二磷酸盐对关节磨屑刺激单核细胞分泌IL-1β的抑制作用

目的:研究二磷酸盐对关节磨屑刺激单核细胞分泌IL-β的影响。方法:分离培养人外周血单核细胞,加入关节磨屑及不同浓度的阿伦膦酸钠,ELISA检测细胞上清中IL-1β的含量,原位杂交检测IL-1β mRNA的表达。结果:关节磨屑组IL-1β及基因的表达显著高于对照组(P<0.01),不同浓度的阿伦膦酸钠组IL-1β及基因的表达明显低于关节磨屑组(P<0.01)。结论:阿伦膦酸钠能通过下调人外周血单核细胞IL-1β3及mRNA的表达,对人工关节松动可能起防治作用。     

阿伦膦酸钠防治人工关节松动的实验研究

目的：评价二膦酸盐阿伦膦酸钠在防治人工关节松动中的作用。方法：36只SD大鼠右膝置入自制人工关节假体,建立人工关节松动的动物模型,分成3组：阴性对照组,阳性对照组和实验组。阴性对照组关节腔内注射磷酸缓冲液与鼠血清混合液,阳性对照组关节腔内注射10^10/ml关节磨屑（超高分子聚乙烯颗粒）,实验组关节腔内注射10^10/ml关节磨屑同时用阿伦膦酸钠灌胃（每日1mg/kg）。术后12周,处死各组动物行组织切片对比观察假体周围骨溶解情况。体外分离培养人外周血单个核细胞并分成5组,A组为单核细胞组,B组为单核细胞和关节磨屑混合培养组,C组为单核细胞和关节磨屑混合培养加入10^-4mol/L阿伦膦酸钠,D组单核细胞和关节磨屑混合培养加入10^-5mol/L阿伦膦酸钠,E组为单核细胞和关节磨屑混合培养加入10^-6mol/L阿伦膦酸钠,检测各组单个核细胞分泌溶骨因子的情况。结果：关节磨屑可引起假体周围骨溶解,刺激单个核细胞分泌溶骨因子,阿伦膦酸钠可阻止这种作用。结论：阿伦膦酸钠可有效防止关节磨屑诱导的人工关节松动,有望用于临床。     

辛伐他汀对钛颗粒刺激人单核细胞形成破骨细胞的影响

目的研究体外辛伐他汀对钛颗粒刺激单核细胞形成破骨细胞的影响,探讨辛伐他汀防治人工关节无菌性松动的可能性。方法体外分离培养人外周血单个核细胞并分成5组,A组为钛颗粒刺激组（单核细胞和磨屑混合培养）,B组为10^-5mol／L辛伐他汀组（单核细胞、磨屑混合培养＋10^-5mol／L辛伐他汀）,C组为10^-6mol／L辛伐他汀组（单核细胞、磨屑混合培养＋10^-6mol／L辛伐他汀）,D组为10^-7mol／L辛伐他汀组（单核细胞、磨屑混合培养＋10^-7mol／L辛伐他汀）,E组为单核细胞组。各组细胞培养24h后取上清液,用ELISA法检测上清液中肿瘤坏死因子（TNF-α）、单核细胞趋化蛋白-1（MCP-1）的含量。分别培养10d、18d后进行TRAP染色阳性细胞计数,采用扫描电镜检测骨磨片的吸收陷窝,观察钛颗粒对破骨细胞形成的影响。结果磨屑刺激单个核细胞分泌溶骨因子,辛伐他汀抑制磨损颗粒刺激单核／巨噬细胞分泌TNF-α及MCP-1;且破骨细胞数明显减少,骨吸收陷窝数减少,与钛颗粒组刺激组比较,差异均有统计学意义（P〈0．05）。结论辛伐他汀通过抑制TNF-α、MCP-1的释放而有效防止磨屑诱导的骨溶解,有望成为防治人工关节无菌性松动的一种有潜力的药物。     

