体外诱导人脐血CD34^+细胞向肝细胞的分化

Differentiation of human umbilical cord blood CD34^+ cells into hepatocyte in vitro

目的探讨在体外培养条件下人脐血CD34+细胞向肝细胞的分化。方法用磁性细胞分选试剂盒(MACS)分离出CD34+细胞后在含50 mL/L FBS,1×ITS,4.7 mg/L亚油酸,10-4mol/L 2-磷酸抗坏血酸的低糖型DMEM中培养,并以FGF4(100μg/L)、HGF(20μg/L)进行诱导。分别于生长因子刺激前和刺激8、16 d时留取细胞。通过RT-PCR对ALB、AFP、GATA-4等mRNA的表达水平进行检测;用免疫细胞化学染色检测ALB蛋白的表达,并收集培养液测定ALB的质量浓度。结果流式细胞仪检测结果显示CD34+细胞的平均分离纯度>90%,达到分离要求。RT-PCR检测到诱导后的CD34+细胞表达ALB、AFP、GATA4 mRNA。免疫细胞化学染色可见诱导16 d的CD34+细胞出现ALB阳性表达细胞,ELISA检测表明诱导组8、16 d培养液中的ALB质量浓度明显增高,与诱导前和未诱导组相比均有显著差异(P<0.01)。结论在体外培养条件下,脐血CD34+造血干细胞可分化为表达白蛋白的类肝细胞。

Objective To investigate the differentiation of CD34^＋ cells derived from human umbilical cord blood into hepatocyte cells in an in vitro system supplemented with growth factors（GF）. Methods CD34^＋ cells were isolated by magnetic cell sorting （MACS）. The purity of acquired cells was determined by FACS analysis. Purified CD34^＋ cells were cultured in DMEM supplemented with 50 mL/L FBS, 1×ITS, 10^4- mol/L ascorbic acid 2-phosphate, 4.7 mg/L linoleic acid , and a combination of FGF4 （100 μg/L） and HGF（20 μg/L）. Cultured cells were collected after 8 d and 16 d to determine the extent of the cell conversion by microscopic observation on the morphology, RT-PCR on the transcription of ALB, AFP and GATA4 mRNA, immunocytochemistry on the expression of ALB and ELISA on the quantification of the ALB product in the medium. Results CD34^＋ cells accounted for more than 90% after MACS. ALB, AFP and GATA-4 mRNA and albumin protein positive cells were found in cultured cells after 16 d. The ALB product in culture medium was significantly increased after culturing with GF in comparison with control groups （P〈0.01）. Conclusion A conversion of CD34^＋ cells derived from human umbilical cord blood into hepatocye-like cells had been observed in this experimental culture system.