Aurora B及其SUMO修飾對小鼠卵泡發(fā)育和顆粒細胞生長的影響及調(diào)控機制研究
發(fā)布時間：2019-05-28 03:59
【摘要】：哺乳動物中,卵泡閉鎖是卵泡發(fā)育過程中一個復雜且不可避免的退化過程,受到激素、生長因子等多種因素的調(diào)控,而顆粒細胞在卵泡的發(fā)育中扮演至關重要的角色,其凋亡是引起卵泡閉鎖的主要原因。Aurora B(絲氨酸/蘇氨酸激酶)作為染色體乘客蛋白的一員,參與調(diào)控染色體的排列、紡錘體的組裝以及胞質(zhì)分裂等有絲分裂過程。此外,SUMO修飾參與蛋白質(zhì)互作、信號轉(zhuǎn)導、核質(zhì)運輸、轉(zhuǎn)錄調(diào)控以及調(diào)節(jié)基因組穩(wěn)定性等方面,雖已有文獻報道Aurora B可以被SUMO修飾,但是對于Aurora B及其SUMO修飾對卵泡發(fā)育的影響方面,仍然是個未解之謎。因此,本課題將以小鼠卵泡及其顆粒細胞為研究模型,主要研究Aurora B及其SUMO修飾對卵泡及其顆粒細胞發(fā)育的影響,初步探索可能存在的調(diào)控機制。本實驗分為兩個部分,具體內(nèi)容和結(jié)果如下:實驗一:Aurora B對小鼠卵泡及其顆粒細胞發(fā)育的影響研究1、Aurora B在卵泡及顆粒細胞中的定位與表達模式。獲取不同發(fā)育階段卵泡及顆粒細胞,檢測Aurora B的定位及表達。研究結(jié)果顯示,Aurora B定位于顆粒細胞核,當胞質(zhì)分裂時,位于圍繞細胞核的胞質(zhì)中,且隨著卵泡的發(fā)育,Aurora B在各級卵泡和顆粒細胞中的表達水平上升,在有腔期達到最大(p0.01);2、Aurora B影響卵泡的發(fā)育。體外分離獲得次級卵泡,添加Aurora B抑制劑培養(yǎng),觀察記錄卵泡的生長情況。研究結(jié)果顯示,抑制Aurora B活性后,卵泡生長速度成下降(p0.05),閉鎖率增加(p0.05),卵泡的發(fā)育受到抑制;3、Aurora B影響顆粒細胞的生長。選取有腔期顆粒細胞(pre-GCs),進行抑制劑培養(yǎng),檢測細胞周期、增殖和凋亡情況。研究結(jié)果顯示,抑制Aurora B后,G0/G1期細胞比例顯著增加(p0.01)、S期細胞比例顯著下降(p0.05),細胞被明顯的阻滯在G1-S期,增殖率顯著下降(p0.05),凋亡率顯著增加(p0.01),致使細胞生長受阻;4、初步探索Aurora B調(diào)控顆粒細胞生長的分子機制。選取pre-GCs進行抑制劑培養(yǎng),檢測調(diào)控細胞增殖與凋亡相關基因和蛋白水平。研究結(jié)果顯示,Aurora B參與調(diào)控p38MAPK與Fas/FasL信號通路,下調(diào)G1期向S期轉(zhuǎn)化的細胞周期蛋白CDK4、增殖相關蛋白PCNA以及抗凋亡蛋白Bcl-2的表達量(p0.05),上調(diào)細胞死亡蛋白Fas、凋亡蛋白caspase-8和caspase-3的表達量(p0.01),最終參與顆粒細胞的生長調(diào)控。實驗二:Aurora B的SUMO修飾驗證及其對卵泡顆粒細胞的影響1、SUMO修飾驗證。成功構(gòu)建幾種真核表達質(zhì)粒,選取pre-GCs體外分離培養(yǎng),利用免疫共沉淀法檢測SUMO修飾。研究結(jié)果顯示,在顆粒細胞中,Aurora B可以被SUMO2修飾,Lys207為主要的SUMO2修飾位點;2、SUMO2修飾影響Aurora B的生物功能。體外分離培養(yǎng)pre-GCs,首先利用免疫細胞化學法檢測Aurora B的定位分布。研究結(jié)果顯示,Aurora B定位于顆粒細胞核內(nèi),而Aurora BK207R主要定位在細胞核,部分定位在細胞質(zhì)中;其次利用Western blot法檢測Aurora B的蛋白穩(wěn)定性。研究結(jié)果顯示,SUMO2可以在一定范圍內(nèi)促進Aurora B的蛋白表達水平。表明,SUMO2修飾可以維持Aurora B在顆粒細胞中的定位與蛋白穩(wěn)定性;3、SUMO2修飾影響顆粒細胞的生長。體外分離培養(yǎng)pre-GCs,分別轉(zhuǎn)染對照組(HA)、正常組(HA-Aurora B)和突變組(HA-Aurora BK207R),檢測細胞的周期、增殖和凋亡情況。研究結(jié)果顯示,突變組G0/G1期細胞比例顯著增加(p0.01)、S期和G2/M期細胞比例均顯著下降(p0.05),細胞被明顯的阻滯在G1-S期,增殖率顯著下降(p0.01),凋亡率顯著增加(p0.01),致使細胞生長受阻。綜上,本研究發(fā)現(xiàn)在小鼠卵泡中,Aurora B定位于顆粒細胞核,胞質(zhì)分裂時,轉(zhuǎn)定位至中間體和圍繞細胞核的胞質(zhì)中,且隨著卵泡的成熟,Aurora B在卵泡和顆粒細胞中的表達量呈上升趨勢,在有腔期達到最大。抑制Aurora B后卵泡生長受阻、凋亡增加,并通過p38MAPK和Fas/FasL信號通路,下調(diào)CDK4、PCNA和Bcl-2的水平,上調(diào)caspase-8、caspase-3水平,影響顆粒細胞生長。此外,我們發(fā)現(xiàn)Aurora B可以被SUMO2修飾,Lys207為主要修飾位點,SUMO2修飾可以維持Aurora B在顆粒細胞中的亞細胞定位和穩(wěn)定性,影響顆粒細胞的生長。本研究結(jié)果將為卵泡的發(fā)育和閉鎖提供一定的理論依據(jù),同時也為更好的治療卵巢疾病奠定基礎。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q492.5
本文編號：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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q492.5
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