【作者】 涂飞；

【导师】 刘守仁； 谢庄；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 哺乳动物卵巢中卵泡闭锁现象是非常普遍的,在卵泡发育过程中,绝大部分卵泡(75%-99%)发生退化现象,只有极少数卵泡能成熟排卵。随着人们对细胞凋亡研究的探索,逐渐明白到颗粒细胞的凋亡是卵泡闭锁的主要原因。近些年来,人们已经发现了许多抑制或促进卵泡闭锁的因素,但是,调控卵泡闭锁的分子机制还未完全阐明,今后仍需要做进一步的研究。研究卵泡发育和闭锁对于人类而言,将为治疗不孕、提高妊娠率有一定的指导意义；对于提高动物繁殖力和胚胎工程发展也有着十分重要的意义。随着人们对nicroRNA(miRNA)的研究,人们发现miRNA是一大类非编码的、有重要调控作用的、功能性RNA家族。miRNA广泛分布于各种真核生物中,参与机体细胞增殖、分化、凋亡、胚胎发育、物质代谢、肿瘤发生与抑制和细胞信号转导等各项生命活动。已有研究证实,在猪卵泡闭锁过程中,有一些miRNA的表达量是显著上调的并且对猪卵巢颗粒细胞的凋亡有促进作用。因此,本文将对miRNA与颗粒细胞的凋亡关系展开研究,以进一步阐明调控卵泡闭锁的分子机制。本研究试验选取猪卵巢颗粒细胞为主要研究对象,基于本课题组研究的基础和国内外文献资料报道,选取miR-26b和miR-34a做为研究卵泡闭锁和颗粒细胞凋亡的调控因子。通过生物信息学网站预测靶基因,结合靶基因的功能选取HAS2和INHBB做为候选靶基因,在HeLa229细胞中,我们利用双荧光素酶报告系统和定点突变试验去验证主效靶基因,然后,转染猪颗粒细胞,利用Real-time PCR技术、流式细胞术和Western Blotting技术检测miR-26b和miR-34a对猪卵巢颗粒细胞的作用以及与其靶基因的调控形式,进一步以阐明对猪卵泡闭锁和颗粒细胞凋亡的调控机制。全文结果如下：(1) RT-PCR发现靶基因HAS2在猪卵巢和颗粒细胞中表达；通过双萤光素酶报告系统和定点突变实验发现,HAS2基因是miR-26b的直接靶基因；Real-time PCR发现转染miR-26b的颗粒细胞发生凋亡；流式细胞仪检测发现miR-26b促进颗粒细胞的凋亡,miR-26b inhibitor能抑制内源性的miR-26b对颗粒细胞的凋亡作用；利用Real-time PCR和Western Blotting技术证实了miR-26b在转录后水平调节靶基因HAS2的表达。(2)在猪卵母细胞体外成熟培养过程中,Real-time PCR检测Oh、12h、24h、36h和44h五个时间点的HAS2表达量发现,其mRNA的表达量显著上升,由此可以推测在卵母细胞成熟过程中,HAS2对卵丘的扩张发挥着十分重要的作用。(3) RT-PCR发现靶基因INHBB在猪卵巢和颗粒细胞中表达；通过双萤光素酶报告系统和定点突变实验发现,INHBB基因是]niR-26b的直接靶基因；利用Real-time PCR和Western Blotting技术证实了miR-26b在转录后水平抑制靶基因INHBB的表达。(4)通过双萤光素酶报告系统和定点突变实验发现,INHBB基因是miR-34a的直接靶基因；Real-time PCR发现转染miR-34a的颗粒细胞发生凋亡；流式细胞仪检测发现miR-34a促进颗粒细胞的凋亡,miR-34a inhibitor能抑制内源性的miR-34a对颗粒细胞的凋亡作用；利用Real-time PCR和Western Blotting技术证实了miR-34a在转录后水平抑制靶基因INHBB的表达。本文证实了HAS2和INHBB基因是miR-26b的靶基因,INHBB基因也是miR-34a的靶基因,miR-34a对靶基因INHBB的调节能力更强；miR-26b和miR-34a都是在转录后水平抑制靶基因的表达,并且促进颗粒细胞的凋亡。从而探索了miRNA促进猪颗粒细胞凋亡的分子机制,这是对：niRNA功能在卵泡闭锁过程中作用的进一步扩展,有助于今后人们深入研究卵泡闭锁分子调控机理。更多还原

【Abstract】 Follicular atresia is a very common phenomenon in the mammalian ovary, during follicle development, most follicles (75%-99%) degradation, only a very small number of follicles to mature and ovulate. With the exploration of the research on apoptosis, we gradually understand that granulosa cell apoptosis is the main cause of follicular atresia. In recent years, it has been found that many of the factors that inhibit or promote follicular atresia; the molecular mechanism of regulation of follicular atresia, however, has not been fully elucidated, the future remains to be done further research. Study of follicular development and atresia in humans, for the treatment of infertility, improve has some guiding significance for pregnancy rate; also has a very important significance for improving animal reproduction and embryonic development.Along with the microRNA (miRNA) researched, it was found that miRNAs are a large class of non-coding, an important role in the regulation of functional RNA family. MiRNA is widely distributed in various eukaryotic organisms, involved in cell proliferation, differentiation, apoptosis, embryonic development, metabolism, and tumor inhibition and cell signal transduction and other life activities. It has been confirmed that some miRNA expression is significantly up-regulated and promote apoptosis of porcine ovarian granulosa cells in porcine follicular atresia. Therefore, this article will research the relationship of miRNA and granulosa