近β钛合金对人成骨细胞骨保护素/核因子-κB受体活化因子配体mRNA表达影响的研究

[目的]探讨近B钛合金Ti-5Zr-3Sn-5Mo-15Nb(TLM)对人成骨样MG63细胞骨保护素(osteoprotegerin,OPG)及核因子-κB受体活化因子配体(receptor activator of nuclear factor kappa B ligand,RANKL)mRNA表达水平的影响.[方法]将人成骨样MG63细胞分别接种于纯钛钛片、TLM及微弧氧化处理的TLM表面,采用荧光实时定量PCR法检测OPG/RANKL mRNA表达水平.[结果]TLM组OPG mRNA表达水平略高于纯钛钛片组,但没有统计学意义(P>0.05),而微弧氧化处理组OPG mRNA水平明显升高,与纯钛钛片组及TLM组相比有显著性差异(P<0.05),三组之间的RANKL mRNA水平没有明显差异(P>0.05).[结论]经过微弧氧化处理的TLM可以上调成骨细胞OPG mRNA水平,从而影响OPG/RANKL mRNA的比值,调节成骨细胞与破骨细胞之间的平衡,进一步促进骨质重建.     

核因子κB受体活化因子对骨质吸收的影响

核因子κB受体活化因子(RANK)的研究多集中在它所参与组成的信号传导通路及其调节破骨细胞增殖分化作用的机制方面。RANK编码基因在转录、翻译最终形成有活性的RANK过程中受到多种因素的调节,其中任何一个环节的异常都会给破骨细胞的增殖分化带来影响,并最终导致出现以骨质吸收异常为特征的疾病。RANK与许多骨质破坏性疾病存在密切关系,近年在治疗这些疾病方面围绕RANK的研究逐步展开,并在体外试验中取得了很大进展,为进一步临床试验奠定基础。该文就近年RANK研究成果作一综述,并探讨控制异常骨质吸收的新途径。     

模拟微重力条件下重组骨保护素融合蛋白(rhOPG-HSA)对破骨前体细胞Raw264.7抑制效应的研究

目的探讨在模拟微重力(Simulated microgravity,SMG)条件下重组骨保护素融合蛋白(rhOPG-HSA)对破骨前体细胞Raw264.7的抑制效应。方法在SMG条件下,分别培养破骨前体细胞Raw264.7与成骨细胞MC3T3-E1,利用ELISA法检测MC3T3-E1细胞培养液中骨保护素活性,用巨噬细胞集落刺激因子(M-CSF)以及可溶性破骨细胞分化因子(sRANKL)诱导Raw264.7分化为破骨细胞,通过抗酒石酸酸性磷酸酶(TRAP)染色法鉴定rhOPG-HSA抑制破骨细胞能力,利用半定量RT-PCR测定破骨细胞中TRAP的表达。结果 SMG条件下,骨保护素活性在72 h后显著降低(P<0.01);Raw264.7细胞经M-CSF及sRANKL诱导3、4、5 d后,与阴性对照组相比,TRAP阳性染色的破骨细胞用rhOPG-HSA处理后明显减少(P<0.05),RT-PCR测定结果表明破骨细胞TRAP的表达降低(P<0.05)。结论 SMG条件下,rhOPG-HSA能够抑制破骨前体细胞Raw264.7的分化。     

核因子KB受体活化因子对骨质吸收的影响

核因子kB受体活化因子（RANK）的研究多集中在它所参与组成的信号传导通路及其调节破骨细胞增殖分化作用的机制方面。RANK编码基因在转录、翻译最终形成有活性的RANK过程中受到多种因素的调节，其中任何一个环节的异常都会给破骨细胞的增殖分化带来影响，并最终导致出现以骨质吸收异常为特征的疾病。RANK与许多骨质破坏性疾病存在密切关系，近年在治疗这些疾病方面围绕RANK的研究逐步展开，并在体外试验中取得了很大进展，为进一步临床试验奠定基础。该文就近年RANK研究成果作一综述，并探讨控制异常骨质吸收的新途径。     

