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

  更多相關文章： 小鼠 胚胎干細胞 誘導 精細胞 Eif2s3y

【摘要】：受精卵的形成在多細胞動物中預示著一個新個體的產(chǎn)生,而兩性生殖細胞作為遺傳物質的攜帶者,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化。然而高等哺乳動物生殖細胞的發(fā)生完全在體內(nèi)進行,因此,很難進行實時動態(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胞發(fā)生異常引起的不育癥呈急劇上升趨勢。因此,生殖細胞的體外發(fā)生就成為生命科學研究的重要課題之一。生殖細胞體外誘導體系的建立,不僅可以為生殖細胞發(fā)生機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染色體關聯(lián)的結構基因(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不僅可以修復正常的精原細胞的增殖,而且還可以促進其分化進入減數(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如何促進精子發(fā)生的調控機理需要進一步探索。本研究利用細胞因子在體外實現(xiàn)小鼠胚胎干細胞(Mouseembryonic stem cell,mESC)經(jīng)原始生殖細胞樣細胞(Primordial germ cell-like cell,PGCLC)階段向精原干細胞樣細胞(Spermatogonia stem cell-like cell,SSCLC)分化并且完成減數(shù)分裂的全過程,并得到精細胞樣細胞(Spermatid-like cell,SLC)。進一步研究證明,Eif2s3y基因不僅可以調控mESC的自我更新,而且可以提高SSCLC以及SLC的誘導效率。并且誘導獲得的SSCLC通過輸出管移植試驗,在小鼠體內(nèi)可以進行正常的精子發(fā)生,并成功得到了轉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體外誘導體系的建立模擬小鼠體內(nèi)雄性生殖細胞的形成過程,結合先前的研究成果,我們成功創(chuàng)建了mESC向SLC的體外誘導體系。首先利用Activin A和bFGF將mESC誘導為外胚層樣細胞(Epiblast-like cell,EpiLC),然后利用飼養(yǎng)層細胞結合細胞因子BMP4,BMP8A,SCF以及Insulin進一步誘導分化為PGCLC。在PGCLC向SSCLC的誘導過程中,我們借助飼養(yǎng)層結合細胞因子GDNF,bFGF,BMP4,LIF,Insulin以及高濃度的KSR共同完成。為了驗證誘導所得細胞的生物學功能,我們將誘導得到的SSCLC移植入無精癥小鼠的曲細精管中。8周后采樣,通過HE切片發(fā)現(xiàn)該無精癥小鼠曲細精管內(nèi)充滿各級雄性生殖細胞,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與低溫誘導相結合的策略,體外成功獲得了可以表達頂體蛋白的的單倍體細胞,而且通過流式細胞儀檢測,單倍體細胞所占的比例大約是9.8%。2.Eif2s3y降低mESC的多能性并促進mESC的增殖鑒于Eif2s3y基因在精子發(fā)生過程中的重要作用,我們在誘導體系中引入了Eif2s3y基因并探究了其對mESC生物學特性的影響。試驗結果證明,Eif2s3y在降低mESC的多能性的同時提高其增殖效率。進一步檢測證明,該基因主要是通過調控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的調控實現(xiàn)的。3.Eif2s3y促進SSC的分化我們對比了生精細胞、支持細胞以及精子中Eif2s3y的表達水平,發(fā)現(xiàn)生精細胞中Eif2s3y的表達水平最高。借助于在建系的小鼠精原干細胞系GC-1中超表達以及干擾Eif2s3y基因,我們發(fā)現(xiàn)Eif2s3y的超表達抑制了維持SSC自我更新相關的基因Plzf和Gfrα1的表達,同時上調了減數(shù)分裂起始因子Stra8和Sycp3的表達。另外,Eif2s3y的敲低下調了Stra8和Sycp3的表達。這些結果表明,Eif2s3y能夠促進SSC的分化。這為我們后續(xù)的試驗奠定了良好的基礎。4.Eif2s3y提高mESC向SLC的誘導效率Eif2s3y的引入對mESC向SLC誘導過程起到了很大的推進作用。我們的研究結果表明,Eif2s3y可以促進SSCLC中DAZL、NGN3以及VASA的表達,即促進SSCLC的分化。通過流式細胞儀檢測發(fā)現(xiàn),SSCLC-Eif2s3y中VASA陽性細胞約為13.3%,而對照組為7.95%左右,這表明Eif2s3y推動了mESC向SSCLC的誘導進程。在進一步向SLC的誘導中,Eif2s3y可以提高VASA、SYCP3、Acrosin以及PRM1的表達。細胞周期分析結果顯示,Eif2s3y超表達的細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PGCLC向SSCLC的誘導分化,而且明顯提高了mESC向SLC的誘導效率。綜上所述,本研究利用明確的細胞因子建立了mESC向SLC的體外誘導體系,并證明了Eif2s3y在整個誘導過程中起到顯著的促進作用。這一誘導體系的建立,不僅為哺乳動物雄性生殖細胞發(fā)生機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。
[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.

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

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