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破骨细胞分化因子可诱导鸡骨髓细胞形成破骨样细胞 总被引：2，自引：0，他引：2
研究了鸡破骨细胞分化因子（Chicken osteoclast differentiation factor，chODF）体外诱导鸡骨髓细胞形成破骨样细胞的能力。在无菌条件下取鸡股骨和胫骨，收集骨髓细胞。试验分设A、B、C、D、E、F、G7组。A组为对照组，不加细胞因子，其他各组为试验组，其中B组仅加chODF，C组只加巨噬细胞集落刺激因子（Macrophage-colony-stimulatingfactor，M-CSF），D、E和F组同时加不同浓度的chODF和M—CSF，G组同时加chODF、M-CSF和鸡护骨素（Chicken osteoprotegerin，chOPG）。通过抗酒石酸盐酸性磷酸酶（Tratrate-resistant acid phosphatase，TRAP）、HE、骨吸收陷窝甲苯胺蓝染色和显微、超微检查，观察和鉴定破骨样细胞（Osteoclastlikecell，OLC）的形成。结果表明，chODF可诱导形成典型的OLC，骨吸收陷窝清晰可见，其陷窝面积大，数量多，且TRAP阳性多核细胞的数目与ODF的浓度呈正相关，多组比较其差异显著（P〈0．05），但若加入chOPG，则阻断了OLC的形成和骨吸收。本试验建立了一种鸡骨髓细胞诱导破骨样细胞方法，既为体外研究鸡破骨细胞的分化发育和功能调节创造了条件，也为进一步研究OPG／ODF／RANK（NF-κB受体，Receptoractivator of NF-κB）在蛋鸡骨质疏松症等骨骼疾病的作用机制提供理论基础。 相似文献
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鸭破骨细胞的培养及鉴定 总被引：1，自引：0，他引：1
健康动物的骨骼处于不断重建的过程,骨重建是旧骨被吸收和新骨形成这一动态平衡过程[1]。20世纪80年代中期,Chambers首先建立了体外破骨细胞的分离培养方法[2],从细胞水平上为骨相关性疾病的研究奠定了基础。已报道的破骨细胞多来源于鼠、兔等实验动物,也有来源于人的组织,但直接从鸭骨髓中分离培养破骨细胞尚未见报道。在规模化养禽生产中,各种因素引起的骨骼疾病造成的经济损失不容忽视[3-4]。因此,建立鸭破骨细胞体外培养体系,将为研究禽类骨营养不良性疾病发生机理提供基础。1材料1.1实验动物7日龄以内番鸭,购自江苏省水禽种质资源基因… 相似文献
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为了探讨肿瘤坏死因子α(TNF-α)/鼠重组白介素-1α(IL-1α)是否能独立于RANKL/RANK/ OPG机制之外直接刺激破骨细胞(OC)的形成和活化.提取4周龄C57雌性小鼠脾细胞,加入鼠巨噬细胞集落刺激因子(M-CSF)TNF-α(±IL-1α)进行体外培养,同时加入骨保护素(OPG)和可溶性NF-κB受体活化子配体(sRANKL)以区别RANKL/RANK/OPG机制.通过OC形态学观察、抗酒石酸酸性磷酸酶(TRAP)染色、骨吸收陷窝方法鉴定OC的形成和活化水平.结果显示:TNF-α(±IL-1α)能诱导睥细胞融合产生TRAP阳性的多核细胞并在象牙片上产生吸收陷窝,加入OPG不能阻断其诱导破骨细胞形成和产生骨吸收陷窝.结果表明:在MCSF存在的情况下,TNF-α以一种独立于RANKL/RANK/OPG之外的机制诱导破骨细胞的形成和活化,而且IL-1α能显著促进TNF-α的诱导作用(P＜0.05). 相似文献
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研究Cdc42在破骨细胞分化成熟过程中的作用及分子机制。采用慢病毒干扰技术构建Cdc42沉默的RAW264.7细胞株,经嘌呤霉素筛选出稳转株。利用荧光定量PCR与Western blot检测其沉默效率。采用TRAP染色观察细胞分化能力。利用激光共聚焦显微镜观察Cdc42沉默经微丝和微管对细胞伪足形成的影响。利用荧光定量PCR与Western blot检测TRAP、PAK4、Cofilin转录和蛋白表达水平。与对照组比较,Cdc42沉默组Cdc42基因的转录极显著下降,蛋白表达水平显著下调,基因沉默效率在50%以上。TRAP阳性破骨细胞数量也急剧减少,细胞呈圆形。丝状伪足和板状伪足减少,TRAP、PAK4、Cofilin转录水平极显著下降,关键蛋白PAK4和Cofilin蛋白表达显著或极显著下调。Cdc42可通过调控细胞骨架蛋白,经丝状伪足和板状伪足,影响破骨细胞的分化。 相似文献
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为获得大量有活力的鸡破骨细胞(Osteoclast,OC),本试验选取18日龄鸡胚,从长骨中提取骨髓细胞,用胰酶消化,40%和70%的percoll梯度离心分离骨髓间质细胞。在此基础上,用60 ng/mL核因子κB受体活化因子配体(RANKL)、50 ng/mL巨噬细胞集落刺激因子(M-CSF)诱导培养。采用抗酒石酸酸性磷酸酶(TRAP)染色、骨吸收陷窝以及标志性蛋白TRAP、基质金属蛋白酶9(MMP-9)、组织蛋白酶K检测鉴定破骨细胞。结果显示,诱导后的TRAP阳性细胞剧增,TRAP、MMP-9和组织蛋白酶K蛋白表达极显著上调,可在牛骨上形成大量的骨吸收陷窝。研究表明,该方法是一种快速、实用、高效的鸡破骨细胞分离培养技术。 相似文献
