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目的:观察不同浓度双酚A(BPA)对原代培养大鼠睾丸支持细胞(SC)糖代谢及乳酸脱氢酶(LDH)表达的影响,探讨BPA致男性不育的机制。方法:采用两步酶消化法分离雄性Wistar大鼠睾丸SC,建立SC原代培养模型,免疫组化鉴定Fas L。传代后SC随机分为对照组和实验组(100 nmol/L、10μmol/L和1 mmol/L BPA)。培养48 h后,CCK-8法测定细胞增殖活性,核磁共振波谱法测定细胞内代谢物浓度,RT-PCR及Western印迹检测LDH的表达。结果:体外分离培养SC的纯度为(96.05±1.28)%(n=10),CCK-8实验结果显示:与对照组相比,暴露于100 nmol/L BPA组[(98±8)%]、10μmol/L BPA组[(96±3)%]和1 mmol/L BPA组[(95±3)%]的细胞增殖率无明显变化(P>0.05);核磁共振波谱显示:与对照组相比,10μmol/L和1 mmol/L BPA作用下SC内葡萄糖及乳酸浓度明显降低(P<0.05);RT-PCR及Western印迹结果显示:LDH mRNA的表达随BPA浓度升高呈现降低趋势(100 nmol/L、10μmol/L及1 mmol/L组P均<0.05),而LDH蛋白的表达只在1 mmol/L组明显降低(P<0.05)。结论:较高浓度BPA降低LDH,影响SC糖代谢过程。推测BPA通过影响SC糖代谢,减少向生殖细胞乳酸的供给,影响精子发生过程。
Objective: To observe the effects of different concentrations of BPA on the glucose metabolism and lactate dehydrogenase (LDH) expression in primary cultured rat sertoli cells and to explore the mechanism of male infertility induced by BPA. Methods: Two-step enzymatic digestion was used to separate the testis SC of male Wistar rats. SC primary culture model was established and FasL was identified by immunohistochemistry. After passage, SC were randomly divided into control group and experimental group (100 nmol / L, 10 μmol / L and 1 mmol / L BPA). After cultured for 48 h, the cell proliferation activity was determined by CCK-8 assay, the intracellular metabolite concentration was determined by nuclear magnetic resonance spectroscopy, and the expression of LDH was detected by RT-PCR and Western blotting. Results: The purity of SC cultured in vitro was (96.05 ± 1.28)% (n = 10). The CCK-8 results showed that the exposure to 100 nmol / L BPA group was (98 ± 8)% compared with the control group (95 ± 3)%] in 10μmol / L BPA group (96 ± 3%) and 1 mmol / L BPA group (95 ± 3%). The results of nuclear magnetic resonance (P <0.05). The results of RT-PCR and Western blot showed that the expression of LDH mRNA decreased with the increase of BPA concentration (P <0.05) 100 nmol / L, 10 μmol / L, and 1 mmol / L P <0.05). However, the expression of LDH protein was only significantly decreased in 1 mmol / L group (P <0.05). Conclusion: Higher concentration of BPA reduces LDH and affects SC glucose metabolism. It is speculated that BPA affects the metabolism of SC, reduces the supply of lactate to germ cells and affects the process of spermatogenesis.