人胚胎干细胞定向诱导分化为肝细胞的实验研究

发布时间：2018-08-02 16:37

【摘要】： 终末期肝衰竭仍然是困扰人类健康的一类顽疾,目前,它的治疗主要依赖于原位肝移植(orthotopic liver transplantation,OLT),但是供体的极度短缺、移植及手术本身相关的死亡、终生服用免疫抑制剂及其所致的致死性并发症等一系列问题极大地限制了这一治疗手段的广泛开展。细胞移植与生物人工肝(bioartificial liver,BAL)作为终末期肝病的替代、补充治疗方法,不仅为患者寻求合适的供肝来源争取到宝贵的时机,甚至可以帮助患者平安渡过危险期,通过肝再生而自然恢复。令人遗憾的是,细胞来源受限、数量不足以及功能欠缺等仍是制约其进一步发展的瓶颈问题。具有高度自我更新和发育全能性的人胚胎干细胞(embryonic stem cells,ES cells)在适宜的环境中可以向各种组织细胞分化,有望为生物人工肝、肝细胞移植、甚至肝组织工程等提供理想的种子细胞。然而,影响人ES细胞向肝细胞有效分化的因素及其调控机制、诱导分化效率及分化细胞的功能状态等均存在许多不确定性,ES细胞应用于临床尚有许多难以逾越的障碍。本研究探讨了诱导人ES细胞分化为具有合成、代谢、分泌及贮存等功能的肝细胞的方法,并通过遗传修饰建立稳定转染pAlb-EGFP的人ES细胞株,提供了纯化目的细胞的手段,为人ES细胞在以细胞治疗和基因治疗为基础的肝脏再生医学中的应用奠定了基础。本研究主要包括三部分内容。一、细胞因子联合胞外基质诱导人ES细胞向肝系细胞分化我们采用以小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)作为饲养层,并摸索了两种传代方法(机械法和胶原酶法),建立了标准的人ES细胞体外培养体系。为进一步探讨人ES细胞肝向分化的潜能,我们在培养初期先将人ES细胞悬浮培养形成包含三胚层结构的拟胚体(embryoid body,EB),然后将其接种到预先包被有Ⅰ型胶原的培养板上,以含有地塞米松、胰岛素等的条件培养液进行肝系诱导。形态学、细胞表型变化及功能检测等多方面结果表明,诱导后的部分细胞基本符合肝细胞的特征,证明人ES细胞具有向肝细胞样细胞分化的潜能。二、分阶段富集联合转基因基质细胞共培养诱导人ES细胞向肝脏细胞分化在前述试验研究中,我们证实了人ES细胞能够在特定环境中向肝细胞样细胞分化,但事实上分化的结果仍存在一定争议。因为与成体干细胞有所不同,ES细胞具有更为特异的全能分化潜能,可以向内、中、外三个胚层乃至胚外组织进行分化,而脏壁内胚层(visceral endoderm)可以表达许多与肝细胞相似的基因,但它只能够参与形成胚外卵黄囊结构,并不能分化发育成肝脏或胰脏等实质性器官,这为获得真正意义上的肝细胞造成障碍。为此,本研究根据人ES细胞全能分化的特点及肝发育机制,设计了阶段性富集联合转基因基质细胞共培养的新策略。首先形成包含了外层的原始内胚层和内层的原始外胚层的早期发育的简单EB,在低血清的条件下,以高浓度的activin A诱导其向定型内胚层的分化。RT-PCR和免疫荧光结果显示,activin A作用3d的细胞高表达(80%)内胚层标志SOX17,而同时脏壁内胚层的标志α-甲胎蛋白(alpha-fetoprotein,AFP)表达缺失,证明所获细胞为定型内胚层细胞。为进一步探讨定型内胚层细胞向肝细胞分化的潜能与机制,我们利用转基因基质细胞共培养系统模拟肝发育时的微环境。共培养11d后,形态学、细胞表型变化及功能检测等多方面结果表明,定型内胚层细胞可以在bFGF和胎肝基质细胞共同创造的环境中向肝系分化,并在HGF、OSM和Dex的作用下进一步分化成熟,这一研究为获得大量真正意义上的肝细胞开创了新的出路。三、建立pAlb-EGFP遗传修饰的人ES细胞株如前所述,新的诱导策略排除了相似细胞的干扰,但所得细胞仍为一混合体,其致瘤、致癌等不可预知的风险不容忽视。在本部分研究中,我们拟通过基因修饰人ES细胞,从而为今后的应用研究提供纯化目的细胞的手段。白蛋白(albumin,Alb)在肝脏发育早期及成体肝脏中均有所表达,是识别肝细胞的一个较为特异的标志。我们设计将含Alb启动子调控增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的质粒pAlb-EGFP转入人ES细胞,由于该载体中含有一段neo抗性基因,所以可以通过G418条件培养进行抗性筛选。通过条件培养基杀灭试验我们首先确定了G418筛选的最佳浓度(200μg/ml)。在此基础上,我们利用EXGen 500转染试剂进行转染并实施抗性筛选,PCR结果证实筛选到的阳性克隆内确实含有Alb-EGFP这段序列,证实为稳定转染,这一研究可能为日后的应用增添新的纯化肝细胞的工具。人ES细胞向成熟的、有生理功能的肝细胞分化,为肝脏疾病的细胞治疗提供了新的选择。对于我们这样一个病毒性肝炎及各种原因导致的终末期肝病高发的国家更具有特殊的意义。具有发育全能性的人ES细胞将为生物人工肝、肝(干/祖)细胞移植和以肝(干/祖)细胞为载体的基因转移治疗等提供优良的种子细胞,借助核移植技术和现有的ES细胞库等资源有望在肝脏疾病的治疗中发挥更大的作用,而现代生物科学及其相关技术在该领域中的应用势必将加快其在临床移植修复治疗中的应用进程。
