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目的:研究大鼠睾丸间质细胞中GnRH激动剂(GnRHa)和拮抗剂(GnRHant)对促分裂原活化蛋白激酶(MAPK)途径的影响。方法:原代培养大鼠睾丸间质细胞24h后,血清饥饿2h。GnRHa(10-7mol/L)或Gn-RHant(10-6mol/L)处理大鼠睾丸间质细胞不同时间(0、5、15、30、60、90min)后,Western印迹检测磷酸化ERK(p-ERK)和磷酸化p38(p-p38)蛋白水平以0min组为对照。此外,不同终浓度(1、5、10、20μmol/L)的PKC抑制剂GF109203X和GnRHa或GnRHant共同作用后,Western印迹检测p-ERK蛋白水平。结果:GnRHa或GnRHant刺激间质细胞不同时间后,p-p38蛋白水平与对照组比较均无显著性差异(P>0.05);加入不同终浓度的PKC抑制剂GF109203X处理细胞20min后,再用GnRHa刺激细胞10min,发现GF109203X在终浓度为10μmol/L和20μmol/L时p-ERK水平显著下降(P<0.05)。GnRHant刺激间质细胞后,p-ERK水平在15min时升高了约65%(P<0.05);30min时升高了约81%,达到最高水平(P<0.05);60min时开始下降,90min时恢复到基础水平。不同浓度GF109203X处理细胞20min后,再用GnRHant刺激细胞30min,p-ERK水平与对照组比较无显著性差异(P>0.05)。结论:GnRHa可能通过PKC途径来诱导ERKMAPK信号通路的活化,而GnRHant对ERKMAPK信号通路的激活作用并非通过PKC途径来实现的;p38可能不参与间质细胞中GnRH类似物作用的MAPK分子信号通路。
AIM: To investigate the effects of GnRHa and GnRHant on the mitogen-activated protein kinase (MAPK) pathway in rat testicular stromal cells. Methods: After primary culture of rat testicular stromal cells for 24 h, serum was starved for 2 h. The rat testicular stromal cells were treated with GnRHa (10-7mol / L) or Gn-RHant (10-6mol / L) for different time (0,5,15,30,60,90min) p-ERK) and phosphorylated p38 (p-p38) protein levels in the 0min group as a control. In addition, PK-inhibitor PK109203X at different final concentrations (1, 5, 10 and 20 μmol / L), together with GnRHa or GnRHant, detected p-ERK protein levels by Western blotting. RESULTS: After stimulated with GnRHa or GnRHant for different time, the level of p-p38 protein had no significant difference compared with the control group (P> 0.05). After the cells were treated with different concentrations of GF109203X, the cells were treated with GnRHa Stimulation of cells 10min, found GF109203X at a final concentration of 10μmol / L and 20μmol / L p-ERK levels were significantly decreased (P <0.05). After GnRHant stimulated mesenchymal cells, the level of p-ERK increased by about 65% (P <0.05) at 15 min and increased by about 81% at 30 min, reaching the highest level (P <0.05) When restored to the basic level. After treated with different concentrations of GF109203X for 20min, the cells were stimulated with GnRHant for 30min. There was no significant difference in the level of p-ERK between the two groups (P> 0.05). CONCLUSION: GnRHa may induce the activation of ERKMAPK signaling pathway through PKC pathway, but GnRHant may not activate PKC pathway through PKC pathway. P38 may not participate in MAPK signaling pathway of GnRH analog in stromal cells.