流体剪应力对大鼠破骨细胞骨吸收活性的影响

探讨流体剪应力对大鼠破骨细胞骨吸收活性的影响。我们采用低温离心法获取6月龄健康雌性SD大鼠椎骨骨髓细胞,以1.6×106细胞密度种植于血盖片上,采用1,25-(OH)2维生素D3体外诱导获取破骨细胞。于破骨细胞诱导的第7d取出细胞爬片,置于流体剪应力装置中,分别加载5.97、11.36、16.08、20.54dyne/cm2大小的流体剪应力,持续30min,以未加载流体剪应力的破骨细胞为对照组。实验结束时,以2.5%戊二醛固定细胞爬片,置于0.25mol/L氢氧化铵液超声处理10min,清除细胞爬片上的破骨细胞,经1%硪酸固定,梯度酒精脱水,醋酸异戊酯置换酒精,CO2临界点干燥,喷金后扫描电镜观察骨吸收陷窝并计数,图像分析法测定骨吸收陷窝的面积;同时分别收集每次灌流液10ml,冻干,用1ml复溶后,紫外分光光度仪检测抗酒石酸酸性磷酸酶(Tartrate-resistant acid phosphatase,TRAP)活性的变化。结果显示:在本实验中所选用的流体剪应力可增强破骨细胞TRAP活性,而骨吸收陷窝的数量和面积也增加,尤其是剪应力在16.08dyne/cm2时,破骨细胞TRAP活性增强以及骨吸收陷窝的数量和面积增高最为明显。结果表明,一定范围内的流体剪应力可以增强破骨细胞的骨吸收活性。

Effects of Fluid Shear Stress on Bone Resorption in Rat Osteoclasts

This study was aimed to assess the effects of fluid shear stress on the bone resorption in rat osteoclasts. The osteoclasts were derived from the lumbar vertebrae marrow cells which were isolated from the 6-month-old female Sprague Dawley rats, cultured on the slide at 1 x 10(6) cell/ml, and induced with 1, 25 (OH)2 Dihydroxyvitamin D3. The slide containing osteoclasts was taken out on day 7 after culture and then put into the flow chamber. The loads of fluid shear stress applied to the osteoclasts were 5.97, 11.36, 16.08 and 20.54 dyne/cm2, respectively, for 30 minutes. The osteoclasts unloading fluid shear stress were used as control. The bone resorptive pits were studied by light microscopy and scanning electron microscopy. The tartrate-resistant acid phosphatase (TRAP) secreted by osteoclasts was detected with ultraviolet spectrophotometry. The results showed that fluid shear stress can increase the activity of TRAP and significantly increase the number and area of bone resorptive pits made by osteoclasts,and the effect of fluid shear stress on the bone resorption of osteoclasts is the same as that on the activity of TRAP. The reaction of the osteoclasts to the fluid shear stress in this study also suggested that the bone resorption of osteoclasts be increased in a magnitude of fluid shear stress-dependent manner, and that the changes of TRAP activity be closely related to the changes of the number and area of resorptive pits of the osteoclasts.