本文關(guān)鍵詞：Eif2s3y促進(jìn)胚胎干細(xì)胞向生精細(xì)胞的分化　出處：《西北農(nóng)林科技大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 小鼠 胚胎干細(xì)胞 誘導(dǎo) 精細(xì)胞 Eif2s3y

【摘要】：受精卵的形成在多細(xì)胞動物中預(yù)示著一個新個體的產(chǎn)生,而兩性生殖細(xì)胞作為遺傳物質(zhì)的攜帶者,其正常發(fā)生不僅保證了物種穩(wěn)定性,且使得遺傳多樣性得以維持,更在一定程度上推動了物種的進(jìn)化。然而高等哺乳動物生殖細(xì)胞的發(fā)生完全在體內(nèi)進(jìn)行,因此,很難進(jìn)行實時動態(tài)地監(jiān)測和研究其中的分子事件。臨床和流行病學(xué)研究表明,全世界有13%-18%的夫婦患生殖障礙疾病,其中由于男性生殖細(xì)胞發(fā)生異常引起的不育癥呈急劇上升趨勢。因此,生殖細(xì)胞的體外發(fā)生就成為生命科學(xué)研究的重要課題之一。生殖細(xì)胞體外誘導(dǎo)體系的建立,不僅可以為生殖細(xì)胞發(fā)生機(jī)理的研究提供一個模型,而且能為人類生殖障礙疾病的治療提供一定的理論基礎(chǔ)和參考價值。在小鼠中,真核翻譯起始因子2,亞基3,Y染色體關(guān)聯(lián)的結(jié)構(gòu)基因(eukaryotic translation initiation factor 2,subunit 3,structural gene Y-linked,Eif2s3y)作為Y染色體短臂上的基因,在精子發(fā)生過程中的關(guān)鍵作用已經(jīng)被證實。在Y染色體短臂缺失的情況下,引入Eif2s3y不僅可以修復(fù)正常的精原細(xì)胞的增殖,而且還可以促進(jìn)其分化進(jìn)入減數(shù)分裂階段。在只有X染色體存在的情況下,僅Eif2s3y和Sry的共同作用就可以進(jìn)行正常的精子發(fā)生,并產(chǎn)生有功能的球形精子。在雄性小鼠中,Eif2s3y的突變會導(dǎo)致睪丸體積明顯變小,并伴隨無精癥的發(fā)生。雖然該基因的功能已經(jīng)得到證實,但是Eif2s3y如何促進(jìn)精子發(fā)生的調(diào)控機(jī)理需要進(jìn)一步探索。本研究利用細(xì)胞因子在體外實現(xiàn)小鼠胚胎干細(xì)胞(Mouseembryonic stem cell,mESC)經(jīng)原始生殖細(xì)胞樣細(xì)胞(Primordial germ cell-like cell,PGCLC)階段向精原干細(xì)胞樣細(xì)胞(Spermatogonia stem cell-like cell,SSCLC)分化并且完成減數(shù)分裂的全過程,并得到精細(xì)胞樣細(xì)胞(Spermatid-like cell,SLC)。進(jìn)一步研究證明,Eif2s3y基因不僅可以調(diào)控mESC的自我更新,而且可以提高SSCLC以及SLC的誘導(dǎo)效率。并且誘導(dǎo)獲得的SSCLC通過輸出管移植試驗,在小鼠體內(nèi)可以進(jìn)行正常的精子發(fā)生,并成功得到了轉(zhuǎn)Eif2s3y基因的健康小鼠后代。本研究的主要內(nèi)容如下:1.mESC向SLC體外誘導(dǎo)體系的建立模擬小鼠體內(nèi)雄性生殖細(xì)胞的形成過程,結(jié)合先前的研究成果,我們成功創(chuàng)建了mESC向SLC的體外誘導(dǎo)體系。首先利用Activin A和bFGF將mESC誘導(dǎo)為外胚層樣細(xì)胞(Epiblast-like cell,EpiLC),然后利用飼養(yǎng)層細(xì)胞結(jié)合細(xì)胞因子BMP4,BMP8A,SCF以及Insulin進(jìn)一步誘導(dǎo)分化為PGCLC。在PGCLC向SSCLC的誘導(dǎo)過程中,我們借助飼養(yǎng)層結(jié)合細(xì)胞因子GDNF,bFGF,BMP4,LIF,Insulin以及高濃度的KSR共同完成。為了驗證誘導(dǎo)所得細(xì)胞的生物學(xué)功能,我們將誘導(dǎo)得到的SSCLC移植入無精癥小鼠的曲細(xì)精管中。8周后采樣,通過HE切片發(fā)現(xiàn)該無精癥小鼠曲細(xì)精管內(nèi)充滿各級雄性生殖細(xì)胞,且附睪中充滿了成熟精子。為了使SSCLC體外實現(xiàn)減數(shù)分裂,本研究采用RA與低溫誘導(dǎo)相結(jié)合的策略,體外成功獲得了可以表達(dá)頂體蛋白的的單倍體細(xì)胞,而且通過流式細(xì)胞儀檢測,單倍體細(xì)胞所占的比例大約是9.8%。2.Eif2s3y降低mESC的多能性并促進(jìn)mESC的增殖鑒于Eif2s3y基因在精子發(fā)生過程中的重要作用,我們在誘導(dǎo)體系中引入了Eif2s3y基因并探究了其對mESC生物學(xué)特性的影響。試驗結(jié)果證明,Eif2s3y在降低mESC的多能性的同時提高其增殖效率。進(jìn)一步檢測證明,該基因主要是通過調(diào)控TET1以及組蛋白甲基化程度來影響mESC的多能性,而對增殖的影響主要是通過對周期蛋白Cyclin A和Cyclin E的調(diào)控實現(xiàn)的。3.Eif2s3y促進(jìn)SSC的分化我們對比了生精細(xì)胞、支持細(xì)胞以及精子中Eif2s3y的表達(dá)水平,發(fā)現(xiàn)生精細(xì)胞中Eif2s3y的表達(dá)水平最高。借助于在建系的小鼠精原干細(xì)胞系GC-1中超表達(dá)以及干擾Eif2s3y基因,我們發(fā)現(xiàn)Eif2s3y的超表達(dá)抑制了維持SSC自我更新相關(guān)的基因Plzf和Gfrα1的表達(dá),同時上調(diào)了減數(shù)分裂起始因子Stra8和Sycp3的表達(dá)。另外,Eif2s3y的敲低下調(diào)了Stra8和Sycp3的表達(dá)。這些結(jié)果表明,Eif2s3y能夠促進(jìn)SSC的分化。這為我們后續(xù)的試驗奠定了良好的基礎(chǔ)。4.Eif2s3y提高mESC向SLC的誘導(dǎo)效率Eif2s3y的引入對mESC向SLC誘導(dǎo)過程起到了很大的推進(jìn)作用。我們的研究結(jié)果表明,Eif2s3y可以促進(jìn)SSCLC中DAZL、NGN3以及VASA的表達(dá),即促進(jìn)SSCLC的分化。通過流式細(xì)胞儀檢測發(fā)現(xiàn),SSCLC-Eif2s3y中VASA陽性細(xì)胞約為13.3%,而對照組為7.95%左右,這表明Eif2s3y推動了mESC向SSCLC的誘導(dǎo)進(jìn)程。在進(jìn)一步向SLC的誘導(dǎo)中,Eif2s3y可以提高VASA、SYCP3、Acrosin以及PRM1的表達(dá)。細(xì)胞周期分析結(jié)果顯示,Eif2s3y超表達(dá)的細(xì)胞群體中單倍體的比例約為11%,而對照組約為8.6%。說明Eif2s3y不僅促進(jìn)PGCLC向SSCLC的誘導(dǎo)分化,而且明顯提高了mESC向SLC的誘導(dǎo)效率。綜上所述,本研究利用明確的細(xì)胞因子建立了mESC向SLC的體外誘導(dǎo)體系,并證明了Eif2s3y在整個誘導(dǎo)過程中起到顯著的促進(jìn)作用。這一誘導(dǎo)體系的建立,不僅為哺乳動物雄性生殖細(xì)胞發(fā)生機(jī)理的研究提供了一個良好的模型,而且為男性不育癥的臨床治療提供了一定的理論基礎(chǔ)。
