体外诱导脐血单核细胞向破骨样细胞的分化

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,1.0 mL/孔,设立空白对照组、10-8mol/L及107mol/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,核的数日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108mol/L 1α,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,107mol/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/L 1α,25-(OH)2D具有最强的生物学效应.

In vitro differentiation of umbilical cord blood-derived mononuclear cells towards osteoclast-like cells

BACKGROUND: Orthodontic tooth movement is dependent on reconstruction of periodontium. Osteoclastic bone resorption is the first step of tooth movement. The present study hotspots focus on signal transduction pathway regarding osteoclast differentiation and functional development under stress and on the relationship between periodontal ligament cells and osteoclasts. OBJECTIVE: To set up a simple method to in vitro culture human osteoclast-like cells and to observe the effects of bone resorption-stimulating factors on differentiation, proliferation, and function of osteoclast-like cells, DESIGN, TIME AND STTING: A cytological in vitro controUod observation was performed at the Central Laboratory,Stomatology Hospital, Xi'an Jiaotong University between October 2007 and May 2008. MATERIALS: Umbilical cord blood was sourced from the healthy puerperae who had not suffered from high-risk pregnancy. Freshly prepared fetal femur provided by Laboratory Animal Center, Xi'an Jiaotong University and were used for preparation of bone flaps at 100-200 μm thickness. 1α ,25-(OH)2D3, macrophage colony-stimulating factor (M-CSF), prostaglandin E2 were purchased from Sigma Company, USA.METHODS: Under aseptic condition, umbilical cord blood was collected. Following Ficoll solution separation and centrifugation, supematant was discarded. Umbilical cord blood-derived mononuclear cells were suspended with o -modified minimal essential medium (α-MEM) solution and then inoculated into a 24-well culture plate, in which, coverslips and femoral slices were pre-placed, at a density of 1×109/L, 1.0 mL per well. Five groups were set, blank control, 108 mol/L 1α ,25-(OH)2D3, 10-7 mol/L 1α ,25-(OH)2D3, macrophage colony stimulating factor (M-CSF), and 1α ,25-(OH)2D3+prostaglandin E2 (PGE2). Each group was cultured for 7 days. MAIN OUTCOME MEASURES: Cellular growth morphology was observed under an inverted microscope; osteoclast-like celt formation was examined by tartrate-resistant acid phosphatase (TRAP) staining; and osteoclastic Howship's lacuna was detected by toluidine blue staining. Any cell with TRAP-positive staining and more than two nuclei was considered osteoclast-like cell and counted. RESULTS: After 3 days of culture, cells from the blank control group did not exhibit apparent changes in morphology and quantity. In the remaining groups, mononuclear cells appeared with confluent tendency. After 7 days of culture, a small number of osteoclast- like cells with 2-3 nuclei were found in the blank control group; a great many of multinucleated osteoclast- like cells with 3-20 nuclei were present in the remaining groups. Through the use of optical microscope, osteoclast-like cells could be found for the presence of red cytoplasm, bright yellow nuclei, and TRAP-positive staining in each inducing factor-treated group, in particular in the 108 mol/L 1α ,25-(OH)2D3 group, which displayed osteoclast- like cells exhibiting 14 nuclei, strong TRAP-positive staining, and a relatively big cell body. But no osteoclastic Howship's lacuna was found in any group. Compared to the blank control group, the numbers of osteoclast-like cells were greater in each inducing factor-treated group (F = 9.78, P < 0.01). There was no significant difference in the numbers of osteoclast-like cells between the 108 mol/L 1α ,25-(OH)2D3 and the 10-7 mol/L 1 a ,25-(OH)2D3 groups (P 0.05). The M-CSF group and the 1α ,25-(OH)2D3+PGE2 group exhibited significantly less numbers of osteoclast-like cells than the 108 mol/L 1α ,25-(OH)2D3 group (F= 7.46, P< 0.01). CONCLUSION: After in vitro culture of 1α ,25-(OH)2D3, M-CSF, and PGE2, umbilical cord blood-derived mononuclear cells can differentiate into TRAP-positive multinucleated osteoclast-like cells, the 10-8 mol/L 1α ,25-(OH)2D3 being the most effective.