雷奈酸锶对钛颗粒刺激单核/巨噬细胞分泌溶骨因子及其RANK表达的影响

目的:研究体外雷奈酸锶对钛(Ti)颗粒刺激单核/巨噬细胞分泌溶骨因子及其膜上RANK表达的影响,探讨雷奈酸锶预防人工关节假体周围无菌性松动的可能性。方法:体外培养单核巨噬细胞白血病细胞RAW264.7,制备Ti颗粒,并采用MTT法检测绘制RAW264.7细胞增殖曲线,寻找雷奈酸锶最佳干预浓度。将RAW264.7分为3组:Ti微粒组(体积分数为0.1%的含Ti微粒+质量分数为10%的胎牛血清DMEM培养基)、Ti+雷奈酸锶组(体积分数为0.1%的含Ti微粒+最佳浓度雷奈酸锶+含质量分数为10%的胎牛血清DMEM培养基)和对照组(含质量分数为10%的胎牛血清DMEM常规培养基)。采用ELISA法检测各组培养液白细胞介素-1(IL-1)、肿瘤坏死因子-α(TNF-α)浓度。采用实时荧光定量SYBR GREEN法检测RANK m RNA表达。结果:Ti微粒组及Ti+雷奈酸锶组的细胞培养液中IL-1、TNF-α的含量及RAW264.7细胞RANK m RNA的表达量明显高于对照组(P0.05)。而Ti+雷奈酸锶组的IL-1、TNF-α的含量及RAW264.7细胞RANK m RNA的表达量明显低于Ti微粒组(P0.05)。结论:Ti颗粒能刺激单核/巨噬细胞分泌IL-1、TNF-α,并能促进单核/巨噬细胞表达RANK。雷奈酸锶能明显抑制Ti颗粒诱导单核细胞分泌IL-1、TNF-α,以及抑制单核/巨噬细胞表达RANK。     

靶向TNF—α的反义寡核苷酸治疗人工关节磨损微粒诱导的骨溶解的实验研究

[目的]研究单次皮下注射靶向肿瘤坏死因子α(tumor necrosis factor,TNF-α)的反义寡核苷酸(anti-sense oligonucleotides,ASO)治疗人工关节磨损微粒诱导的骨溶解的作用,探讨用于防治人工关节置换后假体松动的可能性.[方法] 采用小鼠颅骨建立微粒诱导的骨溶解的动物模型,分为5组:第1组为假手术组,第2组为阳性对照组,第3组为低剂量治疗组,第4组为高剂量治疗组,第5组在第四组处置的基础上再次给予TNF-α.通过甲苯胺蓝染色观察颅骨表面骨吸收陷窝的数量并测量其面积,通过抗酒石酸的酸性磷酸酶(tartrate-resistant acid phospha-tase,TRAP)染色来观察破骨细胞并进行计数,通过定量试剂盒对TRAP的活性进行定量研究.同时采用RT-PCR和ELISA的方法检测TNF-α的表达.[结果]靶向TNF-α的ASO可以显著下调靶基因的表达,对微粒诱导的骨溶解有明显的抑制作用.骨吸收陷窝的数目与面积以及破骨细胞的数量均较阳性对照组有明显下降,定量研究显示TRAP的活性也有明显的下降.这种改变与ASO的用量呈明显的依赖性,高剂量组的治疗结果接近于假手术组.再次给予TNF-α可以逆转这种抑制效果.[结论]靶向炎症因子TNF-α的反义核酸通过抑制TNF-α表达,抑制了由微粒诱导的骨溶解.为防治人工关节置换后假体松动提供了一种很有应用前景的治疗策略.     