[Abstract]:End-stage liver failure is still a kind of stubborn disease that plagued human health. At present, its treatment relies mainly on orthotopic liver transplantation (OLT), but the extreme shortage of donor, the death of transplantation and operation itself, the lifetime taking of immunosuppressant and its fatal complications are a great deal of problems. It restricts the extensive development of this treatment. Cell transplantation and bioartificial liver (BAL) as an alternative to end-stage liver disease, supplemental therapy, not only for patients to seek the appropriate source of liver supply for the precious opportunity, but also help patients safely through the risk period, the natural recovery through liver regeneration. It is regrettable that the limited cell source, insufficient number and lack of function are still the bottlenecks that restrict its further development. Human embryonic stem cells (embryonic stem cells, ES cells), with high self renewal and developmental omnipotent, can differentiate into various tissues and cells in a suitable environment, and are expected to be biological artificial liver and liver cells. Transplantation, even liver tissue engineering provides ideal seed cells. However, there are many uncertainties in the factors affecting the effective differentiation of human ES cells to hepatocytes, the induction of differentiation efficiency and the functional status of differentiated cells. The application of ES cells to the many insurmountable obstacles in clinical Shang Youxu. This study explored the induction of human E S cells differentiate into hepatocytes with functions such as synthesis, metabolism, secretion and storage, and establish a human ES cell line that stably transfect pAlb-EGFP through genetic modification, providing a means to purify the target cells, which lays the foundation for the application of human ES cells in the liver regenerative medicine based on cell therapy and gene therapy.
This study mainly includes three parts.
First, the cytokine combined with extracellular matrix induced human ES cells to differentiate into liver cells. We used mouse embryonic fibroblast (MEF) as a feeder layer, and explored two methods of generation (mechanical and collagenase), and established a standard human ES cell culture system.
