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目的:探讨体外快速大量分离脐带间充质干细胞的新方法。方法:采用复合胶原NB4、dispaseII、透明质酸酶三种酶消化3h,加入PBSA溶液稀释,离心获得脐带间充质干细胞,培养;用流式细胞仪对P3代细胞进行表面标记的鉴定,用化学诱导的方法使第3代细胞向脂肪、骨、软骨细胞分化,2~4周后,分别行oilred、Safranin’O和茜素红染色,倒置显微镜下观察诱导结果。结果:经3种酶消化和PBSA稀释,短时间内从脐带中获得了大量间充质干细胞;伴随着细胞的传代,形态逐渐均一,传至第3代,细胞的形态已基本相似;流式细胞仪鉴定,细胞强表达间充质细胞的特异性标记CD90,CD73,CD105,而不表达造血系或内皮系细胞的标记CD45、CD14、CD11、CD34、CD19,也不表达主要组织相容性抗原HLA-DR;向脂肪细胞诱导后第4周,oilred染色见细胞内大量红染的脂滴;向软骨细胞诱导后第4周,Safranin’O染色见多数切片呈阳性,细胞团块中存在大量软骨特异性的陷窝样结构;向骨细胞诱导后第4周,茜素红染色发现肉眼可见的广泛散在的红色阳性钙结节。结论:本研究所采用的3种酶消化结合PBSA稀释的方法可以快速获得脐带间充质干细胞;
Objective: To explore a new method for ex vivo rapid isolation of umbilical cord mesenchymal stem cells. Methods: The collagenase NB4, dispaseII and hyaluronidase were digested by three enzymes for 3h, diluted in PBSA solution and centrifuged to obtain umbilical cord mesenchymal stem cells. The surface markers of P3 generation cells were identified by flow cytometry. After 3 to 4 weeks, the third generation cells were differentiated into fat, bone and chondrocytes. Oilred, Safranin’O and alizarin red staining were performed respectively. The induced results were observed under an inverted microscope. Results: A large number of mesenchymal stem cells were obtained from the umbilical cord in a short period of time after being digested with three kinds of enzymes and diluted with PBSA. With the passage of cells, the morphology gradually became homogeneous and reached the third generation. The morphology of the cells was similar. Cytometry identified that cells strongly expressed mesenchymal cells specific for markers CD90, CD73, and CD105, but not markers for CD45, CD14, CD11, CD34, and CD19 of hematopoietic or endothelial lineage, nor did they express major histocompatibility Anti-HLA-DR; 4 weeks after the induction of adipocytes, oilred staining showed a large number of lipid-dyed lipid droplets in the cells; in the 4th week after chondrocyte induction, Safranin’O staining showed that most sections were positive, A large number of cartilage-specific lacunar structures; alizarin red staining found widely scattered red-positive calcium nodules visible to the naked eye at 4 weeks after osteoblast induction. Conclusion: The umbilical cord mesenchymal stem cells can be rapidly obtained by three kinds of enzymatic digestion combined with PBSA dilution.