發(fā)布時(shí)間：2018-08-06 15:01

【摘要】：水通道蛋白(Aquaporins, AQPs)是一類位于原核和真核生物細(xì)胞膜上、與水的運(yùn)輸密切相關(guān)的特異膜通道蛋白。對(duì)小鼠及人的研究發(fā)現(xiàn),除水轉(zhuǎn)運(yùn)功能外,部分AQPs參與卵泡形成和發(fā)育,與顆粒細(xì)胞的凋亡、卵泡閉鎖及雌性動(dòng)物生育力密切相關(guān),但對(duì)其作用機(jī)理尚不完全清晰。本研究克隆了水牛AQP9基因的編碼區(qū)序列,分析其在水牛卵泡和早期胚胎發(fā)育過程中的表達(dá)規(guī)律,以及不同閉鎖程度卵泡顆粒細(xì)胞和體外成熟卵母細(xì)胞周圍卵丘細(xì)胞中的AQP9與凋亡相關(guān)基因的表達(dá)水平；其次,通過過表達(dá)AQP9基因及添加PKC抑制劑Staurosporine的方式探索了AQP9基因表達(dá)與水牛顆粒細(xì)胞凋亡的關(guān)系,以及AQP9基因在PKC信號(hào)通路調(diào)控的顆粒細(xì)胞凋亡中的作用。研究可為深入了解水牛卵泡閉鎖發(fā)生機(jī)制、提高水牛繁殖性能奠定基礎(chǔ)。主要研究結(jié)果如下：首先,應(yīng)用RT-PCR方法克隆得到水牛AQP9基因888 bp的編碼區(qū)序列,生物信息學(xué)預(yù)測(cè)其編碼295個(gè)氨基酸。多重序列比較顯示,水牛AQP9基因與牛、豬、綿羊和人相應(yīng)序列相似性分別為99%、90%、97%、88%；氨基酸序列同源性分別為99%、86%、97%、83%,系統(tǒng)進(jìn)化樹分析結(jié)果顯示,AQP9基因在物種進(jìn)化過程中具有高度保守性。蛋白質(zhì)結(jié)構(gòu)分析顯示,水牛AQP9蛋白含有17個(gè)蘇氨酸(Thr),不含有信號(hào)肽,存在潛在的磷酸化位點(diǎn)和水通道蛋白特有的結(jié)構(gòu)域Pfam MIP。其次,對(duì)AQP9基因在水牛雌性生殖細(xì)胞中的表達(dá)規(guī)律研究發(fā)現(xiàn)：AQP9蛋白在水牛原始、初級(jí)、次級(jí)和有腔卵泡顆粒細(xì)胞中均有表達(dá)。qRT-PCR分析結(jié)果顯示,與GV期相比,AQP9基因在體外成熟的MⅡ期卵母細(xì)胞中的表達(dá)水平顯著升高(p0.05),在2-細(xì)胞、4-細(xì)胞胚胎及桑椹胚中未檢測(cè)到其表達(dá),囊胚期有表達(dá)。在不同閉鎖程度水牛卵泡顆粒細(xì)胞中,隨著卵泡閉鎖程度加深,AQP9及凋亡相關(guān)基因Bax、caspase3和Fas的表達(dá)呈上升趨勢(shì),其中AQP9基因的表達(dá)在早期閉鎖卵泡中顯著升高(p0.05)。在不同體外成熟質(zhì)量卵母細(xì)胞周圍卵丘細(xì)胞中的表達(dá)分析發(fā)現(xiàn),與成熟質(zhì)量好的卵母細(xì)胞卵丘細(xì)胞相比,AQP9基因在成熟質(zhì)量差的卵丘細(xì)胞中的表達(dá)顯著升高(p0.01)；同時(shí)凋亡相關(guān)基因caspase3和Fas的表達(dá)也顯著升高(p0.05)。最后,在探討水牛顆粒細(xì)胞凋亡與AQP9表達(dá)關(guān)系的研究發(fā)現(xiàn)：過表AQP9基因的水牛顆粒細(xì)胞經(jīng)無血清培養(yǎng)可顯著上AQP9及凋亡相關(guān)基因caspase3、Fas的表達(dá),顯著下調(diào)Bcl-2的表達(dá)(p0.05)。在分析PKC抑制劑Staurosporine介導(dǎo)的水牛顆粒細(xì)胞凋亡與AQP9基因表達(dá)關(guān)系的實(shí)驗(yàn)中發(fā)現(xiàn),Staurosporine處理的水牛顆粒細(xì)胞,其形態(tài)出現(xiàn)塌陷,細(xì)胞膜出現(xiàn)拉絲樣向周圍分散,胞質(zhì)空泡增多,懸浮死亡細(xì)胞增多,流式分析結(jié)果顯示,Staurosporine以濃度依賴型促進(jìn)顆粒細(xì)胞凋亡,且隨著顆粒細(xì)胞凋亡程度的加深,AQP9及凋亡相關(guān)基因caspase3、Bax和p53的表達(dá)顯著上調(diào)(p0.05), AQP9和Bax蛋白的表達(dá)亦隨著細(xì)胞凋亡程度的加深而升高。以上結(jié)果說明,AQP9基因在不同物種中具有較高的保守性,并參與了水牛卵泡發(fā)育、卵母細(xì)胞成熟、早期胚胎發(fā)育及卵泡閉鎖等過程。表達(dá)于卵丘顆粒細(xì)胞的AQP9可能通過影響卵丘顆粒細(xì)胞的凋亡來影響卵母細(xì)胞成熟,且水牛顆粒細(xì)胞凋亡過程中可能發(fā)揮重要的功能作用。AQP9可能在PKC信號(hào)通路介導(dǎo)的水牛顆粒細(xì)胞凋亡過程中發(fā)揮重要的作用。
[Abstract]:Aquaporins (AQPs) is a kind of specific membrane channel protein which is closely related to the transport of water in the membrane of prokaryotic and eukaryotic cell. In the study of mice and human, it is found that part of AQPs is involved in the formation and development of follicles except for water transport, which is closely related to the apoptosis of granulosa cells, follicle atresia and female fertility. But the mechanism of its action is not completely clear. This study cloned the sequence of the coding region of the buffalo AQP9 gene, analyzed the expression of its expression in the follicular and early embryonic development of buffalo, as well as the expression level of AQP9 and apoptosis related genes in the follicle granulosa cells with different degree of atresia and in the mature oocyte in vitro. Secondly, through the expression of AQP9 gene and the addition of PKC inhibitor Staurosporine, the relationship between the expression of AQP9 gene and the apoptosis of buffalo granulosa cells, as well as the role of AQP9 gene in the apoptosis of granulosa cells regulated by PKC signaling pathway, can be used to understand the mechanism of the closure of the follicle of the buffalo follicle and improve the reproductive performance of buffalo. The main results are as follows: first, the