本文選題：卵巢 + 原始卵泡裝配��； 參考：《南京醫(yī)科大學(xué)》2010年博士論文

【摘要】： 原始卵泡的形成及之后發(fā)育成初級卵泡在卵巢發(fā)育生物學(xué)上是很重要的過程,該過程直接影響了女性一生中能夠提供的卵子數(shù)。一旦原始卵泡庫耗竭,就會出現(xiàn)絕經(jīng),而這一過程調(diào)節(jié)失常可能會出現(xiàn)卵巢早衰(POF)或不孕等相關(guān)的卵巢疾病。然而,到目前為止,原始卵泡形成和發(fā)育的分子機制還不是很清楚。 為了更好的理解原始卵泡形成和發(fā)育的過程,我們運用雙向凝膠電泳(2-D PAGE)和質(zhì)譜(MALDI-TOF/TOF)技術(shù)構(gòu)建了大鼠卵巢在代表原始卵泡形成及發(fā)育的4個特定時間點(出生后0小時,24小時,48小時和72小時)的差異蛋白表達譜。我們共分離鑒定出154個差異蛋白點,對應(yīng)于134種蛋白質(zhì)。進一步的聚類分析將這些差異蛋白點劃歸為4類表達模式(C0-C3),這些不同的表達模式與卵巢早期發(fā)育過程中發(fā)生的特定生物學(xué)事件相關(guān)。為了驗證這些差異蛋白的表達模式,我們隨機選擇了6種蛋白用Western blot的方法進行驗證,并進一步用免疫組化的方法確定其細(xì)胞定位。此外,運用生物信息學(xué)的方法對這些差異蛋白進行了詳細(xì)的功能分析。對這些差異蛋白的鑒定及其功能分析有助于我們進一步理解原始卵泡形成和發(fā)育的分子機制,并為臨床調(diào)節(jié)卵巢功能以及處理卵巢疾病提供了大量潛在的分子靶標(biāo)。 為了進一步研究卵泡早期發(fā)育的分子機制,我們從構(gòu)建的差異蛋白表達譜中挑選了一個轉(zhuǎn)錄因子Hnrnpk展開深入的功能研究。Hnrnpk在出生后24小時(原始卵泡裝配的起始點)表達上調(diào),主要定位于卵母細(xì)胞及顆粒細(xì)胞的細(xì)胞核中。我們運用siRNA干涉的方法來研究Hnrnpk在卵泡早期發(fā)育過程中的功能。結(jié)合新生大鼠卵巢體外培養(yǎng)的技術(shù)平臺,在卵巢早期發(fā)育過程中通過Hnrnpk siRNAs降低Hnrnpk基因的表達。結(jié)果發(fā)現(xiàn),卵巢中卵細(xì)胞的數(shù)目,原始卵泡和初級卵泡的數(shù)目均明顯減少;卵巢的組織結(jié)構(gòu)發(fā)生嚴(yán)重的紊亂伴隨著大量卵母細(xì)胞聚集成巢狀,成簇的體細(xì)胞與卵母細(xì)胞不形成卵泡結(jié)構(gòu),而且許多卵細(xì)胞呈核固縮狀,Tunel結(jié)果證實較多細(xì)胞發(fā)生了凋亡。我們應(yīng)用基因芯片技術(shù)比較分析了Hnrnpk siRNAs干涉組與對照組卵巢中基因的差異表達,篩選出63個共同存在的2倍以上差異表達的基因,其中22個基因表達上調(diào),41個基因表達下調(diào)。生物信息學(xué)分析提示這些差異基因涉及卵巢早期發(fā)育過程中多種關(guān)鍵性的生物事件。以上結(jié)果表明,轉(zhuǎn)錄因子Hnrnpk可以通過調(diào)控下游靶向基因的表達進而影響原始卵泡的形成和發(fā)育,是卵巢早期正常發(fā)育過程中一個重要的調(diào)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346

【參考文獻】

相關(guān)期刊論文 前1條

1 隋旭霞;羅麗莉;傅玉才;郭憲國;許銘炎;許錦階;;新生鼠卵巢FOXO3a表達水平與卵母細(xì)胞凋亡的相關(guān)性初步研究[J];生殖與避孕;2006年09期

，

本文編號：2102339