人脂肪间充质干细胞体外诱导为肝脏样细胞的试验研究

Isolation and identification of human adipose tissue-derived mesenchymal stem cells and differentiation into hepatocyte-like cells in vitro

目的探讨人脂肪间充质干细胞体外分离、培养、鉴定及向肝脏样细胞分化的方法。方法采用胶原酶Ⅰ消化离心法获得人脂肪间充质干细胞(human adipose-derived mesenchymal stem cells,hADSCs),原代培养并采用流式细胞仪检测干细胞相关表面抗原白细胞分化抗原13(Cluster differentiation13,CD13)、CD73、CD34、CD45和人类白细胞DR抗原(human leukocyte antigen DR,HLA-DR)的表达,采用诱导培养基将hADSCs向成脂及成骨方向诱导。采用两阶段细胞因子诱导法诱导hADSCs,并观察其形态变化;采用实时聚合酶链式反应(real-time polymerase chain reaction,RT-PCR)法及细胞免疫组化法检测肝细胞相关基因甲胎蛋白(alpha fetoprotein,AFP)、细胞角蛋白18抗体(cytokeratin18,CK18)、细胞角蛋白19抗体(cytokeratin19,CK19)等在肝脏样细胞中的表达;采用糖元染色法(Periodic acid-Schiff stain,PAS)检测肝脏样细胞糖原合成情况。结果用消化离心法获取的hADSCs大小均匀,呈梭形的成纤维细胞样,体外能长期培养存活,并保持不分化状态;流式细胞仪检测显示hADSCs高表达CD13、CD73,低表达CD34、CD45及HLA-DR;hADSCs体外可被诱导成脂肪细胞及成骨细胞。hADSCs经成肝诱导后,细胞形态由梭形变为多角形的肝脏样细胞,这些肝脏样细胞高表达血清白蛋白(serum albumin,ALB)、AFP、CY3P4等基因,并表达肝细胞相关性蛋白HepPar-1、AFP、CK18、CK19。PAS染色显示肝脏样细胞胞质呈红色表现。结论利用消化离心法在体外成功构建了人原代脂肪干细胞系,可长期存活,反复传代,经诱导后可转化为有功能的肝脏样细胞,为脂肪间充质干细胞作为生物人工肝及肝组织工程的种子细胞提供理论依据。

Objective To explore the method of isolation,culture and identification of human adipose-derived mesenchymal stem cells（ hADSCs） and induce the hADSCs into hepatocyte-like cells in vitro.Methods hADSCs was obtained by collagenase I digestion centrifugation for primary culture and the stem cell surface antigens including CD13,CD73,CD34,CD45 and HLA-DR were determined by flow cytometry（ FCM）. The cells were induced into adipocytes and osteocytes by using induction medium. The hADSCs were induced by using the two-stage cytokine induction method and then the morphological changes were observed. The liver cell-related genes such as AFP,CK18,CK19 and so on were tested in the hepatocyte-like cells by RT-PCR. The expression of the above-mentioned molecules of the hepatocyte-like cells were tested by cell immunohisto-chemical method and glycogen synthesis function of the hepatocyte-like cells was tested by PAS staining. Results hADSCs obtained in the digestion centrifugation are of the uniform size and are fusiform fibroblast-like cells,their proliferation was good. Cells could survive and kept undifferentiated for a long time in vitro. Flow cytometry showed that cells highly expressed CD13,CD73,but the expression of CD34,CD45 and HLA-DR were low; hADSCs could be induced into adipocytes and osteocytes. After being induced into hepatocyte-like cells,the human adipose-derived cells,cellular morphology gradually became polygonal or round,showed high expression of liver cell-related gene,including alpha-fetal protein（ AFP）,albumin（ ALB）,and cytochrome CYP3A4. Besides,the obtained hepatocyte-like cells also expressed liver cell-related proteins incuding HepPar-1,AFP,CK18,CK19. PAS staining showed that the cytoplasm of hepatocyte-like cells were in red.Conclusion hADSCs could be isolated and obtained using digestion centrifugation,the obtained hADSCs could be repeatedly passaged andcould survive for long time,and could be induced into functional hepatocyte-like cells in vitro,which provide experimental basis for ADSCs as seed cells of cell therapy,bioartificial liver and liver tissue engineering.