比较移植及体外培养对小鼠卵巢卵泡早期发育的影响

发布时间：2018-03-03 10:47

本文选题：培养体系　切入点：移植　出处：《华中科技大学》2013年硕士论文　论文类型：学位论文

【摘要】：小鼠模型被广泛应用于卵巢始基卵泡激活（初始募集）基本规律及各种生长分化因子调控作用的研究中。离开体内的自然环境，卵巢必须适应环境的改变，比如二氧化碳浓度、营养供给、温度及酸碱度。更重要的是，离开体内调节系统卵泡的生长会发生改变。近几年，卵巢的孵育方法主要包括体外培养和肾被膜下移植。但没有文献报道不同孵育体系对卵巢发育的影响，及孵育卵巢和在体卵巢间卵泡发育的区别。在本研究中，我们比较了体外培养卵巢、移植卵巢及体内生长卵巢中各级卵泡的数量。用免疫组化技术和半定量逆转录聚合酶链反应（RT-PCR）检测三个重要基因的表达及蛋白定位，包括透明体蛋白3（ZP3）、生长分化因子9（GDF-9）和抗苗勒氏管激素（AMH）。结果显示，，体外培养加速卵泡激活、延缓发育卵泡的生长，影响ZP3、GDF-9和AMH蛋白的表达模式。尽管移植造成少量始基卵泡的丢失，但移植卵巢的特征与体内生长卵巢非常相似，这一结果提示移植可以提供最佳的卵泡孵育环境。在体外培养体系中，alpha-Modifie Eagle Medium （α-MEM）是较好的基础培养基。本实验系统研究了孵育卵巢与体内生长卵巢间的相同及不同，证明了移植可以最好的模拟体内卵巢生长环境。【目的】探讨microRNA-145（miR-145）对小鼠颗粒细胞增殖、分化和甾体激素合成的调节作用及机制。【方法】选择3周龄未性成熟小鼠卵巢，用原位杂交技术检测miR-145的表达。应用机械法获取3周龄小鼠卵巢窦状卵泡中的颗粒细胞进行体外培养。用半定量逆转录聚合酶链反应（RT-PCR）检测颗粒细胞中miR-145、分化相关基因及激素合成酶mRNA的表达。胸腺嘧啶核苷类似物（EdU）方法检测颗粒细胞增殖。用免疫组织化学法检测颗粒细胞分化关键基因蛋白的表达。【结果】miR-145在小鼠卵巢窦状卵泡的颗粒细胞中高表达，始基卵泡、初级卵泡、次级卵泡及卵巢间质中未见明显表达。在体外培养颗粒细胞中，miR-145的表达受卵泡刺激素（FSH）的调节。下调miR-145可显著抑制颗粒细胞的增殖、分化，并且通过降低孕激素合成相关酶表达而抑制孕激素的合成分泌，而对雌激素合成没有影响。【结论】miR-145参与调节颗粒细胞增殖、分化及甾体激素合成，但其具体机制仍有待进一步研究。
[Abstract]:Mouse models are widely used in the study of the basic rules of ovarian primordial follicle activation (initial recruitment) and the regulation of various growth and differentiation factors. Without the natural environment in the body, the ovary must adapt to changes in environment, such as carbon dioxide concentration. Nutrition, temperature, pH. More importantly, follicle growth changes without the regulatory system in the body in recent years, The main methods of ovary incubation include in vitro culture and renal submembrane transplantation. However, there is no literature on the effects of different incubation systems on ovarian development, and the difference between ovarian incubation and follicle development between ovaries in vivo and in vivo. We compared the number of follicles in cultured, transplanted and growing ovaries in vitro. Immunohistochemical technique and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression and protein localization of three important genes. The results showed that culture in vitro could accelerate follicular activation and delay the growth of developmental follicles, including hyalurontin 3 (ZP3), growth differentiation factor 9 (GDF-9) and anti-Mullerian tube hormone (AMHN). The expression pattern of GDF-9 and AMH protein in ZP3 was affected. Although transplantation resulted in the loss of a small number of primary follicles, the characteristics of ovarian transplantation were very similar to those of ovarian growth in vivo. These results suggest that transplantation can provide the best incubation environment for follicles, and alpha-Modifie Eagle Medium (伪 -MEM) is a good basic culture medium in vitro. It is proved that transplantation can best simulate the growth environment of ovaries in vivo. [objective] to investigate the regulatory effect and mechanism of microRNA-145 miR-145) on the proliferation, differentiation and steroid hormone synthesis of mouse granulosa cells. [methods] the ovaries of 3-week-old immature mice were selected. The expression of miR-145 was detected by in situ hybridization. Granulosa cells from antral follicles of 3-week-old mice were obtained by mechanical method and cultured in vitro. MiR-145 in granulosa cells was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The expression of related genes and hormone synthase (mRNA). Thymine nucleoside analogue (Edu) was used to detect the proliferation of granulosa cells and the expression of key gene proteins in granulosa cell differentiation was detected by immunohistochemistry. [results] miR-145 was highly expressed in granulosa cells of mouse ovarian antral follicles, primary follicles, primary follicles, primary follicles, primary follicles, primary follicles, primary follicles, and primary follicles. The expression of miR-145 in granulosa cells was regulated by follicle stimulating hormone (FSH). Down-regulation of miR-145 could significantly inhibit the proliferation and differentiation of granulosa cells. The synthesis and secretion of progesterone were inhibited by reducing the expression of progesterone synthase, but the estrogen synthesis was not affected. [conclusion] miR-145 is involved in the regulation of granulosa cell proliferation, differentiation and steroid hormone synthesis, but the mechanism remains to be further studied.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R321.1

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