小鼠骨髓干细胞体外定向诱导为肝细胞样细胞的实验研究

MOUSE BONE MARROW STEM CELLS DIFFERENTIATE INTO HEPATOCYTE-LIKE CELLS IN VITRO

目的来源于骨髓干细胞的有功能的干细胞,可能成为生物人工肝和肝细胞移植的细胞来源。我们建立恰当的体外诱导培养体系,促使骨髓干细胞转化为肝细胞。方法获取骨髓干细胞,建立以FGF、HGF、OSM、EGF为主的细胞诱导培养体系。在细胞分化过程中,观察细胞形态和数量的变化,RT-PCR检测基因表达的变化,免疫荧光染色技术在蛋白水平检测ALB和CK18表达情况。PAS染色检测其糖代谢功能。结果在诱导过程中,多极性的肝细胞样细胞在12 d内可以观察到,且其随着细胞分化过程逐渐增多、集落增大。诱导细胞在7 d内表达AFPmRNA并维持到第21天,但后期表达减弱;在7天内表达ALBmRNA和CK18mRNA,随着分化时间的延长,其表达不断增强;在14 d内表达TTRmRNA,随着分化时间的延长,其表达不断增强。免疫荧光染色进行定位:在诱导组,诱导21 d的细胞表达ALB和CK18,其中ALB表达于胞浆和胞膜,而CK18表达于胞浆。糖原染色发现诱导组中,细胞胞浆内出现大小不等的红染的糖原颗粒。结论我们建立的以细胞因子FGF、HGF、OSM、EGF为主的细胞诱导培养体系是有效的,通过诱导骨髓干细胞,我们获得了有部分肝细胞形态结构和功能的肝细胞样细胞。

Objective some researchers, may become effective resources of hepatocytes for bioartificial liver and hepatocyte transplantation. We design the appropriate directed differentiation medium in order to gain hepatocyte-like cells through induction of differentiation of bone marrow stem cells. Methods Bone marrow stem cells （BMSCs） were selected from bone marrow, and they were cultured in the directed differentiation media including hepatocyte growth factor （HGF）, fibroblast growth factor-4 （FGF-4）, epidermal growth factor （EGF） and oncostatin M （OSM）. In the course of cell differentiation, cell morphology was observed, and the expression patterns of some genes of the hepatocytes were validated and confirmed by RT-PCR. The albumin-and cytokeratinl8-expressed cells were gone further step to be confirmed by Immunofluorescence. Synthesized glycogen was evaluated by Periodic acid Shift staining. Results Some polygonal cells resembling hepatocytes were observed in the differentiation media on day 12, and the number and size of colonies of polygonal ceils increased in the course of the cell differentiation. AFP mRNA expression appeared within seven days and decreased at the later days. ALB and CK18 mRNA expression appeared wthitin 7 days and increased in the course of later directed differentiation. TrR mRNA expression could be detected within 14 days, and its expression increased at the later days. The ALB and CK18 protein were confirmed to express in the differentiated BMSCs on day 21 by Immunofluorescence. Directed differentiated cells could also synthesize glycogen. Conclusions Bone marrow stem cells could differentiate into hepatocyte-like cells in the directed differentiation media including HGF, FGF-4, EGF, and OSM. Our differentiation media were effective in the differentiation of bone marrow stem cells into hepatocyte-like cells in vitro.