本文選題：胚胎干細(xì)胞 + microRNA�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：論文中包含兩部分的工作。第一部分工作：miR23a～27a～24在胚胎干細(xì)胞分化中的功能研究了解胚胎干細(xì)胞(Embryonic stem cell, ESC)維持自我更新及多向分化潛能的具體機(jī)制,對(duì)于人們未來(lái)更好的將其應(yīng)用于再生醫(yī)學(xué)非常重要。過(guò)去幾十年已經(jīng)對(duì)ESC中轉(zhuǎn)錄因子和信號(hào)通路層面上的調(diào)控機(jī)制開(kāi)展了較全面的研究,也勾畫(huà)出ESC維持其特征的具體整體框架。除了各種轉(zhuǎn)錄因子相互協(xié)調(diào)形成網(wǎng)絡(luò)調(diào)控外,人們也逐漸認(rèn)識(shí)到表觀遺傳修飾,如組蛋白修飾、DNA甲基化及非編碼RNA等,也可以協(xié)同轉(zhuǎn)錄調(diào)節(jié)因子來(lái)共同維持ESC狀態(tài)基因的表達(dá)。其中microRNA在ESC自我更新及分化中發(fā)揮了重要的功能,在ESC中將Dicer和DGCR8敲除均顯示ESC分化存在缺陷,說(shuō)明microRNA在胚胎干細(xì)胞的自我維持尤其是分化中具有非常關(guān)鍵的作用。人們對(duì)于促進(jìn)ESC自我更新和多潛能性,提高iPS細(xì)胞重編程效率的microRNA研究較多。但是,對(duì)于抑制ESC自我更新,促進(jìn)分化的microRNA則研究較少。本課題組在前期的研究工作發(fā)現(xiàn)位于小鼠8號(hào)染色體上的miR-23a～27a～24-2基因簇(miR-23a基因簇)的兩個(gè)成員miR-27a和miR-24在分化細(xì)胞和組織中呈現(xiàn)不同程度的高表達(dá),通過(guò)靶基因Oct4、 Foxo1、 gp130、 Smad3、 Smad4實(shí)現(xiàn)對(duì)ESC自我更新的抑制,并能夠促ESC多胚層分化,抑制二者的表達(dá)能顯著提高三因子介導(dǎo)的iPS細(xì)胞形成的效率,同時(shí)有促進(jìn)胚層特異性分化的相關(guān)報(bào)道�？紤]到上述工作很多是在特定修飾的細(xì)胞系或者體外開(kāi)展,對(duì)miR-27a和miR-24-2在ESC分化中是否不可或缺這一問(wèn)題不能全面回答,miR-23基因家族除了miR-23a基因簇外,還包含有位于13號(hào)染色體上的miR-23b～27b～24-1基因簇(簡(jiǎn)稱miR-23b基因簇),這兩個(gè)基因簇共編碼5個(gè)microRNA:miR-23a,23b,27a,27b,24。 miR-23b基因簇上三個(gè)成員miR-23b,27b,24-1的種子序列與miR-23a基因簇中的三個(gè)成員完全相同,因此它們可能具有相同的靶基因及相應(yīng)的功能,在功能上有代償作用。因此,我們使用CRISPR/Cas9技術(shù)在V6.5細(xì)胞系中分別獲得miR-23a和23b雙基因簇純合敲除(DKO)和miR-23a基因簇純合敲除(KO)細(xì)胞系,而這是第一次由CRISPR/Cas9技術(shù)實(shí)現(xiàn)雙microRNA基因簇的純合敲除。而后,我們將獲得DKO和KO ESC系通過(guò)EB形成實(shí)驗(yàn)、ES細(xì)胞向中內(nèi)胚層定向誘導(dǎo)實(shí)驗(yàn)和畸胎瘤形成實(shí)驗(yàn)確定上述miR-23a/b基因簇敲除ESC系的分化能力。在EB形成實(shí)驗(yàn)中,DKO細(xì)胞顯示導(dǎo)致中胚層分化缺陷而促進(jìn)其它胚層的分化,提示miR-23-27-24基因簇參與ESC自我更新的抑制和早期中胚層的形成；在心肌分化實(shí)驗(yàn)中,DKO ESC 在任意天數(shù)都未觀察到博動(dòng)性cEBs,心肌特異標(biāo)志物Nkx2.5、 Tbx5、a-MHC、 b-MHC和cTnT等的表達(dá)量明顯降低。免疫熒光試驗(yàn)顯示沒(méi)有明顯的肌原纖維形態(tài),三個(gè)心肌標(biāo)志物Actinin、 ANP和Troponin1的表達(dá)量也非常低,提示miR-23～miR-27～miR-24基因簇在心肌細(xì)胞分化中起著非常重要的作用；同過(guò)畸胎瘤體積大小和重量檢測(cè)顯示DKO ESC成瘤能力小于KO和野生型ESC,通過(guò)HE染色,DKO ESC來(lái)源的畸胎瘤中可以觀察到外、內(nèi)胚層的結(jié)構(gòu),而幾乎沒(méi)有中胚層的結(jié)構(gòu),并且存在大量未分化或低分化的區(qū)域；通過(guò)ESC的標(biāo)志物Oct4進(jìn)行免疫組化檢測(cè),發(fā)現(xiàn)DKO ESC來(lái)源的畸胎瘤中存在大片Oct4染色陽(yáng)性區(qū)域,而野生型ESC來(lái)源的畸胎瘤中則幾乎沒(méi)有,KO ESC來(lái)源的畸胎瘤介于兩者之間。