【作者】 罗爱月；

【导师】 王世宣；

【作者基本信息】 华中科技大学 ， 妇产科学， 2010， 博士

【摘要】 背景与目的随着老龄化社会的出现,器官衰老日益受到重视。卵巢衰老所引发的围绝经期症状和诸多老年性疾病严重影响女性的健康。前期探索c-FLIP在宫颈腺癌中的作用中成功建立了一系列分子生物学技术平台。应用已有的肿瘤分子生物学技术平台探索卵巢衰老的内在机制,筛选卵巢衰老过程中的差异表达基因,并分析靶基因在卵泡发育、卵巢功能中的作用。方法激光捕获显微切割技术(Laser Capture Microdissection, LCM)获取卵巢组织中的单个卵泡；提取微量RNA并进行线性扩增；所获得的RNA与Affymetrix GeneChip Mouse Genome 430 2.0 Array杂交并扫描。采用免疫组织化学方法、实时定量聚合酶链反应(Real Time Polymerase Chain Reaction, Real Time PCR)及Weston Blot检测靶基因的表达定位及丰度。应用RNA干扰技术、单克隆抗体特异性封闭靶基因,下调其在组织、细胞中的表达。MTT比色法、流式细胞技术FCS分析下调靶基因对细胞增殖、凋亡的影响；激光共聚焦显微镜、电镜分别观察细胞的大小、形态学及超微结构变化。酶联荧光分析(Enzyme Linked Fluorescence Analysis, ELFA)检测激素表达水平改变。Real Time-PCR及Weston Blot检测相关基因的表达水平改变,探讨基因的相互调节。结果(1)下调cFLIP影响caspase-8蛋白的表达,从而阻抑宫颈腺癌Hela细胞增殖并诱导其凋亡；并且能有效提高Hela细胞对放、化疗的敏感性。(2)LCM获取的卵泡提取RNA经线性扩增后获得的aRNA与表达谱芯片杂交,筛选得到卵巢衰老差异表达基因共334条。(3)其中898-a基因在卵泡中特异性表达,其表达水平随小鼠的发育而降低。下调898-a基因表达,卵泡生长直径变小,卵巢激素分泌水平升高,颗粒细胞体外培养其增殖与激素分泌无明显改变；卵母细胞形态异常,超微结构显著破坏,颗粒细胞无显著改变,透明带呈致密板状,其上突触破坏消失。898-a表达下调,卵母细胞特异性表达基因表达水平升高；甾体激素合成酶类基因及LHR表达显著升高,卵母-颗粒细胞缝隙连接表达下降,颗粒-颗粒细胞缝隙连接表达升高。(4) PRLR表达水平随小鼠不断发育而显著性升高,卵泡募集选择过程中PRLR表达水平上升,排卵过程中表达水平有所下降,但仍高于对照组,黄体形成时期其表达水平再次升高。结论(1)cFLIP对宫颈腺癌生物学行为有重要影响,可能成为宫颈腺癌新的治疗靶点。(2)利用肿瘤分子生物学技术平台成功获得卵巢衰老关键基因并分析其作用机制。(3)LCM联合表达谱芯片是一条可行有效的筛选差异表达基因的技术路线。(4)下调898-a基因抑制卵母细胞的功能,致使颗粒细胞异常分化成熟,可能与卵巢衰老过程卵泡消耗加速相关。(5) PRLR通过调节卵泡细胞的增殖影响小鼠卵泡的募集与发育,并且与黄体的形成和功能维持可能有关。更多还原

【Abstract】 We have payed more attention to the organ aging with the occurrence of the aging society. Perimenopausal symptom and several senium disease resulted from ovarian aging have affected women’s healthy seriously. A series of molecular biotechnology platform were established during the exploration of the c-FLIP function in cervical adenocarcinoma. To screen the functional geens in the process of ovarian aging and analyse the mechanisim of the target gene in the follicular development which help to further investigate the molecular mechanism of ovarian aging by using the molecular biotechnology platform had been established.MethodsLaser capture microdissection was used to obtain the single follicle from the ovary. The amplified RNA which was extracted from the LCM cells was hybridized to the Affymetrix GeneChip Mouse Genome 430 2.0 Array and the signal was scanned. Immunohistochemistry, real time-PCR and Weston blot were used to test the location and expression level of target gene. RNA interference technology and monoclony antibody were used to downregulate the expression level of target genes. MTT and flow cellsorting were taken to test the proliferation and apoptosis ratio of the cells respectively. The size and morphology and its ulturestructure of the cells was observed with confocal microscop and electron microscop. Enzyme Linked Fluorescence Analysis was used to test the hormone level.Results(1) The proliferation of cervical adenocarcinoma cell Hela was inhibited and its apoptosis was induced as the result of cFLIP downregulated with the change of caspase-8 protein level. cFLIP downregulation sensitized the Hela cell to radiotherapy and chemotherapy.(2) 334 functional genes of ovarian aging were abtained by combinging LCM with cDNA array.(3) One of the 334 genes 898-a specifically expressed in ovarian follicle and its expression level reduced with the mouse development. The diameter of follicles cultured was smaller and hormone levels were higher in the proup of 898-a down-regulated compared with that of the control, while the proliferation and hormone secretion level of the granulose cells both were no significantly different between the two groups. The morphology of oocyte was abnormal and its ultrastructure was destroyed and the density of zona pellucida was much thicker and its projections were disappearance with 898-a down-regulated, while there was no change in granulose cells. The expression levels of oocyte specific genes were increased with 898-a downregulated, while that of the steroidogenesis enzyme and LHR were reverse. The gene expression level of the gap junctions between oocyte and granulose cell was decreased while the gene expression level of gap junctions between granulose cells was reverse.(4) PRLR expression level was increased significantly with the mouse development. The expression level of PRLR was increased during the process of follicular recruitment and selection while it was a little lower at the time of ovulation but still much higher than the control, than it increased again during the formation of corpus luteum.Conclusion(1) cFLIP plays an important effection on the biology behaviour and it may become a new target for the cervical adenocarcinoma therapy.(2) It is successful to obtain the key genes of ovarian aging with the tumor molecular biotechnology platform.(3) It is an effectively and feasible technique that combining LCM with cDNA array applied in the screening functional genes.(4) Granulose cells were differentiation to mature abnormally as the result of inhibition of oocyte function with 898-a downregulated which may be associated with the accelerated exhaustion of follicles during the ovarian aging.(5) PRLR may be involved in folliculogenesis through regulating the proliferation of follicular cells and may be associated with the formation and maintenance of corpus luteum in the mouse ovary.更多还原