【摘要】：在哺乳動物中,生殖細(xì)胞及其終末分化形成的精子和卵子均源于原始生殖細(xì)胞(Primordial Germ Cell,PGC),因此闡明PGC發(fā)生分化過程及其機(jī)制,對研究生殖細(xì)胞的發(fā)育過程有著重要的意義。PGC來自于早期胚胎,數(shù)量少,不易獲得,并且利用胚胎進(jìn)行研究會引起倫理道德等諸多方面的問題。而近年干細(xì)胞研究的飛速發(fā)展,給發(fā)育生物學(xué)研究領(lǐng)域帶來了曙光。胚胎干細(xì)胞在適當(dāng)?shù)臈l件下可以自然分化或人為地誘導(dǎo)分化生成各種性能的細(xì)胞,包括生殖細(xì)胞。 我們在以往研究成果的基礎(chǔ)上,完善生殖發(fā)育的研究體系,借助擬胚體作為分化系統(tǒng),用SSEA1和AP染色等方法對一大批與生殖發(fā)育相關(guān)的基因進(jìn)行篩選。研究發(fā)現(xiàn),Asz1在胚胎干細(xì)胞向生殖細(xì)胞分化的過程中起著促進(jìn)作用。本課題對于Asz1的作用機(jī)理進(jìn)行了深入的研究。 我們通過免疫熒光實(shí)驗(yàn)發(fā)現(xiàn)Asz1基因產(chǎn)物特異性地定位于線粒體中。進(jìn)一步研究發(fā)現(xiàn)Asz1由ANK, SAM, ZIP幾個(gè)部分組成,而其C端信號序列對其定位起著至關(guān)重要的作用,并且ASZ1線粒體定位是該基因發(fā)揮促進(jìn)PGC生成功能必不可少的。另外,我們利用質(zhì)譜分析的方法尋找Asz1的作用伴侶,并通過免疫共沉淀和Western-blot的進(jìn)一步驗(yàn)證了Asz1與Daz1的相互作用,揭示了ASZ1通過SAM結(jié)構(gòu)域與DAZL相結(jié)合,雙分子熒光互補(bǔ)(bimolecular fluorescence complementation, BiFC)分析技術(shù)揭示了二者在線粒體的結(jié)合。同時(shí),我們在Asz1過表達(dá)的細(xì)胞中對Daz1進(jìn)行knowdown,發(fā)現(xiàn)Asz1在Daz1減少時(shí)不能促進(jìn)PGC的形成,表明ASZ1是通過與DAZL基因的相互結(jié)合發(fā)揮其促進(jìn)PGC生成的作用。我們在DAZL過表達(dá)的細(xì)胞系中同時(shí)過表達(dá)ASZ1基因,發(fā)現(xiàn)ASZ1和DAZL可以協(xié)同作用發(fā)揮促進(jìn)PGC生成的作用。 綜上所述,ASZ1基因的過表達(dá)可以特異地促進(jìn)胚胎干細(xì)胞向原始生殖細(xì)胞的分化。ASZ1基因特異地定位于線粒體中,并且與其功能的發(fā)揮密不可分。ASZ1可以與DAZL相互結(jié)合并協(xié)同DAZL基因促進(jìn)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.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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