氟咯草酮诱导共培养大鼠睾丸支持-生精细胞凋亡及其可能机制

Potential Mechanism of Co-cultured Sertolli-Germ Cells Apoptosis Induced by Fluorochloridone

[目的]观察氟咯草酮(FLC)对共培养大鼠睾丸支持-生精细胞的影响,探究可能的机制。[方法]采用0.1%胶原酶和0.1%透明质酸酶两步酶消化法,分离得到原代睾丸支持细胞和生精细胞,共培养24 h后,分别用0.01、0.10、1.00μmol/L的FLC染毒24 h。生精细胞脱落试验检测FLC对生精细胞脱落的影响;流式细胞仪检测细胞凋亡率;实时荧光定量-聚合酶链反应检测凋亡通路关键信号分子和p38 MAPK的m RNA表达。[结果]FLC染毒可引起支持-生精细胞共培养体系中生精细胞脱落增加,在0.01、0.10和1.00μmol/L浓度组,生精细胞脱落百分比分别为(238.17±3.78)%,(265.89±9.51)%和(308.73±17.05)%。随着FLC浓度升高,早期凋亡率分别升高至(10.80±1.86)%,(13.52±0.72)%和(16.62±0.35)%;与对照组相比,差异均有统计学意义(P<0.05或P<0.001)。在0.10和1.00μmol/L浓度下,FLC染毒可明显上调凋亡线粒体通路中Bax、Cyt-c、Caspase-9和Caspase-3的m RNA表达(P<0.05或P<0.001),同时下调Bcl-2的m RNA表达(P<0.05);1.00μmol/L的FLC可引起Fas L与p38 MAPK的m RNA表达升高(P<0.05)。[结论]FLC染毒可影响大鼠睾丸支持-生精细胞共培养体系中凋亡通路相关信号分子的m RNA表达,p38 MAPK信号通路可能参与了FLC诱导的细胞凋亡。

［Objective］ To observe the effects and explore potential mechanism of fluorochloridone （FLC） on co-cultured sertolli-germ cells. ［Methods］ Primary sertolli-germ cells were isolated after two-step enzyme digestion using 0.1% collagenase and 0.1% hyaluronidase from rat testes and co-cultured for 24 h. The co-cultured cells were treated with 0.01, 0.10, and 1.00 μmol/L FLC for another 24 h. Detached germ cells induced by FLC was detected by detached germ cell trial. Cell apoptosis was detected by flow cytometry. Real-time polymerase chain reaction was used to measure the mRNA expression of apoptosis related pathway key genes and p38 MAPK. ［Results］ FLC exposure caused dose-dependent increases of the percentages of detached germ cells up to （238.17±3.78）%, （265.89±9.51）%, and （308.73±17.05）% in the sertolli-germ cell co-cultured systems treated with 0.01, 0.10, and 1.00 μmol/L FLC, respectively. The proportions of early apoptotic cells in above three groups were significantly raised to （10.80±1.86）%, （13.52±0.72）%, and （16.62±0.35）%, respectively, and there were statistical differences compared with the control group （P 〈 0.05 or P 〈 0.001）. Up-regulation in the expressions of mitochondrial apoptotic pathway related genes including Bax, Cyt-c, Caspase-9, and Caspase-3 were observed at FLC concentrations of 0.10 and 1.00 μmol/L （P 〈 0.05 or P 〈 0.001）, while the levels of Bcl-2 mRNA expression were decreased （P 〈 0.05）. FLC also raised FasL and p38 MAPK mRNA expression levels at the concentrations of 1.00 μmol/L （P 〈 0.05）. ［Conclusion］ FLC exposure might affect the mRNA expressions of apoptosis related pathway key genes, and the activation of p38 MAPK signaling pathway is probably involved in FLC induced sertolli-germ cell apoptosis.