芹菜素对非洲爪蟾卵母细胞体外成熟的影响及其作用机制

摘要

目的：探讨不同浓度的芹菜素对非洲爪蟾卵母细胞体外成熟的影响及作用机制。方法：将健康的非洲爪蟾卵母细胞移入含不同浓度(0、20、80、160和500μmol/L)的芹菜素培养液中培养，另设阳性对照(孕酮5μg/mL)组。每隔1 h观察生发泡破裂(germinal vesicle breakdown，GVBD)发生情况、并记录发生GVBD的细胞数目；同时用10%的三氯乙酸固定，观察细胞核的变化；采用荧光染色法在激光共聚焦显微镜下观察细胞染色体的变化。收集上述各浓度芹菜素处理8 h后的卵母细胞，采用Western blot法检测卵母细胞成熟相关蛋白p-Erk1、Mos和CyclinB2的表达。结果：与阴性对照组相比，当芹菜素浓度为80~500μmol/L且作用时间在4~8 h时，卵母细胞GVBD发生率均明显升高(P均<0.05)。在80~500μmol/L芹菜素处理细胞8 h后，p-Erk1、Mos和CyclinB2蛋白表达量明显高于阴性对照组(P<0.05)。结论：芹菜素在卵母细胞体外培养过程中促进了第1次减数分裂的开启，其机制可能与MPF和MAPK信号通路的活化有关。%OBJECTIVE: To explore the effect and mechanism of different doses of apigenin on in vitro maturation of Xenopus oocytes. METHODS:Healthy Xenopus oocytes were cultured in different doses (20,80,160 and 500μmol/L) of apigenin. Meanwhile negative and positive control groups were set. Then we observed and recorded the rate of germinal vesicle breakdown (GVBD) per hour,confirmed the transformation of cell nucleus and chromosome by means of trichloroacetic acid fixation and fluorescence staining. Finally oocytes were collected at 8 h in each group,and the expressions of p-Erk1,Mos and CyclinB2 were detected by Western blot. RESULTS:Compared with negative control group,GVBD rates were all highly increased when apigenin-treated dose were 80,160 and 500μmol/L,and time were 4-8 h (P<0.05). The expressions of p-Erk1,Mos and CyclinB2 were all markedly increased when the dose of apigenin were between 80 and 500μmol/L (P<0.05). CONCLUSION:Apigenin may play an important role in promoting the maturation of Xenopus oocytes in vitro through regulation of mature promoting factor (MPF) and mitogen-activated protein kinase (MAKP).