基于基因打靶技术的人胚胎干细胞向功能性肝细胞分化的研究

发布时间：2018-05-18 01:08

本文选题：人胚胎干细胞 + TALEN　；参考：《吉林大学》2014年博士论文

【摘要】：人肝细胞作为检测药物吸收、分布、代谢、排泄及毒性的体外实验金标准模型之一，被广泛应用于临床前药物研究。CYP3A亚家族是细胞色素P450酶系家族中最为重要的成员，它在肝CYP中含量最高，在肝细胞对于药物代谢和解读功能中承担着50%的作用。新鲜分离的人原代肝脏细胞是用于药物研发以及生物人工肝的最理想细胞来源，但是由于肝脏供体的严重稀缺以及体外表型维持困难等特点，极大地限制了其应用；而其他的以细胞为基本单位成分的分析模型，，如其他动物来源的原代肝脏细胞，或人肝癌细胞系，其代谢酶类和表达谱皆不能真实地反应正常人肝细胞的生理表现，例如在一些肝癌细胞系中很多代谢酶的水平与人原代成体肝细胞呈指数级的差异；干细胞体外定向分化研究的进展，使来自于干细胞的肝细胞成为最接近正常人原代肝细胞来源成为可能，然而目前遇到的共性问题是这种在体外定向分化得到的肝细胞，往往在功能上不成熟，表现为参与药物代谢的重要P450蛋白表达量低，不能表达成熟肝细胞特异表达的转录因子或蛋白质；或者这种具备条件的成熟数量极少，很难区分，因此也很难大规模应用。目前尚缺乏能在体外识别信号通路的调节和代谢功能的可靠工具。本研究首次报道了利用TALEN技术构建了打靶的人胚胎干细胞系，在体外直接分化成为具有功能性的肝细胞；这一被标记的干细胞分化体系可以识别能够促进CYP3A4表达的候选者。基于TALEN打靶技术，我们将增强型绿色荧光蛋白eGFP的基因片段插入到CYP3A4基因的外显子区域，使得eGFP能够有效的被CYP3A4的启动子调控。这一敲入CYP3A4:eGFP的细胞系在体外被定向诱导分化为人肝细胞，利用GFP作为CYP3A4的报告基因，沿用前人报道的体外定向诱导分化的方法，验证了这一基因报告细胞系具有和野生型人胚胎干细胞系同样的分化潜能；同时，这个基因标记的人胚胎干细胞系能够正确反映在细胞分化过程中CYP3A4基因的实际表达情况，可以作为筛选影响肝脏细胞成熟过程中关键因子的体系。然后，利用这一被标记的干细胞分化体系，我们在肝脏细胞发育的前体阶段加入了不同的细胞因子，以期筛选出促进功能性肝细胞分化的因子。研究表明表皮生长因子EGF可以作为一个信号蛋白有效地促进和维持人肝细胞在体外CYP3A4的表达。相对于传统的分化体系，添加了EGF的分化体系使得在体外分化的人肝细胞从5.9%增加到40.2%。随后，我们将分化得到的肝细胞移植到肝损伤小鼠URG（Tet-uPA/Rag2-/-/Il2rg-/-）体内，2周后在小鼠的血清中即检测到了人白蛋白的表达，证明分化的细胞具有移植重建肝脏的能力。本研究从实用的角度出发，建立了一种便于操作，直观观察即能评价体外分化的肝脏细胞成熟情况的有效筛选体系；以此为基础，可以对现有的分化体系进行培养条件、诱导时间等方面的优化，以提高分化效率。同时，通过该体系的应用，我们发现EGF作为一个新的调节蛋白在CYP3A4基因表达中起到了至关重要的作用。更为重要的是，用此评价体系得到的肝脏细胞具有和人成体肝脏细胞相类似的蛋白表达和mRNA转录水平，用该体系评价出来的细胞极有可能成为药物开发中检测肝脏毒性、药物相互作用评估的工具。基于TALEN技术的基因打靶，与人胚胎干细胞向肝细胞定向分化体系的结合，可以成为在体外获得稳定代谢功能的人肝细胞的可靠筛选模型，从而为以后提供供体细胞、建立人源化动物模型在体研究和应用分化的干细胞进行体外药物筛选都具有着十分重要的意义。
[Abstract]:Human hepatocytes are one of the gold standard models for the detection of drug absorption, distribution, metabolism, excretion and toxicity in vitro. The.CYP3A subfamily is the most important member of the cytochrome P450 enzyme family, which is the most important member of the cytochrome CYP family. It has the highest content in the liver CYP, and is responsible for the metabolism and interpretation of the liver cells in the liver cells. The role of 0%.
Fresh isolated human primary liver cells are the most ideal source of cell for drug development and bioartificial liver. However, due to the severe scarcity of the liver donor and the difficulty of maintaining the phenotypes in vitro, its application is greatly limited; other analytical models, such as other animal sources, are the basic units of cells. The metabolic enzymes and expression profiles of the liver cells, or human hepatoma cells, can not truly reflect the physiological performance of normal human hepatocytes. For example, many metabolic enzymes in some liver cancer cells are at an exponential level with those of human primary hepatocytes; the progress in the study of stem cell differentiation in vitro is derived from stem cells. Hepatocyte becomes the closest source of primary hepatocyte origin to normal people. However, the common problem is that the liver cells that have been differentiated in vitro are often immature in function, showing the low expression of important P450 proteins involved in drug metabolism, and can not reach a transcriptional factor or protein expressed by the mature hepatocytes. It is difficult to discriminate, and therefore difficult to be used in large-scale applications. There is still a lack of reliable tools to identify the regulatory and metabolic functions of the signaling pathways in vitro.
