新生猕猴肝脏上皮前体细胞无血清培养体系的建立

doi:10.3969/j.issn.1674-5817.2011.01.005

实验动物与比较医学 ›› 2011, Vol. 31 ›› Issue (1): 20-27.DOI: 10.3969/j.issn.1674-5817.2011.01.005

新生猕猴肝脏上皮前体细胞无血清培养体系的建立

孙爱静¹, 黄狄来¹, 茹国美¹, 金立方²

1.绍兴市人民医院＆绍兴文理学院附属第一医院, 医学研究中心动物实验室, 绍兴 312000;
2.绍兴文理学院生命科学学院, 绍兴312000

收稿日期:2010-05-31 出版日期:2011-02-25 发布日期:2011-02-25
作者简介:孙爱静(1963-), 女, 医学博士, 教授, 从事实验动物学研究和肿瘤病理学研究,E-mail: sun_aijing@hotmail.oc.jp
基金资助:
浙江省生物医学重中之重开放基金(SWVX0907)

Culture of Newborn Rhesus Monkey Liver Epithelial Progenitor Cells in Chemical Defined Serum-free Medium

SUN Ai-jing¹, Huang Di-lai¹, RU Guo-mei¹, JIN Li-fang²

1. Department of Pathology, Shaoxing People’s Hospital and the First Affiliated Hospital of Shaoxing University, Shaoxing 312000, China;
2. College of Life Science of Shaoxing University, Shaoxing 312000, China

Received:2010-05-31 Online:2011-02-25 Published:2011-02-25

摘要/Abstract

摘要： 目的建立猕猴肝上皮前体细胞的无血清体外培养体系。方法取健康新生猕猴肝脏, 剪切、消化、离心后在胶原培养体系中用含Ca²⁺的培养基培养, 选取多角形细胞集落, 在不同胞外基质中分离培养, 应用细胞免疫荧光染色、细胞化学染色、RT-PCR、靛青绿吸收染色方法进行细胞表型和双向分化潜能鉴定。结果多角形细胞团块在层粘连蛋白或大鼠鼠尾胶原上增殖传代形成集落, 并特异性地表达肝干细胞的特异性标志物, 不表达成熟肝细胞标志。经地塞米松和抑瘤素诱导分化后表达成熟肝细胞的标志特征, 在小鼠胎儿成纤维饲养层, 表达胆管细胞的特异性标志蛋白并形成胆管样结构。结论利用无血清培养体系从新生猕猴肝组织中分离到具有肝前体细胞特性的多角上皮细胞,这一研究模型的建立可为人类肝脏疾病的研究和细胞治疗提供临床前的评估平台。

关键词: 肝干/前体细胞, 双分化潜能, 无血清, 细胞外基质

Abstract: Objective to establish a chemical defined serum-free culture system for rhesus monkey hepatic progenitor cells. Methods a rhesus monkey liver polygon cell colony was derived from a small piece of newborn livers using chemical defined serum-free medium. Follow by isolated and cultured in different extracellular matrix. And the identification of the cells phenotype and dual-differentiation potential for functional hepatocytes and biliary duct cells in vitro were carry out using immunofluorescence cell staining, Periodic Acid-Shiff Staining, RT-PCR, indocyanine green (ICG) uptake staining method. Result Two types of cell colony were generated: elongated spindle cells and polygonal epithelial cells. Elongated spindle cells were expressed as vimentin and brachyury, and they were disappeared within 5 days in our cultures. The remaining consisted of small polygonal epithelial cells that expressed cytokeratin 7 (CK7), CK8, CK18, nestin, CD49f and E-cad, the markers of hepatic stem cells, but were negative for 〈-fetoprotein, albumin and CK19. They can proliferate and be passaged, if on laminin or rat tail collagen gel, to initiate colonies. When cultured with dexamethasone and oncostatin M, the expression of mature hepatocyte markers, such as 〈-1-antitrypsin, intracytoplasmic glycogen storage, indocyanine green uptake and lipid droplet generation, were induced in differentiated cells. If transferred onto mouse embryonic fibroblasts feeders, they gave rise to CK19 positive cholangiocytes with formation of doughnut-like structure. Conclusion we isolated a polygon epithelial cell with characteristics of live progenitor cells from newborn rhesus monkey live tissue using a chemical defined serum-free culture system, and. may serve as an in vitro model of primate liver development and may facilitate further preclinical research.

Key words: Hepatic progenitor cells, Dual-differentiation potential, Serum free, Extracellullar matrix

孙爱静, 黄狄来, 茹国美, 金立方. 新生猕猴肝脏上皮前体细胞无血清培养体系的建立[J]. 实验动物与比较医学, 2011, 31(1): 20-27.

SUN Ai-jing, Huang Di-lai, RU Guo-mei, JIN Li-fang. Culture of Newborn Rhesus Monkey Liver Epithelial Progenitor Cells in Chemical Defined Serum-free Medium[J]. Laboratory Animal and Comparative Medicine, 2011, 31(1): 20-27.

