阿司匹林体外抑制破骨细胞生成的分子机制研究

目的 探讨阿司匹林对破骨细胞生成的影响及其分子机制。方法 体外培养RAW264.7细胞,以100 ng/ml核激活因子κB( NF-κB)受体配体(RANKL)诱导培养,并同时添加不同溶度的阿司匹林(0,0. 25,0. 5,1. 0,1. 5 mmol/L)培养5天。在不同时间点,通过抗酒石酸酸性磷酸酶(TRAP)染色的方法来观察破骨细胞诱导生成能力,用实时荧光PCR方法检测其破骨细胞系标志基因,包括组织蛋白酶K( CTSK)、TRAP、基质金属蛋白酶9( MMP-9)和降钙素受体(CTR) mRNA的表达。裂解不同培养条件的细胞并提取蛋白上样,行western印迹检测NF-κB通道蛋白的表达以及有丝分裂原激活蛋白激酶(MAPKS)通道蛋白的表达,通过免疫荧光的方法分析确定NF-κB的P65的核易位。结果 阿司匹林抑制RANKL诱导的破骨细胞生成,随着阿司匹林浓度增加,破骨细胞形成数量明显减少;其标志性基因TRAP、CTSK、MMP9及CTR的mRNA表达均有所下调;磷酸化的P65、P50、IKB-a、P38以及氨基端激酶(C-JNK)和胞外信号调节蛋白激酶(ERK)蛋白表达均有所减少,而其非磷酸化的蛋 白表达水平无明显变化。阿司匹林同时对NF-κB P65的核易位也表现出抑制效果。结论 在RAW264. 7细胞系中,阿司匹林通过抑制NF-κB系统(P65、P50、IKB-a)和MAPKS系统(P38、C-JNK和ERK)通道的激活来抑制破骨细胞的生成,且在一定范围内和阿司匹林浓度呈正相关。阿司匹林可能具有临床预防及治疗骨质疏松的潜能。     

阿司匹林对大鼠破骨细胞分化成熟和骨吸收活性的影响

目的 研究不同浓度阿司匹林对体外培养大鼠破骨细胞(Osteoclast,OC)分化成熟及骨吸收活性的影响.方法 建立由核激活因子受体配体(receptor activator of NF-κB ligand,RANKL)和巨噬细胞集落刺激因子(Macrophage colony stimulating factor,M-CSF)共同作用的大鼠破骨细胞骨髓诱导体系,将雌激素(10-6 mmol/L)和不同浓度的阿司匹林(0.25 mmol/L、0.5 mmol/L、1.0 mmol/L、1.5 mmol/L)分别作用于破骨细胞.诱导培养后分别对破骨细胞进行抗酒石酸酸性磷酸酶(The tartrate-resistant acid phosphatase,TRAP)染色,观察细胞形态,并计数破骨样细胞数量;将各组破骨细胞接种于骨磨片上,建立破骨细胞-骨磨片活性分析模型,于不同时间点对骨磨片进行光镜和扫描电镜观察,分析计算骨吸收陷窝面积.结果 与正常对照组相比,雌激素组破骨细胞数量和骨吸收陷窝面积低于正常对照组,差异有统计学意义(P＜0.05);且随着阿司匹林浓度的增加,阿司匹林组TRAP阳性多核破骨细胞数量、骨吸收陷窝面积逐渐减少直至消失,差异有统计学意义(P＜0.05).与雌激素组相比,低浓度阿司匹林组(0.25mmol/L)没有明显差异;但中、高浓度阿司匹林实验组(0.5mmol/L、1.0mmol/L、1.5mmol/L)破骨细胞数量和骨吸收陷窝面积减少,差异有统计学意义(P＜0.05).结论 阿司匹林对破骨细胞的分化成熟及骨吸收功能有抑制作用,且呈剂量依赖性,从而具有抗骨质疏松的作用.     

