Research On Local Regulation Mechanism Of ShRNA Interference Amh In Granulosa Cells Of Mouse In Vitro

Anti-Müllerian hormone(AMH), also referred to as Müllerian inhibiting substance(MIS), is a dimeric glycoprotein connected by two 72 k Da monomeric composed of disulfide, is a member of the transforming growth factor-β super family, which plays an important role in both ovarian primordial follicle recruitment and dominant follicle selection in mice. Amh is produced by the granulosa cells of preantral and small antral follicles. The family including transforming growth factor-β、Activin-Inhibin-Follistain、growth differentiation factor-9 et al. All of the memebers are dimeric glycoprotein, are related to regulate tissue growth and differentiation. It has been confirmed by transforming growth factor-β(TGF-β) superfamily/Smad signaling pathway is an important way of regulating follicular development. In ovary, Anti-Müllerian hormone is produced by the granulosa cells of follicles. Known anti-Mullerian hormone involved in mammalian follicular development, through self/paracrine regulation of granulosa cell proliferation and differentiation. In order to study the anti-Mullerian hormone regulation function of granulosa cells in mice, This research using mice as experimental models to constructed anti-mullerian hormone RNAi interference vector p Silencer4.1-CMV-AMH, Interference of anti-mullerian duct hormone expression in the granular cells, m RNA and protein levels were detected in the expression of anti-Mullerian hormone found in the interference of anti-Mullerian hormone, the following changes occurred in the granulosa cells: 1) Constructed by Western blot analysis of the three anti-Mullerian hormone RNAi interference vectors, the three vectors were significantly down-regulated the expression of anti-Mullerian hormone, which interfere with the effect of interference vector psh-AMH-2 of the best. 2) The cell cycle was verified by flow cytometry. After the anti-Mullerian hormone disturbances in mouse granulosa cells, Find more cell cycle arrest in G1 phase. p21 in cell cycle regulation at the m RNA and protein levels were significantly upregulated.3) Cell apoptosis was detected by flow cytometry and found significantly increased early apoptotic and late apoptotic cells. By Real time PCR and Western blot analysis of pro-apoptotic expression of Bcl-2, Bcl-2 expression was significantly reduced. 4) CCK-8 assay proliferation of granulosa cells, interfering significantly decreased the proliferation of granule cells after anti-Mullerian hormone. 5) To assess an individual’s ovarian reserve, Enzyme-linked immunosorbent assay detected the concentrations of inhibin B and estradiol(E2), inhibin B significantly reduced and estradiol significantly reduced. 6) Real time PCR detected expression of TGF-β superfamily after interference in granulosa cells, include inhibin subunits(inhibin-a 、 inhibin-ba 、 inhibin-bb) 、follistatin、anti-Mullerian hormone receptorⅡ、growth differentiation factor-9. The expression of GDF-9 increased and the others were significantly reduced.