發(fā)布時間：2018-03-03 10:47

  本文選題：培養(yǎng)體系　切入點：移植　出處：《華中科技大學》2013年碩士論文　論文類型：學位論文

【摘要】：小鼠模型被廣泛應用于卵巢始基卵泡激活（初始募集）基本規(guī)律及各種生長分化因子調控作用的研究中。離開體內的自然環(huán)境，卵巢必須適應環(huán)境的改變，比如二氧化碳濃度、營養(yǎng)供給、溫度及酸堿度。更重要的是，離開體內調節(jié)系統(tǒng)卵泡的生長會發(fā)生改變。近幾年，卵巢的孵育方法主要包括體外培養(yǎng)和腎被膜下移植。但沒有文獻報道不同孵育體系對卵巢發(fā)育的影響，及孵育卵巢和在體卵巢間卵泡發(fā)育的區(qū)別。在本研究中，我們比較了體外培養(yǎng)卵巢、移植卵巢及體內生長卵巢中各級卵泡的數(shù)量。用免疫組化技術和半定量逆轉錄聚合酶鏈反應（RT-PCR）檢測三個重要基因的表達及蛋白定位，包括透明體蛋白3（ZP3）、生長分化因子9（GDF-9）和抗苗勒氏管激素（AMH）。結果顯示，，體外培養(yǎng)加速卵泡激活、延緩發(fā)育卵泡的生長，影響ZP3、GDF-9和AMH蛋白的表達模式。盡管移植造成少量始基卵泡的丟失，但移植卵巢的特征與體內生長卵巢非常相似，這一結果提示移植可以提供最佳的卵泡孵育環(huán)境。在體外培養(yǎng)體系中，alpha-Modifie Eagle Medium （α-MEM）是較好的基礎培養(yǎng)基。本實驗系統(tǒng)研究了孵育卵巢與體內生長卵巢間的相同及不同，證明了移植可以最好的模擬體內卵巢生長環(huán)境。 【目的】探討microRNA-145（miR-145）對小鼠顆粒細胞增殖、分化和甾體激素合成的調節(jié)作用及機制。 【方法】選擇3周齡未性成熟小鼠卵巢，用原位雜交技術檢測miR-145的表達。應用機械法獲取3周齡小鼠卵巢竇狀卵泡中的顆粒細胞進行體外培養(yǎng)。用半定量逆轉錄聚合酶鏈反應（RT-PCR）檢測顆粒細胞中miR-145、分化相關基因及激素合成酶mRNA的表達。胸腺嘧啶核苷類似物（EdU）方法檢測顆粒細胞增殖。用免疫組織化學法檢測顆粒細胞分化關鍵基因蛋白的表達。 【結果】miR-145在小鼠卵巢竇狀卵泡的顆粒細胞中高表達，始基卵泡、初級卵泡、次級卵泡及卵巢間質中未見明顯表達。在體外培養(yǎng)顆粒細胞中，miR-145的表達受卵泡刺激素（FSH）的調節(jié)。下調miR-145可顯著抑制顆粒細胞的增殖、分化，并且通過降低孕激素合成相關酶表達而抑制孕激素的合成分泌，而對雌激素合成沒有影響。 【結論】miR-145參與調節(jié)顆粒細胞增殖、分化及甾體激素合成，但其具體機制仍有待進一步研究。
[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 (偽 -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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R321.1

【共引文獻】

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