破骨细胞骨吸收机制研究进展

以绝经后骨量迅速下降为主要特征的绝经后骨质疏松症是生殖健康领域中的重要问题。破骨细胞 (osteoclast)作为骨吸收的主要细胞 ,对于骨量的变化具重要作用。因此 ,研究破骨细胞的生物学特性及骨吸收的机理对于预防和治疗骨质疏松症等代谢性骨病具有十分重要的意义。本综述着重论述破骨细胞的生物学特性及骨吸收的分子生物学机制和机理 ,抑制破骨细胞骨吸收的相关因素 ,以期进一步推动破骨细胞骨吸收功能的研究 ,加强骨质疏松等骨代谢疾病的治疗。一、破骨细胞的生物学特性破骨细胞来源于希腊文“Osteon”(骨 )和“klio”(破坏 )两词的组合…     

破骨细胞RANK激活后的信号通路

破骨细胞属血源性单核-巨噬细胞系统,多核为其重要功能形式,它通过分泌酸和蛋白酶溶解骨组织从而启动骨重建、完成骨吸收。RANKL-RANK-OPG(osteoprotegerin)调节轴是其分化和活化的主要调节形式,其中跨膜受体RANK的激活是该调节轴作用的核心。RANK信号传导通路,以及通路中各因子作用的研究不仅对该调节轴作用机制的阐明具有重要意义,更为在分子水平认识和干预骨代谢疾病提供了理论基础。     

人工关节磨损颗粒对破骨细胞RANK信号转导系统的影响

本文简要介绍了破骨细胞RANK信号转导系统的最新研究进展,人工关节磨损颗粒对破骨细胞RANK信号转导系统的影响及由此造成的破骨细胞分化、功能和凋亡等方面的变化。     

槲皮苷抑制RANKL诱导的破骨细胞形成及骨吸收

[目的]探讨槲皮苷对核因子κB受体激动剂配体(receptor activator of nuclear factor kappa B ligand,RANKL)诱导的破骨细胞形成及骨吸收功能的影响。[方法]通过CCK-8法观察不同浓度槲皮苷(0~800μmol/L)干预不同时间(48 h、96 h)对RAW 264.7细胞的生存影响,确定合适的体外用药浓度;利用体外RANKL诱导RAW 264.7细胞形成破骨细胞体系,通过抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色计数评价槲皮苷(200、400μmol/L)对破骨细胞形成和生存的影响;通过骨片吸收实验对骨凹陷和骨吸收面积统计分析评价槲皮苷(200、400μmol/L)3 d内对成熟破骨细胞骨吸收功能的影响;釆用实时定量(Real-Time)PCR技术,检测槲皮苷(200、400μmol/L)对RANKL诱导的破骨细胞特异性基因NFATc1、TRAP和c-fos表达水平的影响。[结果]细胞生存实验发现槲皮苷干预96 h后,槲皮苷(0~800μmol/L)对RAW 264.7细胞4 d内生存未发现显著影响;通过TRAP染色发现200、400μmol/L槲皮苷能显著抑制体外RANKL诱导的破骨细胞形成;通过骨片吸收实验发现200、400μmol/L槲皮苷3d内能显著降低骨吸收面积,提示其抑制成熟破骨细胞骨吸收功能;同时,槲皮苷能呈剂量依赖性抑制RANKL诱导活化T细胞核因子(nuclear factor of activated T cells,NFAT)c1、TRAP和c-fos基因表达。[结论]槲皮苷通过抑制NFATc1,TRAP和c-fos的表达,来抑制体外RANKL诱导的破骨细胞形成和骨吸收功能,是一种潜在治疗骨质疏松药物。     

薯蓣皂苷通过ERα/miR503/RANK信号通路抑制破骨细胞生成

目的 探究薯蓣皂苷对小鼠破骨细胞前体细胞(RAW264.7)破骨分化的抑制作用以及潜在机制。方法 利用核因子κB受体活化因子配体(RANKL)诱导的RAW264.7破骨分化模型,设置模型对照组、雌激素组、薯蓣皂苷低剂量组、薯蓣皂苷高剂量组,通过CCK8法检测不同浓度薯蓣皂苷对细胞的毒性作用,通过TRAP染色进行破骨细胞计数,qPCR检测细胞中ERα/miR503/RANK信号通路及破骨标志性基因TRAP、MMP9、CTSK基因表达水平,Western blot检测细胞中ERα、RANK蛋白表达水平。结果 薯蓣皂苷对RAW264.7细胞无明显毒性作用。与模型对照组比较,高剂量薯蓣皂苷组及雌激素组的TRAP阳性细胞数目下降(P<0.05),薯蓣皂苷及雌激素上调了ERα、miR-503-5p的表达水平,抑制了RANK的表达水平,下调了破骨标志性基因的表达(P<0.05)。结论 薯蓣皂苷可能通过ERα/miR-503/RANK信号通路下调破骨标志性基因,从而抑制RAW264.7细胞破骨分化。     

