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背景:正畸牙齿移动的基础是牙周组织的改建,其中破骨细胞性骨吸收是牙齿移动的第一步,应力作用下有关破骨细胞分化和功能成熟的信号转导通路,以及牙周膜细胞和破骨细胞之间的关系是目前国内外研究的热点之一。目的:拟建立人破骨样细胞体外培养的简便方法,观察骨吸收刺激因子对破骨样细胞分化、增殖和功能的影响。设计、时间及地点:细胞学体外对照观察,于2007-10/2008-05在西安交大口腔医院中心实验室完成。材料:脐带血来源于非高危妊娠的健康产妇,新鲜牛股骨由西安交通大学动物实验中心提供,用于制备100～200μm厚的骨片,1α,25-(OH)2D3、巨噬细胞集落刺激因子、前列腺素E2等骨吸收刺激因子均为Sigma公司产品。方法:无菌条件下收集脐带血,Ficoll液分离后吸取呈云雾状的白膜层,离心弃上清,加入α-MEM培养液重悬,调整脐血单核细胞浓度为1×109L-1,接种于预置盖玻片和骨片的24孔培养板中,1.0mL/孔,设立空白对照组、10-8mol/L及10-7mol/L1α,25-(OH)2D3组、巨噬细胞集落刺激因子组、1α,25-(OH)2D3+前列腺素E2组,培养7d。主要观察指标:倒置显微镜观察细胞生长形态,TRAP染色法观察破骨样细胞的形成,甲苯胺蓝染色观察骨吸收陷窝情况,以TRAP染色(+)、细胞核≥2个的细胞为破骨样细胞进行计数。结果:培养3d后,空白对照组细胞形态及数量无明显变化,各诱导组单核细胞出现融合趋势;7d时空白对照组出现少量的破骨样细胞,核的数目2～3个,各诱导组可见大量多核破骨样细胞,核的数目3～20个不等。诱导后光镜下可见胞浆呈红色、胞核呈淡黄色的TRAP(+)破骨样细胞,尤其是10-8mol/L1α,25-(OH)2D3组可见含14个核的强阳性破骨样细胞,且胞体较大。各组均尚未形成骨吸收陷窝。与空白对照组比较,各诱导组破骨样细胞数量均明显增多(F=9.78,P<0.01);与10-8mol/L1α,25-(OH)2D3组比较,10-7mol/L1α,25-(OH)2D3组破骨样细胞数量无明显变化(P>0.05),巨噬细胞集落刺激因子组及1α,25-(OH)2D3+前列腺素E2组破骨样细胞数量均明显减少(F=7.46,P<0.01)。结论:脐血单核细胞经骨吸收刺激因子体外诱导培养后,可分化为TRAP(+)的多核破骨样细胞,其中10-8mol/L1α,25-(OH)2D具有最强的生物学效应。
BACKGROUND: The basis of orthodontic tooth movement is the alteration of periodontal tissue, in which osteoclastic bone resorption is the first step in tooth movement, signal transduction pathways related to osteoclast differentiation and function maturation under stress, and periodontal The relationship between membrane cells and osteoclasts is one of the hot spots at home and abroad. OBJECTIVE: To establish a simple and convenient method of culturing human osteoclast-like cells in vitro and observe the effect of bone resorption factor on differentiation, proliferation and function of osteoclast-like cells. DESIGN, TIME AND SETTING: The cytology in vitro control study was performed at the Central Laboratory of Xi’an Jiaotong University Stomatological Hospital from October 2007 to May 2008. MATERIALS: Umbilical cord blood was derived from healthy maternal non-high-risk pregnancies. The fresh bovine femur was provided by the Animal Experimental Center of Xi’an Jiaotong University for the preparation of 100 ~ 200μm bone chips, 1α, 25- (OH) 2D3, macrophage colony Stimulation factor, prostaglandin E2 and other bone resorption factors are Sigma products. Methods: Umbilical cord blood was collected under aseptic conditions. After the Ficoll liquid was separated, the albuginea was cloud-like. The supernatant was removed by centrifugation and resuspended in α-MEM medium. The concentration of cord blood mononuclear cells was adjusted to 1 × 109L-1 Were inoculated into 24-well culture plates with pre-set coverslips and bone slices at 1.0 mL / well to establish blank control group, 10-8 mol / L and 10-7 mol / L1α, 25- (OH) 2D3 groups, Cell colony stimulating factor group, 1α, 25- (OH) 2D3 + prostaglandin E2 group, cultured for 7 days. MAIN OUTCOME MEASURES: The morphological changes of the cells were observed with inverted microscope. The formation of osteoclast-like cells was observed by TRAP staining. The bone resorption lacuna was observed by toluidine blue staining. The osteoclasts were detected by TRAP staining (+), Cells are counted. RESULTS: After 3 days of culture, there was no significant change in morphology and number of cells in the blank control group, and monocytes in each induction group showed a tendency of fusion. On the 7th day, a small amount of osteoclast-like cells appeared in the blank control group with 2 to 3 nuclei in each induction A large number of multinucleated osteoclast-like cells were seen in the group. The number of nuclei ranged from 3 to 20. Under the light microscope, the cytoplasm showed red and the nucleus was light yellow TRAP (+) osteoclast-like cells, especially in 10-8mol / L1α, 25- (OH) 2D3 group, Osteoid cells, and large cell body. No bone resorption lacuna was found in all groups. Compared with the blank control group, the number of osteoclast-like cells in each induction group was significantly increased (F = 9.78, P <0.01). Compared with the 10-8mol / L1α, 25- The numbers of osteoclast-like cells in macrophage colony-stimulating factor group and 1α, 25- (OH) 2D3 + prostaglandin E2 group were significantly decreased (P <0.05) = 7.46, P <0.01). CONCLUSION: The umbilical cord blood mononuclear cells can be differentiated into TRAP (+) multinucleated osteoclast-like cells after osteoblast-stimulating factor induction in vitro. Among them, 10-8mol / L1α, 25- (OH) 2D has the strongest biological effect.