RANKL/RANK/OPG信号通路参与调控磷酸三钙磨损颗粒诱导假体周围骨溶解的研究

目的探讨RANKL/RANK/OPG信号通路在磷酸三钙磨损颗粒诱导假体周围骨溶解中的作用。方法将30只小鼠随机分为假手术组、模型组和OPG组,制备小鼠颅骨溶解模型,OPG组于术后第2天在颅顶局部注射骨保护素(osteoprotegerin,OPG)(3 mg/kg),每隔2 d 1次;假手术组和模型组给予等量生理盐水,共2周。测定破骨细胞数和骨溶解面积,检测白细胞介素-1β(Interleukin-1β,IL-1β)、白细胞介素-6(Interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)、OPG、NF-kappa B的受体激活因子(receptor activator of nuclear factor-κB,RANK)和RANK配体(receptor activator of nuclear factor-κB ligand,RANKL)的水平。结果与假手术组比较,模型组小鼠颅骨破骨细胞数、骨溶解面积均增加,IL-1β、IL-6和TNF-α水平均增加,RANKL和RANK mRNA的相对表达量均增加,OPG mRNA的相对表达量降低(P0.05);与模型组比较,OPG组小鼠颅骨破骨细胞数和骨溶解面积减少,IL-1β、IL-6和TNF-α水平减少,RANKL和RANK mRNA的相对表达量均降低,OPG mRNA的相对表达量增加(P0.05)。结论 RANKL/RANK/OPG信号通路参与调控磷酸三钙磨损颗粒诱导假体周围骨溶解,通过给予外源性OPG能显著抑制磷酸三钙磨损颗粒诱导的骨溶解。     

塞来昔布抑制大鼠颅骨内钛微粒引起骨溶解的实验研究

目的 探讨塞来昔布对关节磨损颗粒诱导的大鼠颅骨骨溶解的影响.方法 取健康雌性大鼠行颅骨手术,实验分为植入钛颗粒组、钛颗粒结合塞来昔布处理组和空白对照组,HE染色后通过测定骨小梁面积比计算骨吸收情况,并用实时荧光定量PCR,Western Blot检测COX-2、TNF-α的表达,ELISA法检测血清IL-1、IL-6、PGE2水平.结果 与对照组比较,钛颗粒组、钛颗粒结合塞来昔布处理组骨吸收明显,而钛颗粒结合塞来昔布处理组骨吸收较钛颗粒组改善;塞来昔布可明显下调钛颗粒植入大鼠颅骨后PGE2、TNF-α、IL-1和IL-6的表达.结论 塞来昔布可抑制钛颗粒诱导的大鼠颅骨骨溶解,有望成为防治人工关节无菌性松动的药物.     

SCID小鼠单次唑来磷酸头皮下注射抑制人假体周围骨溶解

[目的]评价唑来磷酸在新型动物模型中对人关节假体周围界膜组织诱导的骨溶解的抑制效应。[方法]将髋关节翻修病人体内获得的界膜组织移植到SCIDbeige小鼠颅骨上。术后2d将唑来磷酸直接注射至小鼠头皮下,并在术后2、4周处死动物取出带移植组织的颅骨进一步检测。[结果]移植组织在动物体内存活至少4周,HE染色可见界膜组织引起大量的骨胶原纤维碎裂溶解。界膜组织中TNF-α、IL-6、RANKL和CPK的基因表达和蛋白水平均显著高于对照。单次局部注射唑来磷酸显著降低RANKL和CPK的表达但未影响TNF-α和IL-6的水平。[结论]单次局部注射唑来磷酸可有效抑制人界膜组织诱导的骨溶解,且效应持续。该新型动物模型模拟临床程度高,可作为评价假体周围骨溶解药物抑制效应的平台。     

不同浓度钛微粒对护骨素/护骨素配体基因表达影响的体外研究

目的: 探讨磨损微粒引起假体周围骨溶解的机理, 为人工关节假体松动的预防研究打下基础。方法: 采用不同浓度钛微粒 (粒径 <3μm) 对成骨细胞MG 63细胞进行 24h干预, 半定量RT PCR观察其对护骨素 (OPG) 及护骨素配体 (OPG- L) 基因表达的影响。结果:钛微粒能上调MG 63细胞OPG及OPG- L基因表达, 且对后者作用强于前者。结论: OPG- L/OPG比率随着钛微粒浓度的加大而增高, 引起骨溶解大于骨形成, 这可能是磨损微粒引起人工关节假体松动的重要原因, 这表明降低OPG- L/OPG比率有可能预防或逆转此病理过程。     

