中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2011年
25期
1780-1785
,共6页
邓银栓%唐康来%谢美明%曹洪辉%陈磊%常德海%董世武%陶旭%李辉%杨会峰%许建中
鄧銀栓%唐康來%謝美明%曹洪輝%陳磊%常德海%董世武%陶旭%李輝%楊會峰%許建中
산은전%당강래%사미명%조홍휘%진뢰%상덕해%동세무%도욱%리휘%양회봉%허건중
腱%肌动蛋白类%应力,物理
腱%肌動蛋白類%應力,物理
건%기동단백류%응력,물리
Tendons%Actins%Stress,mechanical
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-actln)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、24 h;频率:0.5 H、1.0 Hz.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 5 Hz、牵伸强度4%,牵伸时间2 h时,人肌腱细胞微丝排列紊乱、模糊;牵伸时间4 h时,微丝增粗、减少,出现断裂呈细颗粒状.设定牵伸频率为0 5 Hz、牵伸时间4 h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1.0 Hz,牵伸强度为12%,加载时间分别为2…4 8 12、24 h时,微丝断裂呈递增趋势,牵伸24 h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8 h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,24 h时最低,其中,牵伸频率为0 5l-Iz、牵伸强度4%、牵伸时间4 h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-aetin发生断裂、解聚,F-actin的断裂、解聚与载荷时问及应力强度呈正相关,F-aetin的断裂、解聚诱导了人肌腱细胞生物学行为的改变.
目的 探討不同牽伸載荷條件對體外人肌腱細胞纖維型肌動蛋白(F-actln)的影響,分析不同載荷條件與人肌腱細胞F-actin的變化關繫.方法 取5～7代人肌腱細胞加載不同牽伸載荷,其中牽伸彊度分彆為4%、8%、12%;時間:2、4、8、12、24 h;頻率:0.5 H、1.0 Hz.採用囉丹明標記的鬼筆環肽(rhodamine-phalloidin)及DAPI免疫熒光染色,激光掃描共聚焦顯微鏡觀察F-actin的變化、細胞覈固縮及凋亡小體形成;圖像分析軟件測定單箇細胞平均熒光彊度併進行單因素方差分析.結果 設定牽伸頻率為0 5 Hz、牽伸彊度4%,牽伸時間2 h時,人肌腱細胞微絲排列紊亂、模糊;牽伸時間4 h時,微絲增粗、減少,齣現斷裂呈細顆粒狀.設定牽伸頻率為0 5 Hz、牽伸時間4 h時,牽伸彊度8%微絲解聚、斷裂明顯,F-actin總體分佈減少;牽伸彊度12%胞質微絲全部斷裂,僅有部分胞膜微絲完整.設定牽伸頻率為1.0 Hz,牽伸彊度為12%,加載時間分彆為2…4 8 12、24 h時,微絲斷裂呈遞增趨勢,牽伸24 h肌動蛋白微絲全部斷裂.空白對照組細胞微絲完整熒光彊度整體水平最高.牽伸頻率固定時,隨著牽伸彊度遞增,微絲的熒光彊度呈梯度降低(P＜0.05),且差異有統計學意義;牽伸時間為8 h時,各實驗組微絲熒光彊度增加,但低于對照組,隨著時間的延長,F-actin錶達量逐漸降低,24 h時最低,其中,牽伸頻率為0 5l-Iz、牽伸彊度4%、牽伸時間4 h時,細胞覈固縮最明顯,凋亡小體形成.結論 不同牽伸載荷條件可引起體外培養的人肌腱細胞F-aetin髮生斷裂、解聚,F-actin的斷裂、解聚與載荷時問及應力彊度呈正相關,F-aetin的斷裂、解聚誘導瞭人肌腱細胞生物學行為的改變.
목적 탐토불동견신재하조건대체외인기건세포섬유형기동단백(F-actln)적영향,분석불동재하조건여인기건세포F-actin적변화관계.방법 취5～7대인기건세포가재불동견신재하,기중견신강도분별위4%、8%、12%;시간:2、4、8、12、24 h;빈솔:0.5 H、1.0 Hz.채용라단명표기적귀필배태(rhodamine-phalloidin)급DAPI면역형광염색,격광소묘공취초현미경관찰F-actin적변화、세포핵고축급조망소체형성;도상분석연건측정단개세포평균형광강도병진행단인소방차분석.결과 설정견신빈솔위0 5 Hz、견신강도4%,견신시간2 h시,인기건세포미사배렬문란、모호;견신시간4 h시,미사증조、감소,출현단렬정세과립상.설정견신빈솔위0 5 Hz、견신시간4 h시,견신강도8%미사해취、단렬명현,F-actin총체분포감소;견신강도12%포질미사전부단렬,부유부분포막미사완정.설정견신빈솔위1.0 Hz,견신강도위12%,가재시간분별위2…4 8 12、24 h시,미사단렬정체증추세,견신24 h기동단백미사전부단렬.공백대조조세포미사완정형광강도정체수평최고.견신빈솔고정시,수착견신강도체증,미사적형광강도정제도강저(P＜0.05),차차이유통계학의의;견신시간위8 h시,각실험조미사형광강도증가,단저우대조조,수착시간적연장,F-actin표체량축점강저,24 h시최저,기중,견신빈솔위0 5l-Iz、견신강도4%、견신시간4 h시,세포핵고축최명현,조망소체형성.결론 불동견신재하조건가인기체외배양적인기건세포F-aetin발생단렬、해취,F-actin적단렬、해취여재하시문급응력강도정정상관,F-aetin적단렬、해취유도료인기건세포생물학행위적개변.
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 membrane microfilament 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 24hours. The most obvious phenomenon of nuclear condensation and apoptotic body formation was observed under 4% stretching with a duration of 4 hours at 0.5 Hz. Conclusion Different cyclic mechanical stretching may cause the in vitro breakage and depolymerization of human tenocytic F-actin. Such an effect correlates with stretching force and its duration.
