从人骨巨细胞瘤组织中纯化破骨细胞的简单方法

利用破骨细胞贴壁快以及耐胰蛋白酶地特性,采用0.25%胰蛋白酶和0.2%I型胶原酶来分离纯化骨巨细胞中的破骨细胞,即得到破骨细胞,并进行细胞学和分子生物学鉴定,包括抗酒石酸酸性磷酸酶染色和HE染色,并采用RT-PCR反应方法检测降钙素受体、组织蛋白酶K和破骨细胞分化因子受体的表达。该方法所得细胞纯度可达79.7%,具有破骨细胞表型特征。可用于生化和分子生物学研究,是进行骨细胞学研究的破骨细胞来源。     

绝经后骨质疏松症TNF-α通过激活NF-κB促进RANKL诱导的破骨细胞形成

本研究检测了绝经后骨质疏松症妇女的肿瘤坏死因子-α(TNF-α)和雌激素水平,并探讨了TNF-α对破骨前体细胞RAW264.7中破骨细胞标志物核因子κB受体激活因子(nuclear factor kappa-B, RANK)、组织蛋白酶K (Cathepsin K, CTSK)和凝血酶受体激活肽(thrombin receptor activating peptide, TRAP)以及核因子-κB (NF-κB)亚基(p65)和NF-κB抑制蛋白(IκBα)的影响。研究结果表明,绝经后骨质疏松症患者的TNF-α水平显著升高,而雌二醇水平显著降低。核因子κB受体激活因子配体(receptor activator for NF-κBligand, RANKL)处理1周后,破骨前体细胞RAW264.7中破骨细胞标志物RANK、CTSK和TRAP的mRNA和蛋白高度表达。与RANKL对照组相比,TNF-α处理可上调RANK、CTSK和TRAP m RNA的表达。但是,仅TNF-α不能诱导培养的RAW264.7细胞分化为破骨细胞成。TNF-α以剂量依赖性方式诱导NF-κB亚基p65和IκBα磷酸化,而NF-κB抑制剂处理则有效降低了RANK和TRAP的表达。本研究结论表明,绝经后骨质疏松症中TNF-α通过激活NF-κB来促进RANKL诱导的破骨细胞形成。     

破骨细胞形成抑制因子研究进展

破骨细胞形成抑制因子（OPG/OCIF）是最近发现的一种参与调节骨密度的糖蛋白,是一个肿瘤坏死因子(TNF)受体超家族的新成员,其氨基酸序列中具有TNF受体结构类似区.成熟的OPG/OCIF具有7个结构域,可分为三个功能区,即：TNF受体结构区、致死结构区和肝素结合区.OPG/OCIF基因定位在8q23～24上,由5个外显子和4个内含子组成,其表达受到与骨的形成和破坏有关因子的调控：如TGF-β1、1,25(OH)₂VD₃、TNF-α等等.OPG/OCIF抑制骨的破坏和吸收机制主要是抑制破骨细胞的存活,引起破骨细胞凋亡和抑制破骨细胞形成.     

RANKL在成釉细胞瘤骨吸收机制中的作用

目的:检测RANKL在成釉细胞瘤(ameloblastoma,AM)组织中的表达情况及探讨RANKL在AM骨吸收机制中的作用.方法:通过免疫组化方法检测RANKL在AM组织中的表达情况;通过建立AM细胞/新生大鼠骨细胞共培养体系,观察AM细胞诱导破骨细胞形成的活性,再以OPG(RANKL的抑制剂)进行干预,观察OPG对AM细胞诱导破骨细胞形成活性的影响.结果:RANKL在AM组织中有恒定的表达;AM细胞能够诱导新生大鼠骨细胞分化为成熟的破骨细胞,但此活性可被OPG明显抑制.结论:AM细胞诱导破骨细胞形成可能是AM骨吸收过程中局部破骨细胞形成的重要来源和机制,而RANKL在此过程中发挥重要作用.     

