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目的观察ATM蛋白激酶-检测点激酶2(ATM-Chk2)通路在氟抑制大鼠曲细精管上皮支持细胞增殖中的作用。方法将18日龄SPF级雄性SD大鼠曲细精管上皮支持细胞分别暴露于含0(对照)、0.25、0.50、1.00、2.00、4.00 mmol/L氟化钠的血清培养基。分别于染毒24、48、72 h,采用MTT法测定支持细胞的增殖情况;于染毒24 h,采用实时荧光定量PCR技术检测支持细胞内ATM、Chk2 m RNA的表达水平。结果与对照组比较,染毒24、48、72 h时,4.00 mmol/L氟化钠染毒组大鼠曲细精管上皮支持细胞的抑制率均较高,差异有统计学意义(P<0.05);且随着氟化钠染毒剂量的升高和染毒时间的延长,氟对曲细精管上皮支持细胞的抑制率均呈上升趋势。与对照组比较,1、2 mmol/L氟化钠染毒组曲细精管上皮支持细胞ATM和Chk2 m RNA的表达水平均升高,而4 mmol/L氟化钠染毒组ATM和Chk2 m RNA的表达水平均降低,差异有统计学意义(P<0.05);且随着氟化钠染毒剂量的升高,曲细精管上皮支持细胞ATM、Chk2 m RNA的表达水平均呈下降趋势。结论不同剂量氟抑制生殖细胞增殖可能是通过干扰曲细精管上皮支持细胞中DNA损伤检验点ATM、Chk2的表达而实现的。
Objective To investigate the role of ATM protein kinase-checkpoint kinase 2 (ATM-Chk2) pathway in the inhibition of proliferation of rat seminiferous tubule epithelial cells by fluoride. Methods Sertoli cells of 18-day-old SPF male SD rats were exposed to serum containing 0 (control), 0.25, 0.50, 1.00, 2.00 and 4.00 mmol / L sodium fluoride respectively. The proliferation of support cells was measured by MTT assay at 24, 48 and 72 h after exposure. The expression of ATM and Chk2 m RNA in supportive cells was detected by real-time fluorescence quantitative PCR at 24 h after exposure. Results Compared with the control group, the inhibitory rate of seminiferous tubule epithelial cells in 4.00 mmol / L sodium fluoride exposure group was higher at 24, 48 and 72 h (P < 0.05). With the increase of the dose of sodium fluoride and the prolongation of exposure time, the inhibitory rate of fluoride on the seminiferous tubule epithelial cells showed an upward trend. Compared with the control group, the expression levels of ATM and Chk2 m RNA in the seminiferous tubules of 1,2 mmol / L sodium fluoride exposure group were increased, while those in the 4 mmol / L sodium fluoride exposure group were significantly higher than that of the Chk2 m (P <0.05). And with the increase of sodium fluoride dose, the expression of ATM and Chk2 m RNA in seminiferous tubule epithelial cells showed a decreasing trend . Conclusions Different doses of fluoride inhibit germ cell proliferation may be through interfering with the expression of ATM, Chk2 in DNA damage checkpoint of seminiferous tubule epithelial cells.