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OPG/RANKL/RANK系统的研究进展 总被引：5，自引：0，他引：5
OPG/RANKL/RANK系统是近年来骨科研究领域中的重大突破,研究发现许多激素、细胞因子等均通过直接或间接的调节骨保护素(osteoprotegerin,OPG),核因子-κB受体活化因子配体(receptor activator ofNF-κB ligand,RANKL)的表达,调控OPG、RANKL和核因子-κB受体活化因子(recep-tor activator of NF-κB,RANK)之间的比例,从而介导破骨细胞的分化和功能而达到抗骨质疏松或致骨质疏松的作用。现就OPG/RANKL/RANK系统在骨质疏松中的作用做一综述。 相似文献
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对直接从鸭骨髓腔机械分离的成熟破骨细胞（osteoclasts,OC）和由鸭骨髓来源单核细胞融合成的OC样多核巨细胞（multinucleatedgiantcells,MNGCs）进行培养,分别进行抗酒石酸酸性磷酸酶（tartrate-resistantacidphosphatase,TRAP）染色并计数,扫描电镜观察象牙片吸收陷窝,比较了2种方法获得0C的骨吸收功能。结果显示,2种方法均能分离培养出TRAP阳性且具有骨吸收功能的多核OC,但直接分离获得的成熟OC骨吸收功能更强。 相似文献
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采集ICR小鼠骨髓单核细胞在体外培养过程中加入25.0 μg/L M-CSF+50.0 μg/L RANKL诱导形成破骨细胞,同时选择RAW264.7细胞培养过程中添加100.0 μg/L RANKL诱导分化为破骨细胞.通过检测抗酒石酸碱性磷酸酶(Tartrate resistant acid phosphatase,TRAP)染色阳性数及骨吸收陷窝,比较小鼠骨髓单核细胞和RAW264.7细胞株诱导形成的破骨细胞活性.结果显示,小鼠骨髓诱导的细胞TRAP阳性数显著低于RAW264.7细胞株(P＜0.05),而小鼠骨髓诱导的破骨细胞所形成骨吸收陷窝的面积显著大于RAW264.7细胞株(P＜0.05).结果表明,2种方法均可诱导形成具有典型特征的破骨细胞,骨髓单核细胞诱导形成的破骨细胞活性更强,但破骨细胞的数量和纯度明显低于RAW264.7细胞株. 相似文献
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Immunohistochemical Localization of RANK, RANKL and OPG in Healthy and Arthritic Canine Elbow Joints
ANDREA I. SPAHNI Dr. med. vet. PETER SCHAWALDER Prof. Dr. med. vet. BARBARA ROTHEN PD Dr. sc. nat. ETH DIETER D. BOSSHARDT PD Dr. sc. nat. PhD NIKLAUS LANG Prof. Dr. med. dent. MICHAEL H. STOFFEL Prof. Dr. med. vet. 《Veterinary surgery : VS》2009,38(6):780-786
Objective— To determine if the receptor activator of nuclear factor-κB–receptor activator of nuclear factor-κB ligand–osteoprotegerin (RANK–RANKL–OPG) system is active in bone remodeling in dogs and, if so, whether differences in expression of these mediators occur in healthy and arthritic joints.
Study Design— Experimental study.
Sample Population— Fragmented processus coronoidei (n=20) were surgically removed from dogs with elbow arthritis and 5 corresponding healthy samples from dogs euthanatized for reasons other than elbow joint disease.
Methods— Bright-field immunohistochemistry and high-resolution fluorescence microscopy were used to investigate the distribution of RANK, RANKL, and OPG in healthy and arthritic joints.
