大鼠卵巢原始卵泡形成和发育相关蛋白谱系的构建及功能研究

发布时间：2018-07-06 09:29

本文选题：卵巢 + 原始卵泡装配　；参考：《南京医科大学》2010年博士论文

【摘要】： 原始卵泡的形成及之后发育成初级卵泡在卵巢发育生物学上是很重要的过程,该过程直接影响了女性一生中能够提供的卵子数。一旦原始卵泡库耗竭,就会出现绝经,而这一过程调节失常可能会出现卵巢早衰(POF)或不孕等相关的卵巢疾病。然而,到目前为止,原始卵泡形成和发育的分子机制还不是很清楚。为了更好的理解原始卵泡形成和发育的过程,我们运用双向凝胶电泳(2-D PAGE)和质谱(MALDI-TOF/TOF)技术构建了大鼠卵巢在代表原始卵泡形成及发育的4个特定时间点(出生后0小时,24小时,48小时和72小时)的差异蛋白表达谱。我们共分离鉴定出154个差异蛋白点,对应于134种蛋白质。进一步的聚类分析将这些差异蛋白点划归为4类表达模式(C0-C3),这些不同的表达模式与卵巢早期发育过程中发生的特定生物学事件相关。为了验证这些差异蛋白的表达模式,我们随机选择了6种蛋白用Western blot的方法进行验证,并进一步用免疫组化的方法确定其细胞定位。此外,运用生物信息学的方法对这些差异蛋白进行了详细的功能分析。对这些差异蛋白的鉴定及其功能分析有助于我们进一步理解原始卵泡形成和发育的分子机制,并为临床调节卵巢功能以及处理卵巢疾病提供了大量潜在的分子靶标。为了进一步研究卵泡早期发育的分子机制,我们从构建的差异蛋白表达谱中挑选了一个转录因子Hnrnpk展开深入的功能研究。Hnrnpk在出生后24小时(原始卵泡装配的起始点)表达上调,主要定位于卵母细胞及颗粒细胞的细胞核中。我们运用siRNA干涉的方法来研究Hnrnpk在卵泡早期发育过程中的功能。结合新生大鼠卵巢体外培养的技术平台,在卵巢早期发育过程中通过Hnrnpk siRNAs降低Hnrnpk基因的表达。结果发现,卵巢中卵细胞的数目,原始卵泡和初级卵泡的数目均明显减少;卵巢的组织结构发生严重的紊乱伴随着大量卵母细胞聚集成巢状,成簇的体细胞与卵母细胞不形成卵泡结构,而且许多卵细胞呈核固缩状,Tunel结果证实较多细胞发生了凋亡。我们应用基因芯片技术比较分析了Hnrnpk siRNAs干涉组与对照组卵巢中基因的差异表达,筛选出63个共同存在的2倍以上差异表达的基因,其中22个基因表达上调,41个基因表达下调。生物信息学分析提示这些差异基因涉及卵巢早期发育过程中多种关键性的生物事件。以上结果表明,转录因子Hnrnpk可以通过调控下游靶向基因的表达进而影响原始卵泡的形成和发育,是卵巢早期正常发育过程中一个重要的调控因子。
[Abstract]:Primary follicle formation and subsequent development into primary follicles is a very important process in ovarian development biology, which directly affects the number of eggs that a woman can provide in her lifetime. Once the primordial follicle banks are depleted, menopause occurs, and this process regulation disorder may lead to ovarian diseases such as premature ovarian failure (POF) or infertility. However, the molecular mechanisms of primordial follicle formation and development are unclear. To better understand the process of primordial follicle formation and development, Two dimensional gel electrophoresis (2-D page) and mass spectrometry (MALDI-TOF / TOF) techniques were used to construct differential protein expression profiles of rat ovaries at four specific time points (0 hours after birth, 24 hours and 48 hours and 72 hours after birth), representing the formation and development of primordial follicles. A total of 154 differential protein spots were isolated and identified, corresponding to 134 proteins. Further clustering analysis classified these differentially expressed proteins into four expression patterns (C0-C3), which were related to specific biological events during early ovarian development. In order to verify the expression patterns of these differentially expressed proteins, we randomly selected 6 proteins to be verified by Western blot, and further confirmed their cellular localization by immunohistochemical method. In addition, these differential proteins were analyzed in detail by bioinformatics. The identification and functional analysis of these differential proteins may help us to further understand the molecular mechanism of primordial follicle formation and development, and provide a large number of potential molecular targets for clinical regulation of ovarian function and treatment of ovarian diseases. In order to further study the molecular mechanism of early follicular development, we selected a transcription factor Hnrnpk from the constructed differential protein expression profile to investigate the up-regulation of Hnrnpk expression at 24 hours after birth (the starting point of primordial follicle assembly). Mainly located in the nucleus of oocytes and granulosa cells. We used siRNA interference to study the function of Hnrnpk in early follicular development. The expression of Hnrnpk gene was reduced by Hnrnpk siRNAs during the early stage of ovarian development. The results showed that the number of oocytes, primordial follicles and primary follicles in the ovary decreased significantly, and the serious disorder of the structure of the ovary was accompanied by the accumulation of a large number of oocytes into nests. The cluster of somatic cells and oocytes did not form follicle structure, and many oocytes showed nuclear pyknosis and Tunel, which confirmed that more cells had apoptosis. The differentially expressed genes in the ovary of Hnrnpk siRNAs interference group and control group were compared and analyzed by gene chip technique, and 63 co-existing differentially expressed genes were screened out, of which 22 genes were up-regulated and 41 genes were down-regulated. Bioinformatics analysis suggests that these differential genes are involved in several critical biological events during early ovarian development. These results suggest that the transcription factor Hnrnpk can affect the formation and development of primordial follicles by regulating the expression of downstream target genes and is an important regulatory factor in the early normal development of ovary.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346

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