慢性乙型肝炎患者血清骨保护素和核因子κB受体活化因子配体变化的研究

目的观察慢性乙型肝炎患者血清骨保护素(OPG)和核因子κB受体活化因子配体(RANKL)水平的变化,探讨慢性肝病致骨质疏松的发病机制。方法随机选取300例慢性乙型肝炎患者作为试验组,其中95例不伴有肝硬化,205例伴肝硬化,根据Child-Pugh分级:A级69例,B级62例,C级74例,选取年龄、性别、身高、体重相匹配的100例健康志愿者作为对照组。血清OPG、RANKL应用ELISA方法检测,应用跟骨超声骨密度测定仪测定跟骨硬度指数(SI),对相关数据进行相应的统计学分析。结果各组患者OPG水平差异均具有统计学意义,对照组、不伴肝硬化组、肝硬化A组、肝硬化B组和肝硬化C组患者的血清OPG水平逐渐降低,RANKL值则逐渐升高(P<0.05)。对照组、不伴肝硬化组、肝硬化A组、肝硬化B组和肝硬化C组患者血清OPG/RANKL比值逐渐降低,对照组OPG/RANKL值较其余4组均显著升高(P<0.05)。慢性乙型肝炎组患者的跟骨SI与对照组比较,差异具有统计学意义(P<0.05)。肝硬化A组、B组、C组患者SI值显著低于对照组SI(P<0.05)。结论 OPG、RANKL和OPG/RANKL系统可能参与慢性肝病相关性骨质疏松症的发病过程,慢性乙型肝炎患者可引起OPG、RANKL以及OPG/RANK的变化,上调破骨细胞,使得骨吸收大于骨形成,从而引发骨质疏松。     

核因子κB受体活化因子及其配体在5/6肾切除大鼠肾脏中表达及缬沙坦对其影响

目的 探讨核因子κB受体活化因子(RANK)及其配体(RANKL)在5/6肾切除大鼠(STNx)残肾中表达的意义,以及血管紧张素受体拮抗剂(ARB)缬沙坦对其表达的影响。方法 制备5/6肾切除的SD大鼠动物模型, 分为未给药组、治疗组及假手术组。治疗组在5/6肾切除后即给予缬沙坦治疗,大鼠分别于第4、8、12 周杀检。第12 周时用ELISA方法检测大鼠血清中骨保护蛋白(OPG)及RANKL水平;显微镜观察肾脏病理变化;免疫组化方法及Western印迹方法分析RANK/RANKL/OPG蛋白表达;RT-PCR检测RANK/RANKL/OPG的mRNA表达。结果 (1)第12周时假手术组尿蛋白量为(6.1±0.6) mg/24 h;未给药组为(19.0±1.5) mg/24 h;治疗组为(13.4±1.2) mg/24 h,3 组间的差异有统计学意义(P均 < 0.01)。(2)第12周时未给药组及治疗组肾脏均有明显硬化,但治疗组硬化程度较未给药组轻。(3)未给药组RANK及RANKL蛋白及mRNA的表达均高于治疗组及假手术组,3 组间的差异有统计学意义(P均 < 0.01)。OPG蛋白及mRNA的表达在3 组间无显著性差异。(4)血清中OPG及RANKL含量在3 组间的差异亦无统计学意义。结论 在5/6肾切除大鼠残肾中表达上调的RANK和RANKL可能参与了肾小球硬化的发生发展。抑制RANK和RANKL的表达可能是缬沙坦延缓肾小球硬化的原因之一。     