Results— All 3 molecules were identified by immunostaining of canine bone tissue. In elbow dysplasia, the number of RANK-positive osteoclasts was increased. In their vicinity, cells expressing RANKL, a mediator of osteoclast activation, were abundant whereas the number of osteoblasts having the potential to limit osteoclastogenesis and bone resorption via OPG was few.
Conclusions— The RANK–RANKL–OPG system is active in bone remodeling in dogs. In elbow dysplasia, a surplus of molecules promoting osteoclastogenesis was evident and is indicative of an imbalance between the mediators regulating bone resorption and bone formation. Both OPG and neutralizing antibodies against RANKL have the potential to counterbalance bone resorption.
Clinical Relevance— Therapeutic use of neutralizing antibodies against RANKL to inhibit osteoclast activation warrants further investigation. 相似文献
Study Design— Experimental study.
Sample Population— Fragmented processus coronoidei (n=20) were surgically removed from dogs with elbow arthritis and 5 corresponding healthy samples from dogs euthanatized for reasons other than elbow joint disease.
Methods— Bright-field immunohistochemistry and high-resolution fluorescence microscopy were used to investigate the distribution of RANK, RANKL, and OPG in healthy and arthritic joints.
Results— All 3 molecules were identified by immunostaining of canine bone tissue. In elbow dysplasia, the number of RANK-positive osteoclasts was increased. In their vicinity, cells expressing RANKL, a mediator of osteoclast activation, were abundant whereas the number of osteoblasts having the potential to limit osteoclastogenesis and bone resorption via OPG was few.
Conclusions— The RANK–RANKL–OPG system is active in bone remodeling in dogs. In elbow dysplasia, a surplus of molecules promoting osteoclastogenesis was evident and is indicative of an imbalance between the mediators regulating bone resorption and bone formation. Both OPG and neutralizing antibodies against RANKL have the potential to counterbalance bone resorption.
Clinical Relevance— Therapeutic use of neutralizing antibodies against RANKL to inhibit osteoclast activation warrants further investigation. 相似文献
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Ying-Xiao Fu Jian-Hong Gu Yi-Ran Zhang Xi-Shuai Tong Hong-Yan Zhao Yan Yuan Xue-Zhong Liu Jian-Chun Bian Zong-Ping Liu 《Journal of veterinary science (Suw?n-si, Korea)》2013,14(4):405-412
The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor κB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor κB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions. 相似文献
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de Bruijn ND Kirpensteijn J Neyens IJ Van den Brand JM van den Ingh TS 《Veterinary pathology》2007,44(2):161-169
A retrospective study was performed to characterize 52 new cases of feline epulides between 1995 and 2001, with clinical and pathological results classified according to Head's histopathologic criteria for canine epulides. The incidence of the fibromatous, acanthomatous, ossifying, and giant cell epulis were respectively 57.7% (30/52), 7.7% (4/52), 5.8% (3/52), and 28.8% (15/52). Giant cell epulides presented significant differences in clinical behavior compared with the fibromatous type, including rapid growth (P < .0001), presence of ulcerative changes (P < .01), and rapid recurrence after surgery (P < .01) from which euthanasia was judged necessary in 4 cases. Fifteen giant cell epulides were additionally examined in order to characterize the lesion both histochemically and immunohistochemically and to investigate the origin of the multinucleated giant cells (MGCs). Van Gieson staining showed osteoid and woven bone formation in 11 cases. Both the MGCs and a fraction of the mononuclear cells were positive for vimentin, tartrate-resistant acid phosphatase (TRAP), a commonly accepted marker for osteoclasts, and the polyclonal antibody receptor activator of nuclear factor kappabeta (RANK), a cytokine leading to the differentiation of osteoclast progenitors into mature osteoclasts in presence of its ligand. MGCs were negative for smooth muscle actin, MIB-1, and factor VIII. The giant cell epulis may be a variant of the fibromatous and ossifying epulis in which extensive ulceration and inflammation results in increased osteoclastic activity. The osteoclast-like giant cells are most likely formed from a monocyte/macrophage-like osteoclast precursor that differentiates into osteoclasts under the influence of mononuclear osteoblast-like stromal cells. 相似文献
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Muzylak M Flanagan AM Ingham K Gunn N Price J Horton MA 《Research in veterinary science》2002,73(3):283-290