阿伦膦酸钠对破骨细胞展平的影响

目的 :以破骨细胞展平面积为功能活性指标 ,探讨不同浓度阿伦膦酸钠对破骨细胞功能的影响。方法 :含破骨细胞的骨髓细胞取自出生 2 4h内的新生大鼠 ,以 1× 10 5/ml的浓度种植于 4个 3 5mm培养碟中 ,其中 1个为对照组 ,3个为含有 10 -11M、10 -10 M、10 -9M 3个浓度阿伦膦酸钠的实验组。每组随机选取 6个破骨细胞为观察对象 ,定时观察拍照 ,并将照片扫描输入计算机计算破骨细胞展平面积。结果 :10 -11M阿伦膦酸钠对破骨细胞展平面积无明显影响 (P>0 .0 5 )。 10 -10 M、10 -9M阿伦膦酸钠降低破骨细胞展平面积 (P <0 .0 1)。 10 -9M比 10 -10 M阿伦膦酸钠降低破骨细胞展平面积的程度大 (P <0 .0 1)。结论 :破骨细胞展平面积是与骨吸收功能密切相关的形态指标。展平面积的测量方法简便、迅速。 10 -10 M以上浓度的阿伦膦酸钠能抑制破骨细胞的功能活性 ,随阿伦膦酸钠浓度增加抑制作用增强     

阿仑膦酸钠干预人骨髓基质细胞OPG/RANKL的体外实验研究

目的观察阿仑膦酸钠对人骨髓基质细胞中骨保护素（OPG）核/因子Kappa B受体活化因子配基（RANKL）mRNA表达的影响,探讨阿仑膦酸钠防治骨质疏松的相关机制。方法从24名20~30岁的健康男性志愿者髂前上棘处分别抽取10 m l骨髓,分离培养骨髓基质细胞,取50%融合P2代骨髓基质细胞,混匀后随机分为4组：高、中、低剂量阿仑膦酸钠组和对照组,高、中、低剂量组在细胞培养液中,分别加入1×10-7mol/L、1×10-8mol/L、1×10-9mol/L阿仑膦酸钠;对照组采用普通LG-DMEM培养,不进行特殊处理。采用半定量RT-PCR和W estern blot检测OPG、RANKL mRNA和蛋白表达并计算OPG/RANKL比率。结果在RT-PCR实验中,高、中、低阿仑膦酸钠组和对照组OPG mRNA/RANKL mRNA表达比分别为（8.77±1.16）、（6.68±1.25）、（4.86±0.79）和（0.58±0.13）;W estern blot蛋白印迹实验显示,高、中、低阿仑膦酸钠组和对照组OPG/RANKL蛋白表达之比分别为（1.18±0.47）、（1.09±0.56）、（0.82±0.32）和（0.25±0.12）。经统计学分析,在mRNA和蛋白水平,高、中、低阿仑膦酸钠组OPG/RANKL比值明显高于对照组,差异有统计学意义（P〈0.05）,三组阿仑膦酸钠组比较,差异无统计学意义（P〉0.05）。结论防治骨质疏松药阿仑膦酸钠可以增加骨髓基质细胞中骨保护素的表达,减少核因子Kappa B受体活化因子配基表达。     

黄芪注射液对肾病综合征细胞因子及其基因表达的影响

目的：探讨儿童肾病综合征（NS）外周血白细胞介素－1（IL－1）、白细胞介素－6（IL－6）、白细胞介素－8（IL－8）和肿瘤坏死因子α（TNFα）的变化,糖皮质激素（简称“激素”）加黄芪注射液对IL－1、IL－6、IL－8和TNFα的产生及其基因表达的影响。方法：将观察对象分为四组：①治疗前组;②激素组;③黄芪组;④对照组。应用酶联免疫吸附试验（ELISA）方法检测血清中IL－1、IL－6、IL－8和TNFα的水平。应用原位聚合酶链反应（原位PCR）方法,检测外周血单个核细胞（PBMC）激素加黄芪注射液对IL－1、IL－6、IL－8和TNFαmRNA表达的影响。结果：NS患儿外周血IL－1、IL－6、IL－8和TNFα水平明显高于正常儿童,有显性差异（P＜0．01）;mRNA的表达增加。激素治疗后IL－1、IL－6、IL－8和TNFα水平明显降低（P＜0．05）,mRNA的表达下降。激素加黄芪注射液对上述细胞因子和mRNA表达其降低更加显（P＜0．01）。结论：NS患IL－1、IL－6、IL－8和TNFα水平明显增高,激素对IL－1、IL－6、IL－8和TNFα的产生与mRNA表达有抑制作用,黄芪具有辅助糖皮质激素治疗的作用。     

