周期性张应变对颈椎后纵韧带细胞形态及凋亡影响的体外观察

Morphologic and apoptotic changes of cervical posterior longitudinal ligament fibroblasts in response to in vitro mechanical stretch

目的 研究周期性张应变对体外培养的后纵韧带骨化（OPLL）患者的后纵韧带细胞形态及凋亡的作用.方法 组织块贴壁法培养后纵韧带细胞,选用第3代细胞,长至80%融合时,用含1%血清的培养基同步化24 h后,使用Flexerce114000TM细胞应变加载系统对细胞施加周期性张应变,作用6及24 h,牵拉幅度为10%.运用流式细胞仪对细胞凋亡水平进行检测,β-gal染色评价细胞衰老率.同时,检测细胞碱性磷酸酸（ALP）、Ⅰ型胶原（Col Ⅰ）和骨钙素（OC）mRNA的变化.结果 与不加力的对照组相比,受力组细胞被拉长,并呈垂直作用力的方向取向排列;6 h组较对照组凋亡率略有升高,但差异无统计学意义;24 h组则明显升高（P＜0.05）;6 h组与24 h组较对照组细胞衰老率无明显变化.ALP、Col Ⅰ和OC mRNA表达水平均升高,24 h组升高更明显（P＜0.05）.结论 周期性张应变对后纵韧带细胞的形态具有影响,应变刺激在促进OPLL细胞成骨分化的过程中,同时促进了细胞凋亡和功能的改变.

Objective To investigate the effect of mechanical stretch force on the morphologic and apoptotic changes of fibroblasts derived from the OPLL （ossification of posterior longitudinal ligaments）patients.Methods The third passage cells were collected and subjected to 10% elongations cyclic mechanical stretching for 6 h and 24 h with Flexerce114000TM strain unit.Their morphologic changes were observed and the post-stretching apoptotic rates quantified by flow cytometer.The gene expressions of alkaline phosphatase （ALP）,collagen types Ⅰ （COL Ⅰ） and osteocalcin （OC） were examined.Results The treated cells were arranged along the vertical direction of force.Stretch force led to a slight increase of apoptosis rate at 6 h and a significant increase of apoptosis rate at 24 h.No significant difference in cellular senescence was observed between control group and treated group.The mRNA expressions of ALP,COL Ⅰand OC were positively up-regulated by cyclic stretch at 24 h.Conclusion Stretching force can affect the cellular morphology,promote the osteogenic differentiation and enhance the cellular apoptosis.