牵张应力影响破骨细胞形成的研究

Effect of stretch stress on osteoclast formation

目的 探讨牵张应力对破骨细胞形成的影响,为骨质疏松、骨折延迟愈合等临床疾病的治疗及Ilizarov牵张成骨技术的推广应用奠定理论基础。方法 在体外利用Flex Cell细胞应力加载系统刺激小鼠单核/巨噬细胞系RAW264.7分化为破骨细胞,通过免疫组化、RT-PCR等技术,从蛋白及基因水平探讨牵张应力刺激对诱导破骨细胞形成的作用特点。结果 在M-CSF(30 ng/mL)和RANKL(100 ng/mL)联合诱导的同时加入不同强度的牵张应力刺激,在0、0.5、1.0、2.0 Hz牵张应力刺激后,破骨细胞的形成明显增多,M-CSF阳性细胞面积依次增大(38.36±4.58)%、(42.21±5.74)%、(70.36±9.55)%、(85.42±11.36)%;RANKL阳性细胞面积依次增大(37.15±4.27)%、(40.42±5.25)%、(63.26±9.11)%、(90.25±12.38)%,差异有统计学意义(P<0.05),且随着应力强度的增加,促进显著增强。在体外诱导破骨细胞分化的过程中,给予小鼠单核/巨噬细胞系RAW264.7在0、0.5、1.0、2.0 Hz牵张应力刺激后,TRAP[(0.92±0.22)、(1.44±0.53)、(1.61±0.36)、(2.45±0.67)]、Calcitonin Receptor [(0.94±0.26)、(1.05±0.31)、(1.51±0.42)、(1.97±0.58)]、Cathepsin K[(0.91±0.25)、(1.39±0.35)、(2.11±0.57)、(2.37±0.51)]基因的表达水平明显增高,差异有统计学意义(P<0.05),且随着应力强度的增加,促进作用显著增强。结论 牵张应力环境刺激有利于破骨细胞的形成。

Objective To explore the influence of stretch stress on the formation of osteoclasts,and to lay a theoretical foundation for the treatment of osteoporosis,delayed fracture healing and other clinical diseases and the promotion and application of Ilizarov distraction osteogenesis technology.Methods The differentiation of mouse mononuclear/macrophage Cell line RAW264.7 into osteoclasts was stimulated by Flex Cell augmentation system in vitro.Immunohistochemistry,RT-PCR and other techniques were used to investigate the effect of stretch stress stimulation on osteoclast formation from protein and gene levels.Results The combined induction of M-CSF(30 ng/mL)and RANKL(100 ng/mL)was accompanied by the addition of different intensities of stretch stress stimulation.At 0,0.5,1.0,2.0 Hz,the formation of osteoclasts was significantly increased.The area of M-CSF positive cells was enlarged by(38.36±4.58)%,(42.21±5.74)%,(70.36±9.55)%and(85.42±11.36)%,respectively.RANKL positive cell area was enlarged by(37.15±4.27)%,(40.42±5.25)%,(63.26±9.11)%and(90.25±12.38)%,the difference was statistically significant(P<0.05),and with the increase of the stress intensity,the promotion increased significantly.In the process of inducing osteoclast differentiation in vitro,the mouse monocyte/macrophage line RAW264.7 was subjected to stretch stress stimulation at 0,0.5,1.0 and 2.0 Hz.Expression levels of TRAP[(0.92±0.22),(1.44±0.53),(1.61±0.36)and(2.45±0.67)],Calcitonin Receptor[(0.94±0.26),(1.05±0.31),(1.51±0.42)and(1.97±0.58)]and Cathepsin K[(0.91±0.25),(1.39±0.35),(2.11±0.57)and(2.37±0.51)]genes were significantly increased,the difference was statistically significant(P<0.05),and with the increase of stress intensity,the promoting effect was significantly enhanced.Conclusion The stimulation of the stretch stress environment is beneficial to the formation of osteoclasts.