利塞膦酸钠对绝经后骨质疏松患者外周血单核细胞的影响

目的观察利塞膦酸钠对绝经后骨质疏松症患者外周血单核细胞(PMBC)分泌肿瘤坏死因子(TNFα),白细胞介素1(IL1),白细胞介素6(IL6)水平及PBMC细胞数目的影响。方法采用双盲法将54名绝经后骨质疏松妇女随机分为治疗组和对照组,所有患者在用药前和用药后6月进行外周血单核细胞(PBMC)培养,并以放射免疫方法(RIA)检测培养上清中TNFα、IL1、IL6水平,计录PBMC细胞数目改变。结果利塞膦酸钠治疗组服药6月后,PBMC培养上清中TNFα、IL1水平较治疗前有所下降,IL6较前增加,与对照组无统计学差异,PBMC细胞数目也无明显改变(P>0.05)。结论利塞膦酸钠作为一种骨吸收抑制剂,口服治疗对PBMC分泌细胞因子(IL1、IL6和TNFα)及细胞数目并无明显影响,这些结果提示利塞膦酸钠并未通过影响PBMC参与骨代谢调节。     

康肾Ⅰ号对老年维持性血液透析患者IL-1、IL-2、IL-6mRNA水平的影响

目的 :观察健脾补肾中药康肾Ⅰ号对老年维持性血液透析患者免疫功能紊乱的调节作用。方法 :采取RT -PCR法检测服用康肾Ⅰ号 3个月前后 ,老年维持性血液透析患者外周血单个核细胞IL - 1β、IL - 2、IL - 6mRNA的水平。结果 :将服药前后 ,有同一项白介素表达的相对表达强度 ,进行自身前后对照 ,发现IL - 2mRNA水平升高 ,IL - 1β、IL - 6mRNA水平降低。 结论 :康肾Ⅰ号从提高IL - 2mRNA水平和降低IL - 1β、IL - 6mRNA水平两方面均有益于调节老年维持性血液透析患者免疫功能紊乱     

阿仑磷酸钠对成骨细胞增殖、分泌和成骨功能的影响

目的　观察阿仑膦酸钠对人成骨细胞功能的影响 ,探讨使用此药防治人工关节无菌性松动的可行性。方法　采用体外培养成骨细胞的方法 ,观察不同浓度的阿仑膦酸钠 (1× 10 -11、1× 10 -9、1× 10 -7、1× 10 -5mol/L)对成骨细胞的增殖、碱性膦酸酶活性、分泌骨钙素及成骨能力的影响。结果　阿仑膦酸钠浓度达 1× 10 -5mol/L时抑制成骨细胞增殖 ;浓度为 1× 10 -7、1× 10 -5mol/L时增强成骨细胞的碱性膦酸酶活性和骨钙素分泌 ;对其成骨能力 ,浓度在 1× 10 -11、1× 10 -9mol/L时具有刺激作用 ,1× 10 -5mol/L有抑制作用 ,中间浓度 (1× 10 -7mol/L)无影响。结论　阿仑膦酸钠低浓度时有利于成骨细胞的成骨 ;局部使用有可能成为防治人工关节无菌性松动的方法之一。     

辛伐他汀防治人工关节无菌性松动的实验研究

目的通过观察辛伐他汀对钛颗粒刺激人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)而释放TNF-α及单核细胞趋化蛋白1(monocyte chemoattractant protein1,MCP-1)的影响,分析该过程中p42/p44激酶磷酸化的情况,评价辛伐他汀在防治人工关节无菌性松动中的作用。方法取成年健康志愿者外周血45mL,分层梯度离心法分离获得PBMC,制成密度为5×108个/mL的细胞悬液,根据培养液不同分成5组。A组:加入3mL RPMI-1640培养液及1mL0.01%钛颗粒悬液;B、C、D、E组:同A组试剂外,分别加入2mL浓度为1×10-5、1×10-6、1×10-7mmol/L辛伐他汀和20μmol/L U0126。取培养24h后上清液检测TNF-α及MCP-1含量。取培养2h后PBMC,按A～E组分组方法分别用不同培养液预处理60min,钛颗粒分别刺激培养30、60min,采用Western blot检测细胞外信号调节激酶(extracellular signal-regulated kinases,ERK1/2)的表达水平。结果A、B、C、D、E组TNF-α含量分别为1.1155±0.2436、0.6936±0.3543、0.6957±0.3873、0.7164±0.4789、0.2635±0.1016;MCP-1分别为1.4210±0.1053、0.9151±0.4113、1.0035±0.4642、1.1020±0.3539、0.2713±0.1451。A组TNF-α及MCP-1含量明显高于其他组,差异有统计学意义(P0.05);E组与B、C、D组比较差异有统计学意义(P0.05),B组与C、D组比较差异有统计学意义(P0.05),C、D组间比较差异无统计学意义(P0.05)。Western blot检测显示钛颗粒刺激培养30、60min各组均见ERK1/2表达;刺激培养30min时A、B、C、D、E组ERK1/2含量较低;60min时A、B、C、D、E组ERK1/2含量分别为1.6121±0.0682、1.0781±0.0728、1.2687±0.2231、1.4397±0.1801、0.7320±0.1104,组间比较差异均有统计学意义(P0.05)。结论ERK1/2通路是磨屑诱导巨噬细胞生成TNF-α和MCP-1的主要通路,辛伐他汀可抑制此通路,进而有效防止关节磨屑诱导的人工关节松动。     

Comparison of the response of primary human blood monocytes and the U937 human monocytic cell line to two different sizes of alumina ceramic particles.

