核因子κB受体活化因子配体诱导形成的成熟破骨细胞

背景：破骨细胞作为一种终末细胞,获取困难,而且没有成熟破骨细胞株等因素限制了其应用。先前国内外对破骨细胞的获取一般采用基质细胞诱导培养或共培养,或运用核因子κB受体活化因子配体和巨噬细胞集落刺激因子共同作用诱导形成成熟的破骨细胞。 目的：观察小鼠单核巨噬细胞RAW264.7的一般生物学特征,分析其在核因子κB受体活化因子配体诱导下形成成熟破骨细胞的可行性。 方法：培养RAW264.7后,用核因子κB受体活化因子配体诱导RAW264.7细胞7 d后观察抗酒石酸酸性磷酸酶染色结果,以抗酒石酸酸性磷酸酶染色阳性,细胞核≥3个为破骨细胞。以鬼笔环肽荧光染色观察纤维性肌动蛋白环,甲苯胺蓝染色观察牛骨片表面的吸收陷窝情况。 结果与结论：核因子κB受体活化因子配体可诱导RAW264.7细胞形成抗酒石酸酸性磷酸酶染色阳性的多核细胞,形成纤维性肌动蛋白环,电镜下可见骨片上圆形或椭圆形的吸收陷窝。提示RAW264.7是一种较好的破骨前体细胞模型,可用于破骨细胞分化研究。单用核因子κB受体活化因子配体诱导RAW264.7细胞分化成熟,减少了巨噬细胞集落刺激因子的应用,使培养体系更加简单,易于操作,诱导出的细胞纯度高,适合于破骨细胞的生物学和生化研究。     

不同矫治正畸作用下牙周骨改建过程中压力侧相关因子的变化

背景：近年来研究表明核因子κB受体活化因子配体与正畸牙移动骨改建过程中破骨细胞的形成、分化和功能密切相关。 目的：观察不同矫治器正畸作用下兔牙周组织改建过程中压力侧组织核因子κB受体活化因子配体表达的变化,探讨不同矫治器的正畸效果。 方法：将64只健康大耳白兔随机分为4组：对照组、MBT矫治器组、Begg矫治器组、Damon Ⅲ矫治器组,每组16只。MBT矫治器组、Begg矫治器组、Damon Ⅲ矫治器组分别应用对应的矫治器对兔上颌切牙和第一磨牙进行矫治,近中移动牵引力为80 g;对照组不进行矫治。分别于矫治后第3,7,14,21天每组取4只白兔进行检测。 结果与结论：苏木精-伊红染色显示加力后各矫治器组压力侧牙周膜腔变窄,牙槽骨边缘出现骨吸收陷窝。抗酒石酸酸性磷酸酶染色显示,矫治7 d时,骨改建活跃,破骨细胞数量达到高峰;同时实时荧光定量PCR检测发现加力后压力侧牙槽骨组织中核因子κB受体活化因子配体mRNA的表达明显增高,并于加力后第7天达到最高峰,而后逐渐降低。加力第7天时,Damon Ⅲ矫治器组破骨细胞数量和核因子κB受体活化因子配体mRNA表达水平均明显高于MBT矫治器组和Begg矫治器组(P < 0.05)。提示在骨改建过程中核因子κB受体活化因子配体mRNA表达的变化规律与破骨细胞数量相一致,Damon Ⅲ矫治器的矫治效果优于MBT矫治器、Begg矫治器。     

