食欲素A对绵羊卵巢颗粒细胞分泌孕酮以及cAMP含量的影响

Orexin A influence progesterone secreted and the content of cAMP in sheep ovarian granulosa cells

为了解食欲素A对绵羊卵巢颗粒细胞分泌孕酮以及cAMP浓度的影响,通过食欲素A刺激体外培养的绵羊卵巢颗粒细胞,采用HE染色和免疫荧光观察促卵泡激素受体(folicle-stimulating hormone receptor,FSHR)蛋白表达的方法进行鉴定,并经黄体化处理,添加不同浓度(1 nmol/L,10 nmol/L,58 nmol/L,100 nmol/L或145 nmol/L)的食欲素A分别处理0、24、48、72 h收取细胞上清,运用ELISA方法检测细胞培养上清液孕酮浓度。结果表明,HE染色后镜下可见梭形或不规则星形的卵巢颗粒细胞,细胞核呈深蓝色,胞浆呈淡粉色。免疫荧光结果显示FSHR呈阳性表达,细胞纯度达90%以上。绵羊卵巢黄体化颗粒细胞添加食欲素A,同一时间孕酮分泌量随食欲素浓度的增加呈逐渐升高然后下降的趋势,且添加浓度在10 nmol/L时孕酮分泌量达到最高;添加同一浓度的食欲素A,随着作用时间的延长孕酮分泌量呈逐渐升高然后下降的趋势,且时间在24 h时分泌量达到最高。在进一步的研究中,体外培养绵羊卵巢颗粒细胞,经黄体化处理,添加不同浓度的食欲素A受体阻断剂(HY-10805A)(10 nmol/L,100 nmol/L,1 mol/L)孵育2 h,添加不同浓度(10nmol/L,58 nmol/L)的食欲素A孵育24 h收取细胞上清,运用ELISA方法检测细胞裂解上清中cAMP浓度变化。结果表明:添加食欲素受体阻断剂组与对照组相比c AMP的浓度显著降低(P<0.01)。以上结果说明绵羊卵巢颗粒细胞分泌孕酮受食欲素A的浓度及时间变化影响,并且能够影响细胞中cAMP的含量。

To observe Orexin A influences on sheep ovary granulosa cells secretion of progesterone and the possible mechanisms,sheep ovary granulosa cells were cultured in vitro,and HE and immunohistoflu or-escence staining were used for ovarian granulose cell identification.The experiment group was added with different concentration（1 nmol/L,10 nmol/L,58 nmol/L,100 nmol/L,145 nmol/L）of Orexin A,respectively,pre-incubation for different time（0 h,24 h,48 h,72 h）,and serum P4 levels were measured with an enzyme-linked immunosorbent assay（ELISA）.As different time points progesterone secretion increased,then decreased gradually.The results indicated that gran ulo se cell structure was complete,and the edge was clear,like a triangle cone or irregular star.The cytoplasm was pink,and the nucleus were dyed in blue.The positive rate was more than 90% compared with control group.The sheep ovarian granulosa cells in vitro cultured were more than 90% purity.10 nmol/L subgroups secretion P4 levels were significantly different than other subgroups at the same time（P0.0001）,and a significant rise was observed at 24 h.In the further research,sheep ovary granulosa cells were cultured in vitro and luteinized by FSH,LH and E2.The content of c AMP was measured by ELISA.Compared with added Orexin A groups,the c AMP production significantly reduced in the group treated with Orexin A receptor antagonist.In other words,sheep ovarian granulosa cell secretion of progesterone is affected by Orexin A concentration and time,and can influence the content of cAMP in the sheep ovary granulosa cells.