本文選題：豬 + 腔前卵泡�。� 參考：《安徽農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：卵泡腔形成階段是腔前卵泡發(fā)育最為關(guān)鍵的階段之一,自然狀況下絕大部分卵泡會在此階段發(fā)生閉鎖退化。優(yōu)化豬腔前卵泡體外培養(yǎng)體系,探究卵泡成腔機制,不僅可為豬胚胎工程研究與應(yīng)用提供充足的卵源,而且可為提高人類腔前卵泡體外培養(yǎng)效率提供借鑒。本研究對豬腔前卵泡體外培養(yǎng)體系進行了優(yōu)化,并對可能參與卵泡成腔的基因進行了篩選,試驗內(nèi)容如下:試驗一、豬PFs-OGCs體外培養(yǎng)方式的比較。通過對豬PFs-OGCs體外有血清貼壁培養(yǎng)與無血清懸浮培養(yǎng)16.5 d后,觀察兩種培養(yǎng)方法下的OGCs形態(tài),并比較PFs-OGCs成活率、成腔率,發(fā)現(xiàn)OGCs經(jīng)體外懸浮培養(yǎng)與貼壁培養(yǎng)均能夠形成腔樣結(jié)構(gòu),但貼壁培養(yǎng)獲取OGCs的成活率、成腔率顯著高于懸浮培養(yǎng)(P0.05)。試驗二、豬PFs-OGCs體外貼壁培養(yǎng)培養(yǎng)時間的篩選。通過對豬PFs-OGCs體外貼壁培養(yǎng)16.5 d、18.5 d和20.5 d后,比較PFs-OGCs成活率、成腔率、卵母細胞直徑以及經(jīng)IVM后成熟率,并對體外培養(yǎng)卵母細胞在皮質(zhì)顆粒分布、ROS水平以及卵母細胞超微結(jié)構(gòu)上與體內(nèi)卵母細胞進行對比來評價卵母細胞質(zhì)量。發(fā)現(xiàn)體外貼壁培養(yǎng)16.5 d的PFs-OGCs內(nèi)的卵母細胞直徑顯著小于18.5 d、20.5 d試驗組(P0.05);16.5 d試驗組成活率顯著高于20.5 d組(P0.05);而體外培養(yǎng)18.5 d組的卵母細胞成熟率顯著高于16.5 d與20.5 d組(P0.05);各組間的成腔率無顯著差異(P0.05)。體外培養(yǎng)18.5 d的卵母細胞皮質(zhì)顆粒分布、ROS水平、成熟線粒體的比例及高爾基體形態(tài)、數(shù)量與分布均與體內(nèi)來源的卵母細胞無明顯差異,但其透明帶厚度顯著小于體內(nèi)卵母細胞(P0.05),脂肪堆積較多、脂滴直徑較小(P0.05)。試驗三:參與卵泡腔形成的基因篩選。分離豬PFs-OGCs和EAFs-OGCs并進行轉(zhuǎn)錄組測序(RNA-Seq),通過生物信息學(xué)方法篩選出差異表達基因。發(fā)現(xiàn)EAFs-OGCs上共31個基因上調(diào),13個基因下調(diào);通過GO分析和Pathway分析,它們主要為參與核糖體信號通路、蛋白質(zhì)消化和吸收信號通路上的基因。此外,初步篩選出HPGDS、STMY1、DPPA5、MCT4、FTH1、MHC、γ-TG、fibronectin共8個可能與卵泡囊腔形成相關(guān)的重點候選基因。結(jié)論:1.體外貼壁培養(yǎng)18.5 d是豬PFs-OGCs最優(yōu)培養(yǎng)體系;2.HPGDS、STMY1、DPPA5、MCT4、FTH1、MHC、γ-TG、fibronectin一共8個基因可能參與了豬卵泡腔形成。
[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.

【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S828

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