中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (14): 2503-2507.doi: 10.3969/j.issn.1673-8225.2010.14.007

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

体外诱导人骨髓间充质干细胞向肝样细胞分化：肝细胞生长因子和表皮细胞生长因子的作用

熊剑勇1，陈  斌2，倪  勇2   

  1. 1南方医科大学2007级硕士研究生，广东省广州市 510515；2深圳市第二人民医院肝胆外科，广东省深圳市  518035
  • 出版日期:2010-04-02 发布日期:2010-04-02
  • 通讯作者: 陈 斌， 博士，主任医师，深圳市第二人民医院肝胆外科，广东省深圳市 518035 cbxg@163.com
  • 作者简介:熊剑勇★，男，1982年生，江西省南昌市人，汉族，2010年南方医科大学研究生学院毕业，硕士，医师，主要从事骨髓间充质干细胞方面的研究。 xjyyrx@sina.com
  • 基金资助:

    深圳市卫生局科技基金资助项目（200626），课题名称“骨髓间充质干细胞体内移植治疗肝损伤的实验研究”。

In vitro differentiation of human bone marrow mesenchymal stem cells into hepatocyte-like cells: Effect of hepatocyte growth factor and epidermal growth factor

Xiong Jian-yong1, Chen Bin2, Ni Yong2   

  1. 1 Postgraduate School of Southern Medical University, Guangzhou   510515, Guangdong Province, China; 2 Department of Hepatobiliary Surgery, Shenzhen Second People’s Hospital, Shenzhen   518035, Guangdong Province, China
  • Online:2010-04-02 Published:2010-04-02
  • Contact: Chen Bin, Doctor, Chief physician, Department of Hepatobiliary Surgery, Shenzhen Second People’s Hospital, Shenzhen 518035, Guangdong Province, China
  • About author:Xiong Jian-yong★, Master, Physician, Postgraduate School of Southern Medical University, Guangzhou 510515, Guangdong Province, China xjyyrx@sina.com
  • Supported by:

    Scientific and Technological Foundation of Shenzhen Public Health Bureau, No. 200626*

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摘要：

背景：研究证实人骨髓间充质干细胞能够诱导分化为肝样细胞，但其具体生物学特性及分化机制尚不清楚，且诱导分化培养体系尚不成熟。
目的：探讨肝细胞生长因子和表皮细胞生长因子体外诱导人骨髓间充质干细胞向肝样细胞分化的可行性。
方法：取食管癌手术患者肋骨，采用密度梯度离心联合贴壁筛选法获取人骨髓间充质干细胞，流式细胞仪检测细胞表面分子的表达。取第3代人骨髓间充质干细胞，分为4组，肝细胞生长因子组加入20 μg/L肝细胞生长因子，表皮细胞生长因子组加入20 μg/L表皮细胞生长因子，联合组同时加入上述两种生长因子，空白对照组不加任何生长因子。倒置显微镜下观察细胞形态的变化，诱导7，14 d RT-PCR检测甲胎蛋白与白蛋白mRNA的表达。
结果与结论：人骨髓间质干细胞不表达造血细胞标志CD34，CD45，强表达β1-整合素CD29和基质受体CD44。肝细胞生长因子组、表皮细胞生长因子组、联合组诱导后，细胞形态由长梭形变为类圆形或多角形，诱导第7，14天甲胎蛋白、白蛋白 mRNA均呈阳性表达；空白对照组未见多角形细胞，甲胎蛋白、白蛋白 mRNA均呈阴性表达。证明肝细胞生长因子、表皮细胞生长因子以及二者联合均具有诱导人骨髓间充质干细胞向肝样细胞分化的能力，至于二者联合是否能增强其分化能力尚待进一步免疫细胞化学染色定量分析予以验证。

关键词: 细胞分化, 肝细胞生长因子, 表皮细胞生长因子, 甲胎蛋白, 白蛋白, 肝样细胞, 骨髓间充质干细胞

Abstract:

BACKGROUND: Previous research has demonstrated human bone marrow mesenchymal stem cells (HMSCs) differentiate into hepatocyte-like cells; however, biological characteristics and differentiation mechanism remain unclear, and differentiation system remains immature.

OBJECTIVE: To investigate the feasibility of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) to induce the differentiation of HMSCs into hepatocyte-like cells.
METHODS: HMSCs were obtained from patients with esophageal cancer and were separated by density gradient centrifugation combined with attachment method. The phenotypes of MSCs were identified by flow cytometry. The third-passage HMSCs were divided into four groups: HGF (adding 20 µg/L HGF), EGF (adding 20 µg/L EGF), HGF + EGF, and blank control groups. Morphology was observed using inverted microscope. At days 7 and 14 after induction, α-fetoprotein and albumin mRNA expressions were detected using RT-PCR assay.
RESULTS AND CONCLUSION: The HMSCs did not express hematopoietic cell CD34 and CD35, but strongly expressed β1-integrin CD29 and matrix receptor CD44. HMSCs changed from long fusiform shape to polygon or similar round shape in the HGF, EGF, and HGF + EGF groups. At days 7 and 14 after induction, α-fetoprotein and albumin mRNA expressions were positive. However, polygon cells were not observed in the blank control group, and α-fetoprotein and albumin mRNA expressions were negative. This suggested that HGF, EGF, and HGF + EGF could induce the differentiation of HMSCs into hepatocyte-like cells; however, their differentiation ability still needs to be further semi-quantitatively analyzed using immunohistochemical staining.

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