摘要
目的:观察周期性机械牵张应力对人骨髓间充质干细胞的增殖活性与细胞周期的影响。方法:实验于2006-02/06在同济医院矫形外科实验室完成。①建立体外细胞周期性机械牵张力学装置,并利用三维有限元的方法分析基膜的力学分布。②使用密度梯度离心法分离人骨髓间充质干细胞。③取第3～6代细胞接种于弹性硅胶膜培养板,分组:实验组细胞接受力学装置1.0Hz的频率,不同大小的应力(3%,6%,9%,12%,18%)、不同时间(12,24,36,48,60,72h)的机械信号刺激,对照组细胞不受力学信号刺激,其他培养条件与实验组一致。④细胞行力学干预后,用相差显微镜观察行细胞形态、排列,分别用细胞计数法、四甲基偶氮唑和流式细胞仪测量细胞增殖和细胞周期。结果:①自行建立的力学装置性能稳定,细胞接种硅胶6孔板后12～24h贴壁,贴壁良好,24h后细胞覆盖率约达70%,无细菌污染现象,可以用于应力刺激体外细胞效应的实验研究。②应力干预人骨髓间充质干细胞12h后,细胞形态稍变细长,轮廓鲜明,并沿受力环切线方向规则排列,干预24h变化更明显。③通过细胞计数、四甲基偶氮唑、流式细胞周期检测,发现适当大小的力学刺激对人骨髓间充质干细胞具促细胞增殖作用,增殖指数增大,其中12%的应力作用最明显,其增殖指数约为对照组的1.86倍;过强的机械应力(18%)对细胞不具有促增殖效应,并可能存在毒性作用;早期力学干预可以促进细胞增殖,干预时间过长(48h以后)反而表现为抑制生长趋势。结论:适当的周期性牵张应力可以提高人骨髓间充质干细胞的增殖能力。
AIM： To study the effect of mechanical stretch stress on the cellular proliferation and cell cycle of human bone marrow mesenchymal stem cells （HMSCs）. METHODS： The experiment was carried out in Laboratory of Department of Orthopedic Surgery of Tongji Hospital from February to June 2006. （1） A mechanical stretching loading device was established for cultured cells and a Three-Dimensional Finite Element Model was developed to analyze the mechanic distribution of the membrane. （2） HMSCs were obtained by density gradient centrifugaUon from normal volunteers. （3）3^rd-6^th generational cells were taken and inoculated to elastic silicone membrane plate. Grouping： the experimental group received 1.0 Hz mechanic device and signal stimulus at different stresses （3%,6%,9%, 12%, 18%） and different time points （at hours 12,24,36,48,60, 72）. The control group did not received signal stimulus, and other cultured conditions were the same as the experimental group. （4）After HMSCs were stimulated by mechanical strain, the cellular morphology, distribution and alignment were observed, and cell count, MTT assay and flow Cytometry were applied to measure the cellular proliferation and cell cycle. RESULTS： （1）The mechanical strain unit with stable performance could be used to evaluate cell response sensitivity to mechanical stress in vitro. Cells were adherenced to the wall 12-24 hours after inoculated on the 6-well plate. After 24 hours, the fraction of coverage reached to 70%, and no bacterial contamination. （2）After mechanical stretch stress treatment for 12 hours, the cells had appeared slender with bright outline and regularly aligned and evenly distributed porch stress circumcising line. Changes were more obvious 24 hours after intervention. （3）After appropriate force magnitude/time mechanical stretch stress treatment, HMSCs cellular proliferation had been accelerated and cellular cycle had been changed with proliferation index increasing. When the strain magnitude was 12%, the cellular proliferation was the most obviously effect after treatment. The proliferation index was 1.86 times of that of the control group. Eady mechanical stretch stress treatment could enhance the cell proliferation, but cellular proliferation was suppressed after excessive force magnitude（18%）/time （over 48 hours） mechanical stretch stress treatment, with toxic effect. CONCLUSION： Proper mechanical stretch stress can enhance effect on the cellular proliferation of HMSCs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第7期1247-1251,I0003,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
教育部回国人员基金项目(013456)~~
