人脂肪间充质干细胞体外诱导为肝脏样细胞的试验研究

doi:10.3969/j.issn.1006-7795.2014.05.018

摘要/Abstract

摘要：

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。

关键词: 人脂肪间充质干细胞, 分离, 鉴定, 诱导, 肝脏样细胞

Abstract:

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 and could 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.

Key words: human adipose tissue-derived mesenchymal stem cells (hADSCs), isolation, identification, differentiation, hepatocyte-like cells

中图分类号:

R329

张云巍, 徐丽娟, 王淑芳, 阎丽. 人脂肪间充质干细胞体外诱导为肝脏样细胞的试验研究[J]. 首都医科大学学报, 2014, 35(5): 608-616.

Zhang Yunwei, Xu Lijuan, Wang Shufang, Yan Li. Isolation and identification of human adipose tissue-derived mesenchymal stem cells and differentiation into hepatocyte-like cells in vitro[J]. Journal of Capital Medical University, 2014, 35(5): 608-616.

参考文献

[1] Liu J, Fan D. Hepatitis B in China[J]. Lancet, 2007, 369(9573):1582-1583.
[2] Dhawan A, Puppi J, Hughes R D, et al. Human hepatocyte transplantation: current experience and future[J]. Nat Rev Gastroenterol Hepatol,2010,7(5):288-298.
[3] Okura H, Komoda H, Saga A, et al. Properties of hepatocyte-like cell clusters from human adipose tissue-derived mesenchymal stem cells[J]. Tissue Eng Part C Methods, 2010,16(4):761-770.
[4] Zaret KS, Grompe M. Generation and regeneration of cells of the liver and pancreas[J]. Science,2008,322(5907):1490-1494.
[5] Terry C, Hughes R D, Mitry R R, et al. Cryopreservation-induced nonattachment of human hepatocytes: role of adhesion molecules[J].Cell Transplant,2007,16(6):639-647.
[6] Thomson J A, Itskovitz-Eldor J, Shapiro S S, et al. Embryonic stem cell lines derived from human blastocysts[J].Science,1998,282(5391):1145-1147.
[7] Gallicano G I, Mishra L. Hepatocytes from induced pluripotent stem cells:a giant leap forward for hepatology[J]. Hepatology,2010,51(1):20-22.
[8] Lee K D, Kuo T K, WhangPeng J, et al. In vitro hepatic differentiation of human mesench-ymal stem cells[J]. Hepatology,2004,40(6):1275-1284.
[9] Snykers S, Vanhaecke T, Papeleu P, et al. Sequential exposure to cytokines reflecting-embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells[J]. Toxicol Sci,2006,94(2):330-341,discussion:235-339.
[10] Lee M J, Jung J, Na K H, et al. Anti-fibrotic effect of chorionic plate-derived mesenchymal stem cells isolated from human placenta in a rat model of CCl(4)-injured liver: potential application to the treatment of hepatic diseases[J].J Cell Biochem,2010,111(6):1453-1463.
[11] Shin K S, Lee H J, Jung J, et al. Culture and in vitro hepatogenic differentiation of placenta-derived stem cells, using placental extract as an alternative to serum[J]. Cell Prolif,2010,43(5):435-444.
[12] Lagasse E, Connors H, Al-Dhalimy M,et al. Purified hematopoietic stem cellscandifferentiate into hepatocytes in vivo[J]. Nat Med,2000,6(11):1229-1234.
[13] Zuk P A, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies[J].Tissue Eng,2001,7(2):211-228.
[14] Zuk P A, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells[J]. Mol Biol Cell,2002,13(12):4279-4295.
[15] di Bonzo L V, Ferrero I, Cravanzola C, et al. Human mesenchymal stem cells as a twoedged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential[J]. Gut,2008,57(2):223-231.
[16] Chen Y, Dong X J, Zhang G R, et al. In vitro differentiation of mouse bone marrow stromal stem cells into hepatocytes induced by conditioned culture medium of hepatocytes[J]. J Cell Biochem,2007, 102(1): 52-63.
[17] Matsunaga T, Sakamaki S, Kohgo Y, et al. Recombinant human granulocyte Colony-stimulating factor can mobilize sufficient amounts of peripheral blood stem cells in healthy volunteers for allogeneic transplantation[J]. Bone Marrow Transplant,1993,11(2):103-108.
