本文選題：綿羊 + 卵巢��； 參考：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：NOTCH通路通過相鄰細(xì)胞之間的直接作用,對細(xì)胞的增殖、分化和凋亡等進(jìn)行調(diào)控,從而決定細(xì)胞、組織和器官的分化與發(fā)育。Notch2作為Notch通路最主要受體基因之一,其主要對腫瘤發(fā)生、器官形成等方面起重要作用。2001年Joshua等首次發(fā)現(xiàn)Notch2基因在哺乳動(dòng)物小鼠卵巢上存在表達(dá),隨后研究發(fā)現(xiàn)Notch2基因?qū)υ悸雅莸男纬�、腔前顆粒細(xì)胞的增殖和卵母細(xì)胞發(fā)育也有著重要關(guān)聯(lián)。然而關(guān)于Notch2的研究還未涉及到大型哺乳類動(dòng)物,為此我們想初步探討Notch2在綿羊卵巢上的表達(dá)情況。本研究通過克隆測序首次在綿羊卵巢上獲得Notch2基因部分序列,并使用DNAMAN軟件同其他物種Notch2基因進(jìn)行同源性比對；采用Real-time PCR技術(shù)對綿羊不同大小卵泡的膜細(xì)胞與顆粒細(xì)胞中Votch2 mRNA以及相關(guān)基因Hes1和c-myc mRNA表達(dá)進(jìn)行定量分析,研究其表達(dá)規(guī)律,探討Notch2基因?qū)d羊卵泡發(fā)育的影響；應(yīng)用免疫組化技術(shù)對Notch2蛋白在綿羊卵巢上的表達(dá)進(jìn)行定位研究,主要試驗(yàn)的結(jié)果及分析如下：(1)通過RT-PCR技術(shù),發(fā)現(xiàn)綿羊卵巢有Notch2基因的表達(dá),并克隆測序得到綿羊Notch2基因的部分CDS序列,共527 bp。所得序列利用DNAMAN軟件對比發(fā)現(xiàn)其與藏羚羊、山羊和牛的Notch2基因同源性為99%、99%、98%。(2) Real-time PCR結(jié)果顯示Notch2 mRNA在不同大小卵泡顆粒細(xì)胞內(nèi)表達(dá)差異顯著,且在大卵泡內(nèi)表達(dá)量極顯著高于中卵泡,中卵泡內(nèi)表達(dá)量顯著高于小卵泡,說明Notch2基因可能與顆粒細(xì)胞增殖相關(guān)；Notch2 mRNA在不同大小卵泡顆粒細(xì)胞內(nèi)表達(dá)量與卵泡內(nèi)雌激素的濃度成正相關(guān),表明Notch2可能參與雌激素的合成。(3) Hes1 mRNA在小、中卵泡顆粒細(xì)胞上以及中、大卵泡膜細(xì)胞上的表達(dá)差異顯著,表明Hes1基因可能參與卵泡的發(fā)育過程。Hes1 mRNA在中、小顆粒細(xì)胞間的表達(dá)趨勢同Notch2基因一致,說明從募集前期進(jìn)入募集期這一階段顆粒細(xì)胞內(nèi)Hes1的表達(dá)可能受到Notch2基因的調(diào)控。(4) c-myc mRNA在中卵泡膜細(xì)胞和顆粒細(xì)胞上的表達(dá)量均顯著高于小卵泡和大卵泡,而中卵泡處于卵泡走向閉鎖或發(fā)育成優(yōu)勢卵泡的重要轉(zhuǎn)折點(diǎn),表明c-myc基因可能參與此階段卵泡發(fā)育的重要調(diào)控。c-myc mRNA在中卵泡膜細(xì)胞和顆粒細(xì)胞上表達(dá)量均差異顯著高于小卵泡,且與Notch2 mRNA在兩種卵泡間的表達(dá)趨勢一致,說明Notch2可能通過誘導(dǎo)c-myc的表達(dá)來調(diào)控卵泡募集前期到募集期卵泡的生長。(5)免疫組化結(jié)果表明Notch2蛋白在綿羊腔前卵泡顆粒細(xì)胞、有腔卵泡顆粒細(xì)胞和膜層細(xì)胞內(nèi)均有表達(dá),且腔前卵泡顆粒細(xì)胞上表達(dá)量高于有腔卵泡。
[Abstract]:NOTCH pathway regulates cell proliferation, differentiation and apoptosis through direct action between adjacent cells, which determines the differentiation and development of cells, tissues and organs. Notch2 is one of the most important receptor genes in Notch pathway. Notch2 gene was first found in mammalian ovaries by Joshua in 2001, and then Notch2 gene was found in primordial follicle formation. The proliferation of preluminal granulosa cells is also associated with oocyte development. However, Notch2 has not been studied in large mammals, so we would like to explore the expression of Notch2 in ovaries of sheep. In this study, the partial sequence of Notch2 gene was obtained from ovaries of sheep for the first time by cloning and sequencing, and the homology of Notch2 gene of other species was compared with DNAMAN software. Real-time PCR technique was used to quantitatively analyze the expression of Votch2 mRNA and related genes Hes1 and c-myc mRNA in the membrane cells and granulosa cells of sheep follicles of different sizes, to study the expression pattern of Notch2 gene and to explore the effect of Notch2 gene on ovine follicle development. The expression of Notch2 protein in ovaries of sheep was studied by immunohistochemical technique. The main results and analysis were as follows: (1) the expression of Notch2 gene in ovaries of sheep was found by RT-PCR technique. The partial CDS sequence of sheep Notch2 gene was obtained by cloning and sequencing. The sequence was compared with Tibetan antelope, goat and cow by using DNAMAN software. The homology of Notch2 gene was 99kyun98. The results of Real-time PCR showed that the expression of Notch2 mRNA in granulosa cells of follicles of different sizes was significantly different, and the expression of Notch2 mRNA in large follicles was significantly higher than that in mesofollicles. The expression of Notch2 gene in mesofollicles was significantly higher than that in small follicles, suggesting that the expression of Notch2 gene in granulosa cells may be related to the proliferation of granulosa cells. The expression of Notch2 mRNA in granulosa cells of different sizes was positively correlated with the concentration of estrogen in follicles. These results suggest that Notch2 may be involved in the synthesis of estrogen. The expression of Hes1 mRNA in small, mesofollicular granulosa cells and large follicular membrane cells is significantly different, suggesting that Hes1 gene may be involved in follicular development. Hes1 mRNA may be involved in the development of follicles. The trend of expression between small granulosa cells was consistent with that of Notch2 gene. It was suggested that the expression of Hes1 in granulosa cells was regulated by Notch2 gene from the early recruitment stage to the recruitment stage. The expression of c-myc mRNA in mesofollicular membrane cells and granulosa cells was significantly higher than that in small follicles and large follicles. However, mesofollicles were at an important turning point of follicular atresia or developing into dominant follicles, indicating that the expression of c-myc gene in mesofollicular membrane cells and granulosa cells was significantly higher than that in small follicles. The expression of c-myc mRNA in mesofollicular membrane cells and granulosa cells was significantly higher than that in small follicles. The expression trend of Notch2 mRNA was consistent with the expression of Notch2 mRNA between the two follicles, indicating that Notch2 may regulate the growth of follicles from pre-recruitment to recruitment stage by inducing c-myc expression. The immunohistochemical results showed that Notch2 protein was expressed in preantral follicular granulosa cells of sheep. The expression level of granulosa cells was higher in granulosa cells of antral follicles than in follicles with antral follicles.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S826;Q78

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本文編號(hào)：1797810