母源蛋白RBBP7在小鼠卵母细胞发育成熟过程中的功能研究

发布时间：2018-04-09 15:05

本文选题：RBBP7　切入点：VCP　出处：《南京医科大学》2010年硕士论文

【摘要】：视网膜母细胞瘤结合蛋白7(RBBP7)是一种具有肿瘤抑制作用的核蛋白,有研究表明RBBP7蛋白可以通过干扰正常的细胞周期和增加细胞凋亡进而抑制肿瘤细胞的转化。在本实验室的前期工作中,运用二维电泳和质谱鉴定等蛋白质组学技术建立了小鼠MII期(第二次减数分裂中期)卵细胞的蛋白表达谱。RBBP7就是在该谱系中鉴定得到的蛋白之一。且在激活后其含量下降,表明RBBP7可能在卵母细胞的发育成熟中发挥重要的作用。但RBBP7蛋白在小鼠卵细胞和早期胚胎中的功能研究尚属空白。本研究首先用免疫组化、免疫荧光等方法研究RBBP7在小鼠卵巢组织中的定位。免疫组织化学的结果显示RBBP7蛋白主要定位于各级卵母细胞的细胞核。免疫荧光结果显示RBBP7定位于GV期卵母细胞的细胞核,GVBD期时则弥漫分布于胞浆,MI期定位于纺锤体上,AI期定位于纺锤体的中体上,MII期与纺锤体共定位。为研究RBBP7蛋白在小鼠卵母细胞发育成熟过程中的作用,应用显微注射的方式将RBBP7 siRNA显微注射至GVBD期卵母细胞胞浆中,活细胞工作站观察卵母细胞的发育过程。结果发现:RBBP7蛋白的减少最终不会导致第一极体排放数目的异常,但是在第一极体的排放过程中,RBBP7蛋白的减少会导致卵母细胞胞质分裂的比率增高,同源染色体不分离或不均等分离的比率明显增高,最终分裂沟回缩,产生一个双核细胞,第一极体排放障碍。基于以上实验表型,接下来研究在小鼠卵母细胞中RBBP7影响第一次减数分裂过程中同源染色体分离的机制。应用小鼠卵巢全蛋白免疫共沉淀分析相互作用蛋白,结果显示RBBP7可以与VCP相互作用。VCP的作用是调节有丝分裂后期纺锤体的解聚。RBBP7特异定位于第一次减数分裂后期纺锤体的中体,因此推测RBBP7可能作为一种纺锤体组装因子,通过与VCP的相互作用,进而干扰第一次减数分裂后期纺锤体的解聚,从而影响同源染色体的精确分离,最终产生一个双核细胞。
[Abstract]:Retinoblastoma binding protein 7 (RBBP7) is a kind of nuclear protein with tumor inhibitory effect. It has been shown that RBBP7 protein can inhibit the transformation of tumor cells by interfering with normal cell cycle and increasing apoptosis.In the previous work of our laboratory, proteomic techniques such as two-dimensional electrophoresis and mass spectrometry were used to establish the protein expression profile of mouse MII stage (second meiotic metaphase). RBBP7 was one of the proteins identified in this pedigree.And its content decreased after activation, suggesting that RBBP7 may play an important role in the development and maturation of oocytes.But the function of RBBP7 protein in mouse oocyte and early embryo is still blank.In this study, the localization of RBBP7 in mouse ovary was studied by immunohistochemistry and immunofluorescence.Immunohistochemical results showed that the RBBP7 protein was mainly located in the nuclei of oocytes at all levels.The results of immunofluorescence showed that RBBP7 was located in the nucleus of GV oocytes in the GV phase, and distributed in the cytoplasmic MI phase, located in the upper midbody MII phase of the spindles and co-located with the spindles in the AI phase of the spindles.In order to study the role of RBBP7 protein in the development and maturation of mouse oocytes, RBBP7 siRNA was microinjected into the cytoplasm of GVBD oocytes by microinjection, and the development of oocytes was observed by living cell workstation.The results showed that the decrease of the 1 / RBBP7 protein did not eventually lead to abnormal emission of the first polar body, but the decrease of the RBBP7 protein during the discharge of the first polar body resulted in an increase in the rate of cytokinesis of the oocytes.The rate of homologous chromosome segregation or unequal segregation was significantly increased, and eventually the cleavage groove retracted, resulting in a binuclear cell, the first polar body emission barrier.Based on the above experimental phenotypes, the mechanism of homologous chromosome segregation in mouse oocytes affected by RBBP7 during the first meiosis was studied.The interaction proteins were analyzed by immunoprecipitation of mouse ovary whole protein. The results showed that RBBP7 could interact with VCP. The effect of VCP was to regulate the depolymerization of spindles in late mitotic stage. RBBP7 was specifically located in the midbody of the spindle in the late stage of the first meiosis.It is speculated that RBBP7, as a spindle assembly factor, may interfere with the depolymerization of the spindle in the later stage of meiosis by interacting with VCP, thus affecting the precise separation of homologous chromosomes and eventually producing a binuclear cell.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.7

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