大鼠后牙牙槽骨吸收模型的建立及破骨细胞的鉴定

目的建立大鼠后牙牙槽骨吸收模型并对破骨细胞(OCs)进行鉴定。方法用3-0丝线结扎大鼠上颌第二磨牙牙颈部并喂以高糖软食,行H-E切片常规组织学观察结扎后牙周组织的变化;应用抗CTR抗体通过免疫组化的方法鉴定OCs。结果大鼠后牙结扎后第3天,结扎部位上皮糜烂,结缔组织见大量炎症细胞浸润,牙槽骨表面出现蚕食状吸收陷窝,第7天以后,结扎部位牙槽骨高度降低。在牙槽骨表面形成的骨吸收陷窝内及附近组织,可见CTR阳性的多核和单核细胞。结论丝线结扎和高糖软食的局部刺激成功地诱导了大鼠牙槽骨破骨细胞性骨吸收。应用抗CTR抗体通过免疫组化的方法鉴定OCs可用于探讨牙槽骨吸收机制的研究。     

巴戟天含药血清对原代破骨细胞RANK和CA II mRNA表达的影响

目的 观察巴戟天含药血清对原代破骨细胞RANK和CA II mRNA表达的影响.方法 取SPF级大鼠48只,随机分成正常组12只和去势组36只,正常组切去卵巢周围部分脂肪,去势组切除卵巢.3个月后测定两组雌激素水平并将去势组随机分为骨质疏松组,骨质疏松+雌激素组,骨质疏松+巴戟天含药血清组.采用机械分离法提取各组的破骨细胞,培养5 d后,TRAP染色及电镜扫描骨片等的方法鉴定破骨细胞.最后用雌激素或巴戟天含药血清干预3 d,以RT-PCR法检测各组RANK和CAIImRNA表达.采用单因素方差分析或多样本均数两两比较进行统计分析.结果 去势组后大鼠雌激素水平低于正常组(P＜0.01).骨质疏松组破骨细胞RANK和CAII表达均高于正常组(P＜0.05,P＜0.05);巴戟天含药血清可降低骨质疏松后大鼠破骨细胞RANK和CA II的表达(P＜0.05,P＜0.05).结论 巴戟天和雌激素均可降低骨质疏松大鼠破骨细胞RANK和CAII的表达,从而达到抑制骨质疏松的作用.     

阿仑膦酸钠对0VX大鼠体外培养的破骨细胞的作用

目的研究第三代双膦酸盐类药物阿仑膦酸钠（alendronate，固邦）对体外培养的破骨细胞的作用。方法建立骨质疏松大鼠模型，于0、2、4、8W进行体外骨髓破骨细胞样细胞（OLC）的培养，并进行阿仑膦酸钠干预，观察OLC数量和形态的变化。结果OVX组大鼠OLC数量高于C组和Sham组；在所有组中，阿仑膦酸钠均能使OLC显著减少（P〈0．01）。结论大鼠去卵巢后OLC形成增加；阿仑膦酸钠能显著抑制OVX大鼠体外培养的OLC的形成。     

The origin of osteoclasts: An immunohistochemical study on macrophages and osteoclasts in embryonic rat bone

Summary The origin of osteoclasts was studied in embryonic rat bone primordia using a set of monoclonal antibodies (ED1, ED2, and ED3) that exclusively recognize monocytes and macrophages. ED1 recognizes monocytes and macrophages. Mononuclear phagocytes which were ED1 positive were found in the perichondrium/periosteum of developing bone. These cells started to infiltrate the primordia when the cartilage became hypertrophic. During bone formation, multinucleated ED1-positive cells with the morphological characteristics of osteoclasts were found in the developing bone marrow cavity and against the bone collar. The present findings support the notion that osteoclasts arise by fusion of mononuclear phagocytes derived from blood monocytes.     