核因子κB受体活化因子配体诱导破骨细胞前体的培养与分化

背景：以往的研究多采用长骨机械分离法获得破骨细胞,破骨细胞为终末分化细胞,无法进一步增殖和传代。因此目前常用骨髓细胞诱导培养法和RAW264.7细胞诱导培养法获得大量的破骨细胞以满足实验需要。 目的：探讨细胞核因子κB受体活化因子配体诱导破骨细胞前体细胞分化为成熟破骨细胞的最佳方法。 方法：分离小鼠骨髓细胞后添加核因子κB受体活化因子配体与巨噬细胞集落刺激因子共同诱导或者取RAW264.7细胞单独加入核因子κB受体活化因子配体诱导破骨细胞的形成;分别给予不同浓度的核因子κB受体活化因子配体,观察生成破骨细胞的数量,评价核因子κB受体活化因子配体与破骨细胞生成的量效关系;采用膜联蛋白V-FITC联合PI染色进行流式细胞术分析破骨细胞形成过程中单核巨噬细胞的凋亡情况。 结果与结论：当核因子κB受体活化因子配体浓度为10 μg/L时,破骨细胞形成数量最多的时间点在第六至七天;而核因子κB受体活化因子配体浓度为100 μg/L时,高峰期出现在第四至五天。破骨细胞的形成数量随着核因子κB受体活化因子配体刺激浓度升高而增加,呈浓度依赖性,50 μg/L的核因子κB受体活化因子配体是破骨细胞形成数量与浓度关系曲线的转折点,高于150 μg/L以后破骨细胞形成数量的增幅明显放缓。核因子κB受体活化因子配体即能诱导单核巨噬细胞形成破骨细胞又可以促进其凋亡,通过破骨细胞计数比较发现在同一浓度(104/cm²)接种单核巨噬细胞后以30 μg/L的核因子κB受体活化因子配体诱导后,平均每单位核因子κB受体活化因子配体所获得的破骨细胞数量最多。提示骨髓细胞或RAW264.7细胞诱导破骨细胞的培养方法皆简单可行,细胞接种的最佳浓度为104/cm²;核因子κB受体活化因子配体的适宜刺激浓度为30-50 μg/L。     

补肾接骨中药对骨折修复的成骨作用及机制

背景：骨折愈合是成骨细胞与破骨细胞耦联相互作用相互影响促进骨质生长的过程,其中成骨细胞介导骨的形成,破骨细胞介导骨吸收,使骨重建处于一种动态平衡中,于动态平衡环境中促进骨生长,而目前研究多数倾向于单独的成骨或者破骨机制,但会忽略这两种细胞共同存在的环境下相互作用机制。 目的：观察补肾接骨中药对成骨与破骨细胞耦联中骨护骨素-核因子κB受体激活剂的配基-核因κB受体活化因子的影响及其在骨折治疗中的作用机制。 方法：分离小鼠成骨、破骨细胞并体外细胞培养,建立小鼠“成骨-破骨细胞共育系”作为研究平台,补肾接骨中药1.25,2.5,6.25 g/(kg•d)灌胃,空白对照组给予同等体积的生理盐水灌胃,每日1次,连续7 d。 结果与结论：共育培养24 h后,成骨细胞内碱性磷酸酶水平明显高于单纯培养的成骨细胞(P < 0.05)。实时PCR检测显示,共育体系细胞中碱性磷酸酶、Runx2及骨护骨素表达增加(P < 0.05),呈剂量依赖性(P < 0.05)。Western-blot检测显示,高浓度[6.25 g/(kg•d)]中药能明显促进骨护骨素、核因子κB受体激活剂的配基的表达,抑制核因子κB受体活化因子蛋白表达(P < 0.05)。结果证实,补肾接骨中药可动态调节骨护骨素-核因子κB受体激活剂的配基-核因子κB受体活化因子信号通路,对促进骨组织重建与恢复有着积极的作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

犬切牙压低移动过程中牙周组织骨保护素/核因子κB受体活化因子配体的表达

背景：骨保护素及核因子κB受体活化因子配体是调控破骨细胞生成和活化的关键因子,机械力可影响牙周膜细胞和成骨细胞骨保护素及核因子κB受体活化因子配体的表达。 目的：观察犬切牙压低移动过程中牙周组织中骨保护素/核因子κB受体活化因子配体的表达。 方法：采用微型种植体作为支抗,将犬切牙分别施加牵引力1,2,4,12周,并设置对照组进行比较。牵引后切取犬切牙连同牙龈及牙槽骨组织块,制作组织切片进行骨保护素及核因子κB受体活化因子配体免疫组织化学染色,用Image-Proplus软件半定量分析图像平均吸光度值。 结果与结论：与对照组相比,核因子κB受体活化因子配体和骨保护素分别在在施加牵引力1和2周表达最显著,其平均吸光度值在施加牵引力1和2周时可达到峰值(P < 0.05),随后逐渐下降,施加牵引力12周恢复至对照组水平。结果证实,正畸牙压低移动过程中核因子κB受体活化因子配体及骨保护素的表达变化规律与骨改建过程一致,骨保护素/核因子κB受体活化因子配体系统是牙周组织改建的重要调节因素。     

