猪诱导多能干细胞向雄性生殖细胞样细胞诱导分化的研究

发布时间：2018-08-14 20:27

【摘要】：生殖细胞的分化与发育是一个复杂而有序的过程。建立体外诱导形成生殖细胞的技术方法,对治疗不孕不育、动物优良品种资源的保存及生殖细胞发育机制的研究有重要意义。小鼠和人多能干细胞定向诱导为雄性生殖细胞的研究较为成熟。然而,由于小鼠寿命短等不足之处,人的生殖细胞相关功能探究无法在小鼠体内长期观察;因受到伦理限制也无法直接进行人的体内实验。因此,需要大动物(如猪)模型为医学提供技术及安全性评价体系。与小鼠相比,猪的寿命较长、生理学和解剖学等与人更接近,无苛刻的伦理限制,这使得猪成为医学研究的理想动物模型。另外,猪也是我国畜牧业中重要的家畜之一。目前,猪多能干细胞的相关研究亟待进一步发展,在猪多能干细胞向生殖细胞诱导方面,尚未有相关的报道。因此,本研究的重点为:建立猪诱导多能干细胞(iPSCs)系,进而建立猪ipSCs向雄性生殖细胞样细胞定向分化的诱导体系和技术方法。首先,本研究建立了猪iPSCs系。其形态类似于小鼠胚胎干细胞的形态,呈致密凸起的克隆;该iPSCs的碱性磷酸酶染色呈阳性,并表达OCT4、SOX2、SSEA-1等多能性标记蛋白;在体外,猪iPSCs可以通过拟胚体随机分化为三胚层类型细胞,并可以定向分化为脂类和神经类细胞;在免疫缺陷小鼠体内,猪iPSCs可以形成畸胎瘤,并分化为三胚层类型组织。这说明本研究中的猪iPSCs具有定向分化为生殖细胞的潜力。其次,根据猪iPSCs的特性,借鉴小鼠和人原始生殖细胞样细胞(PGCLCs)诱导方法并进行改进和优化,建立猪生殖细胞样细胞诱导分化的体系及方法。第一,在含有Activin A等细胞因子的培养体系中,将猪iPSCs分化为上胚层干细胞样细胞(EpiLCs),EpiLCs高表达上胚层干细胞标记基因。第二,在含有BMP4等细胞因子的诱导体系中,将EpiLCs定向诱导分化为PGCLCs。PGCLCs高表达原始生殖细胞(PGCs)标记基因Stella等,同时表达PGCs标记蛋白STELLA等;在表观遗传特性方面,PGCLCs中H3K27me3表达上调,H3K9me2表达下调,印记基因DNA甲基化水平呈下降趋势,这与体内PGCs的发育相类似;在转录组水平上,PGCLCs高表达与生殖细胞发育相关的基因。第三,PGCLCs在含有睾酮等试剂的诱导体系中,进一步分化为精原干细胞样细胞(SSCLCs)。SSCLCs表达Stra8、Gsg2等减数分裂标记基因,同时表达DAZL、VASA等精原干细胞标记蛋白;通过流式细胞术检测到SSCLCs中含有单倍体细胞。这些结果表明,本诱导体系可以使猪iPSCs定向诱导分化为雄性生殖细胞样细胞。目前,还没有建立良好的生精细胞损伤不育公猪模型,而生精细胞损伤不育小鼠模型的建立方法及其曲细精管注射技术较成熟,因此选择小鼠作为体内研究的受体。将猪的PGCLCs和SSCLCs移植到小鼠曲细精管中,移植后在不同时间段进行检测,结果显示猪生殖细胞样细胞在曲细精管中形成细胞链和细胞簇。免疫荧光染色结果显示,生殖细胞标记蛋白DAZL、VASA等呈阳性。这说明移植后的细胞具有生殖细胞特性,可能有助于修复生精细胞损伤的睾丸组织。综上所述,本研究得到形态类似于小鼠胚胎干细胞的猪iPSCs系。在此基础上,我们建立了一种将猪iPSCs诱导成雄性生殖细胞样细胞的方法。该研究为进一步探索生殖细胞的发育机制和临床上人多能干细胞向生殖细胞的分化奠定了基础,也为畜牧业育种提供了新的思路。
[Abstract]:Differentiation and development of germ cells is a complex and orderly process. Establishment of a method for inducing germ cells in vitro is of great significance in the treatment of infertility, preservation of fine animal breeds and study of the mechanism of germ cell development. However, due to the short life span of mice and other deficiencies, human reproductive cell related functions can not be observed in vivo for a long time. Due to ethical restrictions, it is not possible to directly carry out human * vivo experiments. Therefore, large animals (such as pig *) models are needed to provide technology and safety evaluation system for medicine. Compared with mice, pigs have long life expectancy. Physiology and anatomy are closely related to human beings, and there is no harsh ethical restriction. * * the pig has become an ideal animal model for medical research. In addition, pig is also one of the important livestock in China's livestock industry. *, at present, the research on pig pluripotent stem cells needs further development, and there is no correlation between porcine pluripotent stem cells and germ cells induction. Therefore, the focus of this study is to establish a pig induced pluripotent stem cell (iPSCs * *) system and further establish the induction system and technical method for directional differentiation of porcine ipSCs into male germ cell like cells. First, * the pig iPSCs line was established in this study, which resembles the morphology of mouse embryonic stem cells, showing a dense convex clone, and the iPSCs base. Expression of