Inhibition of bone resorption by bisphosphonates: Interactions between bisphosphonates,osteoclasts, and bone

Summary Bisphosphonates are nonbiodegradable pyrophosphate analogues that are being used increasingly to inhibit bone resorption in disorders characterized by excessive bone loss. We have previously found that dichloromethylene bisphosphonate (Cl₂MBP) inhibits bone resorption through injury to the cells that resorb Cl₂MBP-contaminated surfaces. 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) is a more potent inhibitor of bone resorptionin vivo, and we have attempted to identify a step in the resorptive pathway that accounts for this increased potency. We found that when osteoclasts, isolated from neonatal rat long bones, were incubated on bone slices in the presence of bisphosphonates, AHPrBP was less, rather than more potent as a resorption-inhibitor than Cl₂MBP. The greater sensitivity of resorption to AHPrBPin vivo could neither be attributed to an effect of AHPrBP on the ability of osteoblastic cells to stimulate resorption in response to calcium-regulating hormonesin vitro nor to an effect on osteoclast generation: osteoclast formation was unaffected by concentrations of AHPrBP 10-fold higher than those of Cl₂MBP which inhibit bone resorption in the bone slice assay. We also found no evidence for impaired osteoclast generationin vivo in AHPrBP-treated rats. These results suggest that the comparisons of potencyin vitro do not include all the factors responsible for determining bisphosphonate potencyin vivo. Because bisphosphonates owe the specificity of their actions to their ability to bind to bone surfaces, we performed experiments using bone slices that had been immersed in bisphosphonates before use. Bone resorption was virtually abolished on bone slices preincubated in 10⁻³ M AHPrBP. Inhibition was associated with degenerative changes in osteoclasts and a more rapid decrease in the number remaining on the bone surface than occurred with Cl₂MBP. The effect was specific for osteoclasts, could be prevented if bone resorption was suppressed by calcitonin, and was not seen in osteoclasts incubated in AHPrBP on plastic coverslips. These observations suggest that AHPrBP inhibits bone resorption through injury to osteoclasts when they solubilize bisphosphonate-contaminated bone. We found that the concentration of AHPrBP used in the preincubation phase could be reduced by an order of magnitude if the volume of the AHPrBP solution was correspondingly increased. This implies that the concentration of bisphosphonate is less relevant to potency comparisons than the density of bisphosphonate on the bone surface. The latter will be strongly influencedin vivo not only by affinity for bone but by the pharmacokinetic and other properties of the compound.     

Hydrochlorothiazide inhibits osteoclastic bone resorption In vitro

Long-term thiazide diuretic use is associated with higher bone mineral density and reduced hip fracture rates, which are attributed to increased serum calcium levels and decreased parathyroid activity that lead to decreased bone resorption. The present study shows that 1–100 M hydro-chlorothiazide (HCTZ) dose dependently inhibits bone resorption by isolated rat osteoclasts in the bone slice assay with an IC₅₀ of 20 M. At these concentrations, HCTZ did not affect osteoclast survival on bone slices and had no effect on the proliferation of UMR-106 rat osteoblasts, indicating that the compound is not cytotoxic. However, such concentrations of HCTZ are unlikely to be achieved in man where therapeutic doses are usually 12.5–100 mg/day. That the in vitro effect of HCTZ on bone resorption may be due to inhibition of osteoclast carbonic anhydrase is discussed.     

Effect of estrogen on MMPs mRNA expression of osteoclasts

目的本实验通过体外分离培养兔破骨细胞，观察不同浓度雌激素对兔破骨细胞基质金属蛋白酶MMPmRNA表达的影响。方法体外分离培养出生24h内的新西兰兔破骨细胞，用含有不同浓度17β-雌二醇（0、10^-5～10^-13mol·L^-1）的M199培养液分别作用于破骨细胞，观察不同浓度雌激素及相同浓度雌激素不同时间对破骨细胞活性的影响，采用半定量逆转录-聚合酶链反应（RT-PCR）方法观察兔破骨细胞MMP-9 mRNA、MMP-8 mRNA表达状况。结果不同浓度17β-雌二醇对破骨细胞的活性有不同程度的抑制，同时对MMP-9 mRNA的表达有明显抑制作用，以10^-5、10^-6、10^-7mol·L^-1（P〈0．05）最为显著。所有破骨细胞均未表达MMP-8mRNA。结论不同浓度的雌激素呈时间和剂量依赖性地抑制破骨细胞的活性，对兔破骨细胞基质金属蛋白酶表达的调控作用随其浓度变化而不同。     