骨代谢中甲状旁腺激素对相关蛋白和核因子κB受体活化因子的调节

背景：甲状旁腺激素是影响骨代谢功能轴最主要的因素。同时也是调控骨保护素和核因子κB受体活化因子配基表达的重要激素。 目的：通过查阅甲状旁腺激素对骨保护素、核因子κB受体活化因子配基调控作用的相关文章,并对其进行综述。 方法：以“甲状旁腺激素,骨保护蛋白,核因子κB受体活化因子”或“Parathyroid hormone, osteoprotegerin, receptor activator of nuclear factor κB ligand”为检索词,由第一作者通过计算机检索 CNKI、HighWire数据库中关于“甲状旁腺激素与骨保护素/核因子κB受体活化因子配基/核因子κB受体活化剂”的相关的论文报告。选择的文章内容与甲状旁腺激素对信号通路的影响有关、近期发表的文献,按纳入和排除标准对文献进行筛选,共纳入31篇文章。 结果与结论：甲状旁腺激素是调节骨代谢最主要的因素,通过增强破骨细胞活动增强而实现的。核因子κB受体活化因子配基是调节骨吸收的关键因子,它通过与核因子κB受体活化剂结合发挥功能。核因子κB受体活化因子配基与核因子κB受体活化剂结合后,启动核因子κB受体活化因子配基信号转导,骨保护素则与核因子κB受体活化因子配基竞争性结合核因子κB受体活化剂,核因子κB受体活化因子配基/骨保护素比值决定破骨细胞分化、成熟及功能。长时间、大强度的运动条件下,机体内甲状旁腺激素的变化是否对于骨保护素、核因子κB受体活化因子配基有影响,进而调节骨代谢的吸收与合成？还有待进一步的研究。     

小分子肽对成骨前体细胞MC3T3-E1骨保护素和核转录因子κB受体活化因子配体表达的影响

背景：体内实验显示,小分子肽能明显增加去卵巢大鼠的骨钙含量,使其骨密度增加,能很好地预防骨质疏松。同时体外实验显示,小分子肽能促进小鼠成骨细胞和成骨前体细胞MC3T3-E1增殖、分化、矿化,并且可能是通过抑制核转录因子 p50和p65的表达来起作用。而小分子肽对骨保护素/核转录因子κB受体活化因子配体的影响尚不明确。 目的：观察小分子肽对MC3T3-E1在增殖、分化、矿化过程中骨保护素和RANKL表达的影响。 方法：以体积分数10%胎牛血清的DMEM培养液为空白对照组,50,100 mg/L质量浓度小分子肽作用小鼠成骨前体细胞MC3T3-E1,分别于作用3,6,12,18,24,30 d后,收集细胞提取蛋白,Western Blot检测骨保护素和核转录因子κB受体活化因子配体蛋白的表达。 结果与结论：50,100 mg/L小分子肽作用MC3T3-E1后能明显促进作用骨保护素的表达(P < 0.01),而对核转录因子κB受体活化因子配体无明显影响。小分子肽作用后MC3T3-E1中骨保护素/核转录因子κB受体活化因子配体的比值要明显高于空白对照组(P < 0.01)。因此,认为小分子肽可以通过增加骨保护素的表达来影响骨保护素/核转录因子κB受体活化因子配体系统,间接地抑制破骨细胞的数量和功能。     

