Eif2s3y促进胚胎干细胞向生精细胞的分化

发布时间：2018-01-04 16:40

  本文关键词：Eif2s3y促进胚胎干细胞向生精细胞的分化　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 小鼠 胚胎干细胞 诱导 精细胞 Eif2s3y

【摘要】：受精卵的形成在多细胞动物中预示着一个新个体的产生,而两性生殖细胞作为遗传物质的携带者,其正常发生不仅保证了物种稳定性,且使得遗传多样性得以维持,更在一定程度上推动了物种的进化。然而高等哺乳动物生殖细胞的发生完全在体内进行,因此,很难进行实时动态地监测和研究其中的分子事件。临床和流行病学研究表明,全世界有13%-18%的夫妇患生殖障碍疾病,其中由于男性生殖细胞发生异常引起的不育症呈急剧上升趋势。因此,生殖细胞的体外发生就成为生命科学研究的重要课题之一。生殖细胞体外诱导体系的建立,不仅可以为生殖细胞发生机理的研究提供一个模型,而且能为人类生殖障碍疾病的治疗提供一定的理论基础和参考价值。在小鼠中,真核翻译起始因子2,亚基3,Y染色体关联的结构基因(eukaryotic translation initiation factor 2,subunit 3,structural gene Y-linked,Eif2s3y)作为Y染色体短臂上的基因,在精子发生过程中的关键作用已经被证实。在Y染色体短臂缺失的情况下,引入Eif2s3y不仅可以修复正常的精原细胞的增殖,而且还可以促进其分化进入减数分裂阶段。在只有X染色体存在的情况下,仅Eif2s3y和Sry的共同作用就可以进行正常的精子发生,并产生有功能的球形精子。在雄性小鼠中,Eif2s3y的突变会导致睾丸体积明显变小,并伴随无精症的发生。虽然该基因的功能已经得到证实,但是Eif2s3y如何促进精子发生的调控机理需要进一步探索。本研究利用细胞因子在体外实现小鼠胚胎干细胞(Mouseembryonic stem cell,mESC)经原始生殖细胞样细胞(Primordial germ cell-like cell,PGCLC)阶段向精原干细胞样细胞(Spermatogonia stem cell-like cell,SSCLC)分化并且完成减数分裂的全过程,并得到精细胞样细胞(Spermatid-like cell,SLC)。进一步研究证明,Eif2s3y基因不仅可以调控mESC的自我更新,而且可以提高SSCLC以及SLC的诱导效率。并且诱导获得的SSCLC通过输出管移植试验,在小鼠体内可以进行正常的精子发生,并成功得到了转Eif2s3y基因的健康小鼠后代。本研究的主要内容如下:1.mESC向SLC体外诱导体系的建立模拟小鼠体内雄性生殖细胞的形成过程,结合先前的研究成果,我们成功创建了mESC向SLC的体外诱导体系。首先利用Activin A和bFGF将mESC诱导为外胚层样细胞(Epiblast-like cell,EpiLC),然后利用饲养层细胞结合细胞因子BMP4,BMP8A,SCF以及Insulin进一步诱导分化为PGCLC。在PGCLC向SSCLC的诱导过程中,我们借助饲养层结合细胞因子GDNF,bFGF,BMP4,LIF,Insulin以及高浓度的KSR共同完成。为了验证诱导所得细胞的生物学功能,我们将诱导得到的SSCLC移植入无精症小鼠的曲细精管中。8周后采样,通过HE切片发现该无精症小鼠曲细精管内充满各级雄性生殖细胞,且附睾中充满了成熟精子。为了使SSCLC体外实现减数分裂,本研究采用RA与低温诱导相结合的策略,体外成功获得了可以表达顶体蛋白的的单倍体细胞,而且通过流式细胞仪检测,单倍体细胞所占的比例大约是9.8%。2.Eif2s3y降低mESC的多能性并促进mESC的增殖鉴于Eif2s3y基因在精子发生过程中的重要作用,我们在诱导体系中引入了Eif2s3y基因并探究了其对mESC生物学特性的影响。试验结果证明,Eif2s3y在降低mESC的多能性的同时提高其增殖效率。进一步检测证明,该基因主要是通过调控TET1以及组蛋白甲基化程度来影响mESC的多能性,而对增殖的影响主要是通过对周期蛋白Cyclin A和Cyclin E的调控实现的。3.Eif2s3y促进SSC的分化我们对比了生精细胞、支持细胞以及精子中Eif2s3y的表达水平,发现生精细胞中Eif2s3y的表达水平最高。借助于在建系的小鼠精原干细胞系GC-1中超表达以及干扰Eif2s3y基因,我们发现Eif2s3y的超表达抑制了维持SSC自我更新相关的基因Plzf和Gfrα1的表达,同时上调了减数分裂起始因子Stra8和Sycp3的表达。另外,Eif2s3y的敲低下调了Stra8和Sycp3的表达。这些结果表明,Eif2s3y能够促进SSC的分化。这为我们后续的试验奠定了良好的基础。4.Eif2s3y提高mESC向SLC的诱导效率Eif2s3y的引入对mESC向SLC诱导过程起到了很大的推进作用。我们的研究结果表明,Eif2s3y可以促进SSCLC中DAZL、NGN3以及VASA的表达,即促进SSCLC的分化。通过流式细胞仪检测发现,SSCLC-Eif2s3y中VASA阳性细胞约为13.3%,而对照组为7.95%左右,这表明Eif2s3y推动了mESC向SSCLC的诱导进程。在进一步向SLC的诱导中,Eif2s3y可以提高VASA、SYCP3、Acrosin以及PRM1的表达。细胞周期分析结果显示,Eif2s3y超表达的细胞群体中单倍体的比例约为11%,而对照组约为8.6%。说明Eif2s3y不仅促进PGCLC向SSCLC的诱导分化,而且明显提高了mESC向SLC的诱导效率。综上所述,本研究利用明确的细胞因子建立了mESC向SLC的体外诱导体系,并证明了Eif2s3y在整个诱导过程中起到显著的促进作用。这一诱导体系的建立,不仅为哺乳动物雄性生殖细胞发生机理的研究提供了一个良好的模型,而且为男性不育症的临床治疗提供了一定的理论基础。
[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.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q132

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