青少年特发性脊柱侧凸患者成骨细胞中核因子κB受体活化子配体和骨保护蛋白的表达

目的:从成骨细胞(osteoblast,OB)水平探讨核因子KB受体活化子配体(receptor activator of NFκBligand,RANKL)、骨保护蛋白(osteopmtegerin,OPG)与青少年特发性脊柱侧凸(adolescent idiopathic scoliosis,AIS)患者骨量降低的关系.方法:AIS患者(AIS组)20例,男5例,女15例,年龄11～19岁,平均14.75岁,Cobb角40°～96°,平均59.65°;同年龄非脊柱畸形患者(对照组)8例,男6例,女2例,年龄10～19岁,平均15.25岁.两组均采用双能X线吸收测量仪(dual-energy X-ray absorptiometry,DEXA)测量骨密度(bone mineral density,BMD),测量部位包括非优势侧股骨近端及腰椎.所有受试者术中取适量髂前上嵴的松质骨,运用植块法培养成骨细胞.培养过程中观察细胞形态学变化,并用碱性磷酸酶染色法、钙结节染色法和RT-PCR检测骨钙素表达,并进行表型鉴定.取P2代细胞应用RT-PCR和Western blotting检测RANKL和OPG的mRNA及蛋白表达情况.结果:植块法培养可以获得较多的原代细胞.碱性磷酸酶染色、钙结节染色及RT-PCR检测骨钙素表达证实所培养的细胞表现成骨细胞特性.AIS组腰椎(L2～L4)及股骨近端的BMD值明显低于对照组;RANKL的核酸及蛋白水平的表达量均较对照组高(P<0.01):OPG的核酸及蛋白水平的表达量与对照组比较无统计学差异(P>0.05);RANKL/OPG比值明显高于对照组(P<0.01).结论:成骨细胞中RANKL及OPG在核酸及蛋白水平的表达异常可能与AIS患者骨量降低的分子机制相关.     

失神经支配在大鼠骨折愈合过程中降钙素基因相关肽对骨保护素/破骨细胞分化因子影响的实验研究

目的 通过建立选择性切断大鼠感觉/运动神经联合胫骨骨折的动物模型,研究感觉/运动神经损伤后对骨折愈合的影响,并检测骨折愈合过程中降钙素基因相关肽(calcitonin gene related peptide,CGRP)对骨保护素(osteoprotegerin,OPG)/破骨细胞分化因子(receptor activator nuclear factor kappa B ligand,RANKL)体系的影响,初步探讨周围神经调节骨折愈合的机制.方法 取60只Wistar大鼠随机分为3组:前根(运动神经)切断+胫骨骨折组(anterior rhizotomy group,ART组);后根(感觉神经)切断+胫骨骨折组(posterior rhizotomy group,PRT组);单纯胫骨骨折组(sham operated group,SO组).分别于骨折术后7、14、21、28d处死大鼠,在骨折处上、下5mm部位取骨痂标本.免疫组织化学检测CGRP、OPG、RANKL的表达;采用软件Image-proplus 6.0进行图片分析;采用SPSS16.0进行统计学分析.结果 CGRP在SO组各时间点均呈强阳性表达,术后14 d和21 d,SO组、ART组和PRT组比较差异均有统计学意义(P＜0.05).OPG在SO组术后7d呈强阳性表达,以后逐渐下降但保持较高水平;术后14 d,PRT组和SO组、ART组比较差异有统计学意义(P＜0.05);术后21 d,SO组和PRT组、ART组比较差异有统计学意义(P＜0.05).RANKL在SO组21 d为表达高峰,以后逐渐下降;术后14 d,PRT组和ART组、SO组比较差异有统计学意义(P＜0.05);术后21 d,SO组和ART组、PRT组比较差异有统计学意义(P＜0.05).结论 在骨折愈合过程中,感觉神经纤维对骨折愈合的影响较运动神经纤维显著.CGRP能够调节OPG/RANKL的表达量的比值,从而影响骨折的愈合过程.失神经支配(尤其是感觉神经)导致CGRP对OPG/RANKL表达的调节作用降低,这不利于骨折的愈合.完整的神经支配是正常骨折愈合的必要条件之一.     

