猪腔前卵泡体外培养体系优化及参与卵泡腔形成的分子筛选

发布时间：2018-04-19 08:55

本文选题：猪 + 腔前卵泡　；参考：《安徽农业大学》2015年硕士论文

【摘要】：卵泡腔形成阶段是腔前卵泡发育最为关键的阶段之一,自然状况下绝大部分卵泡会在此阶段发生闭锁退化。优化猪腔前卵泡体外培养体系,探究卵泡成腔机制,不仅可为猪胚胎工程研究与应用提供充足的卵源,而且可为提高人类腔前卵泡体外培养效率提供借鉴。本研究对猪腔前卵泡体外培养体系进行了优化,并对可能参与卵泡成腔的基因进行了筛选,试验内容如下:试验一、猪PFs-OGCs体外培养方式的比较。通过对猪PFs-OGCs体外有血清贴壁培养与无血清悬浮培养16.5 d后,观察两种培养方法下的OGCs形态,并比较PFs-OGCs成活率、成腔率,发现OGCs经体外悬浮培养与贴壁培养均能够形成腔样结构,但贴壁培养获取OGCs的成活率、成腔率显著高于悬浮培养(P0.05)。试验二、猪PFs-OGCs体外贴壁培养培养时间的筛选。通过对猪PFs-OGCs体外贴壁培养16.5 d、18.5 d和20.5 d后,比较PFs-OGCs成活率、成腔率、卵母细胞直径以及经IVM后成熟率,并对体外培养卵母细胞在皮质颗粒分布、ROS水平以及卵母细胞超微结构上与体内卵母细胞进行对比来评价卵母细胞质量。发现体外贴壁培养16.5 d的PFs-OGCs内的卵母细胞直径显著小于18.5 d、20.5 d试验组(P0.05);16.5 d试验组成活率显著高于20.5 d组(P0.05);而体外培养18.5 d组的卵母细胞成熟率显著高于16.5 d与20.5 d组(P0.05);各组间的成腔率无显著差异(P0.05)。体外培养18.5 d的卵母细胞皮质颗粒分布、ROS水平、成熟线粒体的比例及高尔基体形态、数量与分布均与体内来源的卵母细胞无明显差异,但其透明带厚度显著小于体内卵母细胞(P0.05),脂肪堆积较多、脂滴直径较小(P0.05)。试验三:参与卵泡腔形成的基因筛选。分离猪PFs-OGCs和EAFs-OGCs并进行转录组测序(RNA-Seq),通过生物信息学方法筛选出差异表达基因。发现EAFs-OGCs上共31个基因上调,13个基因下调;通过GO分析和Pathway分析,它们主要为参与核糖体信号通路、蛋白质消化和吸收信号通路上的基因。此外,初步筛选出HPGDS、STMY1、DPPA5、MCT4、FTH1、MHC、γ-TG、fibronectin共8个可能与卵泡囊腔形成相关的重点候选基因。结论:1.体外贴壁培养18.5 d是猪PFs-OGCs最优培养体系;2.HPGDS、STMY1、DPPA5、MCT4、FTH1、MHC、γ-TG、fibronectin一共8个基因可能参与了猪卵泡腔形成。
[Abstract]:Follicular cavity formation stage is the development of preantral follicles is one of the most critical stage, the natural condition will occur at this stage most of follicles atresia. Optimize the culture system of porcine preantral follicles in vitro, explore the follicular cavity mechanism can not only provide enough eggs source for the research and application of pig embryo engineering, but also to improve human preantral follicles cultured in vitro. This study provides reference for the efficiency optimization of porcine preantral follicles in vitro, and may be involved in follicular cavity gene were screened, the test contents are as follows: the first test, the training mode of pig PFs-OGCs in vitro. The serum adherent culture with serum-free medium 16.5 D of porcine PFs-OGCs in vitro, observation of two kinds of training methods under the OGCs form, and compare the PFs-OGCs survival rate, cavity rate, found that OGCs in vitro suspension culture and adherent culture are able to form a cavity like structure But, the adherent survival rate for OGCs, a cavity was significantly higher than that of suspension culture (P0.05). In experiment two, pig PFs-OGCs were cultured in vitro incubation time. Based on the screening of porcine PFs-OGCs in vitro cultured for 16.5 D, 18.5 D and 20.5 d after PFs-OGCs, the survival rate, cavity rate, egg the mother cell diameter and after IVM and in vitro maturation rate of oocytes in the distribution of cortical granules, ROS level and ultrastructure of oocytes and oocyte in vivo were compared to evaluate the quality of oocytes were cultured in vitro. The oocyte diameter of 16.5 D PFs-OGCs was significantly less than 18.5 D 20.5, the D test group (P0.05); D test 16.5 survival rate was significantly higher than that of 20.5 D group (P0.05); and the 18.5 D group in vitro oocyte maturation rate was significantly higher than that of 16.5 D and 20.5 D group (P0.05); the cavity between groups was no significant difference (P0.05). 18.5 d culture in vitro Particle size distribution, oocyte cortical ROS levels, the proportion of mature mitochondria and Golgi morphology, no significant difference between the number and distribution and in vivo derived oocytes, but the zona pellucida thickness was significantly lower than that in oocytes (P0.05), more fat, lipid droplet diameter smaller (P0.05) test in three. In screening of follicular cavity formation. Porcine PFs-OGCs and EAFs-OGCs gene separation and transcriptome sequencing (RNA-Seq), by bioinformatics methods to identify differentially expressed genes. EAFs-OGCs found a total of 31 genes up-regulated and 13 genes were down regulated; through the analysis of GO and Pathway, they are mainly involved in ribosomal protein signaling pathway. The digestion and absorption of signaling pathway genes. In addition, preliminary screening of HPGDS, STMY1, DPPA5, MCT4, FTH1, MHC, -TG y, fibronectin a total of 8 with follicular cyst formation on relevant candidate genes. Conclusion: 1. in vitro Adherent culture of 18.5 D is the best culture system of pig PFs-OGCs; 2.HPGDS, STMY1, DPPA5, MCT4, FTH1, MHC, gamma -TG, fibronectin, a total of 8 genes may be involved in the formation of porcine follicular cavity.

【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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