人脐血源肝干/祖细胞体外定向诱导分化

The Induced Differentiation of Hepatic Stem/Progenitor Cells from Human Umbilical Cord Blood

目的研究具有多分化潜能的脐血单个核细胞,定向诱导分化成肝干/祖细胞的可能性。方法采集32份足月分娩后的新鲜脐带血,密度梯度离心法分离出单个核细胞,随即分为两组:①对照组8份脐血:培养液中无细胞因子;②实验组24份脐血,随机分为4组,分别在培养的当天、7、14、21天进行实验测定:培养液中加入细胞因子SCF、OSM、HGF、LIF、FGF-1、FGF-2;观察细胞形态学的变化,RT-PCR定量测定hALBmRNA,细胞免疫化学检测ALB、CK18、CK19的表达。结果观察到实验组中培养的细胞数目随培养时间的延长逐渐增多,体积增大,折光性增强的形态学变化,RT-PCR检测到hALBmRNA表达逐渐增强,细胞免疫化学显示CK18、CK19、ALB呈阳性表达。结论脐血单个核细胞在定向诱导下有分化为肝干/祖细胞的能力,可以考虑作为肝干/祖细胞和肝细胞新的细胞来源。

Objective To study the possibility of umbilical cord blood mononuclear cells（ UCB MNCs） having polydifferential potention to be induced and differentiated into hepatic stem/progenitor cells. Methods Collecting 32 cases fresh UCB from mature delivery, UCB MNCs were isolated by density - step centrifugal effect and were randomly divided into two groups ： （ 1 ） control group 8 cases ： the basic medium supplemented without cytokines ; （ 2 ） trial group 24 cases, divided into four groups randomly, and the experimental results were detected at 0d ,7d, 14d ,21 d, respectively. The basic medium supplemented with various combinations of growth/differentiation factors （FGF- 1, FGF- 2, LIF, SCF, HGF, and OSM ）;cell morphological change was observed, hALBmRNA was examinated using RTPCR methods,the expressions of ALB.CK18.CK19 were detected by immunohistochemistry. Results In trial group cell number increased,cell volume enlarged, refraction strong, polymorphic cells （mostly oval cells） appeared and obviously attached on the culture dishes. The expression of CK18.CK19 .ALB were positive by immunohistochemistry, and the level of hALBmRNA improved along with the culture time. Con- clusions UCB MNCs can be induced and differentiated into hepatic stem/progenitor cells in vitro. Human umbilical cord blood mononu- clear cells may be a new source of transplantable hepatic stem/progenitor cells and hepatocytes.