This study is the first to report the use of TALEN technology to build a target human embryonic stem cell line, which is directly differentiated into functional liver cells in vitro; this labeled stem cell differentiation system can identify candidates for the promotion of CYP3A4 expression. Based on TALEN targeting, we will enhance the gene for enhanced green fluorescent protein eGFP The fragment was inserted into the exons of the CYP3A4 gene so that eGFP could be effectively regulated by the promoter of the CYP3A4. The cell line that entered CYP3A4:eGFP was directed to differentiate into human hepatocytes in vitro, and GFP was used as a reporter gene for CYP3A4. The gene report was used to verify the gene report. The cell line has the same differentiation potential as the wild human embryonic stem cell line. At the same time, the human embryonic stem cell line marked by this gene can correctly reflect the actual expression of CYP3A4 gene in the process of cell differentiation, and can be used as a system to screen the key factors in the process of liver cell maturation.
Then, using this labeled stem cell differentiation system, we added different cytokines to the precursors of the liver cell development in order to screen out factors that promote functional hepatocyte differentiation. The study shows that epidermal growth factor EGF can effectively promote and maintain human hepatocytes in CYP3A4 as a signal protein. Compared with the traditional differentiation system, the differentiation system of EGF was added to increase the differentiation of human hepatocytes from 5.9% to 40.2%., and the differentiated hepatocytes were transplanted into the liver injury mice URG (Tet-uPA/Rag2-/-/Il2rg-/-). After 2 weeks, the expression of human albumin was detected in the blood albumin of the mice. The transformed cells have the ability to transplant and reconstruct the liver.
From a practical point of view, this research establishes an effective screening system which is convenient for operation and intuitionistic observation that can evaluate the maturation of liver cells differentiated in vitro. Based on this, we can optimize the existing differentiation system and induce time to improve the differentiation efficiency. At the same time, through the application of the system, We found that EGF plays a vital role in the expression of CYP3A4 gene as a new regulatory protein. More importantly, the liver cells obtained by this evaluation system have a similar protein expression and mRNA transcriptional level with human adult liver cells, and the cells evaluated with this system are extremely likely to become drug development. A tool for the detection of liver toxicity and drug interaction. Based on the gene targeting of TALEN technology and the combination of human embryonic stem cells to the directional differentiation system of liver cells, it can be a reliable screening model for human liver cells with stable metabolism in vitro, thus providing donor cells and establishing a humanized animal model for the future. Research and differentiation of stem cells in vitro drug screening are of great significance.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96

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