骨保护蛋白及配体与破骨细胞

破骨细胞对骨量的维持起重要作用,它的发育、成熟信号是由成骨细胞传递的。当受到骨吸收因子作用时,成骨细胞表达骨保护蛋白配体分子,与破骨前体细胞膜上的核因子κB受体激活子结合,使之分化、成熟为破骨细胞。而成骨细胞旁分泌的骨保护蛋白分子,则作为伪受体与核因子κB受体激活子竞争结合骨保护蛋白配体,从而抑制破骨细胞的生成。骨保护蛋白配体-核因子κB受体激活子-骨保护蛋白组成了破骨细胞分化的信号传导通路,对它的认识有助于临床治疗代谢性骨病。     

钛合金颗粒对小鼠成骨细胞增殖及核因子-κB受体活化因子配体的影响

目的 观察钛合金颗粒对体外培养小鼠成骨细胞增殖以及核因子(NF) -κB受体活化因子配体(RANKL)的影响,探讨假体周围骨溶解机制.方法 体外培养小鼠成骨细胞,随机分为空白对照组和钛合金颗粒处理A(0.01％)、B(0.10％)、C(1.00％)组.采用细胞计数试剂盒(CCK)-8法检测培养24、48、96、168 h成骨细胞增殖活性.分别采用逆转录-聚合酶链反应(RT-PCR)、酶联免疫吸附试验(ELISA)检测培养24、48 h后成骨细胞的RANKL mRNA表达及培养上清液的RANKL含量.结果 培养48 h,A组、B组与对照组比较细胞增殖活性差异无统计学意义(P＞0.05),C组与其他各组比较细胞增殖活性明显降低(P＜0.01).培养168 h,B组细胞增殖活性下降,和对照组比较差异有统计学意义(P＜0.01).培养24、48 h,和对照组比较,各实验组RANKL mRNA表达及分泌量均明显升高(P＜0.01),各实验组之间差异有统计学意义(P＜0.01).结论 钛合金颗粒可降低成骨细胞增殖活性,促进成骨细胞RANKL表达与分泌,是假体周围骨溶解机制之一.     

Suramin interacts with RANK and inhibits RANKL-induced osteoclast differentiation

Suramin is a naphthalene trisulfonic acid derivative that inhibits osteoclast differentiation and bone resorption in vitro and in vivo; however, the mechanisms underlying this activity have not been studied. Receptor activator of NF-kB (RANK) ligand (RANKL) is a key regulator of osteoclast differentiation and function and this study evaluated the ability of suramin, which has been shown to disrupt protein-protein interactions, to interfere with RANKL functional activity and binding to RANK. Suramin inhibited osteoclastic bone resorption in a calvarial model and inhibited osteoclast differentiation in RANKL-stimulated murine spleen cells and RAW264.7 cells. RANKL-induced second messenger signaling (AKT and p38 MAP Kinase phosphorylation) was completely blocked by 100 microM suramin. The ability of RANKL to bind to recombinant human RANK-Fc (rhRANK-Fc) was reduced 50% by suramin in an in vitro binding assay. Surface plasmon resonance technology and nuclear magnetic resonance (NMR) were used to evaluate the ability of suramin to bind to rhRANK-Fc. Suramin was found to selectively interact with immobilized rhRANK-Fc chimera in a concentration-dependent manner by Biacore 3000 analysis. Similar results were obtained using saturation transfer difference NMR spectroscopy to demonstrate that suramin binds to rhRANK-Fc, but not IgG1Fc or sRANKL. In summary, these findings demonstrate that suramin inhibits sRANKL-induced osteoclast differentiation and suggest that these effects are mediated by suramin binding to RANK and blocking the ability of sRANKL to induce second messenger signaling.     

