不同细胞因子组合体外诱导人脐血干细胞向胰岛样细胞分化

Differentiation of human umbilical cord blood stem cells into islet-like cells following in vitro induction of various cytokine combination

背景:由干细胞分化而来的胰岛样细胞存在数量少、胰岛素分泌量少以及能否达到临床移植要求等问题。目的:观察不同细胞因子组合对人脐血干细胞体外向胰岛样细胞诱导分化的影响。设计、时间及地点:细胞学体外对照观察,于2007-05/09在辽宁省血液中心输血医学研究所细胞研究中心完成。材料:脐血来源于足月分娩的健康产妇,由沈阳市德济医院提供。方法:密度梯度离心法体外分离人脐血单个核细胞,诱导1组首先以低糖DMEM培养液进行培养,添加20μg/L碱性成纤维细胞生长因子和20μg/L表皮生长因子,培养成巢蛋白阳性细胞后改为高糖DMEM培养液,添加2%B27和10mmol/L尼可酰胺。诱导2组直接以高糖DMEM培养液进行培养,添加20mmol/L尼可酰胺、20μg/L碱性成纤维细胞生长因子、20μg/L肝细胞生长因子、2%B27及0.1mmol/Lβ-巯基乙醇。对照组只添加与诱导组相同的培养液,不加入任何细胞因子。主要观察指标:诱导分化后对贴壁细胞进行形态学观察、胰岛素抗体的细胞免疫荧光染色及双硫腙染色鉴定。结果:体外培养的人脐血干细胞通过细胞因子组合诱导后,可见散在的胰岛样细胞或成群的胰岛样细胞群,呈Insulin阳性反应。诱导1组细胞散在或成群分布,诱导2组则多为散在分布的胰岛样细胞,且细胞相对较小,几乎没有胰岛细胞团。双硫腙染色后诱导1组有少部分较大的胰岛样细胞呈阴性,诱导2组细胞均呈棕红色阳性表达,未诱导的对照组细胞呈阴性。结论:人脐血干细胞通过不同细胞因子组合诱导后具有向胰岛样细胞分化的潜能,但诱导1组中部分细胞可能是非胰岛素细胞,而诱导2组胰岛样细胞均一性较差,可能与胰岛细胞种类较多、大小不一有关。

BACKGROUND： There are still some problems in stem cells-differentiated islet-like cells, such as a low cell number, a low insulin secretion and whether it is up to the required standards of clinical transplantation or not. OBJECTIVE： To observe various cytokine combination effects on the differentiation from human umbilical cord blood stem cells into islet-like cells in vitro. DESIGN, TIME AND SETTING： The cytology in vitro controlled study was performed at the Cell Research Center, Institute of Blood Transfusion Medicine, Liaoning Blood Center from May to September 2007. MATERIALS： Umbilical cord blood from full-term healthy puerperants was supplied by Shenyang Deji Hospital. METHODS： Human umbilical cord blood mononuclear cells were separated in vitro by the density gradient centrifugation. Cells in the induction 1 group were incubated in low-glucose DMEM, supplemented with 20μ g/L basic fibroblast growth factor and 20 μ g/L epidermal growth factor. Following Nestin-positive cells were detected, cells were incubated in high-glucose DMEM, supplemented with 2% B27 and 10 mmol/L nicotinamide. Cells in the induction 2 group were directly incubated in high-glucose DMEM, supplemented with 20 mmol/L nicotinamide, 20 μg/L basic fibroblast growth factor and 20 μg/L hepatocyte growth factor, 2% B27 and 0.1 mmol/L β-mercaptoethanol. Cells in the control group were only treated with the medium as the induction groups, and not treated with any cytokines. MAIN OUTCOME MEASURES： Morphology was observed in adherent cells following differentiation. Insulin antibodies were assessed by cell immunofluorescence staining and dithizon staining. RESULTS： The umbilical cord blood stem cells cultured in vitro were differentiated into dispersed or agminated islet-like cells following induction, with the presence of Insulin-positive reaction. Cells in the induction 1 group were dispersed or agminated. Cells in the induction 2 group were mostly small dispersed islet-like cells and no cell mass. Following Dithizon staining, a few large islet-like cells were negative in the induction 1 group, and cells in the induction 2 group showed brownish red, and cells in the control group were negative. CONCLUSION： Human umbilical cord blood stem cells following induction of various cytokine combinations have the potential of differentiation into islet-like cells. However, cells in the induction 1 group may be not islet-like cells, and islet-like cells in the induction 2 group have a bad homogenicity, which may be associated with the type and size of islet cells.