肝细胞条件培养液对人脂肪问充质干细胞向肝细胞分化和增殖的作用

摘要

目的 探讨人脂肪间充质干细胞在改良的诱导体系下向肝细胞的分化和增殖情况,为肝组织工程提供新的种子细胞来源.方法 从人脂肪组织分离出脂肪间充质干细胞,用含有碱性成纤维细胞生长因子、肝细胞生长因子、成纤维细胞生长因子-4的肝细胞诱导液进行诱导,并于诱导7 d后加入抑瘤素M.用细胞计数试剂盒-8法检测整个诱导过程细胞的增殖情况;通过光学显微镜观察诱导细胞的形态变化;用RT-PCR法和免疫荧光法分别检测肝细胞特异性基因和蛋白的表达;并对多种肝细胞特异性功能进行检测.组间比较采用t-test检验.结果 用改良肝细胞诱导液培养的人脂肪间充质干细胞在培养第5、7、14、21天时,细胞数均明显多于用对照培养液培养的细胞(f值分别为6.59、8.69、15.94和24.64,P值均＜0.05).诱导细胞表现出上皮样肝细胞形态,表达肝细胞特异性基因和蛋白;具有多种肝细胞特异性功能,如靛青绿摄取/排泌、糖原合成以及白蛋白分泌功能.结论 人脂肪间充质干细胞在含碱性成纤维细胞生长因子、肝细胞生长因子、成纤维细胞生长因子-4和抑瘤索M的诱导体系中能够分化为更加成熟的具有多种肝细胞特异性功能的细胞,且此诱导体系同时具有促进细胞增殖的作用.%Objective To induce hepatic differentiation of human adipose-derived stem cells (hADSCs) in vitro. Methods hADSCs were isolated from human adipose tissue and treated with improved hepatic medium containing HGF, bFGF and FGF4. After 7 days of culture, OSM was added to the culture media. Cell growth during hepatic differentiation was evaluated by CCK8 assay. Morphology of differentiation was examined under light microscope. Liver speeific genes and proteins were detected by RT-PCR analysis and immunohistochemical staining, respectively. And functional characteristics of hepatocytes were also examined.Results The number ofhADSCs cultured in the improved hepatic media was increased significantly in comparison to hADSCs cultured in control media from 5 days to 21days (t = 6.59, 8.69, 15.94 and 24.64, respectively, P ＜ 0.05). The hADSCs-derived hepatocyte-like cells exhibited hepatocyte morphology, expressed hepatocyte markers, possessed hepatocyte-specific activities, such as uptake and excretion of indocyanine green, glycogen storage and albumin production. Conclusion hADSCs can be induced into hepatocyte-like cells in this differentiation system. And this differentiation system promoted the growth of hADSCs.