sequence of the coding region of the buffalo AQP9 gene 888 BP was cloned and the bioinformatics predicted its encoding 295 amino acids. The multiple sequence comparison showed that the similarity of the corresponding sequence of the buffalo AQP9 gene and cattle, pigs, sheep and human was 99%, 90%, 97%, 88%; the sequence homology of amino acid sequence was respectively respectively. The results of 99%, 86%, 97%, 83%, phylogenetic tree analysis showed that the AQP9 gene was highly conserved in the evolutionary process. Protein structure analysis showed that Buffalo AQP9 protein contained 17 threonine (Thr), no signal peptide, potential phosphorylation site and Pfam MIP. specific to aquaporin, followed by the AQP9 gene in water AQP9 protein expression in the original Buffalo, primary, secondary and cavity follicle granulosa cells showed that the expression of.QRT-PCR analysis showed that the expression level of AQP9 gene in M II oocyte mature in vitro was significantly higher than that of GV phase (P0.05), in 2- cells, 4- cell embryos and mulberry. The expression in the blastocyst stage was not detected in the embryo. The expression of AQP9 and apoptosis related genes Bax, Caspase3 and Fas increased with the degree of follicle atresia in different degree of atresia, and the expression of AQP9 gene in the early atresia follicle increased significantly (P0.05) in the early atresia follicles. The expression analysis in the cells surrounding the cells showed that the expression of AQP9 gene in the mature oocyte cumulus cells increased significantly (P0.01), and the expression of Caspase3 and Fas of apoptosis related genes increased significantly (P0.05). Finally, the apoptosis of buffalo granulocyte and the AQP9 table were discussed. The study of Da relationship found that the buffalo granulosa cells with over AQP9 gene could significantly increase the expression of AQP9 and apoptosis related genes Caspase3, Fas, and significantly downregulate the expression of Bcl-2 (P0.05). In the analysis of the relationship between the apoptosis of buffalo granulosa cells mediated by PKC inhibitor Staurosporine and the expression of AQP9 gene, Staurosporine, Staurosporine, was found, Staurosporine. The morphology of the treated buffalo granulosa cells collapsed, the membrane of the cells dispersed around the cell membrane, the cytoplasm vacuoles increased, and the suspended dead cells increased. The flow analysis showed that Staurosporine promoted the apoptosis of granulosa cells in a concentration dependent manner, and as the degree of granulosa cells withered, AQP9 and apoptosis related genes were Caspase3, Bax and P. The expression of 53 was significantly up-regulated (P0.05), and the expression of AQP9 and Bax protein increased with the deepening of cell apoptosis. The above results showed that the AQP9 gene had high conservatism in different species, and participated in the process of buffalo follicle development, oocyte maturation, early embryo development and follicle atresia, expressed in the A of cumulus granulosa cells. QP9 may affect oocyte maturation by affecting the apoptosis of cumulus granulosa cells and may play an important functional role in the process of apoptosis of buffalo granulosa cells..AQP9 may play an important role in the process of apoptosis of buffalo granulosa cells mediated by PKC signaling pathway.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823.83

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