It is well recognized that wear particles derived from orthopaedic implants have the potential to induce inflammation, which may eventually lead to aseptic loosening of the artificial joint. We hypothesized that alumina ceramic particles of different sizes cause a differential cytokine response by human monocytes. To test this hypothesis a human monocytic cell line (U937) and primary human blood monocytes obtained from healthy volunteers were exposed to ceramic particles within the range known to be generated in vivo. Cellular responses were measured by quantifying the relative gene expression of 12 different cytokines using TAQman Real-Time Polymerase Chain Reaction (RT-PCR). Our results demonstrate that at a particle to cell ratio of 100:1, 0.5 microm ceramic particles consistently provoked higher amounts of Interleukin-1alpha (IL-1alpha), IL-1beta, IL-8, IL-10 and Tumor necrosis factor-alpha (TNF-alpha) steady state mRNA by U937 cells. As expected, the variability of cytokine expression in primary blood monocytes was much higher compared to the cell line however, a similar trend was observed. These results show a differential response to ceramic particle size, which may imply that 0.5 microm particles are less biocompatible. New ceramic implants can be designed to generate a known particle size range in vivo. Implant materials of this type may induce relatively lower levels of production of inflammatory cytokines resulting in a reduced incidence of failure due to aseptic loosening.     

Inflammatory Response to Implant Particulates in a Macrophage/Osteoblast Coculture Model

The purpose of this study was to further define the cellular response to titanium and polymethylmethacrylate (PMMA) particles in aseptic loosening, and to determine if the use of pamidronate may be effective in inhibiting bone resorption associated with this response. Macrophages and osteoblasts were cocultured to simulate the environment around an aseptically loose prosthesis. Macrophages were plated on the bottom of six well plates and osteoblasts were plated on culture dish inserts, and placed into the wells with the macrophages. Incubation of macrophages with PMMA in this system led to release of prostaglandin E (PGE₂), granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin-6 (IL-6). Incubation with titanium led to release of tumor necrosis factor (TNF) and IL-6. Exposure of calvaria to media from cells exposed to either PMMA or titanium led to release of calcium 45. Incubation of calvaria with pamidronate was able to inhibit release of calcium 45 associated with exposure to the macrophage/osteoblast/particle conditioned medium. Bone resorption at the interface between implant and bone is a consistent feature leading to loosening of orthopedic implants. By inhibiting bone resorption associated with the inflammatory response to implant particulates, pamidronate or other bisphosphonates may have clinical utility in the treatment or prevention or aseptic loosening. Received: 22 December 1995 / Accepted: 3 May 1996     

Cytokine profile of a human bone marrow cell culture on exposure to titanium-aluminium-vanadium particles

INTRODUCTION: The purpose of this study was to prove the effect of wear particles, especially Tivanium, in the mechanism of the aseptic loosening of total joint prostheses. MATERIALS AND METHODS: Therefore, human bone marrow cell cultures were incubated with titanium-aluminium-vanadium particles of different concentrations which were added after the seventh day of culture (10(9), 10(8), 10(7), 10(6) particles per ml medium). From this time starts the real culture period (2 weeks). During these two weeks the medium was changed and the supernatants were sampled. Using an ELISA the cytokine levels of interleukin-6, interleukin-1beta, TNF-alpha and LDH were measured approximately every second day (1, 3, 6, 8, 10, 14). As a marker for toxicity the activity of LDH was determined. RESULTS: Incubation of a human bone marrow cell culture with titanium-aluminium-vanadium particles led to a maximum release of interleukin-6, interleukin-1beta, and TNF-alpha at high particle concentration (10(9) particles per ml medium). An increase of interleukin-1beta was only detectable at particle concentrations of 10(9) per ml medium. Exposure of the human bone marrow cell culture to titanium-aluminium-vanadium particles was toxic for high particle concentrations (10(9) particles per ml medium), as reflected by release of the intracellular enzyme LDH. DISCUSSION: This study shows the ability of tivanium wear particles in a human bone marrow cell culture to induce a signfically higher release of proinflammatory and osteolytic mediators which are responsible for the aseptic loosening of prosthesis and the problem of revisions. In comparison to other cell studies, our results were explained by the human bone marrow cell culture. The human bone marrow is the real effector tissue source "in situ" because the prosthesis is localised intramedullarly.     

