中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (1): 48-52.doi: 10.3969/j.issn.1673-8225.2010.01.011

• 干细胞培养与分化 • 上一篇    下一篇

人脐血间充质干细胞向类肝细胞分化
人肝细胞共培养诱导法的可行性？

韩翠萍1，刘吉勇1，高 蕾2，张晓华1，裴庆山1，孙欣欣1   

  1. 1山东大学附属省立医院消化内科，山东省济南市 250021；
    2深圳市宝安区人民医院，广东省深圳市 51810
  • 出版日期:2010-01-04 发布日期:2010-01-04
  • 通讯作者: 通讯作者：刘吉勇，教授，山东大学附属省立医院消化内科，山东省济南市 250021 liujiyong@medmail.com.cn
  • 作者简介:韩翠萍，女，1984年生，山东省聊城市人，汉族，山东大学医学院在读硕士，主要从事干细胞方面的研究。 hcp0808@163.com
  • 基金资助:

    山东省自然科学基金资助课题(Y2002C10)。

Human umbilical cord blood mesenchymal stem cells differentiate into hepatocypte-like cells: Feasibility of co-culture with human hepatocyte line LO2 in vitro

Han Cui-ping1, Liu Ji-yong1, Gao Lei2, Zhang Xiao-hua1, Pei Qing-shan1, Sun Xin-xin1   

  1. 1Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, Jinan  250021, Shandong Province, China;
    2People’s Hospital of Baoan District, Shenzhen 518101, Guangdong Province, China
  • Online:2010-01-04 Published:2010-01-04
  • Contact: Liu Ji-yong, Professor, Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China 1iujiyong@medmail.com.cn
  • About author:Han Cui-ping, Studying for master’s degree, Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China hcp0808@163.com
  • Supported by:

    the Natural Science Foundation of Shandong Province, No.Y2002C10*

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摘要：

背景：研究表明共培养条件下，骨髓间充质干细胞可向心肌细胞、肝样细胞等方向分化。

目的：探讨人脐血间充质干细胞与人肝细胞在体外共培养条件下，诱导人脐血间充质干细胞分化为类肝细胞的可行性。

方法：无菌条件下采集新生儿脐带血，采用密度梯度离心法与贴壁法分离纯化脐血间充质干细胞，待细胞融合至80%时胰蛋白酶消化传代。应用具有微孔滤膜的插入式细胞培养皿和培养板构建人脐血间充质干细胞-肝细胞共培养模型，脐血间充质干细胞按1×107 L-1密度接种于下层6孔板内，LO2人肝细胞按1×105 L-1密度接种到上层插入式培养皿中。以上、下两层均接种人脐血间充质干细胞作为对照组。采用流式细胞仪检测贴壁细胞的表面标志，倒置相差显微镜观察共培养后细胞形态变化，RT-PCR法检测肝细胞表面抗原mRNA的表达。

结果与结论：贴壁细胞呈长梭形，强表达CD44和CD29，不表达造血细胞表型CD34和CD45。共培养组5 d时长梭形细胞数量减少，随共培养时间的延长，不规则圆形或多角形的细胞逐渐增多，与肝细胞形态相似；对照组培养4周时，人脐血间充质干细胞仍呈形态均一的长梭形。共培养组5 d时甲胎蛋白mRNA呈阳性表达，其余均为阴性；14 d时细胞角蛋白19 mRNA、白蛋白mRNA开始表达，随着时间延长有增强趋势，甲胎蛋白mRNA表达则较前减弱；对照组各抗原的表达均呈阴性。提示与LO2人肝细胞共培养后，人脐血间充质干细胞可诱导分化为类肝细胞。

关键词: 类肝细胞, 共培养, 诱导, 脐血间充质干细胞, 干细胞

Abstract:

BACKGROUND: Some studies have showed that after indirect co-culture, bone marrow mesenchymal stem cells can differentiate into myocardial cells and hepatocypte-like cells.
OBJECTIVE: To investigate the possibility of human umbilical cord blood mesenchymal stem cells (UCB-MSCs) to differentiate into hepatocytes by co-culture with human hepatocyte line LO2 in vitro.
METHODS: Full-term umbilical cord blood samples were obtained sterilely. The UCB-MSCs were isolated by density gradient centrifugation and directly adherence growth, then passaged with trypsin digestion at 80% cell fusion. By utilizing cell culture plate insets with microporous membrane combined with 6-well plate, the LO2-/UCB-MSCs co-culture system was established. UCB-MSCs were plated into the wells of 6-well plate at a density of 1×107/L. LO2 cells were plated into the cell culture plate insert at a density of 1×105/L. UCB-MSCs were plated in both layers in the control group. Surface markers of adhered cells were detected by flow cytometry. Morphological changes of UCB-MSCs were observed by inverted phase contrast microscopy. mRNA expression was detected by reverse transcriptase PCR.
RESULTS AND CONCLUSION: HUCB MSCs expressed CD44 and CD29 strongly, but CD34 and CD45 were expressed negatively. After 5 days, fusiform-shaped cells were reduced in the co-culture group; while, the time passing by, cells shaped irregular round or polygonal were increased, which were similar to hepatocytes. At 4 weeks after culture, UCB-MSCs were still fusiform-shaped in the control group. At day 5 after culture, alpha fetoprotein mRNA expressed positively, but other expressed negatively in the co-culture group; at day 14 after culture, cytokeratin-19 mRNA and albumin mRNA expressions were observed; moreover, with the time passing by, the expression of albumin mRNA was increased, but the expression of alpha fetoprotein-19 mRNA was decreased. Antigenic expressions in the control group were negative. This suggested that UCB-MSCs could differentiate into hepatocypte-like cells by co-culture with human hepatocyte line LO2 in vitro.

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