胚胎干细胞自我更新和分化研究

发布时间：2018-06-07 18:38

  本文选题：胚胎干细胞 + microRNA　； 参考：《北京协和医学院》2016年博士论文

【摘要】：论文中包含两部分的工作。第一部分工作：miR23a～27a～24在胚胎干细胞分化中的功能研究了解胚胎干细胞(Embryonic stem cell, ESC)维持自我更新及多向分化潜能的具体机制,对于人们未来更好的将其应用于再生医学非常重要。过去几十年已经对ESC中转录因子和信号通路层面上的调控机制开展了较全面的研究,也勾画出ESC维持其特征的具体整体框架。除了各种转录因子相互协调形成网络调控外,人们也逐渐认识到表观遗传修饰,如组蛋白修饰、DNA甲基化及非编码RNA等,也可以协同转录调节因子来共同维持ESC状态基因的表达。其中microRNA在ESC自我更新及分化中发挥了重要的功能,在ESC中将Dicer和DGCR8敲除均显示ESC分化存在缺陷,说明microRNA在胚胎干细胞的自我维持尤其是分化中具有非常关键的作用。人们对于促进ESC自我更新和多潜能性,提高iPS细胞重编程效率的microRNA研究较多。但是,对于抑制ESC自我更新,促进分化的microRNA则研究较少。本课题组在前期的研究工作发现位于小鼠8号染色体上的miR-23a～27a～24-2基因簇(miR-23a基因簇)的两个成员miR-27a和miR-24在分化细胞和组织中呈现不同程度的高表达,通过靶基因Oct4、 Foxo1、 gp130、 Smad3、 Smad4实现对ESC自我更新的抑制,并能够促ESC多胚层分化,抑制二者的表达能显著提高三因子介导的iPS细胞形成的效率,同时有促进胚层特异性分化的相关报道。考虑到上述工作很多是在特定修饰的细胞系或者体外开展,对miR-27a和miR-24-2在ESC分化中是否不可或缺这一问题不能全面回答,miR-23基因家族除了miR-23a基因簇外,还包含有位于13号染色体上的miR-23b～27b～24-1基因簇(简称miR-23b基因簇),这两个基因簇共编码5个microRNA:miR-23a,23b,27a,27b,24。 miR-23b基因簇上三个成员miR-23b,27b,24-1的种子序列与miR-23a基因簇中的三个成员完全相同,因此它们可能具有相同的靶基因及相应的功能,在功能上有代偿作用。因此,我们使用CRISPR/Cas9技术在V6.5细胞系中分别获得miR-23a和23b双基因簇纯合敲除(DKO)和miR-23a基因簇纯合敲除(KO)细胞系,而这是第一次由CRISPR/Cas9技术实现双microRNA基因簇的纯合敲除。而后,我们将获得DKO和KO ESC系通过EB形成实验、ES细胞向中内胚层定向诱导实验和畸胎瘤形成实验确定上述miR-23a/b基因簇敲除ESC系的分化能力。在EB形成实验中,DKO细胞显示导致中胚层分化缺陷而促进其它胚层的分化,提示miR-23-27-24基因簇参与ESC自我更新的抑制和早期中胚层的形成；在心肌分化实验中,DKO ESC 在任意天数都未观察到博动性cEBs,心肌特异标志物Nkx2.5、 Tbx5、a-MHC、 b-MHC和cTnT等的表达量明显降低。免疫荧光试验显示没有明显的肌原纤维形态,三个心肌标志物Actinin、 ANP和Troponin1的表达量也非常低,提示miR-23～miR-27～miR-24基因簇在心肌细胞分化中起着非常重要的作用；同过畸胎瘤体积大小和重量检测显示DKO ESC成瘤能力小于KO和野生型ESC,通过HE染色,DKO ESC来源的畸胎瘤中可以观察到外、内胚层的结构,而几乎没有中胚层的结构,并且存在大量未分化或低分化的区域；通过ESC的标志物Oct4进行免疫组化检测,发现DKO ESC来源的畸胎瘤中存在大片Oct4染色阳性区域,而野生型ESC来源的畸胎瘤中则几乎没有,KO ESC来源的畸胎瘤介于两者之间。第二部分工作：单倍体胚胎干细胞突变体文库的建立和应用研究表明转录因子Oct4与Sox2是维持多能性所必须的,它们与其它一些转录因子共同组成了一个调控网络来建立及维持ESC的多能性。当这一调控网络被破坏时,ESC将退出多能性(Exit from Pluripotency)走向分化,但是,对于这一过程的机制,还有很多疑问有待人们研究,目前在国际上已经有几个研究团队利用正向遗传学筛选(Forward Genetics)方法获得了与调控ESC退出多能性相关的候选基因。但这些研究都有自身的局限性,另外,将这些遗传筛选的结果放在一起分析会发现相互间的重复性很差,只有少数几个基因(如Tcf3)在不同研究中都被筛选出来,这也从侧面说明上述研究还远没有全面揭示ESC退出多能性进入分化状态的相关因子及其作用机制。由于多数基因的性状为隐性的,只有两个等位基因都发生突变时,性状的变化才显现出来,因此获得基因组范围的纯合突变细胞文库是进行正向遗传筛选研究必要条件。haESC只具有一套染色体组,并具有ESC的所有特征,因而,在正向遗传筛选工作具有更广泛的优势,目前,在突变ESC基因的多种方法中,利用病毒载体和转座子载体进行的插入突变是使用较为广泛的方法,而转座子载体没有病毒载体那样显著的基因组整合偏好性,可以覆盖更多的基因。因此我们使用piggyBac转座子载体携带的基因诱捕(Gene Trap)元件在haESC中构建了4个纯合突变体文库,约包含60000个突变克隆,通过Southern blot检测其转座子单拷贝插入插入的比例在74%,通过Splinkerette-PCR结合“三引物竞争性PCR”鉴定纯合突变的比例在85%以上,通过Splinkerette-PCR结合高通量测序的方法确定构建的文库中55%以上的插入位点位于基因内,其约涵盖18000个以上的基因。在此基础上,我们构建了一个包含460个突变克隆的小规模矩阵式突变体文库,所谓“矩阵库”就是在单倍体胚胎干细胞混合库的基础上,将每一个突变克隆挑取到96孔细胞培养板中独立培养。随后利用两种分化条件M15 (-LIF)和N2B27对这些细胞进行了分化筛选,共获得了33个仍可以呈ESC样生长的阳性克隆；利用Splinkerette PCR结合常规测序确定了19个阳性克隆在基因组中的插入位点,其中11个插入位点定位于已知基因,包含已知的与分化和发育相关的基因。接着,我们挑选了两个阳性克隆进行回复实验,当转座子捕获载体被切除后,回复克隆在分化的条件下表现出了明显的分化表型,证明了分化缺陷表型的产生是由转座子的插入引起的。这些结果充分证明了我们将建立的矩阵式突变体文库的可应用性以及筛选策略的可行性,为我们下一步扩大矩阵式突变体文库奠定了良好的基础,通过筛选出更多的分化相关基因使我们能更深入全面地了解ESC分化的分子机制。
[Abstract]:The paper contains two parts. The first part: the function of miR23a ~ 27a ~ 24 in the differentiation of embryonic stem cells. It is important to understand the mechanism of Embryonic stem cell (ESC) to maintain self renewal and multidirectional differentiation potential. It is very important for people to use it in regenerative medicine for the past few decades. A more comprehensive study has been carried out on the regulatory mechanisms of transcription factors and signal pathways in ESC, and a specific overall framework for the maintenance of the characteristics of ESC is also outlined. In addition to the coordination of various transcription factors to form a network regulation, epigenetic modification, such as histone modification, DNA methylation and non coded RNA, is also gradually recognized. The expression of ESC state genes can be maintained together with transcriptional regulators, in which microRNA plays an important role in the self renewal and differentiation of ESC. Dicer and DGCR8 knockout