摘要
目的:探讨大鼠颅骨成骨细胞(OB)对机械力刺激的反应及细胞骨架F-actin的变化。方法:体外分离培养大鼠颅骨OB细胞;应用四点弯曲加力系统,对第3代细胞施加2000με的机械牵张力,加力时间分别为0h、2h、6h、12h,应用特异性荧光染料分别标记微丝蛋白F-actin和细胞核;在激光共聚焦显微镜下观察并进行形态计量学测量。应用TUNEL法进行原位凋亡检测。结果:在机械张力刺激下,OB荧光强度减弱,F-actin纤维束变得稀疏而排列紊乱;胞核轮廓模糊,并可观察到凋亡小体。定量分析表明,OB的荧光总强度、绿色荧光强度(F-actin)和红色荧光强度(胞核)均显著下降(9<0.05或P<0.01)。TUNEL检测也表明,随着加力时间延长,凋亡细胞数明显增加,但明显少于F-actin发生改变的细胞数。结论:机械牵张刺激可引起OB细胞骨架F-actin解聚和重排,并诱发部分细胞发生凋亡。
PURPOSE: To explore the response of rat calvarial osteoblasts and the changes of cytoskeleton F-actin under mechanical strain. METHODS: Bone calvarial osteoblasts were isolated from SD rats and cultured in vitro. Mechanical stretch was performed on passage 3 cells at 2000με for 0, 2, 6 and 12 hours using four-point bending system. The response of cells and the distribution of F-actin were observed using fluorescent staining under laser scanning confocal microscope. Terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick-end labeling assay (TUNEL) was also explored to detect apoptotic cells. RESULTS: Under mechanical stretch, the fluorescent staining decreased obviously in most of osteoblasts, and F-actin filaments were rearranged and became tenuous, thinner, and abnormally distributed. The outline of nucleus became unclear and apoptotic changes were observed. Quantity analysis showed that total fluorescent density, green fluorescent density and red fluorescent density (nuclei) all significantly decreased in osteoblasts (P<0.05 or P <0.01).TUNEL detection also showed that apoptotic cells increased significantly with the elongation of mechanical stretch. However, the numbers of apoptotic cells were far less than that of cells in which F-actin bundles were significantly reorganized. CONCLUSION: Mechanical stretch causes extensive response to osteoblasts and leads to the disruption and rearrangement of F-actin filament and thereafter induces apoptosis in some of these cells.
出处
《中国口腔颌面外科杂志》
CAS
2004年第3期181-184,共4页
China Journal of Oral and Maxillofacial Surgery
基金
国家自然科学基金(30371553)
关键词
成骨细胞
机械应力
凋亡
激光共聚焦显微镜
Osteoblasts
Mechanical strain
Apoptosis
Laser scanning confocal microscope
