三种因子联合诱导大鼠骨髓间充质干细胞向肝细胞的分化**

背景：间充质干细胞的生物学特性及影响分化调控因子的研究认为，体外原代培养的间充质干细胞自然分化为肝细胞的比例较低，选择一种合适的诱导剂提高其分化为肝细胞的比例尤为必要。 目的：以肝细胞生长因子、表皮生长因子及成纤维生长因子体外联合，验证其诱导大鼠骨髓间充质干细胞向肝细胞分化的可行性。 设计、时间及地点：细胞学体外观察，于2007-08在潍坊医学院实验中心完成。 材料：Sprague-Dawley大鼠40只，由潍坊医学院实验动物中心提供。 方法：采用贴壁法分离培养大鼠骨髓间充质干细胞，取传至第3代细胞，设立2组：空白对照组加入含体积分数为10%胎牛血清的L-DMEM培养基；联合诱导组在此基础上，另加入10 µg/L成纤维生长因子、8 µg/L肝细胞生长因子、8 µg/L表皮生长因子。 主要观察指标：倒置显微镜观察诱导后细胞形态变化，免疫荧光染色观察甲胎蛋白及白蛋白的表达，PAS检测糖原的表达，靛青绿摄入情况，酶学检测谷丙转氨酶、谷草转氨酶、碱性磷酸酶的水平。 结果：联合诱导组细胞呈多角形、卵圆形或圆形细胞的特征性改变，空白对照组骨髓间充质干细胞仍保持梭形或纺锤形。联合诱导组培养14 d可见白蛋白和甲胎蛋白免疫反应阳性细胞；诱导7 d时偶见PAS阳性细胞与靛青绿阳性细胞，随诱导时间延长，阳性细胞逐渐增多；诱导14 d时开始检测到谷丙转氨酶、谷草转氨酶、碱性磷酸酶的合成，21 d达高峰，之后下降。上述各指标空白对照组细胞均呈阴性。 结论：成纤维生长因子、肝细胞生长因子与表皮生长因子体外联合应用，能够成功诱导大鼠骨髓间充质干细胞向肝样细胞的分化。

Differentiation of rat bone marrow mesenchymal stem cells into hepatocytes using three combined factors

BACKGROUND: Studies of biological characteristics of mesenchymal stem cells (MSCs) and regulatory factors that influenced the differentiation of MSCs have shown that the proportion of the natural differentiation from in vitro primarily cultured MSCs into hepatocytes was low, and to select a suitable inductor is important to enhance the differentiation of MSCs into hepatocytes. OBJECTIVE: To verify the feasibility of induced differentiation of rat bone marrow MSCs (BMSCs) into hepatocytes using the combination of hepatocyte growth factor (HGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF-4). DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Experimental Center, Weifang Medical College in August 2007. MATERIALS: Totally 40 Sprague-Dawley rats were supplied by the Experimental Animal Center, Weifang Medical College. METHODS: Rat BMSCs were incubated by adherent method. BMSCs at passage 3 were assigned to 2 groups. BMSCs in the blank control group were treated with L-DMEM containing 10% fetal bovine serum. BMSCs in the combination group were treated with 10 µg/L FGF, 8 µg/L HGF and 8 µg/L EGF following above-mentioned procedures. MAIN OUTCOME MEASURES: Inverted microscope was used to observe the morphological changes in cells. Immunofluorescence method was used to observe the expression of alpha-fetoprotein (AFP) and albumin (ALB). PAS was employed to detect the expression of glycogen. Fox green intake experiment was conducted. Enzymology was utilized to test the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). RESULTS: BMSCs in the combination group presented polygonal, orbicular or round shape. BMSCs in the blank control group remained spindle. BMSCs in the combination group were positive for AFP and ALB at day 14 following culture, and a few PAS-positive and fox green-positive cells were found at day 7. Positive cells became more over time. Synthesis of ALT, AST and ALP was detected at day 14, reached a peak at 21 days, and then decreased. Above-described indexes were negative in the blank control group. CONCLUSION: After induced by the FGF, HGF and EGF, BMSCs have the ability to differentiate into hepatocytes in vitro.