钛合金颗粒对成骨细胞骨保护素基因表达的影响

目的研究钛合金颗粒对体外培养条件下的成骨细胞骨保护素(OPG)基因表达的影响,探讨人工关节假体松动中磨损颗粒导致的骨溶解机制。方法分别将不同浓度的钛合金颗粒(1、0．1、0．01 mg/mL)与第3～5代成骨细胞共同培养3、6、9d,用RT-PCR方法半定量测定OPG基因mRNA的表达。结果不同浓度的钛合金颗粒均抑制成骨细胞OPG基因表达,各组与空白对照组比较差异有显著性意义(P＜0．01)；存在一定的剂量、时间-效应关系,随时间延长,钛合金颗粒对OPG基因作用逐渐降低。结论铁合金颗粒使成骨细胞OPG基因表达下降,使破骨细胞分化、活化功能增强,这可能是导致假体松动的骨溶解机制之一。     

磨损微粒引起的成骨细胞内质网应激反应在骨溶解中的作用及机制研究

目的：分析内质网应激反应在骨溶解骨组织中成骨细胞凋亡和骨溶解发生发展中的作用,探讨人工关节松动的原因,为人工关节松动的防治提供新的思路和理论依据。方法：采用小鼠颅骨建立磨损微粒诱导骨溶解的动物模型,随机分成4组,每组7只：组1,空白对照组;组2,磨损微粒TiAl6V4纳米合金粉末（TiNPs）组;组3,内质网应激反应阳性对照（TiNPs+Tg）组;组4,内质网应激反应抑制剂（TiNPs+4-PBA）组。通过甲苯胺蓝染色、HE染色和ALP染色观察骨溶解的病理变化;Western Blotting方法检测骨溶解颅骨组织中内质网应激反应标志蛋白的表达变化;TUNEL和Caspase-3免疫组化方法检测骨溶解颅骨组织内成骨细胞的凋亡情况。结果：磨损微粒TiNPs能够在体外诱导骨溶解的发生、加重炎症细胞的浸润以及抑制成骨细胞分化成熟,同时磨损微粒还可以上调成骨细胞内质网应激反应标志蛋白以及促进骨溶解骨组织中成骨细胞的凋亡。在磨损微粒TiNPs的基础上加入内质网应激的抑制剂（4-PBA）后,骨溶解症状明显缓解,骨侵蚀和炎症浸润显著降低,成骨细胞的分化成熟得到改善,凋亡的成骨细胞急剧减少,内质网应激标志蛋白的表达也逐渐减弱。结论：内质网应激参与骨溶解的形成并在骨溶解的发生发展中发挥重要作用。同时,内质网应激可作为一种新的治疗靶点,为临床逆转或治疗骨溶解和无菌性松动提供新的思路和方法。     

RANK、RANKL与人工关节磨损颗粒诱导骨溶解

人工关节磨损颗粒诱导的骨溶解是人工关节置换术后无菌性松动的最主要原因.人工关节各个部件相互摩擦产生的磨损颗粒经体内巨噬细胞反复吞噬、刺激产生多种细胞因子和炎症介质.磨损颗粒与巨噬细胞相互作用产生的促炎症反应使破骨细胞过度生成和激活,人工关节周围正常骨质被吸收破坏,最终形成骨溶解和无菌性松动.研究证实,细胞核因子κB活化因子受体(RANK)及其配体(RANKL)在骨溶解中起重要作用.该文就RANK、RANKL结构,生物学功能及其与磨损颗粒诱导骨溶解的关系等近期研究进展作一综述.     