Musculo-skeletal diseases are a major cause of pain and suffering in cats and several conditions involve increased bone resorption by osteoclasts. However, little is known about the biology of these cells in the cat. In this study we established a method to generate feline osteoclasts from blood mononuclear cells stimulated by macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Cultured osteoclasts are multinucleated, express tartrate resistant acid phosphatase (TRAP), form F-actin rings and resorb bone. They express alpha(v)beta3 vitronectin receptor and osteoclast enzymes, cathepsin K and MMP9; the myeloid antigen, CD18, and the megakaryocyte/platelet integrin, CD41, are absent. This phenotype is typical of osteoclasts from other species. Three resorption inhibitors were examined for activity against feline osteoclasts. Calcitonin, bisphosphonate and RGD integrin inhibitory peptide all reduced bone resorption at doses similar to those efficacious in rabbit or human. We conclude that blood-derived osteoclast cultures are a suitable in vitro system for assessing the ability of drugs to inhibit bone resorption in domestic cats. 相似文献
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Hydroxyapatite, a mineral form of calcium (Ca) and phosphorus (P) that gives bones their rigidity, is the major and essential component of bones and teeth in the human and animal body. A suitable ratio of Ca and P is vital for bone growth. The aim of this study was to explore the effects of dietary calcium to available phosphorus ratios (Ca/AP) on bone metabolism and osteoclast activity of the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa B ligand (RANKL) signalling pathway in piglets. At days 15 and 29, the piglets were assessed for growth performance, blood indicators, cytokines and the OPG/RANK/RANKL signalling pathway. Our results showed that piglets fed a dietary Ca/AP ratio of 2:1 increases growth performance and regulates blood indicators and cytokines (parathyroid hormone (PTH), calcitonin (CT), vitamin D3 (VD3), insulin‐like growth factor‐1 (IGF‐1), transforming growth factor‐β (TGF‐β), interleukin‐1 (IL‐1), interleukin‐6 (IL‐6), carboxyterminal propeptide of type I procollagen (PICP), tartrate‐resistant acid phosphatase (TRACP), alkaline phosphatase (ALP) and osteocalcin (OCN) content). We also demonstrated that this ratio affects hormone secretion and further bone metabolism through the OPG/RANK/RANKL signalling pathway of osteoclasts. These results indicate that a suitable dietary Ca/AP ratio is vital for bone growth and reduce the incidence of bone diseases such as osteoporosis, providing a practical basis for the raising of piglets. 相似文献
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Booij-Vrieling HE de Vries TJ Schoenmaker T Tryfonidou MA Penning LC Hazewinkel HA Everts V 《Research in veterinary science》2012,92(2):311-316
Both vitamin D and inflammatory cytokines can stimulate osteoclast formation and activity. We studied the effect of 1,25-dihydroxycholecalciferol (1,25(OH)(2)D), and interleukin-6 (IL-6), on the formation and activity of feline osteoclasts, using peripheral blood mononuclear cells (PBMCs) from cats with and without tooth resorption (TR(+) and TR(-)) as a source of osteoclast precursors. The formation of osteoclast-like cells (defined as multinucleated, tartrate-resistant acid phosphatase-positive cells) was assessed at 7 and 14 days. In the presence of M-CSF and RANKL, with and without IL-6, more osteoclasts were formed from TR(-) PBMCs than from TR(+) PBMCs on plastic. More osteoclasts were formed from TR(+) PBMCs on bone slices in the presence of M-CSF/RANKL with 1,25(OH)(2)D. This opposite effect may be due to a higher expression of the vitamin D receptor (VDR) in TR(+) osteoclasts and precursors on bone. Formation of resorption pits was analyzed and confirmed with scanning electron microscopy. In conclusion, we propose that TR(+) PBMCs when cultured on bone are sensitive to 1,25(OH)(2)D, whereas the differentiation of TR(-) PMBCs on bone seem more sensitive to IL-6, suggesting that osteoclast precursors from cats with and without tooth resorption respond differently to osteoclast stimulating factors. 相似文献
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Stimulation of osteoclast-like cell formation by Pasteurella multocida toxin from hemopoietic progenitor cells in mouse bone marrow cultures. 
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下载免费PDF全文 The effects of purified Pasteurella multocida toxin (PMT) on osteoclast formation from hemopoietic progenitor cells were studied using an in vitro system. Mononuclear adherent mouse bone marrow cells were cultured for 7 or 14 days in the presence of PMT, or 1,25-dihydroxy-vitamin D3, or both. Mononuclear osteoclast-like cells, which are postmitotic osteoclast precursor cells, were identified as tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells possessing calcitonin receptors. Multinucleated osteoclast-like cells were TRAP-positive multinuclear cells with calcitonin receptors. The results demonstrate that, as does 1,25(OH)2D3, Pasteurella multocida toxin stimulates proliferation of adherent bone marrow mononuclear cells (progenitor cells), and their differentiation into postmitotic mononuclear osteoclast precursor cells. It also causes fusion of the latter into multinuclear osteoclasts; however, the number of osteoclasts obtained with PMT is smaller than with 1,25-dihydroxy-vitamin D3. 相似文献