摘要
目的：探讨体外快速大量分离脐带间充质千细胞的新方法。方法：采用复合胶原NB4、dispasell、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oil red、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 establish a method to isolate human umbilical cord mesenchymal stem cells （HUCMSCs） in vitro. Methods： MSCs were isolated from human umbilical cord with three-enzyme method, digested for 3 hours and diluted with PBSA, then, centrifugated to obtain umbilical cord mesenchymal stem cells in vitro. The surface markers of the passage 3 （P3） cells were identified by flow cytometry; The P3 cells were induced into adipocytes, chondrocytes and osteocytes by chemical-inductive method. After 2-4 weeks, the differentiated cells were stained by oil red, Safranin＇O and alizarin red and observed under inverted microscope to examine the effects of induction. Results： The isolation of human umbilical cord mesenchymal stem cells was fast and efficient ; As the cells were subcultured, the morphology of P3 cells was gradually uniform. The P3 cells expressed CD90,CD73 ,CD44 and CD105, which are known to characterize MSCs, but expressed no cell markers for hematopoietic stem cells or Endothelial cells such as CD45,CD34, CD19, CD14 and human leukocyte antigen HLA-DR, which also lacks in MSCs ;When exposed to lineage-specific differentiation media, HUCMSCs could differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages. Accumulation of adipose lipids within differentiated HUCMSCs was confirmed by staining these cells with Oil red; Chondrogenesis was demonstrated by sulfated proteoglycans stained with saffranin＇ O and osteogenesis by calcium deposition in extracellular matrix stained with Alizarin Red; Conclusion： Three-enzyme digestion and PBSA dilution could fast and efficiently isolate mesenchymal stem cells from human umbilical cord.
出处
《现代生物医学进展》
CAS
2010年第6期1164-1169,共6页
Progress in Modern Biomedicine
基金
国家自然科学基金资助项目(30872630)
关键词
脐带间充质干细胞
三种酶消化法
多向分化
Umbil ical cord mescnchymal stein cells
Three-enzyme digestion
Multiple differentiation
