Aurora B及其SUMO修饰对小鼠卵泡发育和颗粒细胞生长的影响及调控机制研究
发布时间：2019-05-28 03:59
【摘要】：哺乳动物中,卵泡闭锁是卵泡发育过程中一个复杂且不可避免的退化过程,受到激素、生长因子等多种因素的调控,而颗粒细胞在卵泡的发育中扮演至关重要的角色,其凋亡是引起卵泡闭锁的主要原因。Aurora B(丝氨酸/苏氨酸激酶)作为染色体乘客蛋白的一员,参与调控染色体的排列、纺锤体的组装以及胞质分裂等有丝分裂过程。此外,SUMO修饰参与蛋白质互作、信号转导、核质运输、转录调控以及调节基因组稳定性等方面,虽已有文献报道Aurora B可以被SUMO修饰,但是对于Aurora B及其SUMO修饰对卵泡发育的影响方面,仍然是个未解之谜。因此,本课题将以小鼠卵泡及其颗粒细胞为研究模型,主要研究Aurora B及其SUMO修饰对卵泡及其颗粒细胞发育的影响,初步探索可能存在的调控机制。本实验分为两个部分,具体内容和结果如下:实验一:Aurora B对小鼠卵泡及其颗粒细胞发育的影响研究1、Aurora B在卵泡及颗粒细胞中的定位与表达模式。获取不同发育阶段卵泡及颗粒细胞,检测Aurora B的定位及表达。研究结果显示,Aurora B定位于颗粒细胞核,当胞质分裂时,位于围绕细胞核的胞质中,且随着卵泡的发育,Aurora B在各级卵泡和颗粒细胞中的表达水平上升,在有腔期达到最大(p0.01);2、Aurora B影响卵泡的发育。体外分离获得次级卵泡,添加Aurora B抑制剂培养,观察记录卵泡的生长情况。研究结果显示,抑制Aurora B活性后,卵泡生长速度成下降(p0.05),闭锁率增加(p0.05),卵泡的发育受到抑制;3、Aurora B影响颗粒细胞的生长。选取有腔期颗粒细胞(pre-GCs),进行抑制剂培养,检测细胞周期、增殖和凋亡情况。研究结果显示,抑制Aurora B后,G0/G1期细胞比例显著增加(p0.01)、S期细胞比例显著下降(p0.05),细胞被明显的阻滞在G1-S期,增殖率显著下降(p0.05),凋亡率显著增加(p0.01),致使细胞生长受阻;4、初步探索Aurora B调控颗粒细胞生长的分子机制。选取pre-GCs进行抑制剂培养,检测调控细胞增殖与凋亡相关基因和蛋白水平。研究结果显示,Aurora B参与调控p38MAPK与Fas/FasL信号通路,下调G1期向S期转化的细胞周期蛋白CDK4、增殖相关蛋白PCNA以及抗凋亡蛋白Bcl-2的表达量(p0.05),上调细胞死亡蛋白Fas、凋亡蛋白caspase-8和caspase-3的表达量(p0.01),最终参与颗粒细胞的生长调控。实验二:Aurora B的SUMO修饰验证及其对卵泡颗粒细胞的影响1、SUMO修饰验证。成功构建几种真核表达质粒,选取pre-GCs体外分离培养,利用免疫共沉淀法检测SUMO修饰。研究结果显示,在颗粒细胞中,Aurora B可以被SUMO2修饰,Lys207为主要的SUMO2修饰位点;2、SUMO2修饰影响Aurora B的生物功能。体外分离培养pre-GCs,首先利用免疫细胞化学法检测Aurora B的定位分布。研究结果显示,Aurora B定位于颗粒细胞核内,而Aurora BK207R主要定位在细胞核,部分定位在细胞质中;其次利用Western blot法检测Aurora B的蛋白稳定性。研究结果显示,SUMO2可以在一定范围内促进Aurora B的蛋白表达水平。表明,SUMO2修饰可以维持Aurora B在颗粒细胞中的定位与蛋白稳定性;3、SUMO2修饰影响颗粒细胞的生长。体外分离培养pre-GCs,分别转染对照组(HA)、正常组(HA-Aurora B)和突变组(HA-Aurora BK207R),检测细胞的周期、增殖和凋亡情况。研究结果显示,突变组G0/G1期细胞比例显著增加(p0.01)、S期和G2/M期细胞比例均显著下降(p0.05),细胞被明显的阻滞在G1-S期,增殖率显著下降(p0.01),凋亡率显著增加(p0.01),致使细胞生长受阻。综上,本研究发现在小鼠卵泡中,Aurora B定位于颗粒细胞核,胞质分裂时,转定位至中间体和围绕细胞核的胞质中,且随着卵泡的成熟,Aurora B在卵泡和颗粒细胞中的表达量呈上升趋势,在有腔期达到最大。抑制Aurora B后卵泡生长受阻、凋亡增加,并通过p38MAPK和Fas/FasL信号通路,下调CDK4、PCNA和Bcl-2的水平,上调caspase-8、caspase-3水平,影响颗粒细胞生长。此外,我们发现Aurora B可以被SUMO2修饰,Lys207为主要修饰位点,SUMO2修饰可以维持Aurora B在颗粒细胞中的亚细胞定位和稳定性,影响颗粒细胞的生长。本研究结果将为卵泡的发育和闭锁提供一定的理论依据,同时也为更好的治疗卵巢疾病奠定基础。
[Abstract]:In the mammal, the follicular atresia is a complex and inevitable degeneration process in the development of the follicle, and is regulated by various factors such as the hormone and the growth factor, and the granulosa cells play a vital role in the development of the follicle, and the apoptosis is the main cause of the follicular atresia. Aurora B (Serine/ Threonine Kinase), as a member of the chromosomal passenger protein, is involved in the regulation of the arrangement of chromosomes, the assembly of the spindle, and the mitosis processes such as cytokinesis. In addition, SUMO modification is involved in protein interaction, signal transduction, nuclear transport, transcriptional regulation and regulation of genome stability. Although the literature has reported that Aurora B can be modified by SUMO, it is still a mystery to the effect of Aurora B and its SUMO modification on the development of the follicle. Therefore, this subject will study the effect of Aurora B and its SUMO modification on the development of the follicle and its granulosa cells, and explore the possible regulatory mechanism. This experiment is divided into two parts. The specific contents and results are as follows:1. The effect of Aurora B on the development of the follicle and the granulosa cells of the mice is studied. The localization and expression pattern of Aurora B in the follicles and granulosa cells is studied. Follicular and granulosa cells of different stages of development were obtained, and the localization and expression of Aurora B were detected. The results of the study show that Aurora B is located in the nucleus of the particles, and when the cytoplasm is split, the Aurora B is located in the cytoplasm around the nucleus, and with the development of the follicle, the expression level of the Aurora B in the follicles and granulosa cells at all levels is up, reaching the maximum in the cavity (p0.01);2, Aurora B affects the development of the