人工关节假体植入后骨溶解的研究进展

假体植入后松动是许多行关节置换术病人面临的严重问题,解决这一问题对于提高人工关节置换的疗效有重要意义.目前认为导致假体植入后松动的主要原因是关节活动使得假体表面之间长期摩擦,因此产生大量磨损微粒,微粒在骨-假体界面之间迁移,诱发局部环境中细胞分泌各种细胞因子导致假体周围的骨溶解.为此,本文主要研究磨损微粒的产生过程以及微粒的产生对关节假体使用寿命的重要影响,在此基础上探讨如何减少微粒的产生以及限制其在假体周围的迁移,以此来预防骨溶解发生.……     

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。     

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。     

雷尼酸锶对钛颗粒诱导炎性骨溶解的抑制作用

目的观察雷尼酸锶（strontium ranelate,SR）对磨损颗粒诱导炎性骨溶解的影响。方法 30只雄性C57BL/J6小鼠,随机分为空白组、对照组和药物组,每组10只。采用钛（Ti）颗粒诱导的小鼠颅骨溶解模型,药物组建模当日经灌胃予SR[600mg/（kg·d）],空白组和对照组不予处理;持续至建模后10d,处死取材。HE染色观察颅骨溶解程度及骨膜厚度;抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase,TRAP）染色检测成熟破骨细胞;酶联免疫吸附试验（ELISA）检测肿瘤坏死因子（TNF-α）、白细胞介素（IL）-1β和IL-6表达水平。结果HE染色结果,对照组骨膜明显增厚,颅骨溶解区域广;图像分析软件测量结果,对照组骨膜厚度（0.27±0.04）mm,骨溶解率为0.47±0.11,与药物组[（0.11±0.02）mm,0.18±0.05]比较,差异有统计学意义（P〈0.05）;TRAP染色结果,对照组颅骨大片紫红色区域,SR治疗后明显减少;ELISA检测结果,SR加入后,TNF-α、IL-1β和IL-6的表达量分别为[（145.6±14.2）ng/L、（130.2±8.2）ng/L和（137.6±8.2）μg/L],与对照组[（210.2±8.9）ng/L、（159.6±9.7）ng/L、（170.8±9.5）μg/L]比较,差异有统计学意义（P〈0.05）。结论 SR能够减轻Ti颗粒引起的炎症反应、减少炎症因子分泌,抑制骨溶解。     

Polyethylene particle-induced bone resorption in alpha-calcitonin gene-related peptide-deficient mice.

This study investigates the impact of alpha-CGRP on bone metabolism after implantation of polyethylene particles. alpha-CGRP knockout mice showed less osteolysis compared with wildtype mice. The local neurogenic microenvironment might be a crucial factor in particle-induced osteolysis. INTRODUCTION: Periprosthetic osteolysis is the major reason for aseptic loosening in joint arthroplasty. This study aimed to investigate the potential impact of alpha-calcitonin gene-related peptide (alpha-CGRP) deficiency on bone metabolism under conditions of polyethylene particle-induced osteolysis. MATERIALS AND METHODS: We used the murine calvarial osteolysis model based on polyethylene particles in 14 C57BL 6 mice and 14 alpha-CGRP-deficient mice divided into four groups of 7 mice each. Groups 1 (C57BL/J 6) and 3 (alpha-CGRP knockout) received sham surgery, and groups 2 (C57BL/J 6) and 4 (alpha-CGRP knockout) were treated with polyethylene particles. Qualitative and quantitative 3D analyses were performed using microCT. In addition, bone resorption was measured within the midline suture by histological examination. The number of osteoclasts was determined by counting the TRACP(+) cells. Calvarial bone was tested for RANKL expression by RT-PCR and immunocytochemistry. RESULTS: Bone resorption was significantly reduced in alpha-CGRP-deficient mice compared with their corresponding wildtype C57BL 6 mice as confirmed by histomorphometric data (p < 0.001) and microCT (p < 0.01). Osteoclast numbers were significantly reduced in group 3 and the particle subgroup compared with group 1 (p < 0.001). We observed a >3-fold increase of basal RANKL mRNA levels within group 1 compared with group 3. Additional low RANKL immunochemistry staining was noted in groups 3 and 4. CONCLUSIONS: In conclusion, alpha-CGRP knockout mice did not show the expected extended osteolysis compared with wildtype mice expressing alpha-CGRP. One of the most reasonable explanations for the observed decrease in osteolysis could be linked to the osteoprotegerin (OPG)/RANK/RANKL system in alpha-CGRP-deficient animals. As a consequence, the fine tuning of osteoclasts mediating resorption in alpha-CGRP-null mice may be deregulated.     