参考文献

[1] Nussler A, Konig S, Ott M,et al.Present status and perspectives of cell-based therapies for liver diseases[J]. J Hepatol, 2006, 45:144-159.
[2] Suzuki A, Zheng YW, Kaneko S, et al.Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver[J]. J Cell Biol, 2002,156:173-184.
[3] Schmelzer E, Zhang L, Bruce A, et al.Human hepatic stem cells from fetal and postnatal donors[J]. J Exp Med, 2007, 204:1973-1987.
[4] Jin L, Ji S, Tang X, et al.Isolation and characterization of liver epithelial progenitor cells from normal adult rhesus monkeys (Macaca mulatta)[J]. Cell Res, 2009, 19:268-270.
[5] Kano J, Noguchi M, Kodama M, et al.The in vitro differentiating capacity of nonparenchymal epithelial cells derived from adult porcine livers[J]. Am J Pathol, 2000, 156:2033-2043.
[6] Brill S, Zvibel I,Reid, L.M.Expansion conditions for early hepatic progenitor cells from embryonal and neonatal rat livers[J]. Dig Dis Sci, 1999, 44:364-371.
[7] Tsuchiya A, Heike T, Fujino H, et al.Long-term extensive expansion of mouse hepatic stem/progenitor cells in a novel serum-free culture system[J]. Gastroenterology, 2005, 128:2089-2104.
[8] Sasaki K, Kon J, Mizuguchi T, et al.Proliferation of hepatocyte progenitor cells isolated from adult human livers in serum-free medium[J]. Cell Transplant, 2008, 17:1221-1230.
[9] Arends B, Spee B, Schotanus BA, et al.In vitro differentiation of liver progenitor cells derived from healthy dog livers[J]. Stem Cells Dev, 2009, 18:351-358.
[10] Chen Q, Kon J, Ooe H,et al.Selective proliferation of rat hepatocyte progenitor cells in serum-free culture[J]. Nat Protoc, 2007, 2:1197-1205.
[11] Turner WS, Schmelzer E, McClelland R, et al. Human hepatoblast phenotype maintained by hyaluronan hydrogels[J]. J of Biomed Mater Res, 2007, 82:156-168.
[12] Azuma H, Hirose T, Fujii H, et al.Enrichment of hepatic progenitor cells from adult mouse liver[J]. Hepatology, 2003, 37(6):1385-1394.
[13] Dunn JC, Tompkins RG, Yarmush ML.Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration[J]. Biotechnol Prog, 1991, 7(3):237-245.
[14] Koebe HG, Pahernik S, Eyer P.Collagen gel immobilization: a useful cell culture technique for long-term metabolic studies on human hepatocytes[J]. Xenobiotica. 1994 , 24(2):95-107.
[15] Malhi H, Irani AN, Gagandeep S.Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes[J]. J Cell Sci, 2002, 115:2679-2688.
[16] Valdes F, Alvarez AM, Locascio A.The epithelial mesenchymal transition confers resistance to the apoptotic effects of transforming growth factor Beta in fetal rat hepatocytes[J]. Mol Cancer Res, 2002, 1:68-78.
[17] Thiery JP.Epithelial-mesenchymal transitions in development and pathologies[J]. Curr Opin Cell Biol, 2003, 15:740-746.
[18] Paku S, Schnur J, Nagy P.Origin and structural evolution of the early proliferating oval cells in rat liver[J]. Ame J Ppathol, 2001, 158:1313-1323.
[19] Zhang W, Chen XP, Zhang WG.Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration[J]. World J Gastroenterol, 2009, 15:552-560.
[20] Suzuki A, Zheng YW, Kaneko S.Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver[J]. J Cell Biol, 2002, 156:173-184.
[21] Hoppo T, Fujii H, Hirose T.Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver[J]. Hepatology, 2004, 39:1362-1370.
[22] Rao MS, Khan AA, Parveen N.Characterization of hepatic progenitors from human fetal liver during second trimester[J]. World J Gastroenterol, 2008, 14:5730-5737.
[23] 孙晓燕, 安靓. 神经中间丝蛋白nestin在胎肝中的表达[J].第一军医大学学报, 2004, 24:207-209.
[24] Herrera MB, Bruno S, Buttiglieri S.Isolation and characterization of a stem cell population from adult human liver[J]. Stem cells, 2006, 24:2840-2850.
[25] Koenig S, Krause P, Drabent B.The expression of mesenchymal, neural and haematopoietic stem cell markers in adult hepatocytes proliferating in vitro[J]. J Hepatol, 2006,44:1115-1124.
[26] Wiese C, Rolletschek A, Kania G.Nestin expression-a property of multi-lineage progenitor cells?[J]Cell Mol Life Sci, 2004, 61:2510-2522.
[27] Koenig S, Probst I, Becker H.Zonal hierarchy of differentiation markers and nestin expression during oval cell mediated rat liver regeneration[J]. Histochem Cell Biol, 2006, 126:723-734.
[28] Nitou M, Sugiyama Y, Ishikawa K.Purification of fetal mouse hepatoblasts by magnetic beads coated with monoclonal anti-e-cadherin antibodies and their in vitro culture[J]. Exp Cell Res, 2002, 279:330-343.
[29] Nierhoff D, Ogawa A, Oertel M.Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity[J]. Hepatology, 2005, 42:130-139.
[30] Nyamath P, Alvi A, Habeeb A.Characterization of hepatic progenitors from human fetal liver using CD34 as a hepatic progenitor marker[J].World J Gastroenterol, 2007, 13:2319-2323.
[31] Factor VM, Radaeva SA, Thorgeirsson SS.Origin and fate of oval cells in dipin-induced hepatocarcinogenesis in the mouse[J]. Ame J Pathol, 1994, 145:409-422.
[32] Preisegger K, Factor VM, Fuchsbichler A.Atypical ductular proliferation and its inhibition by transforming growth factor beta1 in the 3,5-diethoxycarbonyl- 1,4-dihydrocollidine mouse model for chronic alcoholic liver disease[J]. Lab Invest, 1999,79(2):103-109.

新生猕猴肝脏上皮前体细胞无血清培养体系的建立

Culture of Newborn Rhesus Monkey Liver Epithelial Progenitor Cells in Chemical Defined Serum-free Medium

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