in ESC show that the differentiation of ESC is defective, which indicates that microRNA plays a key role in the self maintenance and differentiation of embryonic stem cells, especially in the differentiation. There are many microRNA studies on promoting self renewal and pluripotency of ESC and improving the efficiency of reprogramming of iPS cells. However, there are few studies on inhibiting the self renewal of ESC and promoting the differentiation of microRNA. The research group found the miR-23a to 27a to 24-2 gene cluster (miR-23a gene cluster) located on chromosome 8 of mice in the previous study. The two members, miR-27a and miR-24, present different degrees of high expression in differentiated cells and tissues. Through target gene Oct4, Foxo1, gp130, Smad3, Smad4, the inhibition of ESC self renewal and the differentiation of ESC multiple germ layers can be promoted. The inhibition of the expression of the two can significantly improve the efficiency of the formation of three factor mediated iPS cells and promote the embryo. The related reports of layer specific differentiation. Considering that much of the work is carried out in specific modified cell lines or in vitro, the problem of whether miR-27a and miR-24-2 are indispensable in ESC differentiation can not be fully answered. The miR-23 family also contains miR-23b to 27b to 24-1 on chromosome 13 besides the miR-23a gene cluster. The gene cluster (miR-23b gene cluster), the two gene clusters co encodes 5 microRNA:miR-23a, 23B, 27a, 27b, and 24. miR-23b gene clusters of miR-23b, 27b, and 24-1 seed sequences are exactly the same as three members of the miR-23a gene cluster, so they may have the same target gene and corresponding function, and have the function of compensatory function. Therefore, we use CRISPR/Cas9 technology to obtain miR-23a and 23B double gene cluster homozygous knockout (DKO) and miR-23a gene cluster homozygous knockout (KO) cell lines in the V6.5 cell line, and this is the first time to realize the homozygous knockout of the double microRNA gene cluster by CRISPR/Cas9 technology. Then, we will obtain the DKO and KO ESC series to form the experiment. In the experiment of EB formation, DKO cells show the differentiation of the mesoderm differentiation and promote the differentiation of other germ layers in the EB formation experiment, suggesting that the miR-23-27-24 gene cluster participates in the inhibition of ESC and the formation of the early mesoderm in the EB formation. In the myocardial differentiation test, DKO ESC did not observe dynamic cEBs at any number of days, the expression of cardiac specific marker Nkx2.5, Tbx5, a-MHC, b-MHC and cTnT decreased obviously. The immunofluorescence test showed no obvious myofibrillar morphology, and the expression of Actinin, ANP and Troponin1 were also very low in the three myocardial markers, suggesting miR-23. The miR-27 ~ miR-24 gene cluster plays a very important role in the differentiation of cardiac myocytes, and the size and weight of the over teratoma show that the DKO ESC is less than KO and the wild type ESC. Through HE staining, the structure of the endoderm can be observed in the teratoma of DKO ESC source, but the structure of the mesoderm is almost without the structure of the mesoderm, and there is a existence of the structure of the mesoderm, and the existence of the structure of the mesoderm, and the existence of the structure of the mesoderm, and the existence of the structure of the mesoderm. A large number of undifferentiated or poorly differentiated regions; detected by immunohistochemical staining of the ESC marker Oct4, it was found that there was a large area of Oct4 staining positive in the teratoma derived from DKO ESC, while the teratoma derived from the wild type ESC was almost not, and the teratoma derived from KO ESC was between the two. The second part of