AFP启动子慢病毒载体的构建及人胎肝干细胞研究

发布时间：2018-06-13 23:43

本文选题：胎肝细胞 + 免疫缺陷鼠　；参考：《华中农业大学》2005年硕士论文

【摘要】：世界上对干细胞的研究已有多年的历史,它已经成为当今生命科学研究的前沿之一。当前,由于各种肝病患者不断增长和肝脏捐献器官来源的缺乏,对肝干细胞的研究已经成为热点,这将有助于更好的了解许多肝脏疾病的产生机理和可能的治疗途径,而胎肝是研究肝干细胞很好的实验材料。本文通过以下三个方面对胎肝细胞的分离,培养,体外诱导分化及体内移植进行了研究: 1 将慢病毒载体plv的CMV启动子替换为人基因组AFP启动子,使该病毒感染胎肝细胞后,在AFP阳性细胞中表达绿色荧光蛋白,从而可利用流式细胞仪分选出表达AFP即GFP阳性的胎肝细胞。该标记筛选系统的建立为进一步研究胎肝细胞的生物学特性和肝干细胞的分离和应用奠定了良好的基础。 2 为了得到大量用于临床治疗和科学研究的细胞,仅靠分离原代细胞,不仅无法得到细胞量的保障,而且样品不稳定,费用昂贵。解决该问题最好的方法就是建立稳定的原代细胞系,但通常原代分离胚胎来源的细胞在培养一段时间后会分化,增殖能力逐渐减弱直到停止分裂。通过两种方法来建立胎肝来源的细胞系:分离胎肝细胞挑取细胞克隆,与饲养层细胞共培养来建立细胞系;分离胎肝细胞并稳定转染hTERT端粒酶基因,使之永生化。 3 为了研究分离的胎肝细胞的分化潜能,将其移植到免疫缺陷鼠体内,经检测发现它能够分化为多个组织的细胞。
[Abstract]:Stem cells have been studied in the world for many years. At present, due to the increasing number of patients with various liver diseases and the lack of donor organ sources, the research on liver stem cells has become a hot topic, which will contribute to a better understanding of the mechanism and possible therapeutic approaches of many liver diseases. Fetal liver is a good experimental material for studying liver stem cells. In this paper, the fetal liver cells were isolated and cultured in the following three aspects. The induction and differentiation in vitro and transplantation in vivo were studied. 1 the plv promoter of lentivirus vector was replaced with human genome AFP promoter, and the virus was infected with fetal hepatocytes. The expression of green fluorescent protein (GFP) in AFP positive cells can be used to select the fetal hepatocytes expressing AFP or GFP positive by flow cytometry. The establishment of the marker screening system laid a good foundation for the further study of the biological characteristics of fetal liver cells and the isolation and application of hepatic stem cells. 2. In order to get a large number of cells for clinical treatment and scientific research, The isolation of primary cells not only can not be guaranteed by the quantity of cells, but also the sample is unstable and expensive. The best way to solve this problem is to establish stable primary cell lines, but usually the cells derived from primary embryos will differentiate after a period of culture, and the ability of proliferation will gradually weaken until the division is stopped. The cell lines derived from fetal liver were established by two methods: isolation of fetal liver cells, isolation of cell clones from fetal liver cells, co-culture with feeder layer cells to establish cell lines, isolation of fetal liver cells and stable transfection of hTERT telomerase gene. To study the differentiation potential of isolated fetal hepatocytes, they were transplanted into immunodeficient mice. It was found to be able to differentiate into cells of multiple tissues.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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