中药骨康调控成骨细胞核内结合因子α1的表达

背景：中药骨康在临床上治疗骨质疏松疗效明确,但其具体作用途径尚不清晰。 目的：假设中药骨康通过调节核内结合因子a1水平,控制其下游基因核因子κB受体活化因子配体和骨保护素表达,起到调控成骨细胞生长发育的作用。 方法：新生24 h内的SD乳鼠用于成骨细胞的分离培养。成年SD雌性大鼠用于制备药物血清,随机分为正常血清组和中药骨康组。2组大鼠按体表面积的方法给予中药骨康方的提取药物和生理盐水灌胃,连续给药1周。最后一次用药后2 h行心脏采血,分离血清。原代培养并传至第3代经碱性磷酸酶鉴定取得的大鼠成骨细胞,消化计数铺板并分为2组,以上述血清处理72 h,MTT法检测成骨细胞的增殖率,ELISA方法检测碱性磷酸酶分泌量并以相应吸光度值进行纠正,运用RT-PCR检测2组成骨细胞核内结合因子α1及其下游核因子κB受体活化因子配体和骨保护素mRNA表达情况。 结果与结论：中药骨康组成骨细胞骨保护素和核内结合因子a1 mRNA水平显著高于正常血清组,核因子κB受体活化因子配体蛋白和mRNA水平显著低于正常血清组(P < 0.01)。实验结果证实,中药骨康可通过影响核内结合因子a1表达,调控其下游基因核因子κB受体活化因子配体和骨保护蛋白表达和分泌,进而发挥治疗骨质疏松的作用。     

双膦酸盐对破骨细胞分化及抗酒石酸酸性磷酸酶的影响

背景:抗酒石酸酸性磷酸酶是破骨细胞分化及骨吸收功能的特异性标志酶,是破骨细胞分化成熟的标志。目的:观察双膦酸盐对破骨细胞分化及骨吸收功能相关因子抗酒石酸酸性磷酸酶的影响。方法:小鼠单核巨噬细胞RAW264.7诱导培养破骨细胞。实验分2组:对照组开始时加入质量浓度100μg/L核因子kB受体活化因子配体进行诱导至收获细胞,双膦酸盐组在对照组的基础上加入10-7 mol/L阿仑膦酸盐处理至收获细胞。培养第7天检测各组破骨细胞生成和骨吸收功能,免疫荧光检测两组抗酒石酸酸性磷酸酶表达的差异,Western blot检测抗酒石酸酸性磷酸酶蛋白表达情况。结果与结论:各组细胞均有抗酒石酸酸性磷酸酶阳性多核破骨细胞生成,并在牙本质磨片上形成吸收陷窝;但对照组抗酒石酸酸性磷酸酶阳性多核细胞数目、吸收陷窝数目及陷窝面积均大于双膦酸盐组(P0.01)。免疫荧光检测显示,对照组抗酒石酸酸性磷酸酶表达均强于双膦酸盐组(P0.01)。Western blot检测显示,双膦酸盐组抗酒石酸酸性磷酸酶蛋白的表达低于对照组(P0.01)。说明双膦酸盐通过抑制抗酒石酸酸性磷酸酶蛋白的表达,阻碍破骨细胞分化生成及骨吸收功能。     

抗骨质疏松药物应用的依据：骨生化代谢标志物及骨组织病理学

背景：目前国际上公认双能X射线吸收测定法为诊断骨质疏松症的金标准,但常由于测量部位异位骨化、骨质增生等因素使得测量结果存在误差。目的：探讨骨代谢标志物在老年骨质疏松骨折诊疗中的临床意义以及它与骨密度和骨组织形态病理学改变的相关性。方法：选取50例需行手术治疗的老年骨质疏松骨折患者,行骨生化4项检测,其中抗酒石酸酸性磷酸酶5b(TRACP 5b)检测值明显增高患者25例(标记为抗酒石酸酸性磷酸酶5b升高组),骨碱性磷酸酶(BAP)检测值明显升高患者25例(标记为骨碱性磷酸酶升高组)。术中抽取两组各8例患者骨折断端部分骨组织,苏木精-伊红染色普通光镜检查和扫描电镜检查病理学改变。术后,抗酒石酸酸性磷酸酶5b升高组患者使用鲑鱼降钙素抗骨质疏松治疗,骨碱性磷酸酶升高组患者使用骨肽注射液抗骨质疏松治疗,6个月后再次检测骨密度和骨生化4项。结果与结论：两组患者术前骨密度和骨生化4项检查结果相比较差异无显著性意义(P > 0.05)。抗酒石酸酸性磷酸酶5b升高组患者骨折断端骨组织病理检查示成骨细胞减少、破骨细胞增多;骨碱性磷酸酶升高组患者骨折断端骨组织病理检查示成骨细胞减少;两组骨小梁/骨面积比值均降低,且抗酒石酸酸性磷酸酶5b升高组较骨碱性磷酸酶升高组降低程度差异有显著性意义(P < 0.05)。扫描电镜检查示两组破骨细胞都较正常组活跃,抗酒石酸酸性磷酸酶5b升高组骨小梁较骨碱性磷酸酶升高组稀松明显,吸收空泡增大。两组于术后使用抗骨质疏松药物治疗,两组治疗前与治疗后骨密度和骨生化4项检测结果差异有显著性意义(P < 0.05)。结果显示：①骨代谢标志物检测能明确患者骨组织是以成骨细胞功能和数量减低还是以破骨细胞功能和数量增加为主,以便指导临床针对性使用抗骨质疏松药物。②骨折断端骨组织形态病理学检查能更好地反映患者骨组织内成骨细胞、破骨细胞和骨小梁等状况。骨质疏松患者针对性使用抗骨质疏松药物治疗能提高疗效、降低相关并发症。     

