周期性压力培养对兔骨髓间充质干细胞增殖的影响

背景：各种外界应力对细胞增殖有重要作用,但涉及骨髓间充质干细胞最佳压力及加载时间的力学研究尚少。目的：探讨周期性压力培养对兔骨髓间充质干细胞增殖活性及细胞周期的影响。设计、时间及地点：细胞学体外对照实验,于2008-01/08在南昌大学重点分子生物实验中心完成。材料：健康6周龄雄性新西兰大白兔4只,由南昌大学实验动物中心提供。自行改制的细胞压力加载装置专利号ZL2006-2-0097266.3。方法：使用密度梯度离心法体外分离培养兔骨髓间充质干细胞,取生长良好的第3代细胞,按4×104个/孔接种于6孔板,设立5组：5.32,10.64,21.28,29.26kPa压力组分别给予对应的压力,每日加压6h×3d;正常培养组细胞只接受正常大气压力学刺激,不予额外加压。主要观察指标：力学干预后,观察细胞形态及生长状况,MTT法检测细胞增殖,流式细胞仪测量细胞周期变化。收集细胞培养上清液,检测基质金属蛋白酶2的质量浓度。结果：6h/d压力干预骨髓间充质干细胞,3d后细胞形态无异常变化,呈集落样生长。随着周期性压力的增加,细胞吸光度值逐渐升高,以21.28kPa压力组为佳,但升高至29.26kPa压力时吸光度值明显降低（F=3.731,P=0.008）。与正常培养组比较,5.32,10.64,21.28kPa压力组细胞周期发生改变,增殖指数均明显升高（P〈0.05或0.01）,但29.26kPa压力组增殖指数则明显下降。细胞培养上清液基质金属蛋白酶2质量浓度21.28kPa压力组最低,29.26kPa压力组最高,组间比较差异有显著性意义（t=213.214,P〈0.001）。结论：5.32～21.28kPa周期性压力可以提高骨髓间充质干细胞的增殖能力,特别是21.28kPa压力刺激促增殖作用尤为明显,但压力过强则抑制细胞增殖。

Effects of cyclical air-pressure culture on proliferation of rabbit bone marrow mesenchymal stem cells

BACKGROUND： Various surrounding stress has important effects on cell proliferation, but mechanics of optimal pressure and loading time on bone marrow mesenchymal stem cells （BMSCs） is few. OBJECTIVE： To explore effects of cyclical air-pressure culture on proliferation, activity and cell cycle of rabbit BMSCs. DESIGN, TIME AND SETTING： The cytology in vitro controlled study was performed at the Key Experimental Center of Molecular Biology, Nanchang University from January to August 2008. MATERIALS： A total of 4 healthy male New Zealand rabbits aged 6 weeks were supplied by the Experimental Animal Center, Nanchang University. Patent number of self-modified cell pressure charger was ZL2006-2-0097266.3. METHODS： Rabbit BMSCs were in vitro isolated by the density gradient centdfugation. At the third passage, cells were incubated in a 6-well plate, 4×10^4 cells per well. Cells were assigned into 5.32, 10.64, 21.28, 29.26 kPa pressure groups, 6 hours per day for 3 days. In the normal group, cells received normal pressure stimulation, without additional pressure. MAIN OUTCOME MEASURES： Following mechanics intervention, cell morphology and growth were observed. Cell proliferation was detected by M-IF assay. Cell cycle changes were measured by flow cytometry. Cell supernatant was collected to detect mass concentration of matrix metalloproteinase-2 （MMP-2）. RESULTS： Pressure to intervene BMSCs 6 hid × 3d, cells had no abnormal changes, showing colony growth. With increase in cyclic pressure, cell absorbance gradually increased, especially in the 21.28 kPa pressure group. When increased to 29.26 kPa pressure, absorbance significantly decreased （F=3.731, P=-0.008）. Compared with the normal group, cell cycle changed in the 5.32, 10.64, 21.28 kPa pressure groups, and proliferation index significantly increased （P 〈 0.05 or 0.01）, but the proliferation index significantly reduced in the 29.26 kPa pressure group. Mass concentration of MMP-2 in supernatant was the lowest in the 21.28 kPa pressure group, and the highest in the 29.26 kPa pressure group. Significant differences were detected among groups （t=213.214, P 〈 0.001）. CONCLUSION： 5.32-21.28 kPa pressure can elevate BMSC proliferation, especially 21.28 kPa pressure, but excessive pressure can inhibit cell proliferation.