follicle. The secondary follicle was isolated in vitro, and the Aurora B inhibitor was added to culture, and the growth of the recorded follicle was observed. The results showed that, after the activity of Aurora B, the growth rate of the follicle was decreased (p0.05), the blocking rate was increased (p0.05), and the development of the follicle was inhibited; and 3, Aurora B affected the growth of the granulosa cells. The cell cycle, proliferation and apoptosis were detected by pre-GCs. The results showed that after the inhibition of Aurora B, the proportion of cells in G0/ G1 phase increased significantly (p0.01), and the proportion of S-phase cells decreased significantly (p0.05). The cells were significantly blocked in the G1-S phase, and the rate of apoptosis was significantly decreased (p0.05), and the apoptosis rate was significantly increased (p0.01), which led to the inhibition of cell growth;4. The molecular mechanism of the control of the growth of granulosa cells by Aurora B was studied. Pre-GCs are selected for inhibitor culture, and the genes and protein levels related to the proliferation and apoptosis of the cells are detected. The results showed that Aurora B was involved in the regulation of p38 MAPK and Fas/ FasL signaling pathway, down-regulation of the cell cycle protein CDK4, the proliferation-related protein PCNA and the expression of anti-apoptotic protein Bcl-2 (p0.05), and up-regulation of the expression of the cell death protein Fas, the apoptosis protein caspase-8 and the caspase-3 (p0.01). And finally participating in the growth regulation of the granulosa cells. Experiment 2: The SUMO modification of Aurora B and its effect on the granulosa cell of the follicle were 1, and the SUMO modification was verified. Several eukaryotic expression plasmids were successfully constructed, and the pre-GCs were selected to be isolated and cultured in vitro, and the SUMO modification was detected by the immunoprecipitation method. The results show that in granulosa cells, Aurora B can be modified by SUM02, and Lys207 is the main SUMO2 modification site;2, SUM02 modification affects the biological function of Aurora B. Pre-GCs were isolated and cultured in vitro, and the localization distribution of Aurora B was first detected by immunocytochemical method. The results show that Aurora B is located in the nucleus of the particles, while Aurora BK207R is located mainly in the nucleus and partially in the cytoplasm; and then the protein stability of Aurora B is detected by Western blot. The results showed that SUM02 could promote the protein expression level of Aurora B in a certain range. It is shown that the modification of SUM02 can maintain the localization and protein stability of Aurora B in the granulosa cells, and the modification of SUM02 affects the growth of granulosa cells. Pre-GCs were isolated and cultured in vitro, and the control group (HA), normal group (HA-Aurora B) and mutation group (HA-Aurora BK207R) were respectively transfected into the control group (HA), normal group (HA-Aurora B) and mutation group (HA-Aurora BK207R), and the cycle, proliferation and apoptosis of the cells were detected. The results showed that the proportion of cells in the G0/ G1 phase of the mutation group was significantly increased (p0.01), the ratio of S-phase and G2/ M cells decreased significantly (p0.05), and the cells were significantly blocked in the G1-S phase. The rate of apoptosis was significantly decreased (p0.01), and the apoptosis rate was significantly increased (p0.01), which led to the inhibition of cell growth. In general, this study found that Aurora B was located in the cytoplasm of the nucleus and the cytoplasm of the mouse, and the expression of Aurora B in the follicle and the granulosa cells was on the rise with the maturation of the follicle, and the maximum in the cavity period. After the inhibition of Aurora B, the growth of the follicle was inhibited, the apoptosis was increased, and the levels of CDK4, PCNA and Bcl-2 