cell apoptosis, to elucidate the molecular mechanism of regulation of follicular atresia.The study of porcine granulosa cells were selected as the main research object. MiR-26b and miR-34a were selected as control factors of follicular atresia and granulosa cells apoptosis based on both our previous research results and abroad references. We selected HAS2and INHBB as the candidate target genes, by the bioinformatics web predicting target genes and the target gene function. In HeLa229cells, we verified the main effect target gene of miRNA by dual luciferase reporter system and site-directed mutagenesis experiments. Then, with transfected porcine granulosa cells, the mechanism of miR-26b and miR-34a regulating their target genes were verified by the Real-time PCR, flow cytometry and Western Blotting, further to clarify porcine follicular atresia and the apoptosis of granulosa cells regulation. The main results achieved were as follows:1.The expression of target gene HAS2was showed by RT-PCR in porcine ovaries and granulosa cells; It was verified that the HAS2gene was a direct target gene of miR-26b by dual luciferase reporter system and site-directed mutagenesis experiments; The apoptosis was found in granulosa cells transfected with miR-26b by Real-time PCR; The phenomenon was found that miR-26b promoted granulosa cells apoptosis, and miR-26b inhibitor can inhibit endogenous miR-26b on granulosa cell apoptosis. It was confirmed that miR-26b regulates HAS2gene expression at the post-transcriptional level by Real-time PCR and Western Blotting.2.HAS2mRNA expression levels were significantly increased with cumulus expansion during IVM, suggesting its important roles in cumulus expansion and oocyte maturation.3.The expression of target gene INHBB was showed by RT-PCR in porcine ovaries and granulosa cells:It was verified that the INHBB gene was a direct target gene of miR-26b by dual luciferase reporter system and site-directed mutagenesis experiments; It was confirmed that miR-26b regulates HAS2gene expression at the post-transcriptional level by Real-time PCR and Western Blotting.4.It was verified that the INHBB gene was a direct target gene of miR-34a by dual luciferase reporter system and site-directed mutagenesis experiments; The apoptosis was found in granulosa cells transfected with miR-34a by Real-time PCR; The phenomenon was found that miR-34a promoted granulosa cells apoptosis, and miR-34a inhibitor can inhibit endogenous miR-34a on granulosa cell apoptosis. It was confirmed that miR-34a regulates INHBB gene expression at the post-transcriptional level by Real-time PCR and Western Blotting.This paper confirmed that the HAS2and INHBB gene were target genes of miR-26b, also INHBB gene was a target gene of miR-34a.The regulation capability of miR-34a for target gene INHBB was more powerful than miR-26b.MiR-26b and miR-34a can both promote granulosa cells apoptosis, inhibit target genes expression at the post-transcriptional level. The study is helpful to explore the molecular mechanisms of miRNA promote pig granulosa cell apoptosis. In the meanwhile, to expands miRNA function, and contributes to in-depth study of follicular atresia molecular regulation mechanism.更多还原