第二部分工作：?jiǎn)伪扼w胚胎干細(xì)胞突變體文庫(kù)的建立和應(yīng)用研究表明轉(zhuǎn)錄因子Oct4與Sox2是維持多能性所必須的,它們與其它一些轉(zhuǎn)錄因子共同組成了一個(gè)調(diào)控網(wǎng)絡(luò)來(lái)建立及維持ESC的多能性。當(dāng)這一調(diào)控網(wǎng)絡(luò)被破壞時(shí),ESC將退出多能性(Exit from Pluripotency)走向分化,但是,對(duì)于這一過(guò)程的機(jī)制,還有很多疑問(wèn)有待人們研究,目前在國(guó)際上已經(jīng)有幾個(gè)研究團(tuán)隊(duì)利用正向遺傳學(xué)篩選(Forward Genetics)方法獲得了與調(diào)控ESC退出多能性相關(guān)的候選基因。但這些研究都有自身的局限性,另外,將這些遺傳篩選的結(jié)果放在一起分析會(huì)發(fā)現(xiàn)相互間的重復(fù)性很差,只有少數(shù)幾個(gè)基因(如Tcf3)在不同研究中都被篩選出來(lái),這也從側(cè)面說(shuō)明上述研究還遠(yuǎn)沒(méi)有全面揭示ESC退出多能性進(jìn)入分化狀態(tài)的相關(guān)因子及其作用機(jī)制。由于多數(shù)基因的性狀為隱性的,只有兩個(gè)等位基因都發(fā)生突變時(shí),性狀的變化才顯現(xiàn)出來(lái),因此獲得基因組范圍的純合突變細(xì)胞文庫(kù)是進(jìn)行正向遺傳篩選研究必要條件。haESC只具有一套染色體組,并具有ESC的所有特征,因而,在正向遺傳篩選工作具有更廣泛的優(yōu)勢(shì),目前,在突變ESC基因的多種方法中,利用病毒載體和轉(zhuǎn)座子載體進(jìn)行的插入突變是使用較為廣泛的方法,而轉(zhuǎn)座子載體沒(méi)有病毒載體那樣顯著的基因組整合偏好性,可以覆蓋更多的基因。因此我們使用piggyBac轉(zhuǎn)座子載體攜帶的基因誘捕(Gene Trap)元件在haESC中構(gòu)建了4個(gè)純合突變體文庫(kù),約包含60000個(gè)突變克隆,通過(guò)Southern blot檢測(cè)其轉(zhuǎn)座子單拷貝插入插入的比例在74%,通過(guò)Splinkerette-PCR結(jié)合“三引物競(jìng)爭(zhēng)性PCR”鑒定純合突變的比例在85%以上,通過(guò)Splinkerette-PCR結(jié)合高通量測(cè)序的方法確定構(gòu)建的文庫(kù)中55%以上的插入位點(diǎn)位于基因內(nèi),其約涵蓋18000個(gè)以上的基因。在此基礎(chǔ)上,我們構(gòu)建了一個(gè)包含460個(gè)突變克隆的小規(guī)模矩陣式突變體文庫(kù),所謂“矩陣庫(kù)”就是在單倍體胚胎干細(xì)胞混合庫(kù)的基礎(chǔ)上,將每一個(gè)突變克隆挑取到96孔細(xì)胞培養(yǎng)板中獨(dú)立培養(yǎng)。隨后利用兩種分化條件M15 (-LIF)和N2B27對(duì)這些細(xì)胞進(jìn)行了分化篩選,共獲得了33個(gè)仍可以呈ESC樣生長(zhǎng)的陽(yáng)性克隆；利用Splinkerette PCR結(jié)合常規(guī)測(cè)序確定了19個(gè)陽(yáng)性克隆在基因組中的插入位點(diǎn),其中11個(gè)插入位點(diǎn)定位于已知基因,包含已知的與分化和發(fā)育相關(guān)的基因。接著,我們挑選了兩個(gè)陽(yáng)性克隆進(jìn)行回復(fù)實(shí)驗(yàn),當(dāng)轉(zhuǎn)座子捕獲載體被切除后,回復(fù)克隆在分化的條件下表現(xiàn)出了明顯的分化表型,證明了分化缺陷表型的產(chǎn)生是由轉(zhuǎn)座子的插入引起的。這些結(jié)果充分證明了我們將建立的矩陣式突變體文庫(kù)的可應(yīng)用性以及篩選策略的可行性,為我們下一步擴(kuò)大矩陣式突變體文庫(kù)奠定了良好的基礎(chǔ),通過(guò)篩選出更多的分化相關(guān)基因使我們能更深入全面地了解ESC分化的分子機(jī)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q25
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本文編號(hào)：1992323