p,p'-DDE,β-BHC单独及其联合对睾丸Sertoli细胞JNK MAPK信号转导通路机制的研究

目的:研究2,2-双-(对氯苯基)-1,1-二氯乙烯(p,p'-DDE)和β-六氯化苯(β-BHC)单独作用及联合作用对离体培养大鼠睾丸Sertoli细胞c-jun氨基末端激酶(JNK)丝裂原活化蛋白激酶(MAPK)信号转导通路的作用机制。方法:从大鼠睾丸组织中分离Sertoli细胞进行离体原代培养2d,设溶剂(DMSO)为对照组,分别以终浓度为10、30、50μmol/L的p,p'-DDE,β-BHC及二者联合染毒Sertoli细胞24h,采用二步RT-PCR法检测Sertoli细胞JNK、c-junmRNA的表达水平。结果:①染毒24h后,对照组及10、30、50μmol/Lp,p'-DDE组Sertoli细胞JNKmRNA灰度比值分别为0.068±0.001、0.164±0.002、0.207±0.006、0.499±0.017;c-junmRNA灰度比值分别为0.122±0.002、0.157±0.006、0.218±0.007、0.289±0.004,随着p,p'-DDE剂量的增加,JNK、c-junmRNA的表达水平均逐渐升高,且10、30、50μmol/L组与对照组差异存在显著性(P<0.05)。β-BHC及联合染毒各组随着染毒剂量的增大,Sertoli细胞JNK、c-junmRNA表达水平也显著增加,且呈剂量依赖性关系(P<0.05)。②p,p'-DDE、β-BHC二者联合作用与单独作用相比,10μmol/L的p,p'-DDE,β-BHC及二者联合染毒组,JNKmRNA灰度比值分别为0.164±0.002、0.149±0.003、0.178±0.004;c-junmRNA灰度比值分别为0.157±0.006、0.131±0.004、0.172±0.002,联合染毒组JNK、c-junmRNA灰度比值均显著高于两者单独作用且存在显著性差异(JNK:P<0.05;c-jun:P<0.01);30、50μmol/L染毒剂量组JNK、c-junmRNA表达水平联合染毒组也显著高于单独染毒组(JNK:P<0.05,c-jun:P<0.01),而各DMSO对照组之间mRNA表达水平无显著性差异(P>0.05)。结论:p,p'-DDE、β-BHC及二者联合作用于睾丸Sertoli细胞后,JNK及其下游的c-jun基因转录水平均明显升高,且联合作用比单独作用更加明显。

Action Mechanism of p,p-DDE and/or β-BHC on JNK and MAPK Signal Transduction Pathways in Rat Sertoli Cells

OBJECTIVE: To study the action mechanism of p,p'-DDE and/or beta-BHC on JNK and MAPK signal transduction pathways in rat Sertoli cells in vitro. METHODS: We cultured the Sertoli cells isolated from rat testicular tissues for 2 days in vitro, divided them into a control group incubated with DMSO and 3 case groups exposed to p,p'-DDE and / or beta-BHC at the final concentration of 10, 30, 50 micromol/L for 24 hours, and then detected the expression levels of JNK and c-jun mRNA by two-step RT-PCR. Results: Twenty-four hours after p,p'-DDE treatment, the grayscale values of JNK mRNA were 0.068 +/- 0.001, 0.164 +/- 0.002, 0.207 +/- 0.006 and 0.499 +/- 0.017, and those of c-jun mRNA were 0.122 +/- 0.002, 0.157 +/- 0.006, 0.218 +/- 0.007 and 0.289 +/- 0.004 respectively in the DMSO control and the 10, 30 and 50 micromol/L groups. The expressions of JNK and c-jun mRNA were elevated with increased concentration of p,p'-DDE, with significant differences between the control and the case groups (P < 0.05), and they were also significantly upregulated in the beta-BHC and p,p'-DDE + beta-BHC groups in a dose-dependent manner. The grayscale values of JNK mRNA in the p,p'-DDE, beta-BHC and p,p'-DDE + beta-BHC groups at the concentration of 10 micromol/L were 0.164 +/- 0.002, 0.149 +/- 0.003 and 0.178 +/- 0.004, and those of c-jun mRNA were 0.157 +/- 0.006, 0.131 +/- 0.004 and 0.172 +/- 0.002, respectively, both significantly higher in the combination group than in the former two (P < 0.05). And the same was the case with the 30 and 50 micromol/L concentrations. Conclusion: Both p,p'-DDE and beta-BHC can enhance the expressions of JNK and c-jun in Sertoli cells, and their combination can produce even more obvious effect.