兔骨髓间充质干细胞体外培养及生物学特性研究

目的:进一步研究兔骨髓间充质干细胞(mesenchymal stem cells,MSCs)分离和培养方法及其生物学特性,为利用MSCs作为种子细胞治疗多种疾病提供实验基础。方法:通过密度梯度离心、贴壁筛选法分离培养兔MSCs。测定其接种贴壁率,在倒置显微镜下连续观察细胞的形态变化,利用甲基噻唑基四唑(methyl thiazdyl tetrazolium,MTT)法测定MSCs生长曲线,并应用流式细胞术测定细胞表面标记物。结果:MSCs接种12h后贴壁率为95%以上,细胞倍增时间约为30.8h,平均传代时间约5～7d;培养的MSCs阳性表达CD29、CD44、CD105,阴性表达CD34。结论:在适宜的实验条件下,体外培养的MSCs能够稳定生存增殖并保持多向分化潜能,可作为组织工程的种子细胞。

The Biological Characteristics and Culture in Vitro of Rabbit Bone Marrow-derived Mesenchymal Stem Cells

Objective To further study the isolation and cultivation procedures of mesenchymal stem cells from rabbit in vitro, and explore their biological properties. To build an experimental basis on applying MSCs as seed cells to treat many diseases. Methods MSCs were obtained from tibias by density gradient centrifugation and were tested the rate of adhesion. The configuration was observed under phase-contrast microscope consecutively and the cell growth was measured by MTT method. Cell cycle and surface antigens were detected through flow cytometer. Results The subcultured cells showed active proliferative ability. More than 95% of subceltured MSCs were adhesive in 12 h. MSCs were positive for CD29, CD44, CD105 and negative for CD34. Conclusion In present culture condion, MSCs showed stable and rapid growth in vitro. The characteristics make it possible to MSCs to be the seed cells for tissue engineering.