骨保护素体外抑制小鼠单核巨噬细胞 成熟分化的实验研究

目的研究骨保护素（Osteoprotegerin, 0PG)抑制核因子NF-KB受体活化因子配体（Receptor activator of nuclear kappa B ligand,RANKL)诱导小鼠单核细胞RAW264. 7成熟分化而导致的溶骨效 应。方法50 ng/mL RANKL诱导RAW264. 7细胞1 d后,加人100 ng/mL 0PG(实验组,即0PG + RANKL组）或不加人0PG(对照组,即RANKL组）分别培养7 d和9 d,经细胞形态学观察其变化,抗 酒石酸酸性碟酸酶（Tartrate resistant acid phosphatase, TRAP)染色法观察TRAP阳性多核细胞,扫描 电镜下观察在骨片上的破骨细胞所致的骨吸收陷窝形成情况。结果对照组培养7 d时,在倒置相 差显微镜、透射电镜、光镜下可见细胞形状为椭圆形或不规则形,胞体明显较KAW264.7细胞增大, 胞核多为6 ~ 10个,扫描电镜下还可见大量伪足形成,而实验组培养7 d后,细胞形状多为圆形,且扫 描电镜下未见明显伪足形成;对照组9 d时可见大量TRAP染色阳性的多核巨细胞（含3个或3个以 上的细胞核）,而实验组中TRAP染色阳性的多核破骨细胞偶见多核巨细胞,培养9 d时很难找到多 核巨细胞;仅用RANKL诱导RAW264.7细胞分化7 d时,对照组中破骨细胞表面可见大量伪足伸出, 并形成明显的骨吸收陷窝,实验组中破骨细胞见少许伪足突出,不能看到明显的骨陷窝形成。结论 单用50 ng/mL RANKL体外连续诱导RAWM4.7细胞7 d时,可以促进成熟的破骨细胞显著分化。 100 ng/mL 0PG培养9 d能有效地抑制破骨细胞的分化,减少破骨细胞的骨吸收效应。     

RANK信号调控破骨细胞分化与成熟的研究进展

破骨细胞来源于微环境造血前体细胞,它的生存、增殖、分化和激活需要巨噬细胞集落刺激因子(M-CSF)和核因子κB受体活化因子配体(RANKL)参与。RANKL与相应的RANK受体结合,从而刺激破骨前体细胞分化成为破骨细胞。这一过程由不同的调节蛋白和激酶来调控,并且依赖于RANKL-RANK信号。本文中,笔者总结了目前已知的在破骨细胞发生过程中调节RANK信号的机制。在早期阶段,RANK信号的调节通过募集调节蛋白如肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6, TRAF6),引起丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)以及转录因子核因子κB(nuclear factor-κB, NF-κB)和激活蛋白-1(activator protein-1, AP-1)的活化。活化的NF-κB进一步激活调节破骨细胞生成的重要因子-T细胞核因子1(nuclear factor of activated T-cells cytoplasmic 1, NFATc1)。在信号传递的中间阶段,共刺激信号通过激活磷脂酶Cγ2(phospholipase Cγ2, PLCγ2)连同c-Fos/AP-1引起钙离子(Ca~(2+))振荡,同时Ca~(2+)信号促进NFATc1的产生。在破骨细胞生成的晚期阶段,NFATc1入核诱导大量的破骨细胞特异性靶基因的表达,从而使细胞融合并发挥其功能。     

Effect of osteoprotegerin in combination with interleukin-6 on inhibition of osteoclast differentiation

Objective： To observe the effect of recombinant interleukin-6 （IL-6） and osteoprotegerin （OPG） on inhibiting bone absorption induced by receptor activa- tor for nuclear factor- rB ligand （RANKL） in murine osteo- clast precursor cells （OCPs） model. Methods： RAW 264.7 cells were solely treated with 50 ng/ml RANKL for 1 day, and then they were divided into three groups： RANKL （control group）, RANKL＋IL-6 （IL-6 group） and RANKL＋IL-6＋OPG （combination group）. These cells were harvested and investigated by means of HE stain- ing under light microscope after consecutive 9 days. Furthermore, staining tartrate-resistant acid phosphatase （TRAP）-positive multinucleated cells were detected by in- verted phase contrast microscope. The absorption pits of bone slices were observed under scanning electron microscope. Results： The number of mature osteoclast cells in control group was more than that in IL-6 alone or IL-6 com- bined with OPG group （P〈0.05）. Interestingly, this experi- ment has also demonstrated that there was a large number of TRAP-positive multinucleated osteoclasts （more than 3 nuclei） and several bone absorption formation in the con- trol group, whereas the outcome was completely different in both IL-6 group and IL-6＋OPG group （P〈0.05）. Conclusion： IL-6 can suppress the differentiation of mature osteoclasts as directly adding it into the RAW 264.7 cells induced by 50 ng/ml RANKL, and further the effect of osteolysis is remarkably reduced. When treatment with IL- 6 combined with OPG, a more effective strategy for the treat- ment of osteoporosis is reached.     