Effects of polyethylene and TiAIV wear particles on expression of RANK,RANKL and OPG mRNA

Background?Wear debris has been associated with periprosthetic osteolysis and loosening of total joint arthroplasties. RANKL (receptor activator of NF-κB ligand), RANK (receptor activator of NF-κB) and OPG (osteoprotegerin) are three key molecules which regulate differentiation, survival, fusion, and activation of osteoclasts.Material and methods?We evaluated the effect of TiAlV and polyethylene particles on expression of RANK, RANKL and OPG mRNA. We used a human monocytic leukemic cell line (THP-1) in this in vitro study. THP-1 monocytes were differentiated into macrophage-like cells and exposed to polyethylene particles and prosthesis-derived TiAlV particles. The supernantant was used for measurement of TNFα protein and total RNA was extracted from the cells. Expression of the genes coding for OPG, RANKL and RANK was analysed at the mRNA level using a semiquantitative RT-PCR method.Results?Both polyethylene and TiAlV particles induced a significant release of TNFα after 6?h of exposure and a significant upregulation of RANK mRNA. OPG mRNA expression was transiently upregulated after exposure to polyethylene and TiAlV particles. These effects were dependent on particle dose. RANKL mRNA was not detectable in our THP-1 model. In contrast, LPS exhibited a different pattern of RANK/RANKL/OPG mRNA expression.Interpretation?Our findings provide evidence that both polyethylene and TiAlV particles induce upregulation of RANK expression in cells of the monocytic lineage, which may be important for periprosthetic osteoclastogenesis.     

葡萄糖对人牙周膜成纤维细胞OPG、RANKLmRNA表达的影响

目的：以体外培养的人牙周膜成纤维细胞（HPDLF）为研究对象,观察不同浓度的葡萄糖对HPDLF中骨保护素（OPG）、核因子KB受体活化剂配体（RANKL）mRNA表达的影响,探讨葡萄糖在牙槽骨吸收中的作用。方法：组织块法体外原代培养HPDLF并鉴定,选择4-8代的HPDLF作为实验的靶细胞,不同浓度的葡萄糖（0、5．5、11．1、16．7、22．2、33．3mmol／1）干预HPDLF,半定量逆转录聚合酶链反应（RT-PCR）检测HPDLF中OPG、RANKL mRNA的表达。结果：高浓度的葡萄糖可下调HPDLF中OPG mRNA的表达,呈剂量依赖性;可上调RANKL mRNA的表达,也呈剂量依赖性,且使RANKL／OPG mRNA的比值随着葡萄糖浓度的增高而呈现持续上升的趋势。结论：糖尿病时,牙周组织中高浓度的葡萄糖,可下调OPG的表达,上调RANKL的表达,使RANKL／OPG的比值升高,调节破骨细胞分化,刺激骨吸收,导致牙槽骨的破坏,加重牙周病的病情。     

Recombinant adeno-associated virus-mediated osteoprotegerin gene therapy inhibits wear debris-induced osteolysis

