Objective:To study the application of the live cell imaging method to observe the whole process of osteoclast formation induced by monocyte macrophages in the blood system in order to clarify the origin of osteoclasts and their cytody-natnics. Methods; Blood samples (8 ml) were collected from the abdominal aorta of male SD rats weighing 280 g. Mononuclear cells were obtained by density gradient centrifugalion and induced by RANKL and M-CSF. The cells were cultured and divided into four groups:inverted phase contrast microscope (IPCM) group,TRAP group,SEM group and live cell imaging (LCI) group. Images of the IPCM group were captured by a digital microscopic imaging system and recorded daily. The TRAP group was identified by enzyme activity staining after a 21-day cultivation period. The SEM group was SEM-observed after a 21-day cultivation period. The LCI group was consecutively and dynamically observed for 35 days. Results; After 2-week cultivation, IPCM observations showed the formation of numerous apocytes. These cells displayed round,fusiform,fan-shaped,elliptic or irregular gibbous profiles. TRAP staining showed that most apocytes and monocytes had positive ( + )reaction. SEM observations showed many bone absorption lacunae .hollows and channels,in which many osteoclasts with absorption activity were observed. Live cell imaging observations found that multinuclear osteoclasts originating from peripheral blood were generated by fusion of monocytes and apocytes and intercross fusion of monocytes and apocytes, which occurred at the adherent stage of the cells. Cy-todynamic observations showed that the cell form of osteoclasts was complex and changeable. Conclusion: RANKL and M-CSF can induce differentiation and formation from monocytes in rat peripheral blood into multinuclear osteoclasts with bone absorption activity. The osteoclasts were formed by various cell fusion processes at the adherent stage. The adherent property of osteoclasts is important for their survival and function. Osteoclasts have phagocytosis and their morphological structure is dynami-(9.57±0.92) |xg/L and the level of tumor necrosis factor-a were (48.47±2.64), (43.46±1.33), (40.96±1.05 )|Ag/L,with sta-tistical differences (P<0.05). The levels of interleukin-1P in synovia fluid of control group were (18.92± 1.83), (2O.25±2.76), (22.13±2.24)u,g/L and the levels of tumor necrosis factor-a were (57.92±2.12), (60.25±1.48), (63.35±2.15) (ig/L. At the same period, the levels of interleukin-1P and tumor necrosis factor-a were lower than those of the control group, which had a statistical significance(P<0.05). Conclusion:Pilose antler polypeptide can inhibit chondrocytes apoptosis,decrease the levels of interleukin-1 p and tumor necrosis factor-a and delay the degeneration of articular cartilage to some extent.%目的:采用活细胞成像技术,观察血系单核细胞诱导形成破骨细胞的全过程,旨在进一步阐明破骨细胞血系起源的发生及其细胞动力学.方法:取成年SPF级纯种雄性SD大鼠1只,体重280 g,腹主动脉采血8ml,经密度梯度离心分离单个核细胞,在RANKL与M-CSF诱导下,分为例置相差显微镜观察组、抗酒石酸酸性磷酸酶染色组、噬骨试验扫描电镜观察组、活细胞成像组4组进行培养.倒置相差显微镜观察组从培养开始,在数字显微成像系统下,每天观察记录1次；抗酒石酸酸性磷酸酶染色组培养21 d作酶活性染色鉴定；噬骨试验扫描电镜观察组培养21d取出骨磨片作扫描电镜观察；活细胞成像组采用多点位缩时电影法对整个培养过程进行长达35 d的连续观察记录.结果:诱导培养2周后,倒置相差显微镜观察可见大量多核细胞形成,外形呈圆形、梭形、扇形、椭圆形及不规则突起状；抗酒石酸酸性磷酸酶染色绝大部分多棱细胞与单核细胞均呈阳性反应；骨磨片扫描电镜观察可见较多骨吸收陷窝、坑洼及沟道,还有位于陷窝及沟道内正在行使骨吸收功能的破骨细胞；活细胞成像观察到起源于周围血的多核破骨细胞是由单核细胞、单核细胞与多核细胞及多核细胞之间相互融合而成,其细胞间的融合均发生在贴壁状态,显微缩时电影观察显示破骨细胞形态表现复杂多变.结论:大鼠周围血单核细胞在RANKL和M-CSF诱导下,可以向破骨细胞分化,形成具有骨吸收功能的多棱破骨细胞.破骨细胞的形成是发生在贴壁状态下多种形式的细胞融合过程,破骨细胞的粘附特性对其存活及功能发挥至关重要.破骨细胞具有吞噬功能,其形态结构动态多变.破骨细胞不仅是一种多核细胞,还可能包括单核破骨细胞.破骨细胞通过融合形成多棱巨细胞的特性,可能是其适应功能需求与骨吸收效率的一种特殊生物学行为.实验结果进一步证实了破骨细胞的血系起源学说,并为深入阐明破骨细胞的细胞动力学与细胞生物学特性提供了新的实验研究依据.
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