苯并(a)芘对大鼠睾丸支持细胞连接蛋白基因表达的影响

Effects of Benzo(a)pyrene on mRNA Expression of Junction Protein Genes in Primary Cultured Sertoli Cells

[目的]探讨苯并(a)芘(BaP)对大鼠原代培养睾丸支持细胞的连接蛋白(connexin 43、occludin、ZO-1、N-cadherin)基因mRNA表达的影响及可能机制。[方法]取出生21d SD大鼠睾丸组织,分离支持细胞,细胞分离后,均按照1.5×106个/mL浓度接种于细胞培养板中。培养48h后,以0、1、10、50、100μmol/L浓度的BaP同时染毒于原代培养的大鼠支持细胞12h,每个剂量设置6个平行孔。检测支持细胞活力、凋亡蛋白caspase-3活性,以及连接蛋白基因的mRNA表达。[结果]在BaP 50μmol/L时,支持细胞活力降低(和对照组相比,P<0.01);在BaP 10μmol/L时caspase-3活性升高,连接蛋白connexin 43、occludin、和ZO-1基因的mRNA表达降低(和对照组相比,P<0.05)。在BaP 100μmol/L时连接蛋白N-cadherin基因的mRNA表达降低(和对照组相比,P<0.05)。加入caspase-3抑制剂后,BaP所诱导的连接蛋白基因表达下降,且随caspase-3活性变化而发生改变。[结论]BaP能诱导大鼠支持细胞连接蛋白mRNA表达下降,BaP诱导的caspase-3活性上升是调节连接蛋白mRNA表达下降的可能机制。

[ Objective ] To investigate the effect of benzo（a）pyrene （BaP） on mRNA expression of junction protein genes in sertoli cells. [ Methods ] Sertoli cells were isolated from 21-day-old male rats and incubated at a density of 1.5×0^6 cells/mL. After incubation for 48 h, the sertoli cells were exposed to varied concentrations of BaP （0, 1, 10, 50, and 100μmol/L） for 12 h. Every group contained 6 samples. Cell viability, caspase-3 activity, and mRNA expression levels of junction protein genes were measured. [ Results ] The cell viability was significantly decreased by the BaP treatment at 50μmol/L compared with the control group （P〈 0.01）. The caspase-3 activity was increased and the mRNA expression levels of connexin 43, occludin, and ZO-1 were decreased after BaP exposure at 10μmol/L compared with the control group （P 〈 0.05）. A down-regulated mRNA expression level of N-cadherin was observed after BaP administration at 100μmol/L compared with control group （P〈 0.05）. The mRNA expression levels of junction protein genes were changed along with the alteration of caspase-3 activity after caspase-3 inhibitor was added. [ Conclusion ] BaP exposure could decrease mRNA expression levels of junction protein genes in primary sertoli cells, in which an up-regulated caspase-3 activity by BaP interference may play an important role.