【作者】 黄伟；

【导师】 李亦平； 邵健忠；

【作者基本信息】 浙江大学 ， 细胞生物学， 2006， 硕士

【摘要】 成体干细胞的可塑性是目前细胞生物学的热点话题，特定的组织干／祖细胞具有多项分化潜能，不仅可以分化为同一胚层来源的细胞，而且可以跨胚层“横向分化”(Trans-differentiation)为不同胚层来源的细胞。但对干细胞的研究还是体内实验为主，没有成熟的体外分离培养肝干细胞的方法。骨髓间充质干细胞具有自我更新和多向分化潜能的特性。一些研究表明经适当处理或是在合适微环境下，骨髓来源的细胞可以在体外mRNA水平上表达多种肝脏特异性基因，表明骨髓可以作为一种肝细胞的祖细胞来源。为探讨体外成年小鼠骨髓间充质干细胞(BM MSCs)是否可通过三种细胞因子组合诱导定向分化为肝细胞，我们从小鼠骨髓中获得间充质干细胞，用添加20ng／ml HGF、10ng／ml FGF-4、10ng／ml OSM和10％NBS的IMDM培养液诱导这些细胞向肝细胞分化。诱导组细胞出现肝细胞样形态变化；培养第10天出现AFP基因表达，其中AFP第20天表达减弱；第15天开始出现ALB、CK18基因表达，而后持续到20天；第20天出现TAT表达。免疫荧光反应表明，培养20天时ALB、CK18表达为阳性；诱导20天的细胞PAS糖元合成反应呈阳性。从而，骨髓间充质干细胞可在HGF，FGF-4和OSM三种细胞因子组合诱导下跨越分化为肝细胞。该体系的建立可能有助于研究间充质干细胞跨越分化的机制，也为肝脏疾病的治疗提供细胞来源。更多还原

【Abstract】 At present, trans-differentiation of adult stem cells is a hot topic in cell biology. Committed tissue stem/progenitor cells possess multipotent differentiation. They can not only differentiated into cell types of the same embryonic layer, but also tran-differentiated into cell types of different embryonic layer. But the majority studies of the hepatic stem cell have been focused on in vivo and minority studies are in vitro. Bone marrow mesenchymal stem cells (BM MSCs) have self-renewal capacity and show multipotency of differentiation. A number of studies have readily demonstrated that, under proper treatments or in a suitable microenvironment, BM-derived cells express various liver specific genes at the mRNA level in vitro, indicating that BM may be an alternative source of hepatocyte progenitors. To establish an effective method for inducing mouse bone marrow mesenchymal stem cells to differentiate into hepatocytes, we isolated MSCs from mouse bone marrow and cultured them in Iscove’s Modified Dulbecco’s medium supplemented with 10% new bovine serum (NBS), 20 ng/mL hepatocyte growth factor (HGF), 10 ng/mL fibroblast growth factor-4 (FGF-4) and 10 ng/ml oncostatin m (OSM) for 20 days’ induction. In groups induced by three cytokines after 20 days, BM MSCs showed characteristic of hepatocytes. On day 10, AFP mRNA of MSCs could be detected by RT-PCR, and the expression of AFP decreased on day 20. CK18 mRNA could also be detected from day 15 to day 20. Albumin and TAT can be detected on day 20 by RT-PCR. Immunofluorescence assay for CK18, albumin showed positive staining reaction on day 20. Differentiated cells further demonstrated these cells also acquired functional characteristics of hepatocytes that they can store glycogen. In conclusion, HGF, FGF-4 and Oncostatin M (OSM) can be successfully employed to induce hepatic transdifferentiation from mouse BM cells. Thus the present work may serve as a novel model for the study of mechanism involved in mesenchymal stem cell transdifferentiation, and a resource for the cytotherapy of liver disease with BM cells.更多还原