体外诱导大鼠骨髓干细胞向肝干细胞分化的特征

Differentiational characteristics of rat bone marrow stem cells into hepatic stem cells in vitro

目的:骨髓干细胞分化为肝干细胞的发现具有重要的临床应用价值,可用其作为生物人工肝或肝细胞移植的供体细胞,以解决供体缺乏,同时自体细胞还可免除异基因或异种细胞所致的许多问题。为此,实验模拟肝脏发育的微环境,观察体外诱导分化大鼠骨髓干细胞为肝干细胞的可行性及特征。方法:实验于2006-10/2007-08在解放军第一二三医院南京军区肝病中心实验室完成。①实验动物:SPF级SD大鼠,2月龄,体质量(200±20)g,雌雄不拘,购于上海斯莱克实验动物公司。实验过程中对动物处置符合动物伦理学标准。②实验方法:采用密度梯度分离法分离培养大鼠骨髓干细胞,并设诱导组和非诱导组,应用25μg/L肝细胞生长因子、10μg/L成纤维生长因子-4、10μg/L干细胞生长因子共同诱导。③实验评估:于诱导第0,7,14,21,28天,观察细胞形态变化,采用放射免疫法检测培养上清液中甲胎蛋白浓度,免疫组织化学染色检测甲胎蛋白、细胞角蛋白19、白蛋白的表达,应用流式细胞仪检测甲胎蛋白、细胞角蛋白19表达阳性细胞比例。结果:①诱导培养的骨髓干细胞呈卵圆形或圆形的肝干细胞样改变。②放射免疫法检测到诱导组细胞甲胎蛋白浓度逐渐升高,第14天达高峰,随后呈下降趋势,与非诱导组相比,除第0,7天无差异,其余各时间点差异均显著(P<0.05)。③免疫细胞化学法检测到诱导组细胞特异性表达甲胎蛋白、细胞角蛋白19及白蛋白,非诱导组不表达3种蛋白。④流式细胞分析诱导组甲胎蛋白阳性细胞比例为66.20%、细胞角蛋白19阳性细胞的比例为44.96%。结论:在实验建立的诱导培养体系中,骨髓干细胞在体外能分化为肝干细胞样细胞,诱导细胞有分泌甲胎蛋白、细胞角蛋白19、白蛋白的特征性功能。

AIM： It is of significance of bone marrow stem cells differentiating into hepatic stem cells, Bone marrow stem cells can be used as donor cells for bioartificial liver or liver cell transplantation. Autogenous cells can avoid many problems induced by allogene or allogenous cells. This study stimulated the liver microenvironment, and explored the feasibility of differentiation of the rat bone marrow stem cells into hepatic stem cells in vitro and the time phase characteristics in the differentiation progress. METHODS： Experiments were performed from October 2006 to August 2007 in the Central Laboratory of Liver Disease, Nanjing Military Area Command, the 123 Hospital of Chinese PLA. ①SD rats （SPF） aged 2 months with the body mass of （200±20） g, male or female were bought from Shanghai SLAKE Experiment Animal Limited Company. Animal intervention met the animal ethical standard. ②Bone marrow stem cells were collected from the aspirates from femurs of SD rats by density gradient centrifugation. The rats were divided into an induction group and a non-induction group. Adult rat bone marrow stem cells were treated with or without 25 μg/L recombinant mouse hepatocyte growth factor, 10 μg/L recombinant human fibroblast growth factor-4 and 10 μg/L recombinant rat stem cell factor. ③On days 0, 7, 14, 21 and 28 after induction, the morphologic characteristics of the cells were observed. The concentration of alpha fetoprotein in culture matrix was determined by radioimmunoassay. The expressions of alpha fetoprotein, cytokeratin 19 and albumin were detected by immunocytochemical technique. The ratio of alpha fetoprotein-positive cells and cytokeratinl9-positive cells were tested by flow cytometer. RESULTS： ①The bone marrow stem cells after being induced showed orbicular-ovate and round transformation of hepatic stem cell-like. ②Radioimmunoassay showed that alpha fetoprotein levels were increased in the induction group, reached a peak at day 14, and then decreased. There were significant differences between the induction group and the non-induction group at days except 0 and 7 （P 〈 0.05）. ③Immunohistochemical method showed that alpha fetoprotein, cytokeratinl9 and albumin were positive in the induction group, but negative in the non-induction group. ④Flow cytometry showed that the ratio of alpha fetoprotein-positive cells was 66.20%, and that of cytokeratin 19-positive cells was 44.96%. CONCLUSION： In the induction culture system, bone marrow stem cells can differentiate into hepatic stem cell-like cells in vitro. Bone marrow stem cells after induction have the characteristic functions of secretion of alpha fetoprotein, cytokeratin 19 and albumin.