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目的探索肝细胞生长因子(HGF)+成纤维生长因子-4(FGF-4)诱导人骨髓来源多能成体祖细胞(hMAPCs)分化为肝细胞的可行性,为肝组织工程提供新的种子细胞来源。方法(1)取志愿者适量骨髓后采用梯度密度离心+贴壁培养获取骨髓间充质干细胞(MSCs),将MSCs通过CD45、GlyA免疫微磁珠负分选得到hMAPCs。(2)将hMAPCs用HGF+FGF-4进行诱导分化。实验分组:A组:HGF (20 ng/ml)+(FGF-4)10ng/ml诱导hMAPCs;B组(阳性对照组):L-02人肝细胞株; C组(阴性对照组):未加任何诱导因素的hMAPCs。(3)免疫细胞化学鉴定不同诱导分化阶段细胞的白蛋白(Alb)、甲胎蛋白(AFP)、细胞角蛋白-18(CK-18)等肝细胞特征的表型变化并计数阳性细胞比率。(4)逆转录-聚合酶链反应检测不同诱导分化阶段细胞的Alb、AFP、CK-18的mRNA转录。(5)Western blot检测诱导分化第21、35天后细胞的Alb表达。结果(1)免疫细胞化学结果:Alb、CK-18在诱导组中不同时间段基本为阳性着色;AFP在诱导分化第7天为阳性着色,在诱导第14、21天为阴性着色。(2)逆转录-聚合酶链反应结果:作为不成熟肝细胞表型的AFP,在诱导分化的第7天有mRNA阳性表达;作为成熟肝细胞表型的Alb及CK-18,在不同时间段mRNA均为阳性表达。(3)Western blot检测诱导分化第21、35天后细胞的Alb表达。结论hMAPCs在一定诱导条件下具有向肝样细胞分化的潜能。
Objective To investigate the feasibility of hMAPCs differentiated into hepatocytes induced by hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) and to provide new seed cells for liver tissue engineering source. Methods (1) BMSCs were harvested by gradient density centrifugation and adherent culture. The MSCs were passaged through CD45 and negatively graded with GlyA immunomagnetic beads to obtain hMAPCs. (2) hMAPCs were induced to differentiate with HGF + FGF-4. Group A: hGAPCs were induced by 10ng / ml HGF (20 ng / ml) + (FGF-4); Group B (positive control): L-02 human hepatocyte cell line; Group C (negative control) Add any inducer of hMAPCs. (3) Immunocytochemistry Identify the phenotypic changes in the characteristics of hepatocytes such as albumin (Alb), alpha-fetoprotein (AFP) and cytokeratin-18 (CK-18) in different stages of differentiation and count the positive cell ratio. (4) Reverse transcriptase-polymerase chain reaction was used to detect the mRNA transcription of Alb, AFP and CK-18 in different stages of differentiation and differentiation. (5) Western blot was used to detect Alb expression on day 21 and 35 after differentiation. Results (1) Immunocytochemistry results: Alb and CK-18 were basically positive staining in different time periods in induction group; AFP positive staining on the 7th day of differentiation and negative staining on the 14th and 21st days of induction. (2) Results of reverse transcription-polymerase chain reaction: AFP, a phenotype of immature hepatocytes, was positive for mRNA expression on the 7th day after induction of differentiation; Alb and CK-18, as mature hepatocyte phenotype, Segment mRNA were positive expression. (3) Western blot was used to detect the expression of Alb in cells after induction of differentiation on the 21st and the 35th day. Conclusion hMAPCs have the potential to differentiate into hepatocyte-like cells under certain inducing conditions.