犬骨髓间充质干细胞的培养及生物学特性

Cultivation and biological characteristics of dog marrow mesenchymal stem cells

目的研究探讨犬骨髓间充质干细胞的分离、培养方法以及生长增殖特性。方法无菌技术抽取比格犬骨髓,密度梯度离心法获取细胞。加入含胎牛血清体积分数为0.1的低糖DMEM培养基中进行原代培养;待细胞生长至80%～90%融合时,1︰3传代培养。倒置显微镜观察细胞形态。MTT法测定3、5和10代细胞生长曲线。流式细胞仪检测第5代细胞的表面抗原分子。结果原代培养24h后看见微少贴壁细胞,初起为小圆形,3d后可见梭形类成纤维细胞呈漩涡样生长。10代以后细胞逐渐有宽大梭形样变,增殖逐渐减缓,偶有细胞飘零。流式细胞仪检测传5代细胞有96%表达CD44,95%表达CD29,而只有5%表达CD34。生长曲线显示,传3、5代骨髓间充质干细胞均有较强的增殖能力,其中传3代细胞更为明显,而传10代细胞的增殖能力较前有所减弱。结论犬骨髓间充质干分离提取操作简便易行,且有较强的生长增殖能力,能够长期培养,符合组织工程种子细胞的基本条件。

【Objective】To investigate the experiment foundation for method of isolation and culture of bone marrow mesenchymal stem cells（BMSCs）,and to evaluate the characteristics of their growth and proliferation.【Methods】Beagle＇s bone marrow sample was taken by puncture in axenic condition and the bone marrow mesenchymal stem cells were isolated by density gradient centrifugation.The original cells were primarily cultured in the low sugar DMEM medium which contained the fetus cow blood-serum with volume fraction of 0.1.While the cells grew to 80%～90% fusion,1︰3 passage culture was done.The characteristics of the cells were observed under inverted microscope,and the P3,P5 and P10 cells growth curves were measured by MTT method.Flow cytometry was used to examine the surface antigen of the P5 cells.【Results】In 24 hours a few adherent cells could be observed after cultivation.The primary BMSCs indicated long or short spindle-shape or multangular shape 48 hours later while it could be observed cell division.Fibroblast-like cells appeared progressively after the 3rd day,and then circinate-like cells could be observed in fusion time.Broad brimmed-like cells appeared after the 10th passage and cell proliferation slowed down.Flow cytometry showed that 95% and 96% of the 5th passage cells expressed CD29 and CD44 respectively,while only 5% of the cells expressed CD34.The growth curve showed that proliferation ability of the 3rd passage and 5th passage cells was more powerful than that of the 10th passage;the 3rd passage cell had the most powerful ability;but the proliferation ability of the 10th passage cells weakened.【Conclusion】The method of isolation and culture of BMSCs is simple and feasible.BMSCs can be cultured in a long period,grow stably and proliferate rapidly,which can meet the requirement of seed cells for tissue engineering.