【摘要】：目的:Notch信號通路是一條在進(jìn)化上十分保守的信號通路,Notch信號能夠傳導(dǎo)相鄰細(xì)胞間的細(xì)胞信號,從而影響細(xì)胞的增殖、分化及凋亡。然而Notch信號通路對哺乳動物卵泡發(fā)育的調(diào)控研究主要集中在對原始卵泡的形成及募集。本實(shí)驗(yàn)?zāi)康臑檠芯縉otch信號通路相關(guān)基因在卵泡優(yōu)勢化中的表達(dá)變化。以探究在牛卵泡優(yōu)勢化過程中具體哪些Notch信號基因表達(dá)會有相應(yīng)的影響。為進(jìn)一步研究Notch信號通路調(diào)控卵泡發(fā)育提供一定的實(shí)驗(yàn)依據(jù)。方法:本實(shí)驗(yàn)取牛優(yōu)勢卵泡和生長卵泡的壁層顆粒細(xì)胞/卵丘顆粒細(xì)胞,應(yīng)用PT-PCR篩選、實(shí)時定量PCR檢測Notch信號通路相關(guān)基因Notch2、Jagged1、RBP/Jκ、hes1和c-myc的表達(dá)變化。在體外條件下培養(yǎng)生長卵泡的顆粒細(xì)胞并通過免疫熒光、細(xì)胞轉(zhuǎn)染、MTT法觀察Notch信號通路與顆粒細(xì)胞增殖之間的關(guān)系。隨后通過篩選牛優(yōu)勢卵泡Marker分子,作為顆粒細(xì)胞優(yōu)勢化的標(biāo)志,觀察刺激/抑制Notch信號通路后其Marker分子的變化情況。結(jié)果:篩選出在牛顆粒細(xì)胞中表達(dá)的Notch家族成員,包括:Notch2、Jagged1、RBP/Jκ、hes1和c-myc。卵泡優(yōu)勢化后壁層顆粒細(xì)胞中的Notch2可能表達(dá)下調(diào),并會影響其靶基因hes1和c-myc的下調(diào)。DAPT(Notch信號通路抑制劑)會抑制顆粒細(xì)胞的增殖,過表達(dá)N2ICD會促進(jìn)顆粒細(xì)胞的增殖。且卵泡優(yōu)勢化以后顆粒細(xì)胞增值速率減緩。成功構(gòu)建了真核重組質(zhì)粒CD513B1-c-myc。并通過體外實(shí)驗(yàn)發(fā)現(xiàn)Notch信號通路可能通過促進(jìn)靶基因c-myc的表達(dá)進(jìn)而促使細(xì)胞增殖。而刺激/抑制Notch信號通路后都不能夠使牛優(yōu)勢卵泡Marker分子上調(diào)。結(jié)論:Notch信號通路與顆粒細(xì)胞增殖呈正向相關(guān),同時體外實(shí)驗(yàn)發(fā)現(xiàn)Notch信號通路可能通過促進(jìn)靶基因c-myc的表達(dá)進(jìn)而促使細(xì)胞增殖。但牛卵泡優(yōu)勢化以后其顆粒細(xì)胞增殖速率放緩且刺激或抑制Notch信號通路后都不能夠使牛優(yōu)勢卵泡Marker分子上調(diào)。
[Abstract]:Objective: Notch signaling pathway is an evolutionarily conserved signal pathway that can transmit cell signals between adjacent cells, thus affecting cell proliferation, differentiation and apoptosis. However, the regulation of Notch signaling pathway on mammalian follicle development is mainly focused on the formation and recruitment of primordial follicles. The aim of this study was to investigate the expression of Notch signaling pathway related genes in follicular dominance. To investigate which Notch signaling genes play a corresponding role in the process of bovine follicle dominance. To further study the regulation of follicle development by Notch signaling pathway. Methods: the parietal granulosa cells / cumulus granulosa cells of bovine dominant follicles and growth follicles were used to detect the expression of Notch signaling pathway related gene (Notch2Jagged1) RBP- J 魏 hes1 and c-myc by PT-PCR screening and real-time quantitative PCR. Granulosa cells of follicular growth were cultured in vitro. The relationship between the Notch signaling pathway and the proliferation of granulosa cells was observed by means of immunofluorescence. Then, Marker molecules of bovine dominant follicles were screened as markers of granulosa cell dominance, and the changes of Marker molecules after stimulation / inhibition of Notch signaling pathway were observed. Results: members of the Notch family expressed in bovine granulosa cells were selected, including the members of the Notch family, including: Notch2, Jagged1, RBP / J / 魏 Hhes1 and c-myc. The expression of Notch2 in granulosa cells may be down-regulated, and the down-regulation of target gene hes1 and c-myc. DAPT (Notch signaling pathway inhibitor) can inhibit the proliferation of granulosa cells, and over-expression of N2ICD can promote the proliferation of granulosa cells. The proliferation rate of granulosa cells slowed down after follicle dominance. The eukaryotic recombinant plasmid CD513B1-c-myc was successfully constructed. It was found that Notch signaling pathway could promote cell proliferation by promoting the expression of target gene c-myc in vitro. Stimulation / inhibition of Notch signaling pathway could not up-regulate Marker molecules in bovine dominant follicles. Conclusion the Notch signaling pathway is positively correlated with the proliferation of granulosa cells, and Notch signaling pathway may promote the proliferation of granulosa cells by promoting the expression of target gene c-myc. However, the proliferation rate of bovine granulosa cells slowed down after predominance of bovine follicles, and neither stimulation nor inhibition of Notch signaling pathway could up-regulate Marker molecules in bovine dominant follicles.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 梁冠生;激素處理控制牛卵泡的發(fā)育[J];國外畜牧科技;1995年04期

，

本文編號：2163929