胚胎干细胞体外分化肝细胞的研究及一种新型祖细胞的鉴定

发布时间：2018-09-04 18:16

【摘要】：目前,胚胎干细胞体外诱导为肝细胞的主要途径是通过拟胚体启动ES细胞分化,再通过细胞因子与胞外基质协同作用等方法完成肝细胞的发育与成熟。由于拟胚体法得到的细胞异质程度高而且分化周期长,不利于细胞移植治疗。因此,通过体外培养的ES细胞直接分化为肝细胞的研究已_丌始受到重视。本实验利用试剂A(药品代号)诱导,在不经过拟胚体的情况下,直接将ES细胞诱导分化为肝细胞,并探讨了该诱导分化过程中出现的一种具有多分化潜能的祖细胞的性质与功能。实验分为两部分。第一部分为ES细胞在试剂A作用下向一种AFp~+/Nestin~+/CKl9~+卵圆形样祖细胞的分化,并对这种祖细胞的性质进行鉴定。ES在2.5mM试剂A作用下,细胞形态发生明显变化,在试剂A处理6d之后,撤去试剂A继续培养7d,可观察到一种特殊类型的卵圆形细胞的出现。利用RT-PCR方法对这种细胞的mRNA表达进行检测,发现这种细胞表达 AFP、CKl9和Nestin。免疫细胞化学检测,发现这种细胞AFP、CKl9和Nestin呈阳性,初步将这种细胞定义为AFP~+/Nestin~+/CKl9~+祖细胞。第二部分为上述试剂A诱导分化而成的AFP~+/Nestin~+/CKl9~+祖细胞多方向分化潜能的鉴定。包括祖细胞向肝细胞的分化,向胆管上皮样细胞的分化和向神经细胞的分化。将 AFP~+/Nestin~+/CKl9~+祖细胞用两种包含不同浓度和不同细胞因子的肝细胞成熟培养液进行培养,一组培养液为低浓度的HGF、Dex和lTS组合,一组培养液为bFGF、Dex和较高浓度的 HGF、OSM的组合。培养15d后,在低浓度因子处理组细胞中检测到TAT、HNF4和ALB等mRNA 的表达,免疫组化和免疫荧光检测显示此细胞表达AFP和微弱的ALB,糖原合成PAS反应也呈阳性。而在高浓度因子处理组细胞中则检测到了ALB的强表达。将祖细胞培养在胞外基质 Matrigel上,以含100ng/m1 HGF的William E培养液培养7d后观察发现祖细胞形成胆管上皮样结构。将祖细胞培养在含RA的培养液中,6d后观察发现具有明显丝状突起的神经样细胞。因此,我们在试剂A诱导的ES细胞体外分化过程中,分离鉴定到了一种 AF})~+/Nestin~+/CKl9~+祖细胞,这种祖细胞在合适的培养条件和培养环境中,能分化为肝细胞、胆管上皮样细胞和神经样细胞,具有多分化潜能。
[Abstract]:At present, the main way of inducing embryonic stem cells into hepatocytes in vitro is to initiate the differentiation of ES cells through embryoid bodies. The development and maturation of hepatocytes were accomplished by the synergistic action of cytokines and extracellular matrix. Because of the high heterogeneity and long differentiation cycle of the cells obtained by the embryoid method, it is not conducive to cell transplantation therapy. Therefore, the study on the direct differentiation of ES cells into hepatocytes in vitro has been paid more and more attention. In this experiment, ES cells were induced to differentiate into hepatocytes directly by reagent A (drug code name) without embryoid. The properties and functions of a kind of progenitor cells with multiple differentiation potential in the process of inducing differentiation were discussed. The experiment is divided into two parts. The first part is the differentiation of ES cells into a kind of AFp~ / Nestin ~ / CKl9oval progenitor cells under the action of reagent A. the properties of the progenitor cells were identified by 2.5mM reagent A. After 6 days of treatment with reagent A, the cell morphology changed obviously. A special type of oval cells was observed after 7 days of culture. RT-PCR method was used to detect the mRNA expression of the cells. It was found that the cells expressed AFP,CKl9 and Nestin.. Immunocytochemistry showed that the cells were positive for AFP,CKl9 and Nestin. In the first step, the cells were defined as AFP~ / Nestin ~ / CKl9- progenitor cells. The second part is the identification of the multidirectional differentiation potential of AFP~ / Nestin ~ / CKl9- progenitor cells induced by reagent A. These include the differentiation of progenitor cells into hepatocytes, to epithelial-like cells of bile duct and to nerve cells. AFP~ / Nestin / CKl9- progenitor cells were cultured in two kinds of hepatocyte maturation medium containing different concentrations and different cytokines. One group was composed of low concentration of HGF,Dex and lTS, one group of medium was combination of bFGF,Dex and higher concentration of HGF,OSM. After 15 days of culture, the expression of mRNA, such as TAT,HNF4 and ALB, was detected in the cells treated with low concentration factor. The expression of AFP and the weak PAS reaction of ALB, glycogen were also positive by immunohistochemistry and immunofluorescence. The high expression of ALB was detected in the cells treated with high concentration of factors. Progenitor cells were cultured on extracellular matrix Matrigel. After being cultured in William E medium containing 100ng/m1 HGF for 7 days, it was observed that the progenitor cells formed epithelial-like structure of bile duct. After the progenitor cells were cultured in the culture medium containing RA for 6 days, the neuron-like cells with obvious filamentous processes were observed. Therefore, during the differentiation of ES cells induced by reagent A in vitro, we isolated and identified a kind of AF} ~ / Nestin ~ / CKl9- progenitor cells, which were cultured under suitable conditions and culture conditions. It can differentiate into hepatocytes, bile duct epithelioid cells and nerve like cells.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329

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