SD大鼠骨髓间充质干细胞分离培养及其生物学特性鉴定

目的:探讨并优化大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cells,rBMSCs)体外分离培养方法,鉴定其生物学特性,为BMSCs应用奠定技术基础。方法:无菌条件下分离SD大鼠股、胫骨,用无血清培养液(α-MEM培养基、100U/mL青霉素,100U/mL链霉素)冲出骨髓,采用密度梯度离心法结合贴壁筛选法分离纯化rBMSCs,通过传代培养,扩增rBMSCs。倒置光学显微镜下观察细胞形态及生长特征,绘制细胞生长曲线。免疫细胞化学染色测定rBMSCs表面标志,成骨诱导试剂盒检测rBMSCs向成骨分化的潜能。结果:镜下rBMSCs呈梭形、多角形成纤维细胞样形态,大小均一,漩涡状生长。生长曲线分析rBMSCs贴壁2d基本无增殖,处于潜伏期,对数增殖期为3～6d,第7天后进入平台期,细胞出现接触抑制现象。rBMSCs表面标志显示第3代rBMSCs纯度可达99%以上,其细胞表型CD44呈阳性表达,CD34呈阴性表达。向成骨诱导rBMSCs10d后细胞表现成骨细胞特性,碱性磷酸酶活性明显增高。结论:本实验从细胞取材、细胞接种密度、条件培养基到细胞微环境等各方面的方法进行了优化,建立了一种理想的体外分离扩增rBMSCs的培养体系。实验结果表明所分离培养的rBMSCs纯度高、扩增迅速、生物学形状稳定,可为组织工程提供比较理想的种子细胞,为进一步研究BMSCs生物学行为及临床研究和应用奠定了坚实的基础。

Isolation,culture and biological characteristics of bone marrow-derived mesenchymal stem cells in SD rats

Objective To establish a more effective and appropriate method to isolate and cultivate ratbone marrow mesenchymal stem cells(rBMSCs) in vitro and identify the biological properties,for further study and clinic application. Methods After the tibias and femurs were dissected from SD rats,the marrow was flushed out with free-serum α-MEM medium under aseptic condition. rBMSCs were isolated and purified by density gradient centrifugation combined with attachment culture method. The growth and morphology of the primary culture and subculture was observed under inverted phasecontrast microscopy and the growth curve was detected by cell counting. rBMSCs membrane antigens were identified with immunocytochemical method. The potential in osteogenous differentiation of rBMSCs be evaluated by the osteogeneic inducer kit. Results After seeded 48 hours ,the adherent cell showed spindle shape,polygonal shape and fibroblast-cell-like shape and the size of rBMSCs was homogeneous.The morphology of subculture was more homogeneous.The rBMSCs were still in latent phase after being adherent to the bottom 2 days; 3 ～ 6 days later,cells were in log phase; 7 days later,and cells came into platform phase and displayed contact inhibition. CD44 memberane antigen could be detected in 99%of the third generation of rBMSCs,but not CD34 membrane antigen. After osteogeneic inducer was added in vitro for ten days,the alkaline phosphatase activities were increased obviously. Conclusion rBMSCs isolated and purified by density gradient centrifugation combined with attachment method had high purity and proliferation activity. The biological properties was stabilization.