不同牵伸载荷对体外培养人肌腱细胞纤维型肌动蛋白的影响  

Effects of different cyclic mechanical stretching loads on human tenocytic cytoskeleton in vitro

作　　者：邓银栓 唐康来 谢美明 曹洪辉 陈磊 常德海 董世武[2] 陶旭 李辉 杨会峰 许建中 

机构地区：[1]第三军医医大学西南医院骨科,重庆400038 [2]第三军大学基础医学部人体解剖学教研室

出　　处：《中华医学杂志》2011年第25期1780-1785,共6页National Medical Journal of China

基　　金：基金项目：国家自然科学基金（30872620,81071464）;全军医学科研“十一五”计划项目（06MB245）;重庆市自然科学基金重点项目（2011JJ0041）

摘　　要：目的探讨不同牵伸载荷条件对体外人肌腱细胞纤维型肌动蛋白（F—actin）的影响，分析不同载荷条件与人肌腱细胞F-actin的变化关系。方法取5～7代人肌腱细胞加载不同牵伸载荷，其中牵伸强度分别为4％、8％、12％；时间：2、4、8、12、24h；频率：0．5H、1．0Hz。采用罗丹明标记的鬼笔环肽（rhodamine—phalloidin）及DAPI免疫荧光染色，激光扫描共聚焦显微镜观察F．actin的变化、细胞核固缩及凋亡小体形成；图像分析软件测定单个细胞平均荧光强度并进行单因素方差分析。结果设定牵伸频率为0．5Hz、牵伸强度4％，牵伸时间2h时，人肌腱细胞微丝排列紊乱、模糊；牵伸时间4h时，微丝增粗、减少，出现断裂呈细颗粒状。设定牵伸频率为0．5Hz、牵伸时间4h时，牵伸强度8％微丝解聚、断裂明显，F-actin总体分布减少；牵伸强度12％胞质微丝全部断裂，仅有部分胞膜微丝完整。设定牵伸频率为1．0Hz，牵伸强度为12％，加载时间分别为2、4、8、12、24h时，微丝断裂呈递增趋势，牵伸24h肌动蛋白微丝全部断裂。空白对照组细胞微丝完整荧光强度整体水平最高。牵伸频率固定时，随着牵伸强度递增，微丝的荧光强度呈梯度降低（P〈0．05），且差异有统计学意义；牵伸时间为8h时，各实验组微丝荧光强度增加，但低于对照组，随着时间的延长，F—actin表达量逐渐降低，24h时最低，其中，牵伸频率为0．5Hz、牵伸强度4％、牵伸时间4h时，细胞核固缩最明显，凋亡小体形成。结论不同牵伸载荷条件可引起体外培养的人肌腱细胞F—actin发生断裂、解聚，F-actin的断裂、解聚与载荷时间及应力强度呈正相关，F—actin的断裂、解聚诱导了人肌腱细胞生物学行为的改变。Objective To investigate the human tenocyte cytoskeleton under different in vitro stretching conditions and analyze the relations between the changes of tenocytic cytoskeleton and different stretching loads. Methods Human tenocytes, cultivated for 5 - 7 passages, were stretched under 4% , 8% and 12% cyclic mechanical stretching with a duration of 2, 4, 8, 12, 24 hours and a frequency of 0. 5 and 1.0 Hz. Laser scanning confocal microscope was used to examine the changes of F-actin and nucleus after immunofluorescent staining at different cyclic mechanical stretching loads on human tenocyte. The uni-cell average fluorescence intensity was measured with an image analysis system by the photos of human tenocyte cytoskeleton and analyzed by the single factor analysis of variance. Results After cyclic stretching under 4% stretching with a duration of 2 hours at 0. 5 Hz, the microfilament of human tenocyte had an irregular and dim alignment. F-actin was thicker and ruptured under 4% stretching with a duration of 4 hours. Under 8% stretching with a duration of 4 hours at 0. 5 Hz, all actin microfilaments ruptured, but part of membranemicrofilament remained intact. There was a rising trend of actin filament fracturing under 12% stretching with a duration of 2, 4, 8, 12, 24 hours at 1.0 Hz. And all actin filaments fractured at 24 hours. In the control group, the fluorescent intensity of F-actin was at the highest and the filament remained intact. Under the same stretching frequency, the fluorescent intensity of F-actin had a declining trend and significant differences existed under different stretching loads with different durations （ P 〈 0.05 ）. The fluorescent intensity of F-actin increased in all experimental groups, but it was lower than that of the control group with a duration of 8 hours. The expression of F-actin decreased with a longer duration and reached its lowest at 24 hours. The most obvious phenomenon of nuclear condensation and apoptotic body formation was observed under 4% stretching with a duration of

关 键 词：腱 肌动蛋白类 应力 物理 

分 类 号：R6[医药卫生—外科学]