本文關(guān)鍵詞： 綿羊 卵泡 顆粒細(xì)胞 Notch信號(hào) DAPT　出處：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：DAPT為γ-分泌酶復(fù)合物抑制劑,Notch蛋白為該酶的主要催化底物,因此,通過影響γ-分泌酶的活性,DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester)可間接阻斷Notch信號(hào)通路。Notch信號(hào)通路廣泛存在于脊椎動(dòng)物和非脊椎動(dòng)物,在進(jìn)化上高度保守,通過相鄰細(xì)胞之間的相互作用調(diào)節(jié)細(xì)胞、組織、器官的分化和發(fā)育。但是,目前關(guān)于Notch信號(hào)通路在綿羊卵泡顆粒細(xì)胞上的研究未見報(bào)道。為了更好地了解Notch信號(hào)對(duì)綿羊卵泡發(fā)育的作用,本研究以晉中綿羊?yàn)檠芯繉?duì)象,采集綿羊卵巢,分離卵泡并收集顆粒細(xì)胞,在無血清體外培養(yǎng)體系中添加不同濃度DAPT (0 μM、5μM、10 μM、20μM)體外培養(yǎng)顆粒細(xì)胞。每個(gè)濃度設(shè)置6個(gè)重復(fù),每48 h換一次液,培養(yǎng)到168 h時(shí),收集培養(yǎng)的顆粒細(xì)胞及其培養(yǎng)液,利用Guava ViaCount(?) Reagent進(jìn)行細(xì)胞計(jì)數(shù),用綿羊Elisa試劑盒測(cè)定雌激素濃度。利用熒光定量PCR技術(shù)檢測(cè)體外培養(yǎng)的綿羊顆粒細(xì)胞的細(xì)胞周期基因CyclinD2、P21,細(xì)胞凋亡相關(guān)基因Bax、 Bcl-2、P53、c-Myc及雌激素合成相關(guān)基因CYP19的相對(duì)表達(dá)量。試驗(yàn)結(jié)果及分析：(1)試驗(yàn)結(jié)果表明,Notch信號(hào)對(duì)綿羊卵泡顆粒細(xì)胞增殖及功能的調(diào)控具有重要作用。未添加DAPT的對(duì)照組,顆粒細(xì)胞的數(shù)目最多,雌激素的產(chǎn)生量最高,與添加DAPT的試驗(yàn)組相比,存在顯著的差異(P0.05)。添加DAPT的各試驗(yàn)組,顆粒細(xì)胞的數(shù)量及雌激素的產(chǎn)生量有不同程度的減少,說明綿羊卵泡顆粒細(xì)胞的增殖及雌激素的產(chǎn)生都受Notch信號(hào)通路調(diào)控。(2)添加DAPT阻斷Notch信號(hào)后,顆粒細(xì)胞雌激素合成相關(guān)基因CYP19及細(xì)胞凋亡相關(guān)基因Bax、Bcl-2、c-Myc的表達(dá)量逐漸降低,細(xì)胞周期基因P21、CyclinD2及凋亡相關(guān)基因P53的表達(dá)量變化不明顯。當(dāng)DAPT濃度為5μM時(shí),Bcl-2、c-Myc基因的相對(duì)表達(dá)量與對(duì)照組相比,顯著降低(P0.05)；與對(duì)照組相比,濃度為20μM的處理組,Bax的相對(duì)表達(dá)量顯著降低(P0.05)。DAPT阻斷Notch信號(hào)通路,對(duì)綿羊顆粒細(xì)胞的增殖及雌激素產(chǎn)生具有一定的負(fù)調(diào)控作用,對(duì)綿羊顆粒細(xì)胞增殖的負(fù)調(diào)控作用可能是通過調(diào)節(jié)細(xì)胞凋亡基因Bax、Bcl-2的表達(dá)實(shí)現(xiàn)的,對(duì)雌激素分泌的負(fù)調(diào)控作用可能是通過調(diào)節(jié)雌激素合成相關(guān)基因CYP19的表達(dá)實(shí)現(xiàn)的。本研究初步探討了DAPT阻斷Notch信號(hào)對(duì)體外培養(yǎng)的綿羊卵泡顆粒細(xì)胞增殖及雌激素產(chǎn)生的影響,為進(jìn)一步研究Notch信號(hào)通路在雌性動(dòng)物卵巢上的功能及其作用機(jī)制奠定理論基礎(chǔ)。
[Abstract]:DAPT is a complex inhibitor of 緯 -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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S826

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