Ibuprofen inhibits localized bone resorption in the middle ear

Summary Localized osteoclastic bone resorption plays a significant role in the pathogenesis of several diseases of the middle ear as well as orthodontic tooth movement and long bone remodeling. The mechanisms of control of localized bone loss and systemic bone resorption may be different but both may be mediated by a final common pathway which includes prostaglandins. Prostaglandins seem to have a predominantly stimulatory effect on bone resorption, although the exact mechanism is poorly understood. Ibuprofen, a nonsteroidal antiinflammatory drug, is known to inhibit the synthesis of prostaglandins. It is likely that ibuprofen, through its inhibition of prostaglandin synthesis, would decrease the localized osteoclastic bone resorption in a previously described animal model system. Mongolian gerbils were divided into three groups: low dose ibuprofen (10 mg/kg per day), high dose ibuprofen (30 mg/kg per day), and a control group. Following surgical implantation of catheters to the right bullae of each gerbil, pressure was applied for 8 days, stimulating osteoclastic bone resorption. After killing the animals and histomorphometric analysis of the bullae from each, comparisons were made between each group using osteoclast surface (percentage of bone area covered by osteoclasts), osteoclast number (number of osteoclasts/mm bone length), and osteoclast profile area (in μm²). Significantly lower osteoclast surface (Oc. S/BS) was found in pressurized bullae from both treatment groups when compared with pressurized bullae from controls (P<0.05) and significantly lower osteoclast number (N.Oc/T.L) in pressurized bullae from both treatment groups when compared with pressurized bullae from controls (P<0.05). These differences were found to be dose-dependent. No significant differences in individual osteoclast profile area were found in either treatment group when compared with controls.     

Promethazine inhibits osteoclastic bone resorption In vitro

Summary Several studies have shown that promethazine can reduce age-related osteopenia in mice. Furthermore, prolonged treatment with promethazine (50 mg/day) increases bone mineral content in the lumbar spine in post-menopausal women with osteopenia. However, the mechanism of action of promethazine has not been elucidated. The present study shows that promethazine HCl (0.01 – 10 M) dose-dependently inhibits bone resorption by isolated rat osteoclasts in the bone slice assay with an IC₅₀ of 1 M. Since these concentrations are likely to be achieved in vivo, it is suggested that the beneficial effect of promethazine on osteopenia is at least partly due to a direct inhibitory effect on osteoclast activity.     

Cytochalasin D reduces osteoclastic bone resorption by inhibiting development of ruffled border-clear zone complex

Summary The osteoclastic cytoskeleton has been demonstrated to be composed of microfilaments. Osteoclastic multinucleated cells were suspended on dentine slices and cultured for 24 hours in the presence or absence of cytochalasin D (CD), a specific and potent inhibitor of actin filament elongation to determine the role of this cytoskeleton. Cultured cells and co-cultured dentine slices were examined ultrastructurally. Unlike those in control cultures without CD, osteoclasts in CD-treated cultures became spherical in shape and lacked microvilli on their basolateral cell surfaces. Most importantly, CD treatment induced a complete disappearance of the ruffled border-clear zone complexes in osteoclasts, which resulted in loss of osteoclast-cytoplasmic polarity. Morphometric analysis of backscattered electron micrographs of co-cultured dentine slices revealed that CD treatment strongly inhibited the formation of resorption lacunae in a dose-dependent manner. These results suggest that the cytoarchitecture, as well as the bone-resorbing function, of the osteoclast is highly regulated by the F-actin-containing microfilamentous cytoskeleton in the ruffled border-clear zone complex.     

Calcitonin gene-related peptide inhibits osteoclastic bone resorption: A comparative study

Summary Besides the calcitonin (CT) precursor, the calcitonin gene also encodes another peptide—calcitonin gene-related peptide (CGRP). We have previously reported that CGRP lowers plasma calcium in the rat. In the present study we have evaluated the effect of CGRP on resorption of bone by isolated rat osteoclasts and have compared these effects to those produced by calcitonins from three species (salmon, pig, and human calcitonins). There was a significant inhibition of bone resorption with rat calcitonin gene-related peptide (rCGRP) at a 1000-fold higher dose than that used for human CT. This effect well explains the CT-like effect of CGRP seen in thein vivo rat CT bioassay. Our results suggest that though CGRP may not be involved in the hormonal control of plasma calcium, the peptide may be an important local regulator of bone cell function.