脐带间充质干细胞向成骨细胞分化的潜能

Potential of human umbilical cord mesenchymal stem cells differentiating into osteoblasts

背景:人脐带间充质干细胞含量丰富,较为原始,分化能力强,免疫原性低,是细胞治疗的理想靶细胞。目的:体外分离培养脐带间充质干细胞并将其定向诱导为成骨细胞。方法:无菌条件下培养脐带间充质干细胞,分为诱导组和对照组,诱导组用成骨诱导液处理、对照组为干细胞培养液处理。倒置光显微镜观察细胞形态,MTT法测细胞增殖,荧光双染法检测细胞活力,流式细胞仪检测细胞周期与细胞表面标记。诱导后:检测碱性磷酸酶,Von Kossa染色分析钙盐沉积,RT-PCR检测骨桥蛋白基因、碱性磷酸酶、骨唾液蛋白mRNA的表达。结果与结论:传代细胞形态稳定、活力好,高标达CD44。诱导后vonKossa染色表现为阳性。碱性磷酸酶活性诱导组比对照组高(P<0.05),不同时间点比较21d最高(P<0.05)。RT-PCR显示:诱导组21,28d碱性磷酸酶mRNA表达均较与对照组增强(P<0.05)。诱导组有骨唾液蛋白和骨桥蛋白基因mRNA表达。提示,人脐带间充质干细胞能够定向分化为成骨细胞。

BACKGROUND：Human umbilical cord mesenchymal stem cells（UCMSCs） were rich and more primordial,had strong differentiation ability and low immunogenicity,suitable for transplanting to different individuals.Therefore,UCMSCs were an ideal target cells for cell therapy.OBJECTIVE：To study the isolation and culture of UCMSCs and to explore the potential of osteogenesis differentiation in vitro.METHODS：UCMSCs were sterilely isolated and cultured.The third passage of cells were divided into induction group and control group.The induction group was treated with ossify induction medium,and control group was treated with stem cell culture medium.The cell morphology was observed under an inverted light microscope.Cell proliferation was determined by MTT assay.The cell viability was detected by double immunofluorescent staining.Cell cycle and cell surface marker were determined using flow cytometry.Following induction,alkaline phosphatase（ALP） activity was measured utilizing microplate reader.Calcium deposition was analyzed using Von Kossa staining.mRNA expression of osteopontin（OPN）,ALP and bone sialoprotein（BSP） was measured by reverse transplantation-polymerase chain reaction（RT-PCR）.RESULTS AND CONCLUSION：The passage cells were steady in the cell shapes,with good viability,and highly expressed CD44.Following induction,the cells were positive in von Kossa staining.ALP activity was higher in induction group compared with control group（P 0.05）.The ALP activity was greatest on day 21（P 0.05）.RT-PCR indicated that ALP mRNA expression was higher in induction group compared with control group（P 0.05）.The BSP and OPN genes were expressed in induction group,which suggested that human UCMSCs can differentiate into osteoblasts.