[Abstract]:The formation of fertilized eggs in multicellular animal indicates that produces a new individual, and sexual cells as carriers of genetic material, which not only ensures the stability of species normally occur, and the genetic diversity is maintained, even to a certain extent promoted the evolution of species. However, germ cells occurs entirely in mammals in vivo, therefore, it is difficult to monitor and study the real-time dynamic molecular events. The clinical and epidemiological studies show that the whole world 13%-18% of couples with reproductive disorders, because of the male germ cell abnormal infertility due to increased rapidly. Therefore, the germ cells in vitro has become one of the important subjects of life scientific research. Establishment of induction system of germ cells in vitro, provide a model for the study can not only the mechanism of germ cells, It can provide a theoretical basis and reference value for the treatment of human reproductive disorders. In mice, eukaryotic translation initiation factor 2, subunit 3, Y chromosome structural gene association (eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked, Eif2s3y) as Y on the short arm of chromosome genes. In the key role in the process of spermatogenesis has been confirmed. The deletion of the short arm of chromosome Y cases, the introduction of Eif2s3y can not only repair the normal spermatogonial proliferation, but also can promote their differentiation into meiosis stage. In the presence of only X chromosomes, the interaction is only Eif2s3y and Sry can for normal spermatogenesis, sperm and produce spherical function. In male mice, Eif2s3y mutations can lead to testicular volume was significantly smaller, with azoospermia. Although the base Because of the function of Eif2s3y has been confirmed, but how to promote the regulation mechanism of spermatogenesis needs further exploration. This study utilizes cytokines in vitro implementation of mouse embryonic stem cells (Mouseembryonic stem cell, mESC) by primordial germ cells (Primordial germ cell-like cell, PGCLC) phase to spermatogonial stem like cells (Spermatogonia stem cell-like cell, SSCLC) differentiation and complete the whole process of the meiosis and fine cell like cells (Spermatid-like cell, SLC). Further studies have shown that Eif2s3y gene can not only regulate mESC self-renewal, and can improve the efficiency of induction SSCLC and SLC. And obtained by SSCLC through output tube grafting test in mice the body can be normal spermatogenesis, and successfully obtained Eif2s3y transgenic mice and healthy offspring. The main contents of this study are as follows: 1 .mESC SLC in vitro induction system to simulate the male germ cells of mice formation, with previous research results, we successfully established the mESC system to induce SLC in vitro. We use Activin A and bFGF mESC will be induced into ectoderm like cells (Epiblast-like cell, EpiLC), and then use the feeder cells with cells BMP8A, factor BMP4, SCF and Insulin further differentiate into PGCLC. to induce the process of SSCLC in PGCLC, we use the feeder cells in combination with factor GDNF, bFGF, BMP4, LIF, Insulin and high concentration of KSR is completed. In