were down-regulated by p38MAPK and Fas/ FasL signaling pathway, and the levels of caspase-8 and caspase-3 were up-regulated, and the growth of granulosa cells was affected. In addition, we find that Aurora B can be modified by SUM02, and Lys207 is the major modification site. The modification of SUM02 can maintain the subcellular localization and stability of Aurora B in the granulosa cells and affect the growth of granulosa cells. The results of this study will provide a certain theoretical basis for the development and locking of the follicle, and also lay the foundation for the better treatment of the ovarian disease.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q492.5
本文编号：2486722
[Abstract]:In the mammal, the follicular atresia is a complex and inevitable degeneration process in the development of the follicle, and is regulated by various factors such as the hormone and the growth factor, and the granulosa cells play a vital role in the development of the follicle, and the apoptosis is the main cause of the follicular atresia. Aurora B (Serine/ Threonine Kinase), as a member of the chromosomal passenger protein, is involved in the regulation of the arrangement of chromosomes, the assembly of the spindle, and the mitosis processes such as cytokinesis. In addition, SUMO modification is involved in protein interaction, signal transduction, nuclear transport, transcriptional regulation and regulation of genome stability. Although the literature has reported that Aurora B can be modified by SUMO, it is still a mystery to the effect of Aurora B and its SUMO modification on the development of the follicle. Therefore, this subject will study the effect of Aurora B and its SUMO modification on the development of the follicle and its granulosa cells, and explore the possible regulatory mechanism. This experiment is divided into two parts. The specific contents and results are as follows:1. The effect of Aurora B on the development of the follicle and the granulosa cells of the mice is studied. The localization and expression pattern of Aurora B in the follicles and granulosa cells is studied. Follicular and granulosa cells of different stages of development were obtained, and the localization and expression of Aurora B were detected. The results of the study show that Aurora B is located in the nucleus of the particles, and when the cytoplasm is split, the Aurora B is located in the cytoplasm around the nucleus, and with the development of the follicle, the expression level of the Aurora B in the follicles and granulosa cells at all levels is up, reaching the maximum in the cavity (p0.01);2, Aurora B affects the development of the follicle. The secondary follicle was isolated in vitro, and the Aurora B inhibitor was added to culture, and the growth of the recorded follicle was observed. The results showed that, after the activity of Aurora B, the growth rate of the follicle was decreased (p0.05), the blocking rate was increased (p0.05), and the development of the follicle was inhibited; and 3, Aurora B affected the growth of the granulosa cells. The cell cycle, proliferation and apoptosis were detected by pre-GCs. The results showed that after the inhibition of Aurora B, the proportion of cells in G0/ G1 phase increased significantly (p0.01), and the proportion of S-phase cells decreased significantly (p0.05). The cells were significantly blocked in the G1-S phase, and the rate of apoptosis was significantly decreased (p0.05), and the apoptosis rate was significantly increased (p0.01), which led to the inhibition of cell growth;4. The molecular mechanism of the control of the growth of granulosa cells by Aurora B was studied. Pre-GCs are selected for inhibitor culture, and the genes and protein levels related to the proliferation and apoptosis of the cells are detected. The results showed that Aurora B was involved in the regulation of p38 MAPK and Fas/ FasL signaling pathway, down-regulation of the cell cycle protein CDK4, the proliferation-related