【摘要】：抗苗勒管激素(Anti-Müllerian hormone,AMH)是由兩個72kDa的單體通過二硫鍵連接構(gòu)成的二聚體糖蛋白。AMH/MIS屬于轉(zhuǎn)化生長因子-β(TGF-β)超家族,該家族包括TGF-β、激活素-抑制素-卵泡抑素(ACT-INH-FST)系統(tǒng)和生長分化因子-9(GDF-9)等。這個家族所有的成員都是二聚體糖蛋白,都與調(diào)節(jié)組織生長與分化有關(guān)�，F(xiàn)已證實轉(zhuǎn)化生長因子-β(TGF-β)超家族/Smad信號轉(zhuǎn)導(dǎo)通路是調(diào)控卵泡發(fā)育的重要途徑。在卵巢組織中,抗苗勒管激素表達(dá)于卵泡的顆粒細(xì)胞。已知抗苗勒管激素參與哺乳動物卵泡發(fā)育,通過自/旁分泌調(diào)節(jié)顆粒細(xì)胞的增殖分化,為了研究抗苗勒管激素對小鼠卵巢顆粒細(xì)胞的調(diào)控作用,本課題針對小鼠基因序列設(shè)計干擾片段,構(gòu)建了抗苗勒管激素RNAi干擾載體pSilencer4.1-CMV-AMH,干擾顆粒細(xì)胞中抗苗勒管激素的表達(dá),分別在mRNA和蛋白水平檢測抗苗勒管激素的表達(dá)情況,發(fā)現(xiàn)在干擾抗苗勒管激素后,顆粒細(xì)胞內(nèi)發(fā)生如下變化:1)通過Western blot檢測構(gòu)建的三個抗苗勒管激素RNAi干擾載體,三個載體都顯著下調(diào)抗苗勒管激素的蛋白表達(dá),其中干擾載體psh-AMH-2的干擾效果最好;2)流式細(xì)胞儀檢測小鼠顆粒細(xì)胞干擾抗苗勒管激素后的細(xì)胞周期,發(fā)現(xiàn)較多的細(xì)胞阻滯在G1期。調(diào)控細(xì)胞周期的p21在mRNA和蛋白水平表達(dá)都顯著上調(diào);3)流式細(xì)胞儀檢測小鼠顆粒細(xì)胞干擾抗苗勒管激素后的細(xì)胞凋亡,發(fā)現(xiàn)細(xì)胞早期凋亡和晚期凋亡都顯著增加。通過Real time PCR和Western blot檢測抑制凋亡的Bcl-2表達(dá)情況,發(fā)現(xiàn)Bcl-2的表達(dá)顯著下調(diào);4)CCK-8法檢測顆粒細(xì)胞的增殖,干擾抗苗勒管激素后顆粒細(xì)胞的增殖降低;5)ELISA檢測顆粒細(xì)胞分泌雌激素和抑制素B的濃度,抑制素B分泌顯著降低,雌激素也顯著降低;6)通過Real time PCR檢測干擾后顆粒細(xì)胞中TGF-β超家族的抑制素各亞基(INHA、INHBA和INHBB)、卵泡抑素(Fst)、抗苗勒管激素受體(AmhRⅡ)和生長分化因子-9(GDF-9)的表達(dá)量,其中GDF-9上調(diào),其他5種繁殖相關(guān)基因全都顯著下調(diào)。
[Abstract]:Anti-M 眉 llerian hormone,AMH is a dimer glycoprotein composed of two 72kDa monomers linked by disulfide bonds. AMH/MIS belongs to the transforming growth factor- 尾 (TGF- 尾) superfamily, which includes TGF- 尾. Activin-inhibin-follicle statin (ACT-INH-FST) system and growth differentiation factor-9 (GDF-9). All members of the family are dimer glycoproteins involved in regulating tissue growth and differentiation. It has been proved that transforming growth factor-尾 (TGF- 尾) superfamily / Smad signal transduction pathway is an important pathway to regulate follicular development. In ovarian tissues, anti-Muller tube hormones are expressed in granulosa cells of follicles. It is known that anti-mullerian hormone is involved in the development of mammalian follicles and regulates the proliferation and differentiation of granulosa cells through autocrine / paracrine. In this study, interference fragments were designed for mouse gene sequence, and anti-Muller tube hormone expression in granulosa cells was interfered with by RNAi interference vector pSilencer4.1-CMV-AMH,. The expression of anti-Muller tube hormone was detected at the level of mRNA and protein respectively. It was found that the following changes occurred in granulosa cells after interfering with anti-Muller tube hormone: 1) three anti-Muller tube hormone RNAi interference vectors were constructed by Western blot detection. The three vectors significantly down-regulated the protein expression of anti-Muller tube hormone, and the interference vector psh-AMH-2 had the best interference effect. 2) flow cytometry was used to detect the cell cycle of mouse granulosa cells which interfered with the effect of anti-Muller tube hormone, and found that more cells were blocked in G1 phase. The expression of p21, which regulates cell cycle, was significantly up-regulated at mRNA and protein levels. 3) flow cytometry was used to detect the apoptosis of mouse granulosa cells after interfering with Muller tube hormone, and it was found that both early and late apoptosis were significantly increased. The expression of Bcl-2 was significantly down-regulated by Real time PCR and Western blot, 4) the proliferation of granulosa cells was detected by CCK-8 assay, and the proliferation of granulosa cells decreased after interfering with the hormone of Muller tube. 5) the concentrations of estradiol and inhibin B in granulosa cells were detected by ELISA. The secretion of inhibin B and estrogen were significantly decreased. 6) Real time PCR was used to detect the expression of TGF- 尾 superfamily in granulosa cells (INHA,INHBA and INHBB), (Fst), anti-Myelian tube hormone receptor (AmhR 鈪，

本文編號：2365861