[18] Kasai M, Kiyama Y, Kawamura A. Application of peripheral blood stem cells(PBSC) mobilized by recombinant human granulocyte colony stimulating factor for allogeneic PBSC transplantation and the comparison of allogeneic PBSC transplantation and bone marrow transplantation[J]. Transfus Apher Sci,2002,26(2):121-127.
[19] Conget P A, Min guell J J. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells[J].J Cell Physiol,1999,181(1):67-73.
[20] Seo M J, Suh S Y, Bae Y C, et al. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem[J]. Biophys Res Commun,2005,328: 258-264.
[21] Safford K M, Hicok K C, Safford S D, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells[J]. BiochemBiophys Res Commun,2002,294:371-379.
[22] Lee R H, Kim B, Choi Iet al. Characterization and expression analysis of mesenchymal stem cells from human bone mar-row and adipose tissue[J]. Cell Physiol Biochem,2004,14(4-6):311-324.
[23] Katz A J, Tholpady A, Tholpady S S, et al.Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells[J]. Stem Cells,2005,23(3):412-423.
[24] Gronthos S, Franklin D M, Leddy H A, et al. Surface protein characterization of human adipose tissue-derived stro-mal cells[J]. J Cell Physiol,2001,189(1):54-63.
[25] Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood or adi-pose tissue[J]. Stem Cells,2006,24(5):1294-1301.
[26] Strem B M, Hicok K C, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells[J]. Keio J Med,2005,54(3):132-141.
[27] Dicker A, Le Blanc K, Astrom G,et al. Functional studies of mesenchymal stem cells derived from adult human adipose tissue[J]. Exp Cell Res,2005,308(2):283-290.
[28] Im G I, Shin Y W, Lee K B. Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells?[J]. Osteoarthritis Cartilage,2005,13(10):845-853.
[29] Safford K M, Hicok K C, Safford S D, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells[J]. BiochemBiophys Res Commun,2002,294(2):371-379.
[30] Secco M, Moreira Y B, Zucconi E, et al. Gene expression profile of mesenchymal stem cells from paired umbilical cord units: cord is different from blood[J]. Stem Cell Rev, 2009,5(4):387-401.
[31] 王晓辉,惠玲,杨霄鹏,等.人脐带间充质干细胞体外培养、鉴定及其诱导分化的研究[J]. 解放军医药杂志,2013,25(10):11-15,30.
[32] Ruhnke M, Ungefroren H, Nussler A, et al. Differentiation of in vitro modified human peripheral blood monocytes into hepatocytelike and pancreatic isletlike cells[J]. Gastroenterology, 2005,128(7):1774-1786.
[33] Izadpanah R, Trygg C, Patel B, et al. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue[J]. J Cell Biochem,2005,99(5): 1285-1297.
[34] He Z, Feng M. Activation, isolation, identification and culture of hepatic stem cells from porcine liver tissues[J]. Cell Prolif,2011,44(6):558-566.
[35] Yan L, Han Y, Wang J, et al. Peripheral blood monocytes from patients with HBV related decompensated liver cirrhosis can differentiate into functional hepatocytes[J]. Am J Hematol,2007,82(11):949-954.
[36] 尹鹏,梁庆威.外源性骨形态发生蛋白7联合血管内皮生长因子诱导大鼠脂肪干细胞向成骨细胞分化的实验研究[J]. 中国医科大学学报,2011,(10):897-901.
[37] Banas A, Teratani T, Yamamoto Y, et al. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes[J]. Hepatology, 2007, 46(1): 219-228.
[38] Aurich H, Sgodda M, Kaltwasser P,et al. Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo[J]. Gut,2009,58(4):570-581.
[39] Fiegel H C, Lioznov M V, Cortes-Dericks L, et al. Liver-Specific gene expression in cultured human hematopoietic stem cells[J]. Stem Cells,2003, 21(1):98-104.
[40] Takeda M, Yamamoto M, Isoda K, et al.Availability of bone marrow stromal cells in three-dimensional coculture with hepatocytes and transplantation into liver damaged mice[J]. J Biosci Bioeng,2005,100(1):77-81.
[41] Wang P P, Wang J H, Yan Z P, et al. Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction[J].Biochem Biophys Res Commun,2004,320(3):712-716.
[42] 张刚庆,方驰华,池达智.肝细胞生长因子诱导骨髓间充质干细胞向肝细胞分化的实验研究[J].中华外科杂志,2005,43(11):716-720.
[43] Schwartz R E, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells[J]. J Clin Invest, 2002, 109(10): 1291-1302.