不同诱导条件对大鼠骨髓间充质干细胞体外诱导类肝细胞的影响

Effect of different induction conditions on hepatocyte-Iike cells induced in vitro by rat bone marrow mesenchymal stem cells

目的:探讨实验性大鼠骨髓间充质干细胞(MSCs)体外培养、诱导的最优条件,为MSCs治疗临床重症肝病患者提供参考依据.方法:采用密度梯度离心法和贴壁法分离大鼠MSCs,经培养传代获得纯化的MSCs。采用析因实验,设不同诱导时间、不同浓度的FBS、不同浓度的细胞因子为3个因素,每个因素设不同水平,对纯化的MSCs进行分组诱导培养.留取15、21、27 d细胞培养液进行白蛋白(Alb)、甲胎蛋白(AFP)检测:于27 d时收集细胞爬片,采用过碘酸希夫(PAS)实验进行糖原染色和免疫细胞化学染色检测MSCs中CK-18和CK-19.结果:诱导15、21、27 d,各MSCs诱导组AFP水平均高于MSCs非诱导组(P<0.01),21 d诱导组AFP水平最高:15 d各MSCs诱导组与MSCs非诱导组白蛋白水平无统计学意义,21、27 d各MSCs诱导组白蛋白水平均高于MSCs非诱导组(P<0.01),27 d诱导组白蛋白水平最高.27 d各MSCs诱导组糖原染色阳性,免疫细胞化学染色CK-18、CK-19均阳性,而MSCs非诱导组糖原染色、CK-18、CK-19均阴性.多因素方差分析表明,MSCs体外最佳诱导条件为含200 mL/L FBS的DMEM+肝细胞生长因子(HGF)20μg/L+成纤维细胞生长因子-4(FGF-4)20μg/L.结论:HGF和FGF-4可在体外诱导实验性大鼠MSCs分化为具有肝细胞样细胞表型和功能的类肝细胞:不同浓度的FBS、HGF和FGF-4影响MSCs的体外分化;MSCs可作为治疗临床重症肝病的一种细胞来源.

AIM： To study the optimal conditions for in vitro induction and culture of rat bone marrow mesenchymal stem cells （MSCs） in order to provide the theoretical basis for the treatment of severe liver disease patients with MSCs. METHODS： MSCs were isolated by gradient density centrifugation and plastic adherence and purified. MSCs were induced by different concentrations of FBS and cytokines. Levels of AFP and albumin in the supernatant were measured by radioimmunoassay on days 15, 21 and 27. On day 27, cells were collected.Glycogen store of heoatocytes was determined by periodic acid-Schiff staining and the expression of CK-18 and CK-19 in MSCs were detected by immunocytochemical analysis. RESULTS： The level of AFP was higher in induced MSCs than in non-induced MSCs on days 15, 21, 27, and reached its highest on day 21. There was no significant difference in albumin levels on day 15, between induced and non- induced MSCs. However, on days 21 and 27, the albumin level was higher in induced MSCs than in non-induced MSCs. Glycogen storage in induced MSCs was observed on day 27 but not in non-induced MSCs. The induced MSCs expressed CK-18 and CK-19 while the non-induced MSCs did not. Multiple factor analysis by ANOVA showed that MSCs should be cultured in Dulbecco＇s modified Eagle＇s medium supplemented with 200 mL/L FBS, 20 μg/L hepatocyte growth factor （HGF） and 20 μg/L fibroblast growth factor 4 （FGF-4）, which might be the best induction conditions for MSCs. CONCLUSION： Rat MSCs can differentiate into hepatocyte-like cells with hepatic phenotypes and functions in the presence HGF and/or FGF-4 in vitro. The concentrations of fetal bovine serum, HGF and FGF-4 affect the differentiation of rat MSCs into hepatocyte-like cells. MSCs can be used in the treatment of severe hepatic diseases.