金雀异黄素抑制IL-1α刺激破骨样细胞的组织蛋白酶K表达

从人骨巨细胞瘤组织中纯化破骨样细胞,用不同浓度的金雀异黄素温育48h,观察IL-1α刺激后1h后组织蛋白酶K表达。结果发现:与阴性对照组相比,IL-1α明显刺激破骨样细胞表达组织蛋白酶K(P<0.01);而金雀异黄素抑制IL-1α刺激后组织蛋白酶K转录及表达,且呈剂量依赖关系(P<0.01);加用雌激素受体拮抗剂ICI182.780后,金雀异黄素作用被部分拮抗。金雀异黄素通过雌激素受体部分抑制IL-1α刺激破骨样细胞的组织蛋白酶K表达。     

类风湿关节炎破骨细胞的活化机制及其预测指标

类风湿关节炎(rheumatoid arthritis, RA)是一种系统性、慢性、炎性的自身免疫性疾病。骨破坏在RA的发生和发展中占有重要地位,是RA致残致畸的主要原因。破骨细胞(osteoclast, OC)的异常增生与活化对RA骨侵蚀的发病进展具有重要作用。近年来,RA患者骨破坏的研究逐渐增多。本文结合国内外研究对核因子-κB受体活化因子配体(receptor activation for nuclear factor-κB ligand, RANKL)/核激活因子受体(receptor activator for nuclear factor-κB, RANK)/骨保护素(orthopantomography, OPG)信号通路、Wnt信号通路、抗瓜氨酸蛋白抗体(anti-citrulline protein antibody, ACPA)、基质金属蛋白酶(matrixmetalloproteinases,MMPs)、14-3-3η蛋白、基质细胞衍生因子-1(stromalcellderivedfactor-1,SDF-1)、小泛素样修饰物蛋白(smallubiquitin-likemodifierprotein,SUMO)、分泌型卷曲相关蛋白(secreted frizzled-related protein, SFRP)、骨转换标志物等作一阐述,旨在为RA早期骨破坏诊断提供相关依据。     

HIF-1α在骨组织细胞代谢及骨疾病中的调控作用

低氧诱导因子-1α(HIF-1α)是调节细胞对低氧应答的关键因子,可在氧含量降低时被激活,能够调节氧代谢、糖酵解等多种生理活动。骨代谢主要包括骨形成和骨吸收作用,均受到氧浓度等多种因素的调控。HIF-1α在细胞代谢、骨组织生理及病理过程的调控中起着重要的作用,能够增加骨组织的低氧耐受能力,调节骨形成和矿化过程。该文主要综述了HIF-1α对成骨细胞、破骨细胞、骨髓间充质干细胞、软骨细胞等骨组织细胞的调控,对骨血管形成过程的影响,以及对肿瘤骨转移、股骨头坏死、异位骨化等病理过程的调节作用,为探讨HIF-1α对骨代谢的调控和相关疾病的治疗提供参考。     

自噬对骨疾病调控作用的研究进展

骨疾病是指机体因先天或后天性因素破坏正常骨代谢,导致骨代谢障碍而发生的一类疾病。骨主要由负责骨吸收的破骨细胞和负责骨重建的成骨细胞以及骨细胞构成。正常成人的骨形成量基本等于骨吸收量,两者处于动态平衡状态,保证了骨结构和功能的完整性。自噬是一种重要的细胞内清除机制,通过形成自噬溶酶体降解其所包裹的受损细胞器或蛋白质,实现细胞代谢和细胞器的更新。自噬相关基因的缺失能够抑制破骨细胞的骨吸收和成骨细胞的骨重建,而药物、肿瘤坏死因子等能够使自噬相关基因过表达导致骨吸收异常增加,造成骨吸收和骨形成之间的动态平衡失调,从而引起骨代谢障碍,形成骨疾病。该文分别就自噬与破骨细胞、成骨细胞以及骨疾病之间的研究进展进行综述,希望可以为骨疾病的靶向治疗提供新的思路。     

热作用下成骨细胞HSP70 mRNA的表达变化及其对破骨细胞增殖的影响

目的:研究不同温度热作用对小鼠成骨细胞系中HSP70 mRNA表达水平的影响及其培养液对小鼠破骨细胞增殖的影响.方法:取小鼠成骨细胞系MC3T3,不同温度(37℃-39℃-41℃-42.5℃)作用其一周,RT PCR方法观察其HSP70表达水平变化,同时取其培养液上清与小鼠破骨前体细胞RAW264.7共培养,MTT法观察其增殖情况.结果:经不同温度热(37℃-39℃-41℃-42.5℃)处理后,MC3T3细胞系中HSP70表达水平明显增加,并且呈现与温度梯度依赖性,其处理后的上清液分别与小鼠破故前体细胞系RAW264.7共培养,MTT法测定其增值水平,结果其增殖受到抑制,且抑制水平与温度呈梯度依赖关系.结论:热刺激可以促进成骨细胞中HSP70的表达,促使成骨细胞抑制破骨细胞增殖,HSP70可能是参与调控成骨细胞与破骨细胞平衡的重要因子.     