protein PCNA and the expression of anti-apoptotic protein Bcl-2 (p0.05), and up-regulation of the expression of the cell death protein Fas, the apoptosis protein caspase-8 and the caspase-3 (p0.01). And finally participating in the growth regulation of the granulosa cells. Experiment 2: The SUMO modification of Aurora B and its effect on the granulosa cell of the follicle were 1, and the SUMO modification was verified. Several eukaryotic expression plasmids were successfully constructed, and the pre-GCs were selected to be isolated and cultured in vitro, and the SUMO modification was detected by the immunoprecipitation method. The results show that in granulosa cells, Aurora B can be modified by SUM02, and Lys207 is the main SUMO2 modification site;2, SUM02 modification affects the biological function of Aurora B. Pre-GCs were isolated and cultured in vitro, and the localization distribution of Aurora B was first detected by immunocytochemical method. The results show that Aurora B is located in the nucleus of the particles, while Aurora BK207R is located mainly in the nucleus and partially in the cytoplasm; and then the protein stability of Aurora B is detected by Western blot. The results showed that SUM02 could promote the protein expression level of Aurora B in a certain range. It is shown that the modification of SUM02 can maintain the localization and protein stability of Aurora B in the granulosa cells, and the modification of SUM02 affects the growth of granulosa cells. Pre-GCs were isolated and cultured in vitro, and the control group (HA), normal group (HA-Aurora B) and mutation group (HA-Aurora BK207R) were respectively transfected into the control group (HA), normal group (HA-Aurora B) and mutation group (HA-Aurora BK207R), and the cycle, proliferation and apoptosis of the cells were detected. The results showed that the proportion of cells in the G0/ G1 phase of the mutation group was significantly increased (p0.01), the ratio of S-phase and G2/ M cells decreased significantly (p0.05), and the cells were significantly blocked in the G1-S phase. The rate of apoptosis was significantly decreased (p0.01), and the apoptosis rate was significantly increased (p0.01), which led to the inhibition of cell growth. In general, this study found that Aurora B was located in the cytoplasm of the nucleus and the cytoplasm of the mouse, and the expression of Aurora B in the follicle and the granulosa cells was on the rise with the maturation of the follicle, and the maximum in the cavity period. After the inhibition of Aurora B, the growth of the follicle was inhibited, the apoptosis was increased, and the levels of CDK4, PCNA and Bcl-2 were down-regulated by p38MAPK and Fas/ FasL signaling pathway, and the levels of caspase-8 and caspase-3 were up-regulated, and the growth of granulosa cells was affected. In addition, we find that Aurora B can be modified by SUM02, and Lys207 is the major modification site. The modification of SUM02 can maintain the subcellular localization and stability of Aurora B in the granulosa cells and affect the growth of granulosa cells. The results of this study will provide a certain theoretical basis for the development and locking of the follicle, and also lay the foundation for the better treatment of the ovarian disease.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q492.5
【参考文献】
相关期刊论文 前6条
1 张月;张斌;冯炜红;李媛媛;曹旭晨;;ZM447439对乳腺癌T47D细胞生长和细胞周期的影响[J];中国肿瘤临床;2011年22期
2 任利容;肖兰;梁铭霖;王泽华;;RNAi抑制Aurora B激酶对卵巢癌A2780细胞增殖和周期的影响[J];华中科技大学学报(医学版);2011年03期
3 卢锦;朱艳;;卵泡发育及其调节因素的研究进展[J];医学综述;2008年04期
4 ;Function and regulation of Aurora/Ipllp kinase family in cell division[J];Cell Research;2003年02期
5 马永臻,柏建安;卵泡生长发育的调节因素[J];临沂医学专科学校学报;2002年02期
6 刘家慧,张桂华,彭如心,王启发,王京花,程治平;表皮生长因子对黄体细胞孕酮生成的影响[J];生殖与避孕;1998年01期
相关博士学位论文 前3条
1 倪俊;Aurora-A激酶自身调节机制及其受Ajuba上调激活机制的研究[D];复旦大学;2008年
2 柯玉文;Aurora激酶生化通路对细胞有丝分裂可塑性的调节作用[D];中国科学技术大学;2007年
3 闫晓梅;细胞有丝分裂相关激酶Aurora C的功能研究[D];复旦大学;2005年
，本文编号：2486722
本文链接：https://www.wllwen.com/shoufeilunwen/jckxbs/2486722.html
