人胎肝干细胞的分离培养、鉴定及mRNA转录分析  

Isolation, Culture, Identification and mRNA Transcription Analysis of Human Fetal Hepatic Stem Cells

作　　者：郭向飞[1] 金敏[1] 陈照立[1] 李君文[1] 

机构地区：[1]军事医学科学院卫生学环境医学研究所,天津300050

出　　处：《生物技术通讯》2009年第3期346-349,共4页Letters in Biotechnology

摘　　要：目的:探讨体外大量扩增培养人胎肝干细胞的方法,研究其形态、特性及mRNA转录情况。方法:采用两步灌流法结合链霉蛋白酶消化及Percoll密度梯度离心法分离12～20周胎龄的胎肝细胞,采用免疫细胞化学染色及RT-PCR方法对分离培养的细胞进行鉴定分析。结果:刚分离的细胞活力在80%以上,原代培养3d开始出现小细胞团,2周后即形成肉眼可见的细胞集落,细胞体积小,核质比大;原代、传代培养的胎肝细胞甲胎蛋白(AFP)、细胞角蛋白19(CK19)、卵圆标记蛋白OV-6、细胞分化抗原34免疫染色阳性;RT-PCR分析证明胎肝干细胞中AFP、白蛋白、CK19、CK18和八聚体结合蛋白(OCT)-4mRNA的表达。结论:分离了胎肝干细胞,具有肝细胞、胆管细胞及干细胞表面标志及相应的基因表达,为进一步的基础研究奠定了基础。Objective： To establish a practical method of isolation, culture and identification of fetal hepatic stem cells for study of the morphous characterization and mRNA transcription of fetal hepatic stem cells. Methods： Aborted fetuses of 12-20 weeks were used for the isolation of fetal hepatic stem cells. Fetal liver was digested by two-step perfusion of 0.1% collagenase IV and pronase E, then the obtained heterogeneous liver cells were isolated and purified by percoll density gradient centrifugation. Immunocytochemical stain and RT-PCR were used to examine special protein and mRNA expression in hepatic stem cell. Results： The survival rate of the newly isolated hepatic stem cells were more than 80%. The hepatic stem cells have become clusters after 3 days primary culture. Two weeks after isolation, there were many stem-cell clones with high nuclear-cytoplasmic ratio. Protein marker of ot-fetoprotein（AFP）, cytokeratin-19（CK19）, oval cell protein marker OV-6, and cell differentiation antigen 34（CD34） were positive stained by immunocytochemistry. The transcriptions of mRNA of the hepatic stem cell such as AFP, albumin, CK19, CK18 and octamer-binding protein 4（OCT-4） were all detected by RT-PCR. Conclusion： The successful development of the feasible isolation and purification procedure as well as the identification method for hepatic stem cells with special surface marker of hepatic, ductal and stem cells that may provide a fundamental for further studies.

关 键 词：胎肝干细胞 分离鉴定 MRNA转录 

分 类 号：Q813[生物学—生物工程]