不同细胞接种浓度影响人脐带间充质干细胞生长及多向分化

Effects of different implantation densities on the growth and multi-directional differentiation of human umbilical cord mesenchymal stem cells

背景:在干细胞培养过程中寻找合理的细胞接种浓度,从而得到能稳定、高效生长的干细胞非常重要。目的:观察不同接种浓度对人脐带间充质干细胞原代培养及分化为神经元样、胰岛样、脂肪样细胞的影响。方法:分离人脐静脉内皮及内皮下层细胞,按不同接种浓度分组进行原代培养,记录原代培养时间并进行传代。取第1代人脐带间充质干细胞,体外诱导其向神经元样、胰岛样、脂肪样细胞分化。结果与结论:原代培养发现以5×105～1×106/cm2细胞浓度接种的人脐带间充质干细胞生长状态最佳。经β-巯基乙醇诱导6h,细胞即表达Nestin、神经丝蛋白及胶质纤维酸性蛋白;经尼克酰胺,活化素A,胰高血糖素样肽1诱导21d可见明显的胰岛样细胞团,双硫腙染色呈砖红色;经成脂诱导培养基诱导后细胞油红O染色阳性,有明显脂滴出现。可见选取合适的细胞浓度可以快速获得大量的人脐带间充质干细胞,且该细胞经体外诱导可向神经元样、胰岛样及脂肪样细胞分化。

BACKGROUND：It is important to search a rational cell implantation density to achieve stably and high efficiently growing stem cells during the culture of stem cells.OBJECTIVE：To investigate the effects of different implantation densities on primary culture of human umbilical cord mesenchymal stem cells and their differentiation into neuron-,islet-,and adipose-like cells.METHODS：Human umbilical cord venous endothelial and subendothelial layer cells were isolated and grouped according to different implantation densities for primary culture.The time for primary culture was recorded and cell passage was performed.Passage 1 human umbilical cord mesenchymal stem cells were in vitro induced to differentiate into neuron-,islet-,and adipose-like cells.RESULTS AND CONCLUSION：Human umbilical cord mensenchymal stem cells at an implantation density of 5×10 5-1 ×10 6 /cm 2 cell density showed optimal growth.After induced by β-sulfhydryl alcohol for 6 hours,human umbilical cord mesenchymal stem cells expressed nestin,neurofilament protein and glial fibrillary acidic protein.After induced by niacinamide,activin A,glucagon-like peptide 1 for 21 days,obvious islet-like cell masses appeared and were stained red by dithizone.After induced by adipogenic culture medium,cells were positive for oil red O staining,with appearance of obvious liquid drops.A large number of human umbilical cord mesenchymal stem cells could be quickly harvested at a proper implantation density and the cells could be induced to differentiate into neuron-,islet-,and adipose-like cells.