CCN2 enhances RANKL-induced osteoclast differentiation via direct binding to RANK and OPG

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) is a multi-potent factor for mesenchymal cells such as chondrocytes, osteoblasts, osteoclasts, and endothelial cells. CCN2 is also known as a modulator of other cytokines and receptors via direct molecular interactions with them. We screened additional factors binding to CCN2 and found receptor activator of NF-kappa B (RANK) as one of them. RANK is also known as TNF-related activation-induced cytokine (TRANCE) receptor, and its signaling plays a critical role in osteoclastogenesis. Notable affinity between CCN2 and RANK was confirmed by using surface plasmon resonance (SPR) analysis. In fact, CCN2 enhanced the RANK-mediated signaling, such as occurs in NF-kappa B, p38 and JNK pathways, in pre-osteoclastic RAW264.7 cells; whereas CCN2 had no influence on RANK–RANK ligand (RANKL) binding. Moreover, CCN2 also significantly bound to osteoprotegerin (OPG), which is a decoy receptor of RANKL. Of note, OPG markedly inhibited the binding between CCN2 and RANK; and CCN2 canceled the inhibitory effect of OPG on osteoclast differentiation. These findings suggest CCN2 as a candidate of the fourth factor in the RANK/RANKL/OPG system for osteoclastogenesis, which regulates OPG and RANK via direct interaction.     

RANK/RANKL在假体松动中的分子机制研究与探讨

人工关节置换是20世纪骨科领域具有革命性的飞跃之一.理想的人工关节置换术后,能够达到解除患者关节疼痛、重建活动功能的根本目的 ,然而,我们却面临了一个严峻的问题,就是术后晚期假体的松动.据相关资料统计,在假体置换的15～20年后,大约有10%～15%的人工关节将发生假体松动继而失效.假体长期磨损产生的颗粒诱发假体周围的破骨细胞大量生成,从而导致骨的溶解和骨质疏松,是人工关节晚期松动主要的原因之一.本综述重点分析RANK/RANKL信号途径中酶的激活对破骨细胞分化和激活重要性.     

Pulsed electromagnetic fields stimulation affects osteoclast formation by modulation of osteoprotegerin, RANK ligand and macrophage colony-stimulating factor

Electromagnetic stimulation has been documented to treat recalcitrant problems of musculoskeletal system. Yet, the underlying mechanisms are not completely understood. In this study, we investigated effect of pulsed electromagnetic fields (PEMF) with parameters modified from clinical bone growth stimulator on osteoclast formation, bone resorption, and cytokines associated with osteoclastogenesis. Marrow cells were harvested from both femora and tibiae of 6 week-old mice and cultured in 8-well chamber slides or 16-well calcium phosphate apatite-coated multitest slides. After 1-day incubation, marrow cells were exposed to PEMF at different electric field intensities for 2h/day and continued for 9 days. Osteoprotegerin (OPG), receptor activator of NFkappaB-ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) concentrations of each group were determined after PEMF stimulation. Osteoclast identity was confirmed by both tartrate resistant acid phosphatase (TRAP) stain and bone resorption assay. A statistically significant increase and decrease of osteoclastogenesis and bone resorption areas were found when exposed to PEMF with different intensities. Besides, consistent correlations among OPG, RANKL, M-CSF, osteoclast numbers, and bone resorption after exposure to different intensities of PEMF were observed. These data demonstrated that PEMF with different intensities could regulate osteoclastogenesis, bone resorption, OPG, RANKL, and M-CSF concentrations in marrow culture system.     

