【作者】 冯涛；

【导师】 吴森；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2015， 博士

【摘要】 猪因为与人更为相似的身体结构和生理特征而成为新兴的模式动物,关于其胚胎干细胞(Embryonic stem cells,ESCs)和诱导多能性干细胞(Induced pluripotent stem cells,iPSCs)的研究自然也受到人们的广泛关注。但是目前为止,猪ES细胞尚未建系成功,iPS细胞也没有生殖嵌合的报道。这严重制约了药物筛选、细胞治疗、器官移植和大动物基因修饰方面的研究。为了获得高质量的猪iPS细胞,本研究分别采用piggyBac(PB)转座子质粒和episome非整合质粒诱导得到了猪iPS细胞。所得iPS细胞碱性磷酸酶(Alkalinephosphatase,AP)染色呈阳性,多能性因子的免疫荧光染色呈阳性,核型正确,能够在SCID鼠体内形成有三胚层分化的畸胎瘤。进一步对所得细胞进行体外嵌合能力的检测时发现,利用胚胎注射和囊胚聚合两种方法均能成功获得有iPS细胞参与发育的嵌合囊胚。嵌合囊胚可以在代孕母猪体内发育到期生成仔猪,但是在这些仔猪体内均检测不到iPS细胞来源的成分。本研究通过进一步分析发现,内源多能性基因不激活导致了体细胞重编程不完全,这是猪iPS细胞生成嵌合体失败的主要原因之一。为了简单方便地观察内源OCT4的启动情况,本研究分别构建了 PB系统和非PB系统的猪源、鼠源和人源的OCT4-EGFP报告系统。结果发现,猪源的OCT4-EGFP报告系统在体细胞克隆(Somatic cell nuclear transfer,SCNT)胚胎中能够被很好地被激活,但是在iPS细胞中不能被激活。这进一步证实了猪iPS细胞内源多能性基因沉默的推测;同时发现,曲古柳菌素A(Trichostatin A,TSA)能够解除再克隆胚胎中OCT4-EGFP沉默的现象。为了更加全面地研究多能性基因在猪早期胚胎中的表达,本研究分别构建了猪源的SOX2-tdTomato和REX1-tdTomato报告系统,结果发现SOX2在早期胚胎中能被很好地激活,而同时期的REX1表达水平相对较低。另外,为了找到阻碍猪体细胞重编程的关键基因,本研究引入了基于CRISPR/Cas文库的筛选系统,并且发现它在猪iPS细胞中能很好地发挥作用,这为解决目前猪iPS细胞研究的困境提供了强大的支持。猪ES细胞建系一直未能成功,并且导致失败的最主要原因是什么,并不确定。为了探索不同因素对猪ES细胞体外建系的影响,本研究分别使用了猪孤雌激活(Parthenogenetic activation,PA)胚胎、体外受精(In vitro fertilization,IVF)胚胎、带有荧光报告系统的SCNT胚胎和体内胚胎为原始材料,向培养基中分别加入碱性成纤维细胞生长因子(Basic fibroblast growth factor,bFGF)、白血病抑制因子(Leukemia inhibitory factor,LIF)、胎猪血清、猪卵泡液以及小分子抑制剂PD0325901和CHIR99021(2i)等尝试分离培养猪ES细胞。结果发现,多能性基因OCT4和SOX2在类ES细胞克隆生成之初就已经被沉默;bFGF和胎猪血清利于类ES细胞克隆的快速增长,而猪卵泡液和2i/LIF却抑制类ES细胞克隆的生成。虽然在含bFGF和胎猪血清的培养基中类ES细胞克隆能够快速生长,但是在传代之后这些细胞均发生分化或者死亡。综上所述,PB质粒和episome质粒诱导得到的猪iPS细胞能在体外参与形成嵌合囊胚,但是都不能形成嵌合个体。利用OCT4-EGFP报告系统,本研究发现猪iPS细胞中内源多能性基因未被激活。另外,本研究分别用了不同的培养条件,从不同来源的猪囊胚中分离培养得到了类ES细胞克隆,结果发现,OCT4和SOX2在类ES细胞克隆生长之初就已经被沉默,bFGF和胎猪血清对类ES细胞的生长有利。这些结果为大动物中多能性细胞建系奠定了坚实的基础。更多还原

