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目的通过检测孕酮合成相关酶的基因表达水平探讨重金属铅对睾丸间质瘤细胞的毒理作用,为揭示重金属导致男性性功能低下的毒性作用机制提供实验依据。方法采用睾丸间质细胞的肿瘤细胞株R2C作为受试对象,暴露于含重金属铅的无血清培养环境,在1、3、6、12和24 h测定细胞活性,收集细胞上清液,利用放射免疫法,检测孕酮合成的变化;应用RT-RCR的方法观察孕酮合成相关酶在基因水平上的表达差异。结果通过CCK-8检测发现,100μmol/L重金属铅作用于R2C细胞12和24 h后,细胞存活率未显著降低(P>0.05);放射免疫检测各个时间点孕酮的变化发现,12和24 h能够显著降低孕酮的合成(P<0.05)。细胞暴露于含铅培养基后,尽管在作用12和24 h后类固醇激素合成酶基因水平的表达都基本恢复到正常水平,但是在铅暴露3 h后,St AR、P450scc基因的表达显著降低(P<0.05),6 h后St AR基因的表达降低更明显(P<0.01)。结论重金属铅能够抑制R2C细胞合成孕酮的功能,主要通过下调孕酮合成通路中胆固醇转运酶St AR和限速酶P450scc的基因表达水平发挥作用。
Objective To investigate the toxic effects of heavy metal lead on testicular stromal tumor cells by detecting the gene expression level of enzymes involved in progesterone synthesis, providing experimental evidence for revealing the toxic mechanism of heavy metal induced male sexual dysfunction. Methods The tumor cell line R2C of testicular stromal cells was exposed to serum-free culture environment containing heavy metal lead. Cell viability was measured at 1, 3, 6, 12 and 24 h. Cell supernatants were harvested by radioactive Immunoassay was used to detect the changes of progesterone synthesis. RT-RCR method was used to observe the gene expression differences of progesterone synthesis related enzymes. Results CCK-8 assay showed that cell viabilities of R2C cells were not significantly decreased after exposure to 100μmol / L lead for 12 and 24 h (P> 0.05). The changes of progesterone at different time points were detected by radioimmunoassay. h can significantly reduce the progesterone synthesis (P <0.05). After exposing cells to lead-containing medium, the gene expression of St AR and P450scc was significantly decreased 3 h after exposure to lead (P <0.05), although the expression of steroid hormone synthetase gene returned to normal level at 12 and 24 h P <0.05). After 6 h, the expression of St AR gene decreased more obviously (P <0.01). Conclusion Heavy metal lead can inhibit the function of progesterone synthesis in R2C cells and mainly through down-regulating the gene expression of cholesterol transport enzyme St AR and rate-limiting enzyme P450scc in the progesterone synthesis pathway.