BACKGROUND: Aseptic loosening of orthopaedic implants secondary to wear debris-induced osteolysis is a serious problem. Osteoprotegerin (OPG) is a natural decoy protein that inhibits osteoclast activation and bone resorption. This study investigated whether gene therapy using a recombinant adeno-associated viral vector that expresses OPG can inhibit wear debris-induced osteolysis. METHODS: A recombinant adeno-associated virus (rAAV) vector co-expressing OPG (rAAV-OPG-IRES-EGFP) was generated. A control vector expressing b-galactosidase (rAAV-LacZ) was also prepared. In vitro validation experiments were performed to determine rAAV-OPG-IRES-EGFP transduction efficiency, OPG expression level and function in bone wafer, and osteoclastic activity. The effect of rAAV-OPG-IRES-EGFP in vivo gene therapy on wear debris-induced osteolysis was then evaluated in a mouse calvarial model in which a single intramuscular injection of the vector was administered prior to the introduction of the wear debris. The effects of the rAAV-OPG-IRES-EGFP gene therapy on wear debris-induced osteoclastogenesis and bone resorption were determined by histomorphometry on day 10. RESULTS: In vitro experiments revealed that 100% of human embryonic kidney 293 cells were transduced at a multiplicity of infection of 1000 with both rAAV-OPG-IRES-EGFP and rAAV-LacZ. At a rAAV-OPG-IRES-EGFP multiplicity of infection of 1000, an OPG concentration of 135 ng/mL of culture media was achieved after four days. Using a bone-wafer assay for osteoclast activity, we found that treatment with rAAV-OPG-IRES-EGFP reduced resorption sevenfold compared with parathyroid hormone-stimulated controls and elevenfold compared with rAAV-LacZ controls. Furthermore, a seventeenfold decrease in RANKL and macrophage colony-stimulating factor-induced splenocyte osteoclastogenesis was observed in co-cultures containing rAAV-OPG-IRES-EGFP-infected fibroblasts. In vivo administration of rAAV-OPG-IRES-EGFP resulted in detectable transduction of myocytes at the injection site and a significant increase in expression of serum OPG levels by the second day (p < 0.05). Maximal concentrations were obtained on day 6 and then leveled off throughout the observation period. In contrast, serum OPG could not be detected in the sham-treated, uninfected titanium-stimulated, or rAAV-LacZ-infected mice. In the control mice, titanium implantation resulted in a threefold increase in the mean number of osteoclasts adjacent to the sagittal suture as well as a twofold increase in the mean area of soft tissue in the sagittal suture compared with the sham-treated mice. In contrast, osteoclast numbers remained at basal levels, and the area of soft tissue in the sagittal suture was markedly reduced in titanium-implanted animals that received rAAV-OPG-IRES-EGFP treatment, demonstrating a complete inhibition of osteolysis in response to titanium particles. CONCLUSIONS: A single intramuscular injection of the rAAV-OPG-IRES-EGFP vector can efficiently transduce myocytes to produce high levels of OPG. The OPG effectively inhibits wear debris-induced osteoclastogenesis and osteolysis. Clinical Relevance: Currently, there is no approved drug therapy to prevent or inhibit periprosthetic osteolysis. Although preclinical studies have identified potential drug therapies (i.e., bisphosphonates), there is no evidence that these drugs can effectively treat aseptic loosening in patients. This is the first evidence that in vivo OPG gene therapy can be used to prevent wear debris-induced osteolysis.     

