【摘要】：水通道蛋白(Aquaporins, AQPs)是一類位于原核和真核生物細胞膜上、與水的運輸密切相關的特異膜通道蛋白。對小鼠及人的研究發(fā)現(xiàn),除水轉運功能外,部分AQPs參與卵泡形成和發(fā)育,與顆粒細胞的凋亡、卵泡閉鎖及雌性動物生育力密切相關,但對其作用機理尚不完全清晰。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)育過程中的表達規(guī)律,以及不同閉鎖程度卵泡顆粒細胞和體外成熟卵母細胞周圍卵丘細胞中的AQP9與凋亡相關基因的表達水平；其次,通過過表達AQP9基因及添加PKC抑制劑Staurosporine的方式探索了AQP9基因表達與水牛顆粒細胞凋亡的關系,以及AQP9基因在PKC信號通路調控的顆粒細胞凋亡中的作用。研究可為深入了解水牛卵泡閉鎖發(fā)生機制、提高水牛繁殖性能奠定基礎。主要研究結果如下：首先,應用RT-PCR方法克隆得到水牛AQP9基因888 bp的編碼區(qū)序列,生物信息學預測其編碼295個氨基酸。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)進化樹分析結果顯示,AQP9基因在物種進化過程中具有高度保守性。蛋白質結構分析顯示,水牛AQP9蛋白含有17個蘇氨酸(Thr),不含有信號肽,存在潛在的磷酸化位點和水通道蛋白特有的結構域Pfam MIP。其次,對AQP9基因在水牛雌性生殖細胞中的表達規(guī)律研究發(fā)現(xiàn)：AQP9蛋白在水牛原始、初級、次級和有腔卵泡顆粒細胞中均有表達。qRT-PCR分析結果顯示,與GV期相比,AQP9基因在體外成熟的MⅡ期卵母細胞中的表達水平顯著升高(p0.05),在2-細胞、4-細胞胚胎及桑椹胚中未檢測到其表達,囊胚期有表達。在不同閉鎖程度水牛卵泡顆粒細胞中,隨著卵泡閉鎖程度加深,AQP9及凋亡相關基因Bax、caspase3和Fas的表達呈上升趨勢,其中AQP9基因的表達在早期閉鎖卵泡中顯著升高(p0.05)。在不同體外成熟質量卵母細胞周圍卵丘細胞中的表達分析發(fā)現(xiàn),與成熟質量好的卵母細胞卵丘細胞相比,AQP9基因在成熟質量差的卵丘細胞中的表達顯著升高(p0.01)；同時凋亡相關基因caspase3和Fas的表達也顯著升高(p0.05)。最后,在探討水牛顆粒細胞凋亡與AQP9表達關系的研究發(fā)現(xiàn)：過表AQP9基因的水牛顆粒細胞經無血清培養(yǎng)可顯著上AQP9及凋亡相關基因caspase3、Fas的表達,顯著下調Bcl-2的表達(p0.05)。在分析PKC抑制劑Staurosporine介導的水牛顆粒細胞凋亡與AQP9基因表達關系的實驗中發(fā)現(xiàn),Staurosporine處理的水牛顆粒細胞,其形態(tài)出現(xiàn)塌陷,細胞膜出現(xiàn)拉絲樣向周圍分散,胞質空泡增多,懸浮死亡細胞增多,流式分析結果顯示,Staurosporine以濃度依賴型促進顆粒細胞凋亡,且隨著顆粒細胞凋亡程度的加深,AQP9及凋亡相關基因caspase3、Bax和p53的表達顯著上調(p0.05), AQP9和Bax蛋白的表達亦隨著細胞凋亡程度的加深而升高。以上結果說明,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、卵母細胞成熟、早期胚胎發(fā)育及卵泡閉鎖等過程。表達于卵丘顆粒細胞的AQP9可能通過影響卵丘顆粒細胞的凋亡來影響卵母細胞成熟,且水牛顆粒細胞凋亡過程中可能發(fā)揮重要的功能作用。AQP9可能在PKC信號通路介導的水牛顆粒細胞凋亡過程中發(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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S823.83

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