OCT4, SOX2, SSEA-1 and other pluripotent labelled proteins were positive in vitro. * in vitro, pig iPSCs could be randomly differentiated into three germ layer type cells through embryoid bodies, and could differentiate into lipid and neural cells. In immunodeficient mice, pig * iPSCs could form teratoma and differentiate into three germ layer type tissues. It indicates that the pig iPSCs has the potential to differentiate into germ cells in the * * *. Secondly, according to the characteristics of pig iPSCs, we can learn from mouse and human primordial germ cells * (PGCLCs) induction methods and improve and optimize them, and establish the system and methods of inducing differentiation of porcine germ cell like cells. First, in the presence of cytokines such as Activin A, etc. In the culture system, the porcine iPSCs was differentiated into EpiLCs *, and EpiLCs expressed the gene of the upper embryonic stem cells. Second, in the induction system containing cytokines such as BMP4, EpiLCs was induced to differentiate into PGCLCs.PGCLCs high expression primordial germ cell (PGCs) marker gene Stella, and PGCs expression protein S was expressed at the same time. In terms of epigenetic characteristics, the expression of H3K27me3 was up-regulated, the expression of H3K9me2 was down-regulated, and the level of DNA methylation of imprinted gene was down-regulated, which was similar to the development of PGCs in vivo. Further differentiated into spermatogonial stem like cells (SSCLCs),.SSCLCs expressed Stra8, Gsg2 and other meiotic marker genes, and expressed DAZL, VASA and other spermatogonial stem cell marker proteins. Flow cytometry detected SSCLCs containing haploid cells. These results showed that the * induced system could induce pig iPSCs to differentiate into male reproduction. At present, there is no established model of spermatogenic cell injury male sterility boar *, and the establishment method of infertile mouse model with spermatogenic cell injury and the injection technique of seminiferous tubules are relatively mature. Therefore, * mice are selected as the recipients of in vivo studies. The porcine PGCLCs and SSCLCs are transplanted into the seminiferous tubules of mice, and after transplantation, they are different. The results showed that porcine germ cells like * cells formed cell chains and clusters in the seminiferous tubules. Immunofluorescence staining showed that DAZL and VASA were positive. This indicates that the transplanted cells have germ cell characteristics and may help repair testicular tissue damaged by spermatogenic cells. In this study, we obtained a porcine iPSCs strain similar to mouse embryonic stem cells. Based on this * *, we established a method to induce porcine iPSCs into male germ cell like cells. This study laid the foundation for further exploring the developmental mechanism of germ cells and the differentiation of human pluripotent stem cells into germ cells in clinic. Animal husbandry breeding provides a new way of thinking.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q813

【参考文献】