肝细胞生长因子与成纤维生长因子联合诱导人脐带间充质干细胞向肝样细胞的分化

背景：成纤维生长因子可促进间充质干细胞增殖、贴壁生长，但对其诱导间充质干细胞向肝细胞分化的实验报道为数不多。当肝细胞生长因子质量浓度达1μg／L时，可促进肝细胞有丝分裂，它是正常肝细胞最强的促有丝分裂剂。 目的：体外分离培养人脐带间充质干细胞，拟揭示其生物学特性及在细胞因子联合诱导下向肝样细胞分化的能力。 设计、时间及地点：细胞学体外观察，于2008—08／2009-04在暨南大学血研所完成。 材料：脐带取自健康足月胎儿，产妇对实验知情同意，由广州华侨医院提供。肝细胞生长因子、成纤维生长因子为美国Peprotech产品。 方法：Ⅳ型胶原酶消化＋差速贴壁法分离培养人脐带间充质干细胞，取传至第3代细胞，进行细胞表面抗原分析、细胞周期测定，检测其成脂、成骨能力。取第5代细胞，调整细胞密度为5×10^9L^-1，分为2组：对照组用含体积分数为5％胎牛血清的DMEM／F12培养液培养：诱导组在其基础上，添加20μg／L肝细胞生长因子、10μg／L成纤维生长因子联合诱导其向肝样细胞分化。 主要观察指标：人脐带间充质干细胞的生物学特性，人脐带间充质干细胞体外向肝样细胞的分化情况。 结果：成功从人脐带中分离并纯化得到间充质干细胞，第3代细胞92．2％处在G0/G1期：表达CD29，CD44，CD105，不表达造血细胞标志CD34，CD45．油红O染色后胞浆中呈现红色颗粒，碱性磷酸酶染色后细胞质呈黑色，具有成脂、成骨能力。经肝细胞生长因子、成纤维生长因子联合诱导10d后，RT-PCR及Western blot检测结果显示细胞表达肝细胞特异性抗原甲胎蛋白、白蛋白，对照组均呈阴性表达。 结论：人脐带中含有丰富的间充质干细胞,其具有较强的多向分化潜能，经肝细胞生长因子与成纤维生长因子联合诱导后,易向肝样细胞分化。

Differentiation of human umbilical cord mesenchymal.stem cells into hepatocyte-like cells with the combined induction of hepatocyte growth factor and fibroblast growth factor

BACKGROUND： Fibroblast growth factor can promote proliferation of mesenchymal stem cells （MSCs）, grew along the wall. However, there are few reports on the differentiation of MSCs into hepatocytes following fibroblast growth factor induction. When mass concentration of hepatocyte growth factor was 1 μg/L, it can promote mitosis of hepatocytes, and is the strongest mitogenic agent for normal hepatocytes.
OBJECTIVE： To explore the biological characteristics of human umbilical cord MSCs in vitro and their differentiation ability to hepatocyte-like cells under the induction of chemical factors.
DESIGN, TIME AND SETTING： The cytological in vitro study was conducted at the Institute of Blood, Jinan University from August 2008 to April 2009.
MATERIALS： Umbilical cord was obtained from healthy full-term fetus, which was provided by the Guangzhou Huaqiao Hospital. The parturient signed informed consent. Hepatocyte growth factor and fibroblast growth factor were bought from Peprotech, USA. METHODS： The MSCs from human cord were isolated and cultured by type IV colagenase digestion ＋ differential adherence. At the third passage, MSCs received cell surface antigen analysis and cell cycle determination to detect their ability to differentiate into adipocytes and osteoblasts. At the fifth passage, MSCs were adjusted into 5× 10^9/L and assigned into 2 groups. BMSCs in the control group were incubated in DMEM/F12 containing 5% fetal bovine serum. BMSCs in the induction group were treated with above-mentioned medium supplemented with 20 pg/L hepatocyte growth factor and 10 IJg/L fibroblast growth factor.
MAIN OUTCOME MEASURES： The following parameters were measured： biological characteristics of human umbilical cord MSCs and differentiation of human umbilical cord MSCs into hepatocyte-like cells in vitro.
RESULTS： At the third passage, MSCs derived from human cord expressed CD29, CD44, CD105, but not antigens of hematopoietic CD34, CD45, and 92.2% of them were in G0/G1 phase. Oil red O staining showed cytoplasm presented red granules. Alkaline phosphatase staining demonstrated that cytoplasm was black, with the differentiation ability into adipocytes and osteoblasts. Following 10 days of combined induction of hepatocyte growth factor and fibroblast growth factor, RT-PCR and Western blot results confirmed that cells expressed alpha fetoproteln and albumin. Negative expression was found in the control group.
CONCLUSION： Human umbilical cord contained plenty of MSCs, with strong potential of multi-differentiation. Umbilical cord MSCs can differentiate into hepatocyte-like cells following combined induction of hepatocyte growth factor and fibroblast growth factor.