AQP9基因在水牛卵泡颗粒细胞中的表达及其与细胞凋亡的初步研究

发布时间：2018-08-06 15:01

【摘要】：水通道蛋白(Aquaporins, AQPs)是一类位于原核和真核生物细胞膜上、与水的运输密切相关的特异膜通道蛋白。对小鼠及人的研究发现,除水转运功能外,部分AQPs参与卵泡形成和发育,与颗粒细胞的凋亡、卵泡闭锁及雌性动物生育力密切相关,但对其作用机理尚不完全清晰。本研究克隆了水牛AQP9基因的编码区序列,分析其在水牛卵泡和早期胚胎发育过程中的表达规律,以及不同闭锁程度卵泡颗粒细胞和体外成熟卵母细胞周围卵丘细胞中的AQP9与凋亡相关基因的表达水平；其次,通过过表达AQP9基因及添加PKC抑制剂Staurosporine的方式探索了AQP9基因表达与水牛颗粒细胞凋亡的关系,以及AQP9基因在PKC信号通路调控的颗粒细胞凋亡中的作用。研究可为深入了解水牛卵泡闭锁发生机制、提高水牛繁殖性能奠定基础。主要研究结果如下：首先,应用RT-PCR方法克隆得到水牛AQP9基因888 bp的编码区序列,生物信息学预测其编码295个氨基酸。多重序列比较显示,水牛AQP9基因与牛、猪、绵羊和人相应序列相似性分别为99%、90%、97%、88%；氨基酸序列同源性分别为99%、86%、97%、83%,系统进化树分析结果显示,AQP9基因在物种进化过程中具有高度保守性。蛋白质结构分析显示,水牛AQP9蛋白含有17个苏氨酸(Thr),不含有信号肽,存在潜在的磷酸化位点和水通道蛋白特有的结构域Pfam MIP。其次,对AQP9基因在水牛雌性生殖细胞中的表达规律研究发现：AQP9蛋白在水牛原始、初级、次级和有腔卵泡颗粒细胞中均有表达。qRT-PCR分析结果显示,与GV期相比,AQP9基因在体外成熟的MⅡ期卵母细胞中的表达水平显著升高(p0.05),在2-细胞、4-细胞胚胎及桑椹胚中未检测到其表达,囊胚期有表达。在不同闭锁程度水牛卵泡颗粒细胞中,随着卵泡闭锁程度加深,AQP9及凋亡相关基因Bax、caspase3和Fas的表达呈上升趋势,其中AQP9基因的表达在早期闭锁卵泡中显著升高(p0.05)。在不同体外成熟质量卵母细胞周围卵丘细胞中的表达分析发现,与成熟质量好的卵母细胞卵丘细胞相比,AQP9基因在成熟质量差的卵丘细胞中的表达显著升高(p0.01)；同时凋亡相关基因caspase3和Fas的表达也显著升高(p0.05)。最后,在探讨水牛颗粒细胞凋亡与AQP9表达关系的研究发现：过表AQP9基因的水牛颗粒细胞经无血清培养可显著上AQP9及凋亡相关基因caspase3、Fas的表达,显著下调Bcl-2的表达(p0.05)。在分析PKC抑制剂Staurosporine介导的水牛颗粒细胞凋亡与AQP9基因表达关系的实验中发现,Staurosporine处理的水牛颗粒细胞,其形态出现塌陷,细胞膜出现拉丝样向周围分散,胞质空泡增多,悬浮死亡细胞增多,流式分析结果显示,Staurosporine以浓度依赖型促进颗粒细胞凋亡,且随着颗粒细胞凋亡程度的加深,AQP9及凋亡相关基因caspase3、Bax和p53的表达显著上调(p0.05), AQP9和Bax蛋白的表达亦随着细胞凋亡程度的加深而升高。以上结果说明,AQP9基因在不同物种中具有较高的保守性,并参与了水牛卵泡发育、卵母细胞成熟、早期胚胎发育及卵泡闭锁等过程。表达于卵丘颗粒细胞的AQP9可能通过影响卵丘颗粒细胞的凋亡来影响卵母细胞成熟,且水牛颗粒细胞凋亡过程中可能发挥重要的功能作用。AQP9可能在PKC信号通路介导的水牛颗粒细胞凋亡过程中发挥重要的作用。
[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.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823.83

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