【摘要】：目前,胚胎干細(xì)胞體外誘導(dǎo)為肝細(xì)胞的主要途徑是通過擬胚體啟動(dòng)ES細(xì)胞分化,再通 過細(xì)胞因子與胞外基質(zhì)協(xié)同作用等方法完成肝細(xì)胞的發(fā)育與成熟。由于擬胚體法得到的細(xì)胞 異質(zhì)程度高而且分化周期長,不利于細(xì)胞移植治療。因此,通過體外培養(yǎng)的ES細(xì)胞直接分 化為肝細(xì)胞的研究已_丌始受到重視。本實(shí)驗(yàn)利用試劑A(藥品代號)誘導(dǎo),在不經(jīng)過擬胚體 的情況下,直接將ES細(xì)胞誘導(dǎo)分化為肝細(xì)胞,并探討了該誘導(dǎo)分化過程中出現(xiàn)的一種具有 多分化潛能的祖細(xì)胞的性質(zhì)與功能。 實(shí)驗(yàn)分為兩部分。第一部分為ES細(xì)胞在試劑A作用下向一種AFp~+/Nestin~+/CKl9~+卵圓形 樣祖細(xì)胞的分化,并對這種祖細(xì)胞的性質(zhì)進(jìn)行鑒定。ES在2.5mM試劑A作用下,細(xì)胞形態(tài) 發(fā)生明顯變化,在試劑A處理6d之后,撤去試劑A繼續(xù)培養(yǎng)7d,可觀察到一種特殊類型的 卵圓形細(xì)胞的出現(xiàn)。利用RT-PCR方法對這種細(xì)胞的mRNA表達(dá)進(jìn)行檢測,發(fā)現(xiàn)這種細(xì)胞表達(dá) AFP、CKl9和Nestin。免疫細(xì)胞化學(xué)檢測,發(fā)現(xiàn)這種細(xì)胞AFP、CKl9和Nestin呈陽性,初 步將這種細(xì)胞定義為AFP~+/Nestin~+/CKl9~+祖細(xì)胞。 第二部分為上述試劑A誘導(dǎo)分化而成的AFP~+/Nestin~+/CKl9~+祖細(xì)胞多方向分化潛能的鑒 定。包括祖細(xì)胞向肝細(xì)胞的分化,向膽管上皮樣細(xì)胞的分化和向神經(jīng)細(xì)胞的分化。將 AFP~+/Nestin~+/CKl9~+祖細(xì)胞用兩種包含不同濃度和不同細(xì)胞因子的肝細(xì)胞成熟培養(yǎng)液進(jìn)行培 養(yǎng),一組培養(yǎng)液為低濃度的HGF、Dex和lTS組合,一組培養(yǎng)液為bFGF、Dex和較高濃度的 HGF、OSM的組合。培養(yǎng)15d后,在低濃度因子處理組細(xì)胞中檢測到TAT、HNF4和ALB等mRNA 的表達(dá),免疫組化和免疫熒光檢測顯示此細(xì)胞表達(dá)AFP和微弱的ALB,糖原合成PAS反應(yīng)也 呈陽性。而在高濃度因子處理組細(xì)胞中則檢測到了ALB的強(qiáng)表達(dá)。將祖細(xì)胞培養(yǎng)在胞外基質(zhì) Matrigel上,以含100ng/m1 HGF的William E培養(yǎng)液培養(yǎng)7d后觀察發(fā)現(xiàn)祖細(xì)胞形成膽管 上皮樣結(jié)構(gòu)。將祖細(xì)胞培養(yǎng)在含RA的培養(yǎng)液中,6d后觀察發(fā)現(xiàn)具有明顯絲狀突起的神經(jīng)樣 細(xì)胞。 因此,我們在試劑A誘導(dǎo)的ES細(xì)胞體外分化過程中,分離鑒定到了一種 AF})~+/Nestin~+/CKl9~+祖細(xì)胞,這種祖細(xì)胞在合適的培養(yǎng)條件和培養(yǎng)環(huán)境中,能分化為肝細(xì)胞、 膽管上皮樣細(xì)胞和神經(jīng)樣細(xì)胞,具有多分化潛能。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R329

【共引文獻(xiàn)】

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