The cause of loosening in cemented hip joint prostheses. Light and electron microscopy study and laser microprobe mass analysis

Aseptic loosening of joint prostheses is a quantitatively increasing problem. For evaluation of the pathogenesis of prosthesis loosening joint capsules and tissue membranes taken from the femoral and acetabular bone-cement-interface of 23 hips revised for aseptic loosening were examined. In all cases we found an excessive inflammatory histiocytic reaction with intracytoplasmatic incorporation of a small granular foreign material. This could be identified to be zirconium oxide by LAMMA-analysis. Zirconium oxide and polymethylmethacrylate (PMMA) wear particles could be shown in the cytoplasm of histiocytes by transmission electron microscopy. By quantitative grading of the histiocytic reaction and the intracellular granular wear particles we found that both were evenly distributed around the loosened prosthesis. Chronic inflammation due to bone cement abrasion is regarded as a leading cause of aseptic prosthesis loosening.     

磨损微粒诱导细胞凋亡与无菌性松动的研究进展

无菌性松动是关节置换术后最常见的远期并发症之一,限制了关节假体的使用寿命。近年来人们对假体周围界膜组织内的巨噬细胞、成骨细胞、破骨细胞及成纤维细胞作了大量的研究发现,人工关节置换产生的磨损颗粒在与界膜组织周围的细胞接触或被吞噬后可诱导假体周围界膜的慢性炎症反应并引起细胞凋亡,最终导致假体周围骨溶解以及无菌性松动的发生,提示细胞凋亡在无菌性松动中起着重要作用,细胞凋亡有可能成为无菌性松动新的治疗方法。本文就磨损颗粒引起细胞凋亡与无菌松动之间关系做一综述。     

Cytokine receptor profile of arthroplasty macrophages, foreign body giant cells and mature osteoclasts

In the arthroplasty pseudomembrane surrounding a loose prosthesis there is a marked macrophage and foreign body giant cell (FBGC) response to implant-derived wear particles. These cells contribute to the osteolysis of loosening by releasing cytokines and growth factors which influence the formation and activity of osteoclasts. Using a panel of monoclonal antibodies directed against known cytokine/growth factor receptors, we have determined by immunohistochemistry whether arthroplasty macrophages, FB-GCs and osteoclasts express receptors for cytokines and growth factors that are known to modulate osteolysis. All these cell types reacted with antibodies directed against the following cytokine/growth factor receptors: gp130, IL-1R type 1, IL-2R, IL-4R, IL-6R, TNFR, M-CSFR, GM-CSFR and SCFR but not with antibodies directed against IL-3R and IL-8R. Arthroplasty macrophages, FBGCs and osteoclasts thus show a similar pattern of cytokine/growth factor receptor expression. This reflects the fact that arthroplasty macrophages are capable of osteoclast differentiation and that these cell types form part of the mononuclear phagocyte system. As regards the osteolysis of aseptic loosening, it also indicates that these cells are targets for numerous cytokines and growth factors which influence osteoclast formation and bone resorption.     

Cytokine receptor profile of arthroplasty macrophages, foreign body giant cells and mature osteoclasts

In the arthroplasty pseudomembrane surrounding a loose prosthesis there is a marked macrophage and foreign body giant cell (FBGC) response to implant-derived wear particles. These cells contribute to the osteolysis of loosening by releasing cytokines and growth factors which influence the formation and activity of osteoclasts. Using a panel of monoclonal antibodies directed against known cytokine/growth factor receptors, we have determined by immunohis-tochemistry whether arthroplasty macrophages, FB-GCs and osteoclasts express receptors for cytokines and growth factors that are known to modulate osteolysis.

All these cell types reacted with antibodies directed against the following cytokine/growth factor receptors: gp130, IL-1R type 1, IL-2R, IL-4R, IL-6R, TNFR, M-CSFR, GM-CSFR and SCFR but not with antibodies directed against IL-3R and IL-8R. Arthroplasty macrophages, FBGCs and osteoclasts thus show a similar pattern of cytokine/growth factor receptor expression. This reflects the fact that arthroplasty macrophages are capable of osteoclast differentiation and that these cell types form part of the mononuclear phagocyte system. As regards the osteolysis of aseptic loosening, it also indicates that these cells are targets for numerous cytokines and growth factors which influence osteoclast formation and bone resorption.