小分子化合物介导谱系重编程获取内胚层祖细胞及其相关机制研究

发布时间：2018-04-09 15:34

本文选题：小分子化合物　切入点：胃上皮细胞　出处：《中国人民解放军军事医学科学院》2016年博士论文

【摘要】：干细胞是一类具有自我更新和多向分化潜能的细胞,由于能够生成功能成熟的终末细胞,因此在器官组织再生、疾病模型建立、发育生物学及药物研发等领域具有非常广阔的应用前景。这其中又以胚胎干细胞(ESCs)最早受到关注,ESCs具有无限的自我更新和向成体所有细胞进行分化的潜能,因此是细胞治疗的良好种子细胞。然而ESCs应用面临伦理学的困境,免疫排斥和致畸胎瘤的问题。2006年Yamanaka使用多能性转录因子OCT4,SOX2,KLF4和c-MYC(OSKM)成功的将成纤维细胞重编程(Reprogramming)为诱导多能性干细胞(iPSCs)。iPSCs避免了ESCs的伦理学争议、免疫排斥问题,但依然难以回避致畸胎瘤的风险。近年来使用多种不同的诱导方式使细胞命运发生转换的谱系重编程技术(Lineage Reprogramming)在整个干细胞与再生医学领域,吸引了广泛的关注并成为获取干细胞和功能成熟细胞的前沿和极具价值的技术策略。与iPSCs及其来源的细胞相比,lineage reprogramming获取的细胞具有多方面的优势和特点。Lineage reprogramming获取细胞不经过多能性阶段,直接实现胚层内或胚层间的细胞类型转换,因此不具有成畸胎瘤的潜在风险。由于细胞发生直接的命运转换,不需要多步的诱导分化,由此获得的细胞类型更为均一可控,临床应用也更加安全。此外,Lineage reprogramming同样具有iPSCs个体特异性,移植后不具有免疫排斥的优势。长期以来谱系重编程最常用的策略是在成体细胞中过表达靶细胞谱系特异性的转录因子,结合相应的诱导培养条件,建立靶细胞的基因调控网络,从而实现细胞命运转化。利用这种方式在内,中,外三个胚层的多个细胞类型之间实现了命运转换。然而,目前谱系重编程的主要策略仍然是依赖于病毒载体介导转录因子过表达的方式,因此会不可避免的存在病毒载体基因在目的细胞基因组中的整合,进而导致目的细胞基因组的不稳定,从而造成细胞移植入体内后带来长期的安全性隐患。因此,寻求非整合方式的谱系重编程策略,来替代病毒介导转录因子的过表达,将是谱系重编程走向临床应用的必经之路。小分子化合物是一类可自由通透细胞,性状稳定,质量可控,可调和作用时间和作用强度,非整合性及无免疫源性的化合物,因此是一类非常安全的具有巨大临床应用前景的谱系重编程方式。近期,完全使用小分子组合已成功的将小鼠成纤维细胞重编程到诱导多能性干细胞、神经细胞、神经干细胞;将人的成纤维细胞、星状细胞重编程为神经元,心肌细胞。然而目前还没有将人或鼠的体细胞重编程到内胚层祖细胞的先例。而且小分子化合物诱导iPSCs产生的机制才初步阐明,小分子化合物介导谱系重编程的机制尚不清楚。针对这些关键的科学问题,我们针对起始细胞的选择、小分子化合物及滋养层细胞的筛选进行了系统研究,最终成功的使用确定的小分子组合在特定的滋养层细胞的支持下,将人的胃上皮细胞(hgecs)重编程到诱导内胚层祖细胞阶段(hiendopcs),并对hiendopcs的生物学特性以及hgecs向hiendopcs谱系重编程的机制进行了深入系统的研究。基于以上内容,本课题分为三部分,第一部分建立小分子化合物诱导人胃上皮细胞重编程为内胚层祖细胞的体系;第二部分鉴定诱导内胚层祖细胞的表型及分化潜能;第三部分是对人胃上皮细胞向诱导内胚层祖细胞重编程过程中的机制进行深入研究,揭示各影响因素的作用机制。一、小分子化合物诱导人胃上皮细胞向内胚层祖细胞转换的体系建立我们分离培养人胃和十二指肠来源的上皮细胞来作为谱系重编程的起始细胞,通过初步筛选的八个小分子组合:sb431542+vpa+pd0325901+rg108+bix01294+bayk8644+ps48+fbp,并在人消化道来源的肌成纤维细胞作为滋养层的条件下可以将胃上皮(hgecs)或十二指肠上皮细胞(hdecs)重编程为内胚层祖细胞样的克隆。我们以能够出现内胚层祖细胞样克隆为标准,然后对重编程的体系—小分子化合物和滋养层细胞进行了优化,最终确定四个小分子组合:bix01294+bayk8644+rg108+sb431542(bbrs)为最优的小分子组合。由于滋养层细胞—人胃肌成纤维细胞(gsemfs)的获取最为便利,因此我们确立了重编程最优的体系是bbrs+gsemfs,在这样的体系中重编程的效率为~5%。我们也发现能够实现重编程的最少小分子组合是sb431542。此外,我们还证实了hiendopcs来源于ncam阳性的hgecs,实现了起始细胞的精确定位。二、诱导内胚层祖细胞的表型鉴定及分化潜能研究我们首先检测了常见的内胚层祖细胞特征性的转录因子foxa2、sox9、hnf1b、pdx1、gata4,其他干/祖细胞特征性蛋白包括cxcr4、epcam、lgr5、ck19以及胃上皮特异性的标志muc6、gastrin等在hiendopcs中的表达情况,发现与hgecs相比内胚层标志性的基因在hiendopcs中明显上调,而胃特异性的基因则不表达,初步证实了hgecs向内胚层祖细胞重编程的成功。表观遗传分析也显示重编程后细胞发生了显著改变,具备了内胚层祖细胞的分子特征。对hiendopcs的发育阶段进行精确定位,深度测序的结果显示hiendopcs处于hescs来源的原始消化管(pgt)和前肠后段(pfg)这两个内胚层发育阶段之间,且更接近pfg。此外,hiendopcs具有干细胞微观水平的特征并能进行4-6次的传代扩增。由于发育过程中内胚层祖细胞具有向胰腺、肝脏、肠、肺和甲状腺等内胚层器官进行分化的潜能,因此我们使用相应的诱导分化条件发现hiendopcs同样具有形成功能性胰腺β细胞、肝细胞、肠道细胞、肺细胞和甲状腺细胞的能力。通过对hiEndoPCs的表型分析、生物信息学分析以及内胚层分化潜能的评价,我们明确证实了hiEndoPCs的内胚层祖细胞特性以及hGECs向hiEndoPCs重编程的成功。三、胃上皮细胞向诱导内胚层祖细胞重编程的机制研究由于在我们的重编程体系中,小分子化合物和滋养层细胞这两个因素是不可或缺的,因此我们首先对小分子可能的作用机制进行了探究,发现TGF-β信号通路的抑制是必须的,也是能够保证重编程成功的最少小分子组合,也就是说TGF-β信号通路抑制介导的上皮特性的维持在重编程的过程中是必须的,然而我们发现早期的上皮间质转换(EMT)和随后的间质上皮转换(MET)也存在于重编程的过程中。重编程的另一个关键因素是滋养层细胞的作用,对滋养层细胞的分泌蛋白表达谱分析显示促进细胞周期加速的蛋白高表达,在滋养层细胞的作用下hGECs细胞周期明显加快,提示了细胞周期的加速有助于重编程的发生。此外Wnt信号通路被证明在重编程的过程中处于激活的状态,抑制Wnt信号通路明显的抑制或取消了重编程。因此,TGF-β信号通路的抑制,细胞周期的促进及Wnt信号通路的激活等多因素协同作用是重编程发生的重要机制。综上所述,我们首次成功的实现了小分子化合物介导谱系重编程从胃上皮细胞中获取内胚层祖细胞,全面证实了所获得细胞的内胚层祖细胞属性并揭示了胃上皮细胞向内胚层祖细胞转换过程中的具体机制。