阿达木单抗的临床应用进展

阿达木单抗是一种抗肿瘤坏死因子α的全人源单克隆抗体,可特异性的结合人肿瘤坏死因子α,阻止其与效应细胞结合而发挥生物学效应,在风湿性关节炎、银屑病、强直性脊柱炎,克罗恩病以及一些自身免疫性疾病的治疗上取得了广泛的应用,本文仅就在这些疾病的临床治疗进展进行综述。     

Studies on Correlation Between Single-Nucleotide Polymorphisms of Tumor Necrosis Factor Gene and Different Stages of Ankylosing Spondylitis

To examine if there is any correlation between ankylosing spondylitis (AS) and TNF-α gene promoter single-nucleotide polymorphisms (SNP) and their associated haplotypes. Using restriction fragment length polymorphism—polymerase chain reaction method, the polymorphism of TNF-α-238, -308, -850, -857, -863 locus, and TNF-β +252 were analyzed in patients with progressive AS, stable AS and control. (1) Neither the genotypes nor the allele frequencies of TNF-α (-308), (-238), (-863), and TNF-β +252 showed differences in each group. TNF-α (-850) CC genotype and C allele frequency distribution was significantly higher in healthy controls group than in the stable and progressive groups. TNF-α (-857) CT, CC genotype, and C, T allele frequency showed differences in all groups. (2) Polymorphism linkage equilibrium test revealed that association of six TNF-α, β gene SNPs with haplotype GACTCG in progressive group is significantly higher than in the stable group and healthy control group (P < 0.05). TNF-α (-857), (-850) gene polymorphism may increase the susceptibility to AS, but do not reflect the disease active state. The CC genotype and C allele may play a protective role in the pathogenesis of AS. TNF-α (-308) may be a weak indicator reflecting the active state of AS. Haplotype GACTCG may indicate both the susceptibility and the activity of AS.     

Association of cytokine and matrix metalloproteinase profiles with disease activity and function in ankylosing spondylitis

Introduction

The pathology of ankylosing spondylitis (AS) suggests that certain cytokines and matrix metalloproteinases (MMPs) might provide useful markers of disease activity. Serum levels of some cytokines and MMPs have been found to be elevated in active disease, but there is a general lack of information about biomarker profiles in AS and how these are related to disease activity and function. The purpose of this study was to investigate whether clinical measures of disease activity and function in AS are associated with particular profiles of circulating cytokines and MMPs.

Methods

Measurement of 30 cytokines, five MMPs and four tissue inhibitors of metalloproteinases was carried out using Luminex^® technology on a well-characterised population of AS patients (n = 157). The relationship between biomarker levels and measures of disease activity (Bath ankylosing spondylitis disease activity index (BASDAI)), function (Bath ankylosing spondylitis functional index) and global health (Bath ankylosing spondylitis global health) was investigated. Principal component analysis was used to reduce the large number of biomarkers to a smaller set of independent components, which were investigated for their association with clinical measures. Further analyses were carried out using hierarchical clustering, multiple regression or multivariate logistic regression.

Results

Principal component analysis identified eight clusters consisting of various combinations of cytokines and MMPs. The strongest association with the BASDAI was found with a component consisting of MMP-8, MMP-9, hepatocyte growth factor and CXCL8, and was independent of C-reactive protein levels. This component was also associated with current smoking. Hierarchical clustering revealed two distinct patient clusters that could be separated on the basis of MMP levels. The high MMP cluster was associated with increased C-reactive protein, the BASDAI and the Bath ankylosing spondylitis functional index.

Conclusions

A profile consisting of high levels of MMP-8, MMP-9, hepatocyte growth factor and CXCL8 is associated with increased disease activity in AS. High MMP levels are also associated with smoking and worse function in AS.     

来氟米特联合依那西普对佐剂性关节炎大鼠免疫功能的影响

目的：研究来氟米特和依那西普联合使用对佐剂性关节炎（AA）大鼠的治疗作用及其可能的作用机制。方法：建立AA大鼠关节炎模型,分为正常对照组、模型组、来氟米特组、依那西普组、来氟米特联合依那西普配伍组;采用关节炎指数评分法评价大鼠关节炎症程度,半定量RT-PCR和放射免疫法检测滑膜组织及血清中IL-1β、TNF-α表达水平,免疫组化方法检测滑膜组织中MMP－3含量。结果：①相较于AA模型组,来氟米特组、依那西普组和配伍组中大鼠的AI评分均显著下降（P〈0．01）,其中以配伍组关节炎指数为最低（P〈0．05）。②模型组大鼠血清及滑膜组织的IL-1β和TNF-α水平明显高于正常对照组（P〈0．01）,用药后各组的IL-1β和TNF-α水平均有所下降,并以配伍组降低最为明显（P〈0．01或P〈0．05）;③模型组大鼠滑膜组织MMP-3表达阳性密度显著高于正常对照组（P〈0．01）,用药后备组的MMP-3阳性密度降低（P〈0．01）,其中配伍组下降程度明显高于来氟米特组和依那西普组（P〈0．01）。结论：来氟米特和依那西普联合使用可明显减轻AA大鼠的关节炎症,降低血清和滑膜组织中IL-1β和TNF-α平,减少滑膜中MMP-3的表达,疗效优于单独使用来氟米特或依那西普。     

Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification. In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.     

