p38MAPK抑制剂SB202190对猪颗粒细胞活力的影响

Effect of p38MAPK-SB202190 Inhibitor on the Activity of Porcine Granulosa Cells

p38MAPK是丝裂原活化蛋白激酶(Mitogen-activated potein kinase,MAPK)家族成员,是细胞内重要的信号转导系统之一,对细胞的生长、分化、凋亡具有重要影响及作用。为探究p38MAPK信号对猪卵巢颗粒细胞增殖和凋亡的影响及其可能作用机制,研究以体外培养的猪卵巢颗粒细胞为模型,在培养液中分别添加浓度为0、1、10、20μmol/L的p38MAPK特异性抑制剂SB202190。以免疫细胞化学染色法鉴定体外培养猪卵巢颗粒细胞,通过CCK-8法检测其增殖活性,应用Annwxin V-FITC/PI及细胞周期试剂盒染色检测细胞凋亡及细胞周期状况,以实时定量PCR检测猪卵巢颗粒细胞中凋亡、周期相关基因的相对表达水平。结果表明,添加p38APK信号抑制剂SB202190可抑制体外培养猪卵巢颗粒细胞的增殖活性,20μmol/L处理组与其他组相比呈显著性差异(P<0.05);20μmol/L SB202190添加处理未显著影响颗粒细胞的凋亡水平,而该浓度SB202190可将大部分细胞阻滞在G1/G0期,与对照组相比差异显著(P<0.05);基因相对表达水平检测显示SB202190添加可抑制Fas、FasL、CDK-4的表达,而同时促进Bcl-2、Bax、P27的表达(P<0.05),对Caspase-3的表达没显著影响(P>0.05)。综上所述,SB202190通过调控猪卵巢颗粒细胞的周期来影响其增殖作用,但对其凋亡没有影响。

p38MAPK is a member of Mitogen-activated potein kinase （MAPK）, which is one of the im- portant intracellular signaling pathways and it plays important roles in cell growth, differentiation and ap- optosis. To explore the possible role of p38MAPK on the proliferation and apoptosis of porcine granulosa cells, the porcine ovarian granulosa cells were used as in vitro model in this study, and different concentra- tions（0,1, 10, 20 μmol/L） of SB202190 which is the specific inhibitor of p38MAPK were added into the media of in vitro cultured porcine granulosa cells. The granulosa cells cultured in vitro were identified via immunocytochemical staining,the viability of porcine granulosa cells was assessed by CCK-8 assay, the cell apoptosis and cell cycle retardation were determined by Annexin V-FITC/PI staining and cell cycle staining Kit, the relative mRNA levels of apoptotic-related and cell cycle related genes were detected by real-time qPCR. The results showed that the addition of SB202190 inhibited the growth activity of granulosa cell and the concentration of 20 μM was significant to other groups（P〈0.05）. However, it could not accelerate the apoptosis, and most cells were retarded at G1/G0 phase with significant difference between two groups （P〈0.05）. qPCR results showed that after SB202190 was added, the expression of Fas, FasL and CDK- 4 were suppressed, and the expression of Bax, Bcl, P27 were promoted（P〈0.05）, with no effect on the expression of Caspase-3（P〈0 05）. In conclusion, SB202190 could affect the cell growth of ovarian granu- losa cells by regulating the cell cycle, with no effect on cell apoptosis.