全骨髓细胞贴壁法分离与培养大鼠骨髓间充质干细胞的多向分化能力

背景：分离培养纯度较高的骨髓间充质干细胞是对其进行深入研究的前提。目的：观察全骨髓贴壁法分离培养大鼠骨髓间充质干细胞的生物学特征。方法：选取体质量为80-100g雄性SD大鼠,采用全骨髓细胞贴壁培养法获取骨髓间充质干细胞。在取材过程中需严格掌握大鼠处死至将细胞悬液放入培养箱内的时间,一般控制在40min以内。使用基础培养基冲洗骨髓腔时力度适中且在骨髓腔内旋转数次,可使骨髓细胞充分脱落,保证骨髓细胞的获得量。结果与结论：原代骨髓间充质干细胞为贴壁生长的成纤维样细胞,传代后的细胞形态均一,呈漩涡状排列;第3代骨髓间充质干细胞的生长曲线呈S形,经历3个生长时期：潜伏期、对数生长期和停滞期;流式细胞仪检测骨髓间充质干细胞表面抗原结果示：CD29＋99.45%、CD34＋1.45%、CD44＋99.52%、CD45＋1.41%;成骨、成脂诱导分化后,矿化结节被茜素红染成橘红色,脂滴被油红O染成红色。说明骨髓间充质干细胞能向成骨、成脂分化,具有多向分化潜能,符合国际细胞治疗学会间充质及组织干细胞委员会提出的鉴定动物来源骨髓间充质干细胞的最低标准。

Multiple differentiation potential of bone marrow mesenchymal stem cells isolated and cultured by whole bone marrow adherence method

BACKGROUND：Isolation and culture of highly purified bone marrow mesenchymal stem cells are the premise to study their function.OBJECTIVE：To investigate the biological characteristics of rat bone marrow mesenchymal stem cells isolated and cultured by whole bone marrow adherence method.METHODS：Sprague-Dawley rats weighing 80-100 g were selected,and the bone marrow mesenchymal stem cells were isolated from Sprague-Dawley rats by whole bone marrow adherence method.During isolation,the time period from sacrificing to putting the cell suspension into the incubator should be controlled strictly,generally less than 40 minutes.Moderate intensity was preferred to flush the bone marrow cavity with basal culture medium,and rotation for several times could make the bone marrow cells fully dropped off and ensure the quantity of cells.RESULTS AND CONCLUSION：The primary cultured bone marrow mesenchymal stem cells were the adherent fibroblast-like cells,and after passage,the cells were uniform and showed spindle shaped and typical swirl morphology.The growth curve of passage 3 bone marrow mesenchymal stem cells resembled S shape and had three periods：latent period,logarithmic growth phase and lag phase.Flow cytometry detection showed the content of surface antigen：CD29 ＋（99.45%）,CD34 ＋（1.45%）,CD44 ＋（99.52%）,CD45 ＋（1.41%）.After osteogenic and adipogenic differentiation,mineralized nodules were orange under alizarin red staining,and lipid droplets were red under oil red O staining.Bone marrow mesenchymal stem cells have the multiple differentiation potential and can differentiate into osteoblasts and adipocytes.The multiple differentiation potential of bone marrow mesenchymal stem cells corresponds to the minimum standards for identifying the animal bone marrow mesenchymal stem cells raised by International Society of Cell Therapy and Mesenchymal and Tissue Stem Cells Committee.