Role of the A20-TRAF6 axis in lipopolysaccharide-mediated osteoclastogenesis

Bacterial lipopolysaccharide (LPS) has long been suggested as a potent inducer of bone loss in vivo despite controversial effects on osteoclast precursors. Recently, the role of the deubiquitinating protease A20 in regulating the LPS response in various organs was reported. In the present study, we investigated whether A20 is expressed in osteoclast cultures in response to RANKL or LPS and whether this protein plays a role in osteoclast formation and activation. Human peripheral blood mononuclear cells were cultured in the presence of M-CSF ± RANKL ± LPS. Although LPS induced the formation of multinucleated TRAP-positive cells expressing OSCAR, cathepsin K, and the calcitonin receptor, these cells were not capable of lacunar resorption. Release of TNF-α was noted in LPS-treated cultures, and the addition of a neutralizing anti-TNF-α antibody abrogated osteoclast formation in these cultures. A20 appeared to be a late-expressed gene in LPS-treated cultures and was associated with TRAF6 degradation and NF-κB inhibition. Silencing of A20 restored TRAF6 expression and NF-κB activation and resulted in increased bone resorption in LPS-treated cultures. A20 appeared important in the control of bone resorption and could represent a therapeutic target to treat patients with bone resorption associated with inflammatory diseases.     

Expression of CD147 on monocytes/macrophages in rheumatoid arthritis: its potential role in monocyte accumulation and matrix metalloproteinase production

Monocytes/macrophages play an important role in rheumatoid arthritis (RA) pathogenesis. They can activate fibroblasts through many molecules, including IL-1 and tumor necrosis factor-alpha, but there have been very few reports on the role of CD147 in RA. In our study, the results of flow cytometry reveal that the mean fluorescence intensity (MFI) of CD147 expression on CD14+ monocytes of peripheral blood from RA patients was higher than that in normal control and ankylosing spondylitis (AS) patients. The MFI of CD147 expression on the CD14+ monocytes in RA synovial fluid was higher than that in RA peripheral blood. Immunohistochemical staining shows that CD147 expression in RA synovium correlated with matrix metalloproteinase (MMP)-1 expression. A double immunofluorescent assay shows that CD147 was expressed on CD68+ cells in RA synovium. The potential role of CD147 in cyclophilin A (CyPA)-mediated cell migration was studied using a chemotaxis assay in vitro and it was found that the addition of anti-CD147 antibody or a CD147 antagonistic peptide significantly decreased the chemotactic index of the mononuclear cells. The role of CD147 in MMP production and cell invasion in vitro were studied through the co-culture of human CD14+ monocytes or monocytic line THP-1 cells and human fibroblasts, as well as by gel zymography and an invasion assay. Significantly elevated release and activation of MMP-9 and/or MMP-2 were seen in the co-culture of human monocytes/THP-1 cells and fibroblasts compared with cultures of the cells alone. An increased number of cells invading through the filters in the invasion assays was also observed in the co-cultured cells. The addition of CD147 antagonistic peptide had some inhibitory effect, not only on MMP production but also on cell invasion in the co-culture. Our study demonstrates that the increased expression of CD147 on monocytes/macrophages in RA may be responsible for elevated MMP secretion, cell invasion and CyPA-mediated cell migration into the joints, all of which may contribute to the cartilage and bone destruction of RA. These findings, together with a better understanding of CD147, CyPA and RA, will help in the development of innovative therapeutic interventions for RA.     