[Abstract]:Stem cells are self-renewal and pluripotent cells, due to the end cell generation function is mature, therefore in organ regeneration, disease model, and has very broad application prospects in developmental biology and drug development and other fields. The embryonic stem cell (ESCs) is the earliest concern. ESCs has unlimited self-renewal and into all cell differentiation potential, so it is a good seed cells for cell therapy. However, ESCs applications are facing an ethical dilemma, immune rejection and teratoma problem.2006 Yamanaka using pluripotent transcription factor OCT4, SOX2, KLF4 and c-MYC (OSKM) will be successful fiber cell reprogramming (Reprogramming) induced pluripotent stem cells (iPSCs).IPSCs ESCs to avoid the ethical controversy, problem of immune rejection, but still difficult to avoid the risk of fetal teratogenic tumors. In recent years To use a variety of different ways to induce cell fate changes, lineage reprogramming Technology (Lineage Reprogramming) in the field of stem cell and regenerative medicine, has attracted wide attention and become a source of stem cells and mature cells in the forefront and valuable technical strategy. Compared with iPSCs and lineage cells, reprogramming the cell has many advantages and characteristics of.Lineage reprogramming cells after obtaining pluripotent stage, directly within or between cells derived from mesoderm type conversion, therefore does not have the potential risk into a teratoma. Because the cell fate of direct conversion, no differentiation of multiple steps, the resulting cells type for the uniform, clinical application is more safe. In addition, Lineage reprogramming also has iPSCs individual specificity, after transplantation is not free For a long time. The advantage of disease rejection lineage reprogramming most commonly used strategy in a target cell lineage specific transcription factor expression in somatic cells, and induce the corresponding culture conditions, the construction of gene regulation network of target cells, so as to realize the transformation of cell fate. And by this way, between multiple cell types the three layers of the fate of conversion. However, at present the main strategy of lineage reprogramming is still dependent on viral vector mediated transcription factor expression way, so there will be the integration of viral vector gene in the target cell genome in the inevitable, leading to genomic instability, resulting in safety the risks of long-term cell transplantation in vivo. Therefore, seeking the lineage reprogramming strategy of non conformity way, instead of over expression of virus mediated transcription factor, is the pedigree The only way which must be passed reprogramming to clinical application. Small molecular compound is a kind of free transparent cells, stable, controllable, adjustable time and intensity, non conformity and compound no immunogenicity, therefore is a kind of very safe has great clinical application prospect of recent lineage reprogramming. Completely, have successfully reprogrammed mouse fibroblasts into induced pluripotent stem cells, using small molecule combination neural cells, neural stem cells; the human fibroblasts, stellate cell