【摘要】：生殖細(xì)胞的分化與發(fā)育是一個復(fù)雜而有序的過程。建立體外誘導(dǎo)形成生殖細(xì)胞的技術(shù)方法,對治療不孕不育、動物優(yōu)良品種資源的保存及生殖細(xì)胞發(fā)育機制的研究有重要意義。小鼠和人多能干細(xì)胞定向誘導(dǎo)為雄性生殖細(xì)胞的研究較為成熟。然而,由于小鼠壽命短等不足之處,人的生殖細(xì)胞相關(guān)功能探究無法在小鼠體內(nèi)長期觀察;因受到倫理限制也無法直接進行人的體內(nèi)實驗。因此,需要大動物(如豬)模型為醫(yī)學(xué)提供技術(shù)及安全性評價體系。與小鼠相比,豬的壽命較長、生理學(xué)和解剖學(xué)等與人更接近,無苛刻的倫理限制,這使得豬成為醫(yī)學(xué)研究的理想動物模型。另外,豬也是我國畜牧業(yè)中重要的家畜之一。目前,豬多能干細(xì)胞的相關(guān)研究亟待進一步發(fā)展,在豬多能干細(xì)胞向生殖細(xì)胞誘導(dǎo)方面,尚未有相關(guān)的報道。因此,本研究的重點為:建立豬誘導(dǎo)多能干細(xì)胞(iPSCs)系,進而建立豬ipSCs向雄性生殖細(xì)胞樣細(xì)胞定向分化的誘導(dǎo)體系和技術(shù)方法。首先,本研究建立了豬iPSCs系。其形態(tài)類似于小鼠胚胎干細(xì)胞的形態(tài),呈致密凸起的克隆;該iPSCs的堿性磷酸酶染色呈陽性,并表達OCT4、SOX2、SSEA-1等多能性標(biāo)記蛋白;在體外,豬iPSCs可以通過擬胚體隨機分化為三胚層類型細(xì)胞,并可以定向分化為脂類和神經(jīng)類細(xì)胞;在免疫缺陷小鼠體內(nèi),豬iPSCs可以形成畸胎瘤,并分化為三胚層類型組織。這說明本研究中的豬iPSCs具有定向分化為生殖細(xì)胞的潛力。其次,根據(jù)豬iPSCs的特性,借鑒小鼠和人原始生殖細(xì)胞樣細(xì)胞(PGCLCs)誘導(dǎo)方法并進行改進和優(yōu)化,建立豬生殖細(xì)胞樣細(xì)胞誘導(dǎo)分化的體系及方法。第一,在含有Activin A等細(xì)胞因子的培養(yǎng)體系中,將豬iPSCs分化為上胚層干細(xì)胞樣細(xì)胞(EpiLCs),EpiLCs高表達上胚層干細(xì)胞標(biāo)記基因。第二,在含有BMP4等細(xì)胞因子的誘導(dǎo)體系中,將EpiLCs定向誘導(dǎo)分化為PGCLCs。PGCLCs高表達原始生殖細(xì)胞(PGCs)標(biāo)記基因Stella等,同時表達PGCs標(biāo)記蛋白STELLA等;在表觀遺傳特性方面,PGCLCs中H3K27me3表達上調(diào),H3K9me2表達下調(diào),印記基因DNA甲基化水平呈下降趨勢,這與體內(nèi)PGCs的發(fā)育相類似;在轉(zhuǎn)錄組水平上,PGCLCs高表達與生殖細(xì)胞發(fā)育相關(guān)的基因。第三,PGCLCs在含有睪酮等試劑的誘導(dǎo)體系中,進一步分化為精原干細(xì)胞樣細(xì)胞(SSCLCs)。SSCLCs表達Stra8、Gsg2等減數(shù)分裂標(biāo)記基因,同時表達DAZL、VASA等精原干細(xì)胞標(biāo)記蛋白;通過流式細(xì)胞術(shù)檢測到SSCLCs中含有單倍體細(xì)胞。這些結(jié)果表明,本誘導(dǎo)體系可以使豬iPSCs定向誘導(dǎo)分化為雄性生殖細(xì)胞樣細(xì)胞。目前,還沒有建立良好的生精細(xì)胞損傷不育公豬模型,而生精細(xì)胞損傷不育小鼠模型的建立方法及其曲細(xì)精管注射技術(shù)較成熟,因此選擇小鼠作為體內(nèi)研究的受體。將豬的PGCLCs和SSCLCs移植到小鼠曲細(xì)精管中,移植后在不同時間段進行檢測,結(jié)果顯示豬生殖細(xì)胞樣細(xì)胞在曲細(xì)精管中形成細(xì)胞鏈和細(xì)胞簇。免疫熒光染色結(jié)果顯示,生殖細(xì)胞標(biāo)記蛋白DAZL、VASA等呈陽性。這說明移植后的細(xì)胞具有生殖細(xì)胞特性,可能有助于修復(fù)生精細(xì)胞損傷的睪丸組織。綜上所述,本研究得到形態(tài)類似于小鼠胚胎干細(xì)胞的豬iPSCs系。在此基礎(chǔ)上,我們建立了一種將豬iPSCs誘導(dǎo)成雄性生殖細(xì)胞樣細(xì)胞的方法。該研究為進一步探索生殖細(xì)胞的發(fā)育機制和臨床上人多能干細(xì)胞向生殖細(xì)胞的分化奠定了基礎(chǔ),也為畜牧業(yè)育種提供了新的思路。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q813

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相關(guān)期刊論文 前1條

1 Yimei Cong;Jing Ma;Ruizhen Sun;Jianyu Wang;Binghua Xue;Jiaqiang Wang;Bingteng Xie;Juan Wang;Kui Hu;Zhonghua Liu;;Derivation of Putative Porcine Embryonic Germ Cells and Analysis of Their Multi-Lineage Differentiation Potential[J];Journal of Genetics and Genomics;2013年09期

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本文編號：2184027