Incidence of toxoplasma retinochoroiditis in patients with ankylosing spondylitis after using TNF-α blockers

TNF-α blockers are associated with reactivation of latent granulomatous infections and almost 6% of the world population has some chorioretinitis (CR) caused by Toxoplasma gondii. Thus, the blockade of TNF-α could reactivate a latent toxoplasmosis infection (LTxI). This study was conducted to evaluate the prevalence and incidence of chronic and active CR related to T. gondii in patients with ankylosing spondylitis (AS). A total of 74 eyes from 37 active AS outpatients starting TNFα blockers were compared with 35 AS patients, matched to age and sex, under conventional therapy in a prospective and controlled trial. All patients underwent serological tests for T. gondii, as well as periodic ophthalmologic examination during 12 months. Active CR was defined if a white, focal retinochoroidal lesion with overlying vitreous inflammation had been found. Retinochoroidal lesions with sharp edges, hyperpigmented borders and atrophic center were defined as CR scars. At baseline, no patient had active CR. From the 144 eyes examined, almost 6% had CR scars and only 2.1% had a typical toxoplasmic CR scar and all of them were negative for HLA-B27. During 12 months of follow-up, no recurrence or new CR were observed. AS patients using TNF-α blockers do not have a higher risk of acute or chronic CR caused by T. gondii.     

吉林人群强直性脊柱炎6号染色体短臂上的HLA区域遗传易感基因定位研究

为了寻找中国人群中与强直性脊柱炎相关的新的易感基因及其所在位置, 在与强直性脊柱炎强连锁的6 号染色体短臂上的HLA基因区域内选取11个SNPs多态位点, 通过对中国吉林地区79名AS患者和132名正常对照者进行case-control分析, 发现TNF-a -850处TT突变基因型在AS组中的分布高于正常对照组（P=0.027）, 突变型T等位基因在AS组和正常对照组中的分布差异更为显著（P=0.002）。通过多位点之间的连锁不平衡分析发现, LTA基因、TNF-a基因、LST1基因和NCR3基因中的 5个SNPs多态位点之间存在连锁不平衡, 范围是15 kb, 在这5个SNPs多态位点组成的单体型中, TCTTC单体型在AS组和正常对照组中的分布有显著差异（c2=7.406, P=0.0065）,并且该单体型中含有具有统计学意义的TNF-a –850的突变型等位基因T。提示在LTA、TNF-a、NCR3和LST1 这4个基因构成的15 kb范围内可能存在增加AS患病易感性的位点, 可能是TNF-a –850 C→T突变, 也可能是在TNF-a –850附近的其他位点。     

Does protein kinase R mediate TNF-α- and ceramide-induced increases in expression and activation of matrix metalloproteinases in articular cartilage by a novel mechanism?

We investigated the role of the proinflammatory cytokine TNF-α, the second messenger C₂-ceramide, and protein kinase R (PKR) in bovine articular cartilage degradation. Bovine articular cartilage explants were stimulated with C₂-ceramide or TNF-α for 24 hours. To inhibit the activation of PKR, 2-aminopurine was added to duplicate cultures. Matrix metalloproteinase (MMP) expression and activation in the medium were analysed by gelatin zymography, proteoglycan release by the dimethylmethylene blue assay, and cell viability by the Cytotox 96^® assay. C₂-ceramide treatment of cartilage explants resulted in a significant release of both pro- and active MMP-2 into the medium. Small increases were also seen with TNF-α treatment. Incubation of explants with 2-aminopurine before TNF-α or C₂-ceramide treatment resulted in a marked reduction in expression and activation of both MMP-2 and MMP-9. TNF-α and C₂-ceramide significantly increased proteoglycan release into the medium, which was also inhibited by cotreatment with 2-aminopurine. A loss of cell viability was observed when explants were treated with TNF-α and C₂-ceramide, which was found to be regulated by PKR. We have shown that C₂-ceramide and TNF-α treatment of articular cartilage result in the increased synthesis and activation of MMPs, increased release of proteoglycan, and increased cell death. These effects are abrogated by treatment with the PKR inhibitor 2-aminopurine. Collectively, these results suggest a novel role for PKR in the synthesis and activation of MMPs and support our hypothesis that PKR and its activator, PACT, are implicated in the cartilage degradation that occurs in arthritic disease.     

Structural damage in rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis: traditional views, novel insights gained from TNF blockade, and concepts for the future

Structural changes of bone and cartilage are a hallmark of inflammatory joint diseases such as rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS). Despite certain similarities - in particular, inflammation as the driving force for structural changes - the three major inflammatory joint diseases show considerably different pathologies. Whereas RA primarily results in bone and cartilage resorption, PsA combines destructive elements with anabolic bone responses, and AS is the prototype of a hyper-responsive joint disease associated with substantial bone and cartilage apposition. In the present review we summarize the clinical picture and pathophysiologic processes of bone and cartilage damage in RA, PsA, and AS, we describe the key insights obtained from the introduction of TNF blockade, and we discuss the future challenges and frontiers of structural damage in arthritis.