Notch2基因在绵羊卵巢表达的研究

发布时间：2018-04-24 18:53

  本文选题：绵羊 + 卵巢　； 参考：《山西农业大学》2015年硕士论文

【摘要】：NOTCH通路通过相邻细胞之间的直接作用,对细胞的增殖、分化和凋亡等进行调控,从而决定细胞、组织和器官的分化与发育。Notch2作为Notch通路最主要受体基因之一,其主要对肿瘤发生、器官形成等方面起重要作用。2001年Joshua等首次发现Notch2基因在哺乳动物小鼠卵巢上存在表达,随后研究发现Notch2基因对原始卵泡的形成、腔前颗粒细胞的增殖和卵母细胞发育也有着重要关联。然而关于Notch2的研究还未涉及到大型哺乳类动物,为此我们想初步探讨Notch2在绵羊卵巢上的表达情况。本研究通过克隆测序首次在绵羊卵巢上获得Notch2基因部分序列,并使用DNAMAN软件同其他物种Notch2基因进行同源性比对；采用Real-time PCR技术对绵羊不同大小卵泡的膜细胞与颗粒细胞中Votch2 mRNA以及相关基因Hes1和c-myc mRNA表达进行定量分析,研究其表达规律,探讨Notch2基因对绵羊卵泡发育的影响；应用免疫组化技术对Notch2蛋白在绵羊卵巢上的表达进行定位研究,主要试验的结果及分析如下：(1)通过RT-PCR技术,发现绵羊卵巢有Notch2基因的表达,并克隆测序得到绵羊Notch2基因的部分CDS序列,共527 bp。所得序列利用DNAMAN软件对比发现其与藏羚羊、山羊和牛的Notch2基因同源性为99%、99%、98%。(2) Real-time PCR结果显示Notch2 mRNA在不同大小卵泡颗粒细胞内表达差异显著,且在大卵泡内表达量极显著高于中卵泡,中卵泡内表达量显著高于小卵泡,说明Notch2基因可能与颗粒细胞增殖相关；Notch2 mRNA在不同大小卵泡颗粒细胞内表达量与卵泡内雌激素的浓度成正相关,表明Notch2可能参与雌激素的合成。(3) Hes1 mRNA在小、中卵泡颗粒细胞上以及中、大卵泡膜细胞上的表达差异显著,表明Hes1基因可能参与卵泡的发育过程。Hes1 mRNA在中、小颗粒细胞间的表达趋势同Notch2基因一致,说明从募集前期进入募集期这一阶段颗粒细胞内Hes1的表达可能受到Notch2基因的调控。(4) c-myc mRNA在中卵泡膜细胞和颗粒细胞上的表达量均显著高于小卵泡和大卵泡,而中卵泡处于卵泡走向闭锁或发育成优势卵泡的重要转折点,表明c-myc基因可能参与此阶段卵泡发育的重要调控。c-myc mRNA在中卵泡膜细胞和颗粒细胞上表达量均差异显著高于小卵泡,且与Notch2 mRNA在两种卵泡间的表达趋势一致,说明Notch2可能通过诱导c-myc的表达来调控卵泡募集前期到募集期卵泡的生长。(5)免疫组化结果表明Notch2蛋白在绵羊腔前卵泡颗粒细胞、有腔卵泡颗粒细胞和膜层细胞内均有表达,且腔前卵泡颗粒细胞上表达量高于有腔卵泡。
[Abstract]:NOTCH pathway regulates cell proliferation, differentiation and apoptosis through direct action between adjacent cells, which determines the differentiation and development of cells, tissues and organs. Notch2 is one of the most important receptor genes in Notch pathway. Notch2 gene was first found in mammalian ovaries by Joshua in 2001, and then Notch2 gene was found in primordial follicle formation. The proliferation of preluminal granulosa cells is also associated with oocyte development. However, Notch2 has not been studied in large mammals, so we would like to explore the expression of Notch2 in ovaries of sheep. In this study, the partial sequence of Notch2 gene was obtained from ovaries of sheep for the first time by cloning and sequencing, and the homology of Notch2 gene of other species was compared with DNAMAN software. Real-time PCR technique was used to quantitatively analyze the expression of Votch2 mRNA and related genes Hes1 and c-myc mRNA in the membrane cells and granulosa cells of sheep follicles of different sizes, to study the expression pattern of Notch2 gene and to explore the effect of Notch2 gene on ovine follicle development. The expression of Notch2 protein in ovaries of sheep was studied by immunohistochemical technique. The main results and analysis were as follows: (1) the expression of Notch2 gene in ovaries of sheep was found by RT-PCR technique. The partial CDS sequence of sheep Notch2 gene was obtained by cloning and sequencing. The sequence was compared with Tibetan antelope, goat and cow by using DNAMAN software. The homology of Notch2 gene was 99kyun98. The results of Real-time PCR showed that the expression of Notch2 mRNA in granulosa cells of follicles of different sizes was significantly different, and the expression of Notch2 mRNA in large follicles was significantly higher than that in mesofollicles. The expression of Notch2 gene in mesofollicles was significantly higher than that in small follicles, suggesting that the expression of Notch2 gene in granulosa cells may be related to the proliferation of granulosa cells. The expression of Notch2 mRNA in granulosa cells of different sizes was positively correlated with the concentration of estrogen in follicles. These results suggest that Notch2 may be involved in the synthesis of estrogen. The expression of Hes1 mRNA in small, mesofollicular granulosa cells and large follicular membrane cells is significantly different, suggesting that Hes1 gene may be involved in follicular development. Hes1 mRNA may be involved in the development of follicles. The trend of expression between small granulosa cells was consistent with that of Notch2 gene. It was suggested that the expression of Hes1 in granulosa cells was regulated by Notch2 gene from the early recruitment stage to the recruitment stage. The expression of c-myc mRNA in mesofollicular membrane cells and granulosa cells was significantly higher than that in small follicles and large follicles. However, mesofollicles were at an important turning point of follicular atresia or developing into dominant follicles, indicating that the expression of c-myc gene in mesofollicular membrane cells and granulosa cells was significantly higher than that in small follicles. The expression of c-myc mRNA in mesofollicular membrane cells and granulosa cells was significantly higher than that in small follicles. The expression trend of Notch2 mRNA was consistent with the expression of Notch2 mRNA between the two follicles, indicating that Notch2 may regulate the growth of follicles from pre-recruitment to recruitment stage by inducing c-myc expression. The immunohistochemical results showed that Notch2 protein was expressed in preantral follicular granulosa cells of sheep. The expression level of granulosa cells was higher in granulosa cells of antral follicles than in follicles with antral follicles.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826;Q78

【参考文献】

相关期刊论文 前4条

1 李大卫;吴晴;彭志海;杨兆瑞;王一;;Notch1和PTEN在胃癌组织中的表达及其意义[J];癌症;2007年11期

2 金艳梅;;颗粒细胞对卵泡发育的影响[J];中国畜牧兽医;2010年08期

3 刘春梅;常立文;李文斌;汪鸿;张谦慎;;Notch2、Notch4在大鼠早期肺发育过程中的动态表达[J];实用儿科临床杂志;2006年02期

4 颜君;郭安源;贾海波;刘剑峰;;HES转录因子的研究现状[J];现代生物医学进展;2013年05期

相关硕士学位论文 前1条

1 陈五妍;p21、c-myc和S100蛋白在小鼠卵泡及黄体中的表达[D];西北农林科技大学;2009年

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