大鼠骨髓间充质干细胞分离培养方法的实验研究

Study on Isolation and Cultivation of Rat Bone Marrow-derived Mesenchymal Stem Cells

目的 建立一种体外分离纯化、培养扩增大鼠骨髓间充质干细胞 (Mesenchymalstemcells,MSCs)的方法 ,为MSCs的进一步诱导分化和应用奠定基础。方法用密度梯度离心结合贴壁培养法分离纯化大鼠骨髓MSCs,筛选合适培养基及适宜血清含量 ,传代扩增 ,测定生长曲线和贴壁率 ,形态学观察 ,免疫细胞化学法鉴定细胞膜抗原和细胞基质蛋白属性。结果MSCs属骨髓中的单个核细胞 ,密度梯度离心结合贴壁培养法能有效分离纯化大鼠骨髓MSCs,MSCs在含 10 %小牛血清的L DMEM培养液中生长性状相对稳定 ,1、3、5代细胞生长曲线、贴壁率基本相同 ,增殖速度快 ,群体倍增时间约为 3 4小时 ,贴壁存活率高 ,适应性强 ,传代后 12小时贴壁率达 89%。细胞呈均一的成纤维细胞样 ,表达CD44、CD5 4、纤维粘连蛋白 (Fibronectin ,FN)、Ⅰ型胶原 (CollagenI)。结论 密度梯度离心结合贴壁培养法能有效分离纯化大鼠骨髓MSCs,在含 10 %小牛血清的L DMEM培养液中 。

Objective To establish a method for isolation and cultivation of mesenchymal stem cells (MSCs) from rat bone marrow for offering an experimental foundation for their further differentiation and actual application.MethodsSacrifice the SD rats, separate and purify the marrow MSCs by density gradient centrifugation and by adhering to the culture plastic, select suitable culture medium and concentration of serum. After the in vitro subculture and expansion, observe the living characteristic of the cells, draw the growth curve ,measure the adhesive rate, study the morphology of the cells with phase contrast microscope, and examine the cell surface antigen and matrix protein by immunocytochemistry technique.Results The isolated MSCs belonged to the mononuclear cells of marrow, the living characteristic was quite stable in L-DMEM medium containing 100ml/L newborn bovine serum, the growh curves and adhesive rate of passage 1?3 and 5 were much similar, about 89% subcultured cells adhered the wall in 12hs, exhibiting a large expansive potential and high suitability, a population doubling time of 34h, a typical fibroblastlike morphology,and uniformly expressing CD44?CD54?fibronectin and collagen I.ConclusionThe marrow MSCs could be effectively isolated and purified by density gradient centrifugation and adhering to the culture plastic and expanded satisfactorily in L-DMEM with 10% newborn bovine serum.