| Objective:1. Rat ovarian granulosa cells in this experiment in vitro culture as the carrier, the effects of BSHX containing serum on cell ActA/Smads signal transduction pathway in rat ovarian granulosa, and determine their related targets, clarify its promote micro mechanism of follicular development, so as to lay the experimental research on the foundation for the new drug of tonifying kidney Chinese medicine research gui.Methods:1. Ovarian granulosa cells cultured in vitro grew well,12h grow pseudopodia,3-4d entered the proliferation stage, about14d cell apoptosis significantly, FSHR immunohistochemical staining:the nucleus was blue purple, pulp brown yellow, and the positive rate of more than95%, HE staining:nuclei was dark blue, cytoplasm is pink;2.Randomly divided into8groups, each group had12holes, i.e.1%,5%,10%,15%,20%different concentrations of serum containing medium group (dose group containing serum in FSH25ng/ml,10%), fetal bovine serum medium group, serum free medium group,100ul/hole, OD values after48h to determine the most suitable concentration, E2and P secretion of the other measured under different concentrations of supernatants were collected after48h;3. Were randomly divided into6groups, namely:15%BSHX serum containing high, medium and low dose group,15%normal saline containing serum group,25ng/mlFSH group, serum containing and serum-free DMEN/F12medium respectively in the blank group, lh,2h, 6h,12h,24h,48h after the ELISA method for the determination of ActA secretion and SP immunohistochemistry method for determination of ActR â…¡ A, Smad2, Smad3expression.Results:1.Ovarian granulosa cells cultured in vitro grew well,12h grow pseudopodia,3-4d entered the proliferation stage, about14d cell apoptosis significantly, FSHR immunohistochemical staining:the nucleus was blue purple, pulp brown yellow, and the positive rate of more than95%, HE staining:nuclei was dark blue, cytoplasm is pink;2.he serum containing BSHX optimal concentration of15%, with statistical significance, the differences between each group p<0.05;48h E2, serum15%BSHX secretion is most obvious, there is statistical significance, the differences between each group p<0.05; the secretion of P showed no significant change, p>0.05;3.ActA secretion and ActR â…¡ ASmad2, the expression level of both Smad3and time exist certain dependence, the peak of each BSHX containing serum group, after12h, the optimal dose for middle dose group.Conclusion:BSHX serum can promote the proliferation and differentiation of ovarian granulosa cells cultured in vitro, promote the secretion of the E2. To the effect of ActA/Smads signaling conduction through the secretion and upregulation of ActR â…¡ A, Smad2, Smad3, promote the expression of ActA. |