order to verify the biological function of induced cells, we induced SSCLC transplanted into song fine azoospermia mice in.8 weeks after sampling, through the HE section found the azoospermia mouse seminiferous tubules with all levels of male germ cells, and full of mature sperm in the epididymis. In order to make the implementation of SSCLC in vitro meiosis, this study adopts RA and low temperature induced by the combination of strategy, success can be expressed in vitro acrosome proteins of haploid cells, and by flow cytometry, proportion of haploid cells ratio is about 9.8%.2.Eif2s3y lower mESC pluripotency and promote the proliferation of mESC in Eif2s3y gene in spermatogenesis an important role in the process, we introduced the system in the induction of Eif2s3y gene and explore its effect on the biological characteristics of mESC. Experimental results show that Eif2s3y can reduce mESC in the more at the same time improve the proliferation efficiency. Further analysis showed that this gene is mainly regulated by TET1 and group protein methylation affects the pluripotent nature of mESC, and the effect on proliferation is mainly through the regulation of cyclin Cyclin A and Cyclin E to achieve the.3.Eif2s3y promote SSC points We compared the spermatogenic cells, Sertoli cells and the expression level of sperm Eif2s3y, found that the expression level of Eif2s3y in sperm cells. With the help of the highest fine lines in mouse spermatogonial stem cell line and the expression of GC-1 in the interference of Eif2s3y gene, we found that Eif2s3y overexpression inhibited the expression of SSC related to maintain self-renewal gene Plzf and Gfr alpha 1, also up-regulated the expression of meiosis initiation factor Stra8 and Sycp3. In addition, Eif2s3y knockdown reduced the expression of Stra8 and Sycp3. These results indicate that Eif2s3y can promote the differentiation of SSC. This has laid the foundation to improve the.4.Eif2s3y good mESC SLC into the Eif2s3y on the induction efficiency mESC to SLC induced process has played a great role in promoting. The test results of our study suggest that Eif2s3y can promote the SSCLC in DAZL, NGN3 and VASA expression, namely to promote SSCLC Differentiation by flow cytometry showed that VASA positive cells was about 13.3% SSCLC-Eif2s3y, while the control group was 7.95%, which indicated that Eif2s3y promoted mESC induced SSCLC process. Further to the induction of SLC, Eif2s3y SYCP3, Acrosin VASA can be improved, and the expression of PRM1. The results of cell cycle analysis show that the haploid Eif2s3y over expression in the cell population ratio is about 11%, while the control group is about 8.6%. Eif2s3y not only promote PGCLC to induce differentiation of SSCLC, but also improves the efficiency of mESC differentiating into SLC. In summary, this study clearly established using cytokines induced by mESC system to SLC in vitro, and that Eif2s3y play a significant role in the process of induction. The induction system, provides a good model to study not only for mammalian male germ cell genesis mechanism It provides a theoretical basis for the clinical treatment of male infertility.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q132

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