In order to further explore the potential of human ES cells to liver differentiation, we first suspended human ES cells in the early stage of culture to form a embryoid body (embryoid body, EB) containing the structure of the germ layer, and then inoculated them into a culture plate with type I collagen in advance, and induced the liver system with the conditioned medium containing dexamethasone and insulin. Multifaceted results, such as state of state, cell phenotype change and functional detection, show that the induced part of the cells basically conforms to the characteristics of hepatocytes, proving that human ES cells have the potential to differentiate into hepatocyte like cells.
Two, the enrichment of human ES cells into hepatocytes by co culture of co cultured transgenic stromal cells in different stages.
In the previous experimental study, we have confirmed that human ES cells can differentiate into hepatocyte like cells in a specific environment, but the result of differentiation is still in dispute. Because different from adult stem cells, ES cells have a more specific omnipotent differentiation potential and can differentiate into three outer and outer tissues from the inner, middle and outer cells. The dirty wall endoderm (visceral endoderm) can express many genes similar to the liver cells, but it can only participate in the formation of the outer yolk sac structure, and can not differentiate into liver or pancreas and other parenchymal organs, which can cause obstacles to the real liver cells. Therefore, this study is based on the characteristics of the total differentiation of human ES cells. As well as the mechanism of liver development, a new strategy for co culture of phase enrichment and transgenic stroma cells was designed. First, a simple EB was formed for the early development of the primitive ectoderm containing the outer layer of the endoderm and the inner layer. Under the condition of low serum, the differentiation of.RT-PCR and immunofluorescence to the stereotypical endoderm was induced by high concentration of A. The results showed that the cells of activin A expressed high expression of 3D (80%) the endoderm marker SOX17, while the expression of alpha fetoprotein (alpha-fetoprotein, AFP) in the inner endoderm was missing, which proved that the cells were stereotypical endoderm cells. In order to further explore the potential and mechanism of the differentiation of the endoderm cells to the liver cells, we use the transgenic cells. The matrix cell co culture system simulates the microenvironment in the development of liver. After co culture of 11d, the morphology, cell phenotype changes and functional detection results show that the stereotypical endoderm cells can differentiate into the liver in the environment created by bFGF and fetal liver stromal cells, and further differentiate and mature under the action of HGF, OSM and Dex. The research has opened up a new way to get a lot of real hepatocytes.
Three, the establishment of pAlb-EGFP genetically modified human ES cell line
As mentioned earlier, the new induction strategy excludes the interference of similar cells, but the cells are still a mixture, and the unpredictable risks such as tumorigenesis and carcinogenesis can not be ignored. In this part, we intend to modify human ES cells by gene modification to provide a means to purify the target cells for future application. Albumin (albumin, Alb) is in the present study. In the early stage of liver development and in the adult liver, it is a specific marker to identify the liver cells. We designed plasmid pAlb-EGFP containing the Alb promoter to regulate the enhanced green fluorescent protein (EGFP) gene into human ES cells, because the vector contains a segment of the Neo resistance gene. Resistance screening can be carried out through G418 conditions. We first identified the optimum concentration of G418 screening (200 mu g/ml) through the conditional culture medium killing test. On this basis, we used EXGen 500 transfection reagent to transfect and carry out resistance screening. PCR results confirmed that the screened positive clones did contain Alb-EGFP sequence. Stable transfection may provide new tools for purify hepatocytes in future applications.
The differentiation of human ES cells to mature and physiological liver cells provides a new choice for the treatment of liver disease cells. It is of great significance for a country with high incidence of end-stage liver disease caused by viral hepatitis and various causes. Human ES cells with developmental omnipotence will be bioartificial liver, liver (dry / progenitor). Cell transplantation and gene transfer therapy for liver (stem / progenitor) cells provide excellent seed cells. Nuclear transplantation and existing ES cell banks are expected to play a greater role in the treatment of liver diseases. The application of modern biological science and its related technologies in this field will certainly accelerate its clinical migration. The application process in plant repair.
【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】：2008
【分类号】：R329

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