SHRNA干扰沉默AMH基因对体外培养小鼠卵巢颗粒细胞的局部调节机制研究
发布时间：2018-12-06 10:02
【摘要】：抗苗勒管激素(Anti-Müllerian hormone,AMH)是由两个72kDa的单体通过二硫键连接构成的二聚体糖蛋白。AMH/MIS属于转化生长因子-β(TGF-β)超家族,该家族包括TGF-β、激活素-抑制素-卵泡抑素(ACT-INH-FST)系统和生长分化因子-9(GDF-9)等。这个家族所有的成员都是二聚体糖蛋白,都与调节组织生长与分化有关。现已证实转化生长因子-β(TGF-β)超家族/Smad信号转导通路是调控卵泡发育的重要途径。在卵巢组织中,抗苗勒管激素表达于卵泡的颗粒细胞。已知抗苗勒管激素参与哺乳动物卵泡发育,通过自/旁分泌调节颗粒细胞的增殖分化,为了研究抗苗勒管激素对小鼠卵巢颗粒细胞的调控作用,本课题针对小鼠基因序列设计干扰片段,构建了抗苗勒管激素RNAi干扰载体pSilencer4.1-CMV-AMH,干扰颗粒细胞中抗苗勒管激素的表达,分别在mRNA和蛋白水平检测抗苗勒管激素的表达情况,发现在干扰抗苗勒管激素后,颗粒细胞内发生如下变化:1)通过Western blot检测构建的三个抗苗勒管激素RNAi干扰载体,三个载体都显著下调抗苗勒管激素的蛋白表达,其中干扰载体psh-AMH-2的干扰效果最好;2)流式细胞仪检测小鼠颗粒细胞干扰抗苗勒管激素后的细胞周期,发现较多的细胞阻滞在G1期。调控细胞周期的p21在mRNA和蛋白水平表达都显著上调;3)流式细胞仪检测小鼠颗粒细胞干扰抗苗勒管激素后的细胞凋亡,发现细胞早期凋亡和晚期凋亡都显著增加。通过Real time PCR和Western blot检测抑制凋亡的Bcl-2表达情况,发现Bcl-2的表达显著下调;4)CCK-8法检测颗粒细胞的增殖,干扰抗苗勒管激素后颗粒细胞的增殖降低;5)ELISA检测颗粒细胞分泌雌激素和抑制素B的浓度,抑制素B分泌显著降低,雌激素也显著降低;6)通过Real time PCR检测干扰后颗粒细胞中TGF-β超家族的抑制素各亚基(INHA、INHBA和INHBB)、卵泡抑素(Fst)、抗苗勒管激素受体(AmhRⅡ)和生长分化因子-9(GDF-9)的表达量,其中GDF-9上调,其他5种繁殖相关基因全都显著下调。
[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，
本文编号：2365861
[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，
本文编号：2365861
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2365861.html
