【摘要】：Annexin A2(ANXA2)是一種鈣依賴性膜磷脂結合蛋白,存在于生物體的多種細胞類型中。脊椎動物細胞的Annexins被命名為Annexin A家族。其中Annexin A2由339個氨基酸組成,分子量為36 kD,主要表達于內皮細胞、單核細胞及某些腫瘤細胞中,主要參與細胞膜的形成、細胞膜的轉運、胞吞胞吐作用、細胞增殖活動、細胞信號轉導、分化及凋亡過程以及血管生成等重要的細胞生命活動。ANXA2基因在多種腫瘤中經(jīng)常上調表達,如卵巢癌。根據(jù)先前的研究結果,我們也發(fā)現(xiàn)ANXA2基因在高產(chǎn)蛋率雞卵巢中表達上調,該結果表明ANXA2基因與禽類卵泡發(fā)育成熟相關。本研究利用熒光定量PCR技術,我們研究了ANXA2 mRNA在雞卵巢成熟和卵泡發(fā)育過程中的表達變化,結果顯示,在未開產(chǎn)雞卵巢(6周齡)中ANXA2 mRNA的表達較低,隨著年齡的增長,在開產(chǎn)雞卵巢(23周齡)中ANXA2 mRNA的表達極顯著升高(P0.01)。本實驗還收集了產(chǎn)蛋高峰期蛋雞的各級卵泡,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),熒光定量PCR檢測到不同卵泡中ANXA2 mRNA的表達情況如下:從等級前卵泡到排卵前卵泡,隨著卵泡的發(fā)育,ANXA2 mRNA的表達逐漸增加(P0.01);多次重復實驗顯示F1卵泡的ANXA2 mRNA表達水平最高,表明ANXA2基因很可能與參與排卵過程;POF1卵泡的ANXA2 mRNA表達水平顯著下降(P0.01),并且和SWF卵泡中的ANXA2 mRNA表達水平相當。我們分離了F1~F4卵泡的膜細胞和顆粒細胞,熒光定量PCR結果表明ANXA2 mRNA在膜細胞中的表達極顯著高于顆粒細胞(P0.01)。在卵泡膜細胞中,FSH和雌激素可促進ANXA2基因的表達,而LH和孕酮對ANXA2基因的表達無顯著作用。另外,本研究進一步證明了FSH誘導ANXA2基因表達的調控區(qū)為核心啟動子區(qū)。本實驗還采用真核基因過表達技術研究了ANXA2基因的表達調控機制,將構建好的真核過表達載體轉染到分離培養(yǎng)的雞卵泡膜細胞中,結果表明,過表達膜細胞ANXA2基因可促進血管生成因子及其受體的轉錄表達。而且,過表達ANXA2增加了血管內皮生長因子A(VEGFA)的分泌量,促進了膜細胞的增殖。我們目前的研究不僅為ANXA2基因在雞卵巢中的表達調控提供了首要證據(jù),而且表明了ANXA2和卵泡血管生成有關,并有助于卵泡的發(fā)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.
【學位授予單位】：曲阜師范大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831

【共引文獻】

相關期刊論文 前4條

1 李秀凌;梁萌;劉清;劉濤;林仁勇;盧曉梅;鄭樹濤;;過表達AnnexinA2抑制食管癌Eca109細胞增殖和遷移[J];疾病預防控制通報;2013年01期

2 張海健;姚登福;秦呈林;;膜聯(lián)蛋白A2過表達在肝細胞癌發(fā)生發(fā)展中的作用[J];臨床肝膽病雜志;2011年07期

3 徐倩楠;王宏林;鄭捷;;膜聯(lián)蛋白A2在銀屑病中的表達及意義[J];實用皮膚病學雜志;2011年03期

4 奉艷;肖麗;何慧敏;高寧;石紅燕;馬樂樂;達苗苗;胥謹慧;許楠;劉雨恩;宋喜貴;侯穎春;;ANXA2對Caco2細胞凋亡的影響[J];陜西師范大學學報(自然科學版);2015年01期

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1 王猛;ANXA2在視網(wǎng)膜新生血管形成過程中的表達及作用機制[D];青島大學;2012年

2 丁滔;膜聯(lián)蛋白II的表達與前列腺癌臨床病理特征及進展的關系研究[D];中南大學;2011年

3 張付;甲基苯丙胺對PC12細胞的毒性損傷、蛋白質硝基化、L-257抗損傷等作用的機制研究[D];南方醫(yī)科大學;2012年

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1 王蕾;肝細粒棘球蚴周圍纖維囊壁形成機制的初步探索[D];石河子大學;2010年

2 李鵬鴿;膜聯(lián)蛋白A2對人胃癌細胞生物學行為影響的研究[D];陜西師范大學;2011年

3 楊麟珂;CD147、MMP-2、AnnexinⅡ在甲狀腺乳頭狀癌組織中的表達及意義[D];鄭州大學;2011年

4 曲艷紅;5-FU干擾下膜聯(lián)蛋白-Ⅱ、VEGF在HepG2肝癌細胞系中的表達及意義[D];鄭州大學;2011年

5 黃波;AnnexinⅡ、P19在膽管癌中的表達及其意義的研究[D];寧夏醫(yī)科大學;2011年

6 薛雷喜;硼替佐米對內皮細胞的細胞毒性效應和細胞遷移的影響[D];蘇州大學;2010年

7 劉小花;Annexin A2、CD147、MMP-2在宮頸鱗癌組織中的表達及意義[D];鄭州大學;2012年

8 畢超;膜聯(lián)蛋白A2在糖基化終末產(chǎn)物誘導的內皮細胞骨架重構及遷移中的作用初探[D];濟南大學;2012年

9 董小榮;急性早幼粒細胞白血病中Annexin II高表達的分子機制[D];大連醫(yī)科大學;2012年

10 王宇晴;Annexin a2與Stat3的相互作用在人乳腺癌中的功能研究[D];天津醫(yī)科大學;2013年

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本文編號：2388912