reprogramming into neurons, cardiomyocytes. However there is still no reprogramming of somatic cells to human or mouse endoderm progenitor cells and mechanism of precedent. Small molecular compounds induce the production of iPSCs was preliminarily elucidated, mechanism of small molecule mediated lineage reprogramming is not clear. In view of these key scientific problems, according to the US The initial cell selection, screening of small molecule compounds and trophoblast cells were studied using the ultimate success of the determination of small molecules in the specific combination of trophoblast cells under the support of human gastric epithelial cells (hgecs) induced reprogramming to endoderm progenitor cell stage (hiendopcs), and the biological characteristics of hiendopcs and hgecs to the hiendopcs lineage reprogramming mechanism is studied. Based on the above content, this paper is divided into three parts, the first part of the establishment of small molecule induced human gastric epithelial cell reprogramming into endoderm progenitor cell system; the second part identified by phenotype and differentiation potential of endodermal progenitor cells; the third part is the further research of human gastric epithelial cells to induce endodermal progenitor cell reprogramming mechanism in the process of the mechanism of various factors. A small molecular compound Induction of human gastric epithelial cells of endoderm progenitor cell transformation system we isolated from human gastric and duodenal epithelial cells as a source of initial cell lineage reprogramming, through eight small molecular screening: sb431542+vpa+pd0325901+rg108+bix01294+ bayk8644+ps48+fbp, and in the human digestive tract from fibroblasts as feeder conditions can be gastric epithelial cells (hgecs) or duodenal epithelial cells (hdecs) clone reprogramming into endoderm progenitor cells. We can appear endoderm progenitor like cloning as a standard, then the reprogramming system of small molecules and trophoblast cells were optimized, and ultimately determine the four small molecular bix01294+bayk8644+rg108+sb431542 (BBRs) is: the optimal combination of small molecules. Because trophoblast cells - human gastric fibroblast (gsemfs) to obtain the most For convenience, we established the optimal weight programming system is bbrs+gsemfs, the efficiency of reprogramming in this system is ~5%. we also found that the minimum combination of small molecules can realize reprogramming is sb431542. in addition, we also demonstrated that hiendopcs derived from NCAM positive hgecs, to achieve the precise positioning of the starting cell. Two. Study on phenotypic identification and differentiation induction of endoderm progenitor cells we first examined the characteristics of endodermal progenitor cells of transcription factor Foxa2, common Sox9, HNF1B, PDX1, GATA4 and other stem / progenitor cells characteristic proteins including CXCR4, EpCAM, Lgr5, CK19 and mark MUC6 gastric epithelial specific expression of gastrin. In the hiendopcs, found that compared with hgecs, the iconic endoderm gene in hiendopcs was significantly increased, while the stomach is not the