the work: haploid embryonic stem cells The study of the establishment and application of the mutant library shows that the transcription factor Oct4 and Sox2 are necessary to maintain the pluripotent activity. They form a regulatory network with some other transcription factors to build and maintain the pluripotent of ESC. When this regulatory network is destroyed, ESC will withdraw from the pluripotent (Exit from Pluripotency) to differentiate, but, however, There are many questions to be studied about the mechanism of this process. At present, several research teams in the world have used the Forward Genetics method to obtain the candidate genes related to the regulation of ESC withdrawal, but these studies have their own limitations. In addition, the results of these genetic screening are placed. Only a few genes, such as Tcf3, are screened in different studies. This also shows that the above studies are far from fully revealing the related factors and the mechanism of ESC withdrawal from pluripotent pluripotent differentiation. Because most genes are recessive, only two of the genes are recessive. When the allele is mutated, the change of the trait appears. Therefore, the acquisition of the genome range of the homozygous mutant cell library is the necessary condition for the study of the positive genetic screening..haESC has only a set of chromosomes and has all the characteristics of ESC. In a variety of methods for changing the ESC gene, the insertion mutation using viral vectors and transposon vectors is the more widely used method, while the transposon vector has no significant genomic integration preference like a viral vector and can cover more genes. Therefore, we use the gene trap (Gene Trap) element carried by the piggyBac transposon carrier. 4 homozygous mutant libraries were constructed in haESC, including 60000 mutant clones. The proportion of the insertion insertion of the single copy of the transposon was detected by Southern blot in 74%. The proportion of homozygous mutations was identified by Splinkerette-PCR combined with "three primer competitive PCR", and the proportion of the homozygous mutation was above 85%, and by Splinkerette-PCR combined with high throughput sequencing. More than 55% insertion sites in the constructed library are located in the gene, which covers more than 18000 genes. On this basis, we have constructed a small matrix mutant library containing 460 mutant clones. The so-called "matrix library" is based on the hybrid library of haploid embryonic stem cells. The 96 cell culture plates were isolated and cultured independently. Then two differentiation conditions M15 (-LIF) and N2B27 were used to differentiate these cells. A total of 33 positive clones that still showed ESC like growth were obtained. The insertion sites of 19 positive clones in the genome were determined by Splinkerette PCR combined with conventional sequencing, and 11 of them were inserted in the genome. The insertion site was located in the known gene, including the known genes related to differentiation and development. Then, two positive clones were selected for recovery experiments. When the transposon capture vector was excised, the replying clones showed a distinct differentiation form under the condition of differentiation, proving that the differentiation defect phenotype was produced by the transposing. These results fully demonstrate the applicability of the matrix mutant library we will establish and the feasibility of the screening strategy. It lays a good foundation for the next step of expanding the matrix mutant library. By screening more differentiation related groups, we can understand the ESC differentiation in a more thorough and comprehensive way. Molecular mechanism.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q25
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本文编号：1992323