不同分离及培养方法对大鼠骨髓间充质干细胞生长增殖和生物学特性的影响

Effects of the Different Culture and Isolation Methods on the Growth,Proliferation and Biology Characteristics of Rat Bone Marrow Mesenchymal Stem Cells

目的探讨获得大量、高纯度、高活性骨髓间充质干细胞(BMSCs)的有效途径。方法用密度梯度离心法和贴壁法分离纯化BMSCs,在无血清、含小牛或胎牛血清以及不同体积分数的胎牛血清的培养基中培养,传代扩增,观察各组细胞形态、生长特性,测定生长曲线,免疫细胞化学染色鉴定表面抗原,电镜观察细胞显微结构。结果密度梯度离心法可获得较纯的原代细胞,但细胞增殖缓慢,容易老化;贴壁法分离的细胞生长增殖迅速,经传代换液,第4代细胞基本纯化;小牛和胎牛血清均能促进BMSCs生长增殖,但在体积分数为0.12的胎牛血清中集落形成率最高(46.50%);细胞表达CD44、CD90,而CD14、CD45阴性;电镜下BMSCs符合干细胞的一般特性。结论经多次换液、严格控制传代消化时间(2～3min)和酶浓度(2.5g/L),采用贴壁纯化法在含体积分数为0.12的胎牛血清L-DMEM培养液中培养可获得大量、高纯度、高活性的BMSCs。

Objective To explore a suitable method for isolation, purification and multiplication of bone marrow mesenchymal stem cells （BMSCs）. Methods Density gradient centrifugation and adherence separation methods were applied for isolation of BMSCs from Wistar rats. The cells were cultured and proliferated in culture medium containing calf serum （CS）, fetal bovine serum （FBS）, free of serum or different volume fraction of FBS. The characteristic and the morphology of BMSCs were observed under inverted microscope every day. The growth curves were draw and the surface antigen of BMSCs were detected by immunocytochemistry technique. The microstructure was observed by transmission electron microscope （TEM）. Results The pure primary cells can be procured by density gradient centrifugation. But the primary cells cultured by adherence separation methods demonstrated higher cytoactive, more rapid proliferation, earlier colony confluence and shorter time for passage than that cultured by density gradient centrifugation method. The cells by adherence separation methods were essentially purified at passage 4. Both CS and FBS can promote the growth and proliferation of BMSCs, but the colony forming efficacy of cells （46.50%） cultured in medium containing 0. 12 volume fraction FBS was the highest. The cells surface markers CD44, CD90 were positive and CD14, CD45 were negative. BMSCs were observed by TEM and possessed the characteristic of stem cells. Conclusion BMSCs with high quality and activity can be obtained with adherence separation by suitable method and culture conditions. L-DMEM medium containing 0.12 volume fraction FBS showed more profitable for the growth and proliferation of BMSCs.