体外诱导小鼠骨髓干细胞转化为肝细胞的实验研究

目的模拟体内肝脏发生发育的环境和条件，建立以细胞因子为主的体外诱导培养体系，探讨骨髓干细胞体外转化肝细胞的可行性。方法获取小鼠骨髓干细胞，建立以细胞因子为细胞诱导的培养体系。在细胞培养过程中，观察细胞形态和数量，逆转录-聚合酶链反应（RT—PCR）检测肝细胞特异性基因的表达。Western blot和流式细胞仪检测ALB和CK18在蛋白水平的表达情况。糖元染色法行细胞糖原染色、尿素合成试验检测细胞的合成和代谢功能。结果在诱导培养12d，可以观察到多极性的肝细胞样细胞。且细胞逐渐增多、集落不断增大。诱导细胞在培养7d开始表达AFP mRNA并维持到第21天。此后表达逐渐减弱；培养7天开始表达ALB mRNA和CK18 mRNA。随着培养时间的延长表达不断增强；培养14d开始表达TTR mRNA，随着培养时间的延长表达不断增强。通过Western blot检测，诱导21d的细胞表达ALB和CK18蛋白，流式细胞术分析ALB阳性细胞的比例为60．45％，CK18阳性细胞的比例为67％。诱导培养21d，细胞胞浆内可见红染的糖原颗粒；诱导培养6d，细胞开始合成尿素，尿素合成功能随诱导时间的延长而增强，于第15天达到高峰。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能促使骨髓干细胞定向转化为肝细胞。

In vitro experimental study on the differentiation of mouse bone marrow stem cells into hepatocytes

To design the effective directed differentiation medium to differentiate bone marrow stem cells (BMSCs) into hepatocytes. Methods FGF-4, OSM, HGF and EGF were selected as differentiation factors, and the appropriate directed differentiation medium was designed. Moue BMSCs were cultured in the directed differentiation media including FGF-4, OSM, HGF and EGF. During 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 ALB-, and CK18- expressed cells were gone further step to be confirmed by Western blot analysis and flow cytometric analysis (FACS). Synthesis and metabolism functions of hepatocytes from the directed differentiational cells were investigated by Periodic acid Shiff staining and urea assay. Results Some hepatocyte-like cells appeared in the differentiation media on the culture day 12. The AFP mRNA expression appeared on the culture day 7 and decreased in the later days; Th ALB and CK18 mRNA expression appeared on the culture day 7, and increased with the differentiation of BMSCs. The TTR mRNA expression appeared on the culture day 14, and also increased with the differentiation of BMSCs. ALB and CK18 were confirmed to exit in the differentiated BMSCs by Western blot analysis. The ratio of ALB-positive cells was 60.45%, and that of CK18-positive cells was 67% . The directed differentiated cells synthesized urea in a time-dependent manner. They could also synthesize glycogen. Conclusion BMSCs could differentiate into hepatocytes in the differentiation media including HGF, FGF-4, EGF, and OSM.