Denosumab与破骨细胞RANKL/RANK通路

Denosumab(狄诺塞麦)是一种人工合成、完全人源化、可与RNAKL结合的单克隆抗体(IgG2抗体),对人源RANKL具有很高的亲和力和特异性~([1]),具有较好的骨吸收抑制作用,是以破骨细胞RANKL/RANK信号调控通路为靶点的骨质疏松靶向治疗药物,并可降低恶性肿瘤骨转移患者骨骼相关事件(SRE)的发生、延缓骨痛的进展。本文综述了狄诺塞麦抑制骨吸收的生理作用机制,狄诺塞麦治疗骨质疏松和恶性肿瘤骨转移的研究进展,为狄诺塞麦在临床的应用提供更好的循证医学证据。     

重组人骨保护素对体外培养兔破骨细胞的影响

目的观察重组人骨保护素(recombinanthumanosteoprotegerin,rhOPG)对体外培养兔破骨细胞(osteoclast,OC)生存和功能的影响。方法将rhOPG用于干预从新生兔分离出的OC,于干预后1、3、7d取细胞玻片、皮质骨片进行HE、Giemsa、甲苯胺蓝染色,并观察抗酒石酸酸性磷酸酶(TRAP)染色阳性细胞和骨片吸收陷窝,扫描电镜进一步观察吸收陷窝形态。结果分离培养的兔OC多核形态明显;rhOPG对TRAP阳性OC生存的影响,1、3d结果差别不明显,7d的结果差异有显著性(P<0.05);rhOPG能够明显地抑制OC在骨片上形成吸收陷窝,三个时点计数结果差异均有显著性(P<0.01)。结论rhOPG可明显地抑制体外培养兔OC的骨吸收功能。     

RANK Expression as a cell surface marker of human osteoclast precursors in peripheral blood, bone marrow, and giant cell tumors of bone.

RANK expression in vivo on hematopoietic subsets including pre-osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14(+)RANK(high) cells constitute a circulating pre-osteoclast pool. INTRODUCTION: The expression of RANK by subsets of hematopoietic cells has not been adequately studied in humans. While attributed to the monocytoid lineage, the phenotype of the pre-osteoclast (pre-OC) with respect to RANK expression in vivo remains unclear. We tested monoclonal antibodies (MAbs) raised against the extracellular domain of recombinant human RANK for reactivity with normal peripheral blood (PB) and bone marrow (BM) mononuclear cells (PBMNCs and BMMNCs, respectively). We also tested reactivity with giant cell tumor cells (GCT), a confirmed source of pre-OC and mature OCs. MATERIALS AND METHODS: Human PBMNCs, BMMNCs, and GCT cells were analyzed for reactivity with anti-RANK MAbs by flow cytometry in combination with hematopoietic lineage restricted markers. GCTs were also analyzed by immunofluorescence. CD14+ monocytoid cells were sorted by fluorescence-activated cell sorting (FACS) based on their relative RANK expression and cultured under OC-forming conditions. RESULTS: RANK+ cells were detected similarly by three independent anti-RANK MAbs. One MAb (80736) immunoprecipitated RANK-RANKL complexes from surface-biotinylated GCT lysates. Using dual-color flow cytometry, RANK was detected on CD14+ (monocytoid), CD19+ (B-lymphoid), CD56+ (NK cell), and glycophorin A+ erythroid progenitors. Minor populations of both CD3+ T lymphocytes and BM CD34+ hematopoietic progenitors also expressed cell surface RANK. In GCTs, RANK expression was identified on mononuclear CD45(+)CD14(+)alphaVbeta3(+)c-Fms+ cells, likely to be committed pre-OC, and on multinucleated CD45(+)alphaVbeta3(+)TRACP(+) OCs. Importantly, sorted CD14(+)RANK(high) PBMNCs treated with recombinant RANKL and macrophage-colony stimulating factor (M-CSF) gave rise to approximately twice the number of osteoclasts than RANK(mid) or RANK(low) cells. CONCLUSIONS: These results suggest that committed monocytoid RANK+ pre-OCs are represented in the marrow and circulate in the periphery, forming a pool of cells capable of responding rapidly to RANKL. The ability to reliably detect committed pre-OC in peripheral blood could have important clinical applications in the management of diseases characterized by abnormal osteoclastic activity.