唑来膦酸钠在抑制钛颗粒诱导骨溶解中作用的体外实验研究

目的：研究唑来膦酸钠（ ZOL）对钛颗粒诱导的骨溶解的影响。方法分离6～8周C57BL/6J小鼠长骨中的前体破骨细胞（ OCP）并分为6组,A组：OCP＋细胞培养液,B组：OCP＋巨噬细胞集落刺激因子（M-CSF）＋NF-κB 受体活化因子配体（RANKL）＋细胞培养液,C组：OCP＋钛颗粒＋细胞培养液,D组：OCP＋上清液（钛颗粒刺激巨噬细胞24 h后上清液）＋细胞培养液,E组：OCP＋M-CSF＋RANKL＋ZOL＋细胞培养液,F组：OCP＋上清液＋ZOL＋细胞培养液。每组细胞分别接种在玻璃盖玻片、皮质骨磨片和含骨检测表面的96孔板上,10 d后检测玻璃盖玻片上细胞抗酒石酸磷酸酶（ TRAP）的表达及皮质骨磨片上骨吸收陷窝的形成,并以骨检测表面的骨吸收面积为指标比较各组破骨细胞的骨吸收活性。结果 B组、D组、E组和F组的OCP均能分化为能被TRAP染色成阳性的破骨细胞并形成骨吸收陷窝,其余组均未发现TRAP染色阳性的破骨细胞和骨陷窝。加入ZOL的F组骨吸收面积（5.54％±1.25％）较D组（10.34％±1.69％）明显减少,差异具有统计学意义（t＝5.61,P＜0.01）。结论在体外实验中钛颗粒并不能直接刺激前体破骨细胞向破骨细胞转化;唑来膦酸钠可以抑制钛颗粒诱导的骨溶解作用。     

激素性股骨头坏死患者骨髓基质细胞骨保护素/核因子kappa B受体活化因子配基蛋白表达的研究

目的观察激素性股骨头坏死患者骨髓基质细胞（bone marrow stroma cells,BMSCs）骨保护素（osteoprotegerin,OPG）/核因子kappa B受体活化因子配基（receptor activator of nuclear factor kappa B ligand,RANKL）蛋白表达情况,探讨长期应用激素导致股骨头坏死的另一病理机制。方法 2007年3月至2008年3月,取激素性股骨头坏死患者骨髓及股骨头骨组织35例（实验组）,股骨颈骨折患者骨髓及股骨头骨组织21例（对照组）。两组男女比例均为4：3;年龄41～70岁,实验组平均55.34岁,对照组平均55.33岁;实验组最近2年内接受过皮质激素治疗超过3周或超过1周的大剂量冲击治疗,对照组从未接受过超过1周的激素治疗。骨组织标本行多聚甲醛固定后石蜡包埋,HE染色。所取骨髓采用贴壁法分离培养骨髓基质细胞,采用蛋白免疫印迹（Western blot）技术检测骨髓基质细胞OPG和RANKL蛋白表达水平,并得出OPG/RANKL比值。结果 HE染色：实验组未见完整的骨小梁和骨单位,可见不连续的骨碎片,碎片骨陷窝内骨细胞大部分消失,周围大量炎性肉芽组织。对照组可见完整骨单位由板层骨构成,板层骨连续完整,围绕血管呈同心圆排列,小梁骨陷窝内可见骨细胞。骨髓基质细胞Western blot检测：实验组和对照组OPG/RANKL蛋白表达比分别为1.13±0.65和2.54±0.35,实验组明显低于对照组,有统计学差异（P〈0.05）。结论长期应用糖皮质激素致股骨头坏死可能与其调控骨髓基质细胞OPG和RANKL表达有关。     

流体剪切力对MC3T3-E1细胞OPG、RANKL蛋白表达的实验研究

目的探讨流体剪切力(fluid shear stress,FSS)作用下,两种调节骨骼重建的重要分子骨保护素(osteoprotegerin,OPG)和细胞核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)的蛋白表达情况。方法采用体外模型对MC3T3-E1细胞加载流体剪切力,细胞经不同时间加力后(0,30,60,90,120min),对细胞分别进行染色和裂解,运用免疫荧光和蛋白印迹法对OPG和RANKL的蛋白表达水平进行定量分析。结果 FSS作用30,60,90,120min后能够显著增加OPG的蛋白表达(P0.05),减少RANKL的蛋白表达(P0.05)。两者共同作用使得OPG/RANKL值显著增高(P0.05)。结论流体剪切力刺激提示OPG/RANKL的比值可能在成骨细胞和破骨细胞联合调节骨骼形成和吸收的过程中起着重要的调节作用。