expression of specific genes, confirmed that hgecs endoderm progenitor cell weight Programming success. Epigenetic reprogramming after cell analysis also showed markedly changed, with the molecular characteristics of endodermal progenitor cells. The accurate positioning of the development stage of the hiendopcs, the depth of sequencing results show the original hiendopcs in hESCs derived digestive tube (PGT) and the posterior segment (PFG) between the two the endoderm development stage, and more close to the pfg. in addition, hiendopcs has the characteristics of stem cells in the micro level and can be passaged 4-6 times amplification. Because of endodermal progenitor cells during development to pancreas, liver, intestine, lung and thyroid gland and other organs endoderm differentiation potential, so we use induction and differentiation condition of the corresponding discovery hiendopcs also has the formation of functional pancreatic beta cells, liver cells, intestinal cells, ability of lung cells and thyroid cells. Through phenotypic analysis of hiEndoPCs, bioinformatics analysis to Evaluation and endoderm differentiation potential, we confirmed endodermal progenitor cells characteristic of hiEndoPCs and hGECs to hiEndoPCs reprogramming success. Three, gastric epithelial cells to study the mechanism of induction of endoderm progenitor cell reprogramming by reprogramming in our system, the two factors of small molecules and trophoblast cells is indispensable. So we may be the first mechanism of small molecules was investigated, found that inhibition of TGF- beta signaling pathway is necessary, also can guarantee the minimum combination of small molecule reprogramming success, i.e. epithelial characteristics of TGF- beta signaling pathway mediated by inhibition of the maintenance in the process of reprogramming is a must, but we the early detection of epithelial mesenchymal transition (EMT) and subsequent mesenchymal to epithelial transition (MET) process also exists in the reprogramming. Another key factor in reprogramming of trophoblast Cells of trophoblast cells secreting protein expression profile analysis showed that the high expression of acceleration of cell cycle protein, significantly accelerate the cell cycle of hGECs in trophoblast cells under the effect of that cell cycle contributes to accelerated reprogramming. In addition the Wnt signaling pathway was shown in the activated state in the process of reprogramming in the inhibited or abolished the reprogramming of the inhibition of Wnt pathway. Therefore, inhibition of TGF- beta signaling pathway, Wnt signaling pathway and promote cell cycle activation of multiple factors is mechanism of reprogramming occurred. In summary, we successfully realized the small molecule mediated lineage heavy programming for endodermal progenitor cells from gastric epithelial cells, fully confirmed endodermal progenitor cells and reveal the properties of cells in gastric epithelial cells of endoderm progenitor cell conversion The specific mechanism in the process.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R329.2

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