 5.0 μmol / The expression of PAR was detected by immunofluorescence, the cell cycle was observed by flow cytometry, and the expression of ATM and P53 mRNA was analyzed by RTPCR. Results S phase of normal 16HBE cells was prolonged after exposure to Cr (Ⅵ). The proportion of S phase cells in the 0, 0.3, 0.6, 1.2, 2.5 and 5.0 μmol / L groups was 18.3%, 21.6% and 26.0% 30.9%, 38.8% and 43.2%, respectively. G2 phase was significantly shortened. S phase of sh PARG cells was prolonged but the rate of increase was much smaller than that of normal 16HBE cells. S phase cells of 0, 0.3, 0.6, 1.2, 2.5 and 5.0μmol / The proportions were 17.0%, 19.0%, 20.1%, 21.2%, 24.5% and 31.3% respectively. The expression of P53 gene in normal 16HBE cells increased gradually with the increasing dose of Cr (Ⅵ), while the expression of P53 gene in PARG-deficient cells changed insignificantly (P> 0.05). Cr (Ⅵ) , The expression of ATM gene in both cells increased, but the expression of ATM gene in normal 16HBE cells increased up to 6.67 times when added with 5.0μmol / L Cr (Ⅵ) (P <0.01 compared with the control group), while the expression of ATM gene in sh PARG cells increased by only 2.27 times (P <0.01 compared with the control group). Conclusion PARG gene silencing can antagonize the changes of cell cycle phases induced by Cr (Ⅵ).