【Abstract】 Since pigs are important animal models because of their similar anatomic and physiological features with human,study of their embryonic stem cells(ESCs)and induced pluripotent stem cells(iPSCs)is increasingly making itself the mainstream research in the field.To date,however,neither porcine ES cell lines nor germline-competent iPS cell lines have been established.This fact is impeding researches on cell fate determination,drug discovery,cell therapies and genetic engineering in large animals.To get high-quality porcine iPSCs,we derived pig iPSCs by two methods with piggyBac(P8)transposon and episomal plasmids respectively.These iPS cells were positive for alkaline phosphatase(AP),expressed the pluripotent markers,OCT4,SOX2,NANOG,KLF4 and c-MYC.They had normal karyotype,and formed teratomas that consisted of tissue types from entoderm,mesoblast and ectoderm in SCID mice.Furthermore,by embryo injection or 4-cell embryos aggregation method,we could produce chimeric blastocysts with pig iPS cells in vitro.But we failed to detect any iPSCs-derived cells in piglets which developed from chimeric blastocysts,suggesting that iPSCs we obtained lack the ability to form chimeras.We found the inactivation of endogenous pluripotency genes of iPS cells is a critical reason for the failure to produce chimeras.To visually track endogenous OCT4 expression,we constructed PB and non-PB reporter vectors containing EGFP driven by mouse,human and pig OCT4 promoter,respectively.The results showed that the pig OCT4-EGFP could be activated in somatic cell nuclear transfer(SCNT)embryos,but could not be activated in iPS cells.This confirmed qPCR results revealing the inactivation status of endogenous OCT4 in pig iPS cells.Meanwhile,we found that Trichostatin A(TSA)could activate the silenced OCT4-EGFP in re-cloned embryos by promoting epigenetic reprogramming during somatic nuclear transfer.To furthermore study the expression pattern of pluripotent genes in pig preimplantation embryos,we constructed non-PB reporter vectors containing tdTomato driven by pig SOX2 and REX1 promoter,respectively.The results showed that SOX2 was in a high expression level in SCNT embryos,however,REX 1 was activated weakly.Additional,to identify gene(s)hindering the activation of endogenous pluripotent genes,we constructed a screening system based on CRISPR/Cas library.We found that this system is a very powerful tool.To explore reasons why it failed to establish pig ESCs,we have used pig embryos of different source to isolate ESCs,including parthenogenetic activation(PA)embryos,in vitro fertilization(IVF)embryos,SCNT embryos carrying OCT4-EGFP or SOX2-tdTomato and in vivo embryos.Medium was optimized by screening for various supplements,such as basic fibroblast growth factor(bFGF),Leukemia inhibitory factor(LIF),fetal pig serum(FPS),pig follicular fluid(PFF),and small molecular inhibitors.ES-like cells could be derived in early passages.We found that bFGF and pig fetal serum could benefit the growth of ES-like cells,but pig follicular fluid and small molecular inhibitors used here inhibited the ES-like cell clones formation.Further,expression of OCT4 and SOX2 lost rapidly after formation of ES-like cell clones.In this study,we demonstrated that pig iPS cells could contribute to chimeric blastocysts in vitro,but no chimeric piglet could be obtained.The inactivation of endogenous OCT4 genes of iPS cells was confirmed by using an OCT4-EGFP reproter.We also optimized culture condition for pig pluripotent stem cells and ESC isolation and found that ES-like cells could grow fast in medium supplenmented with bFGF and FPS,while formation of ES-like cell clones was inhibited by PFF and small molecular inhibitors.Our ESCs/iPSCs study could serve as a systematic start point and benefit further researches in this field.更多还原