鸡ANXA2基因在卵泡发育中的表达及调控研究

发布时间：2018-12-21 12:27

【摘要】：Annexin A2(ANXA2)是一种钙依赖性膜磷脂结合蛋白,存在于生物体的多种细胞类型中。脊椎动物细胞的Annexins被命名为Annexin A家族。其中Annexin A2由339个氨基酸组成,分子量为36 kD,主要表达于内皮细胞、单核细胞及某些肿瘤细胞中,主要参与细胞膜的形成、细胞膜的转运、胞吞胞吐作用、细胞增殖活动、细胞信号转导、分化及凋亡过程以及血管生成等重要的细胞生命活动。ANXA2基因在多种肿瘤中经常上调表达,如卵巢癌。根据先前的研究结果,我们也发现ANXA2基因在高产蛋率鸡卵巢中表达上调,该结果表明ANXA2基因与禽类卵泡发育成熟相关。本研究利用荧光定量PCR技术,我们研究了ANXA2 mRNA在鸡卵巢成熟和卵泡发育过程中的表达变化,结果显示,在未开产鸡卵巢(6周龄)中ANXA2 mRNA的表达较低,随着年龄的增长,在开产鸡卵巢(23周龄)中ANXA2 mRNA的表达极显著升高(P0.01)。本实验还收集了产蛋高峰期蛋鸡的各级卵泡,包括POF1(the first post-ovulatory follicle)、F1(the first large follicle)、F3(the third large follicle)和小白卵泡(SWF,the small white follicle),荧光定量PCR检测到不同卵泡中ANXA2 mRNA的表达情况如下:从等级前卵泡到排卵前卵泡,随着卵泡的发育,ANXA2 mRNA的表达逐渐增加(P0.01);多次重复实验显示F1卵泡的ANXA2 mRNA表达水平最高,表明ANXA2基因很可能与参与排卵过程;POF1卵泡的ANXA2 mRNA表达水平显著下降(P0.01),并且和SWF卵泡中的ANXA2 mRNA表达水平相当。我们分离了F1~F4卵泡的膜细胞和颗粒细胞,荧光定量PCR结果表明ANXA2 mRNA在膜细胞中的表达极显著高于颗粒细胞(P0.01)。在卵泡膜细胞中,FSH和雌激素可促进ANXA2基因的表达,而LH和孕酮对ANXA2基因的表达无显著作用。另外,本研究进一步证明了FSH诱导ANXA2基因表达的调控区为核心启动子区。本实验还采用真核基因过表达技术研究了ANXA2基因的表达调控机制,将构建好的真核过表达载体转染到分离培养的鸡卵泡膜细胞中,结果表明,过表达膜细胞ANXA2基因可促进血管生成因子及其受体的转录表达。而且,过表达ANXA2增加了血管内皮生长因子A(VEGFA)的分泌量,促进了膜细胞的增殖。我们目前的研究不仅为ANXA2基因在鸡卵巢中的表达调控提供了首要证据,而且表明了ANXA2和卵泡血管生成有关,并有助于卵泡的发育和成功排卵。
[Abstract]:Annexin A 2 (ANXA2) is a calcium-dependent membrane phospholipid binding protein found in many cell types of organisms. The Annexins of vertebrate cells is named Annexin A family. Among them, Annexin A2 is composed of 339 amino acids and its molecular weight is 36 kD, which is mainly expressed in endothelial cells, monocytes and some tumor cells. It is mainly involved in cell membrane formation, cell membrane transport, endocytosis and cell proliferation. Cell signal transduction, differentiation and apoptosis, angiogenesis and other important cellular life activities. ANXA2 gene is often up-regulated in many kinds of tumors, such as ovarian cancer. Based on previous studies, we also found that the expression of ANXA2 gene was up-regulated in the ovaries of high laying rate hens, which suggested that ANXA2 gene was associated with follicular maturation in poultry. In this study, we studied the expression of ANXA2 mRNA during ovarian maturation and follicle development by using fluorescence quantitative PCR technique. The results showed that the expression of ANXA2 mRNA was lower in the ovaries (6 weeks of age) than that in the control group, and the expression of ANXA2 mRNA increased with age. The expression of ANXA2 mRNA in the ovary (23 weeks old) was significantly increased (P0.01). The follicles of layers with peak laying period were collected, including POF1 (the first post-ovulatory follicle), F1 (the first large follicle), F3 (the third large follicle) and small white follicles (SWF,the small white follicle),). The expression of ANXA2 mRNA in different follicles was detected by fluorescence quantitative PCR as follows: from pre-grade follicles to pre-ovulation follicles, the expression of ANXA2 mRNA increased with the development of follicles (P0.01). Multiple repeat experiments showed that the expression of ANXA2 mRNA was the highest in F1 follicles, indicating that ANXA2 gene might be involved in ovulation, and ANXA2 mRNA expression in POF1 follicles was significantly lower than that in SWF follicles (P0.01), and the expression level of ANXA2 mRNA in POF1 follicles was similar to that in SWF follicles. The membrane cells and granulosa cells of F1~F4 follicles were isolated. The results of fluorescence quantitative PCR showed that the expression of ANXA2 mRNA in membrane cells was significantly higher than that in granulosa cells (P0.01). In follicular membrane cells, FSH and estrogen could promote the expression of ANXA2 gene, but LH and progesterone had no significant effect on the expression of ANXA2 gene. In addition, this study further demonstrated that the regulatory region of FSH induced ANXA2 gene expression is the core promoter region. The expression and regulation mechanism of ANXA2 gene was studied by using eukaryotic gene overexpression technique. The constructed eukaryotic expression vector was transfected into chicken follicular membrane cells. Overexpression of ANXA2 gene can promote the transcriptional expression of angiogenic factor and its receptor. Moreover, overexpression of ANXA2 increased the secretion of vascular endothelial growth factor A (VEGFA) and promoted the proliferation of membrane cells. Our current study not only provides primary evidence for the regulation of ANXA2 gene expression in chicken ovary, but also suggests that ANXA2 is involved in follicular angiogenesis and contributes to follicle development and successful ovulation.
【学位授予单位】：曲阜师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

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