Notch信号通路相关基因在牛卵泡发育中表达情况的初步研究
发布时间：2018-08-04 12:47
【摘要】：目的:Notch信号通路是一条在进化上十分保守的信号通路,Notch信号能够传导相邻细胞间的细胞信号,从而影响细胞的增殖、分化及凋亡。然而Notch信号通路对哺乳动物卵泡发育的调控研究主要集中在对原始卵泡的形成及募集。本实验目的为研究Notch信号通路相关基因在卵泡优势化中的表达变化。以探究在牛卵泡优势化过程中具体哪些Notch信号基因表达会有相应的影响。为进一步研究Notch信号通路调控卵泡发育提供一定的实验依据。方法:本实验取牛优势卵泡和生长卵泡的壁层颗粒细胞/卵丘颗粒细胞,应用PT-PCR筛选、实时定量PCR检测Notch信号通路相关基因Notch2、Jagged1、RBP/Jκ、hes1和c-myc的表达变化。在体外条件下培养生长卵泡的颗粒细胞并通过免疫荧光、细胞转染、MTT法观察Notch信号通路与颗粒细胞增殖之间的关系。随后通过筛选牛优势卵泡Marker分子,作为颗粒细胞优势化的标志,观察刺激/抑制Notch信号通路后其Marker分子的变化情况。结果:筛选出在牛颗粒细胞中表达的Notch家族成员,包括:Notch2、Jagged1、RBP/Jκ、hes1和c-myc。卵泡优势化后壁层颗粒细胞中的Notch2可能表达下调,并会影响其靶基因hes1和c-myc的下调。DAPT(Notch信号通路抑制剂)会抑制颗粒细胞的增殖,过表达N2ICD会促进颗粒细胞的增殖。且卵泡优势化以后颗粒细胞增值速率减缓。成功构建了真核重组质粒CD513B1-c-myc。并通过体外实验发现Notch信号通路可能通过促进靶基因c-myc的表达进而促使细胞增殖。而刺激/抑制Notch信号通路后都不能够使牛优势卵泡Marker分子上调。结论:Notch信号通路与颗粒细胞增殖呈正向相关,同时体外实验发现Notch信号通路可能通过促进靶基因c-myc的表达进而促使细胞增殖。但牛卵泡优势化以后其颗粒细胞增殖速率放缓且刺激或抑制Notch信号通路后都不能够使牛优势卵泡Marker分子上调。
[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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
本文编号：2163929
[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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
【参考文献】
相关期刊论文 前1条
1 梁冠生;激素处理控制牛卵泡的发育[J];国外畜牧科技;1995年04期
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