DAPT阻断Notch信号通路对绵羊卵泡颗粒细胞功能的影响

发布时间：2018-01-30 03:26

本文关键词： 绵羊卵泡颗粒细胞 Notch信号 DAPT　出处：《山西农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：DAPT为γ-分泌酶复合物抑制剂,Notch蛋白为该酶的主要催化底物,因此,通过影响γ-分泌酶的活性,DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester)可间接阻断Notch信号通路。Notch信号通路广泛存在于脊椎动物和非脊椎动物,在进化上高度保守,通过相邻细胞之间的相互作用调节细胞、组织、器官的分化和发育。但是,目前关于Notch信号通路在绵羊卵泡颗粒细胞上的研究未见报道。为了更好地了解Notch信号对绵羊卵泡发育的作用,本研究以晋中绵羊为研究对象,采集绵羊卵巢,分离卵泡并收集颗粒细胞,在无血清体外培养体系中添加不同浓度DAPT (0 μM、5μM、10 μM、20μM)体外培养颗粒细胞。每个浓度设置6个重复,每48 h换一次液,培养到168 h时,收集培养的颗粒细胞及其培养液,利用Guava ViaCount(?) Reagent进行细胞计数,用绵羊Elisa试剂盒测定雌激素浓度。利用荧光定量PCR技术检测体外培养的绵羊颗粒细胞的细胞周期基因CyclinD2、P21,细胞凋亡相关基因Bax、 Bcl-2、P53、c-Myc及雌激素合成相关基因CYP19的相对表达量。试验结果及分析：(1)试验结果表明,Notch信号对绵羊卵泡颗粒细胞增殖及功能的调控具有重要作用。未添加DAPT的对照组,颗粒细胞的数目最多,雌激素的产生量最高,与添加DAPT的试验组相比,存在显著的差异(P0.05)。添加DAPT的各试验组,颗粒细胞的数量及雌激素的产生量有不同程度的减少,说明绵羊卵泡颗粒细胞的增殖及雌激素的产生都受Notch信号通路调控。(2)添加DAPT阻断Notch信号后,颗粒细胞雌激素合成相关基因CYP19及细胞凋亡相关基因Bax、Bcl-2、c-Myc的表达量逐渐降低,细胞周期基因P21、CyclinD2及凋亡相关基因P53的表达量变化不明显。当DAPT浓度为5μM时,Bcl-2、c-Myc基因的相对表达量与对照组相比,显著降低(P0.05)；与对照组相比,浓度为20μM的处理组,Bax的相对表达量显著降低(P0.05)。DAPT阻断Notch信号通路,对绵羊颗粒细胞的增殖及雌激素产生具有一定的负调控作用,对绵羊颗粒细胞增殖的负调控作用可能是通过调节细胞凋亡基因Bax、Bcl-2的表达实现的,对雌激素分泌的负调控作用可能是通过调节雌激素合成相关基因CYP19的表达实现的。本研究初步探讨了DAPT阻断Notch信号对体外培养的绵羊卵泡颗粒细胞增殖及雌激素产生的影响,为进一步研究Notch信号通路在雌性动物卵巢上的功能及其作用机制奠定理论基础。
[Abstract]:DAPT is a complex inhibitor of 纬 -secretase and Notch protein is the main catalytic substrate of this enzyme. [N-3. (5-Difluorophenophenacetyl-L-alanyl] -S-phenylglycine t-butyl esteresterl). Notch signaling pathway. Notch signaling pathway is widely existed in vertebrates and non-vertebrates. Highly conserved in evolution, regulating the differentiation and development of cells, tissues, and organs through the interaction of adjacent cells. In order to better understand the effect of Notch signal on ovine follicle development, Jinzhong Sheep was studied in this study. Ovaries were collected, follicles were isolated and granulosa cells were collected, and 10 渭 M of DAPT was added in serum-free culture system. 20 渭 M) granulosa cells were cultured in vitro. The granulosa cells and their culture medium were collected at 168 h after each concentration was set up 6 repetitions and changed once every 48 hours. Using Guava ViaCountl? Reagent was used to count cells. The concentration of estrogen was determined by sheep Elisa kit, and the cell cycle gene CyclinD2OP21 was detected by fluorescence quantitative PCR technique in sheep granulosa cells cultured in vitro. The relative expression of apoptosis-related genes (Bax, Bcl-2P53C- Myc and estrogen biosynthesis related gene CYP19). Notch signal plays an important role in the regulation of the proliferation and function of sheep follicular granulosa cells. In the control group without DAPT, the number of granulosa cells is the largest and the quantity of estrogen is the highest. Compared with the experimental group supplemented with DAPT, there was a significant difference between the two groups (P 0.05). The number of granulosa cells and the amount of estrogen in the experimental group supplemented with DAPT decreased in varying degrees. The results showed that the proliferation of sheep follicular granulosa cells and the production of estrogen were regulated by the Notch signaling pathway. (2) DAPT was added to block the Notch signal. The expression of estrogen synthesis-associated gene CYP19 and apoptosis-related gene Bax-2c-Myc in granulosa cells decreased gradually, and the cell cycle gene P21. The expression of CyclinD2 and apoptosis-related gene p53 was not significantly changed. When the concentration of DAPT was 5 渭 M, the relative expression of Bcl-2c-Myc gene was higher than that of the control group. P0. 05% decreased significantly; Compared with the control group, the relative expression of Bax in the 20 渭 M treatment group was significantly lower than that in the control group. DAPT blocked the Notch signaling pathway. The negative regulation of sheep granulosa cell proliferation and estrogen production, the negative regulation of sheep granulosa cell proliferation may be through the regulation of apoptosis gene Bax. The expression of Bcl-2 is realized. The negative regulation of estrogen secretion may be achieved by regulating the expression of estrogen synthesis-associated gene CYP19. In this study, we studied the effect of DAPT blocking Notch signal on sheep eggs in vitro. Effects of granulosa cell proliferation and estrogen production. It provides a theoretical basis for further study on the function and mechanism of Notch signaling pathway in female ovaries.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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