Aszl促进胚胎干细胞向原始生殖细胞分化的分子机制研究
发布时间：2019-06-09 12:28
【摘要】：在哺乳动物中,生殖细胞及其终末分化形成的精子和卵子均源于原始生殖细胞(Primordial Germ Cell,PGC),因此阐明PGC发生分化过程及其机制,对研究生殖细胞的发育过程有着重要的意义。PGC来自于早期胚胎,数量少,不易获得,并且利用胚胎进行研究会引起伦理道德等诸多方面的问题。而近年干细胞研究的飞速发展,给发育生物学研究领域带来了曙光。胚胎干细胞在适当的条件下可以自然分化或人为地诱导分化生成各种性能的细胞,包括生殖细胞。 我们在以往研究成果的基础上,完善生殖发育的研究体系,借助拟胚体作为分化系统,用SSEA1和AP染色等方法对一大批与生殖发育相关的基因进行筛选。研究发现,Asz1在胚胎干细胞向生殖细胞分化的过程中起着促进作用。本课题对于Asz1的作用机理进行了深入的研究。 我们通过免疫荧光实验发现Asz1基因产物特异性地定位于线粒体中。进一步研究发现Asz1由ANK, SAM, ZIP几个部分组成,而其C端信号序列对其定位起着至关重要的作用,并且ASZ1线粒体定位是该基因发挥促进PGC生成功能必不可少的。另外,我们利用质谱分析的方法寻找Asz1的作用伴侣,并通过免疫共沉淀和Western-blot的进一步验证了Asz1与Daz1的相互作用,揭示了ASZ1通过SAM结构域与DAZL相结合,双分子荧光互补(bimolecular fluorescence complementation, BiFC)分析技术揭示了二者在线粒体的结合。同时,我们在Asz1过表达的细胞中对Daz1进行knowdown,发现Asz1在Daz1减少时不能促进PGC的形成,表明ASZ1是通过与DAZL基因的相互结合发挥其促进PGC生成的作用。我们在DAZL过表达的细胞系中同时过表达ASZ1基因,发现ASZ1和DAZL可以协同作用发挥促进PGC生成的作用。 综上所述,ASZ1基因的过表达可以特异地促进胚胎干细胞向原始生殖细胞的分化。ASZ1基因特异地定位于线粒体中,并且与其功能的发挥密不可分。ASZ1可以与DAZL相互结合并协同DAZL基因促进PGC的生成。
[Abstract]:In mammals, spermatozoa and eggs formed by germ cells and their terminal differentiation originate from primordial germ cells (Primordial Germ Cell,PGC), so the process of differentiation of PGC and its mechanism are clarified. PGC comes from early embryos, which is small in number and difficult to obtain, and the use of embryos for research will lead to many problems, such as ethics and morality, etc., PGC comes from early embryos, the number of PGC is small, it is not easy to obtain, and the use of embryos for research will cause ethical and moral problems. In recent years, the rapid development of stem cell research has brought dawn to the field of developmental biology. Embryonic stem cells can differentiate naturally or artificially into cells of various properties, including germ cells, under appropriate conditions. On the basis of the previous research results, we improved the research system of reproductive development, and screened a large number of genes related to reproductive development by SSEA1 and AP staining with the help of embryoid as differentiation system. It is found that Asz1 plays an important role in the differentiation of embryonic stem cells into germ cells. In this paper, the mechanism of Asz1 is deeply studied. We found that Asz1 gene products were specifically located in mitochondria by immunofluorescence assay. It is found that Asz1 is composed of several parts of ANK, SAM, ZIP, and its C-terminal signal sequence plays an important role in its localization, and ASZ1 mtDNA localization is essential for the gene to promote PGC generation. In addition, we used mass spectrometry to find the chaperone of Asz1, and further verified the interaction between Asz1 and Daz1 by immunoprecipitation and Western-blot, and revealed that ASZ1 was combined with DAZL through SAM domain. Bimolecule fluorescence complementary (bimolecular fluorescence complementation, BiFC) analysis revealed the binding of the two in mitochondria. At the same time, we knowdown, Daz1 in cells overexpressed by Asz1. It was found that Asz1 could not promote the formation of PGC when Daz1 decreased, indicating that ASZ1 played an important role in promoting PGC production by binding with DAZL gene. We overexpressed ASZ1 gene in DAZL overexpressed cell lines at the same time. It was found that ASZ1 and DAZL could play a synergistic role in promoting PGC production. In conclusion, the overexpression of ASZ1 gene can specifically promote the differentiation of embryonic stem cells into primordial germ cells. ASZ1 gene is specifically located in mitochondria. ASZ1 can combine with DAZL and cooperate with DAZL gene to promote PGC generation.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
[Abstract]:In mammals, spermatozoa and eggs formed by germ cells and their terminal differentiation originate from primordial germ cells (Primordial Germ Cell,PGC), so the process of differentiation of PGC and its mechanism are clarified. PGC comes from early embryos, which is small in number and difficult to obtain, and the use of embryos for research will lead to many problems, such as ethics and morality, etc., PGC comes from early embryos, the number of PGC is small, it is not easy to obtain, and the use of embryos for research will cause ethical and moral problems. In recent years, the rapid development of stem cell research has brought dawn to the field of developmental biology. Embryonic stem cells can differentiate naturally or artificially into cells of various properties, including germ cells, under appropriate conditions. On the basis of the previous research results, we improved the research system of reproductive development, and screened a large number of genes related to reproductive development by SSEA1 and AP staining with the help of embryoid as differentiation system. It is found that Asz1 plays an important role in the differentiation of embryonic stem cells into germ cells. In this paper, the mechanism of Asz1 is deeply studied. We found that Asz1 gene products were specifically located in mitochondria by immunofluorescence assay. It is found that Asz1 is composed of several parts of ANK, SAM, ZIP, and its C-terminal signal sequence plays an important role in its localization, and ASZ1 mtDNA localization is essential for the gene to promote PGC generation. In addition, we used mass spectrometry to find the chaperone of Asz1, and further verified the interaction between Asz1 and Daz1 by immunoprecipitation and Western-blot, and revealed that ASZ1 was combined with DAZL through SAM domain. Bimolecule fluorescence complementary (bimolecular fluorescence complementation, BiFC) analysis revealed the binding of the two in mitochondria. At the same time, we knowdown, Daz1 in cells overexpressed by Asz1. It was found that Asz1 could not promote the formation of PGC when Daz1 decreased, indicating that ASZ1 played an important role in promoting PGC production by binding with DAZL gene. We overexpressed ASZ1 gene in DAZL overexpressed cell lines at the same time. It was found that ASZ1 and DAZL could play a synergistic role in promoting PGC production. In conclusion, the overexpression of ASZ1 gene can specifically promote the differentiation of embryonic stem cells into primordial germ cells. ASZ1 gene is specifically located in mitochondria. ASZ1 can combine with DAZL and cooperate with DAZL gene to promote PGC generation.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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