Local injection/induction of osteoclasts for the treatment of calcified tendinitis

Calcified tendinitis is characterized by calcification in the rotary cuff tendon of the shoulder. The rationale of therapeutic methods is mainly removal of the calcification. Osteoclasts are the principle cells capable of resorbing bone and calcified tissues. Therefore, we hypothesized local injection of cultured human osteoclasts or induction of osteoclasts by recombinant RANKL in vivo may be effective for the treatment of calcified tendonitis. Human osteoclasts cultured in vitro is technically feasible and the osteoclasts are capable of active bone resorption. Thus, injection of osteoclasts may help remove the calcified tendonitis. In addition, human RANKL is commercially available. Therefore, local RANKL injection can recruit peripheral monocytes and macrophages. In the presence of RANKL, these monocytes and macrophages can subsequently differentiate into osteoclasts that can directly resorb calcification via their bone resorbing machinery. Different from the other treatments, the advantage of this therapeutic method includes: (1) less invasive because only local injection/induction of osteoclasts into the calcified lesion is conducted; (2) more efficient by direct osteoclasts injection or using RANKL to recruit osteoclasts to efficiently resorb calcification. Therefore, we proposed local injection/induction of osteoclasts as a potential biological method for clinical treatment of calcified tendinitis.     

Stimulatory effect of hydrothermally synthesized biodegradable hydroxyapatite granules on osteogenesis and direct association with osteoclasts

Calcium-deficient hydroxyapatite (HA) granules with a unique spherical shape were prepared using an applied hydrothermal method. Spherical stoichiometric HA granules were also prepared by normal sintering and both granules were used for implantation into rat tibiae to compare the biological responses to each implant. Twelve and 24 weeks after implantation, the volume of calcium-deficient HA granules was significantly less than that of stoichiometric HA granules, and the biodegradability of calcium-deficient HA granules was confirmed. The larger number of osteoclasts, larger osteoblast surface and larger bone volume in the implanted area of calcium-deficient HA than those of stoichiometric HA suggested that osteoclastic resorption of calcium-deficient HA affected osteogenesis in that area. To analyze the direct contribution of osteoclasts to osteogenesis, C2C12 multipotent myoblastic cells, which have the potential to differentiate into osteoblasts in the presence of bone morphogenetic protein 2, were cultured with supernatants of osteoclasts cultured on calcium-deficient HA, stoichiometric HA, β-tricalcium phosphate disks or plastic dishes, or bone marrow macrophages cultured on plastic dishes. Supernatants of osteoclasts but not bone marrow macrophages stimulated the expression of Runx2 and osteocalcin in C2C12 cells in concert with bone morphogenetic protein 2. The expression of alkaline phosphatase was stimulated with supernatants of osteoclasts cultured on ceramic disks. These results suggested that osteoclasts produced certain soluble factors which stimulated osteoblastic differentiation and they were thought to be associated with the induction of a larger osteoblast surface and bone volume in the animals implanted with calcium-deficient HA granules.     

Ultrastructural study of cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro

Many biochemical reports support cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro, however there have been few morphological studies supporting this. Details of cell-cell interaction between osteoclasts and osteoblasts/stroma cells remain unclear. The present study examined cell-cell interaction between osteoclasts and osteoblasts/stroma cells by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Osteoclasts, osteoblasts/stroma cells, and bone marrow cells obtained from 10-day-old ddY mice were cultured on dentin slices for 72 hr. Specimens were fixed, and some were examined by SEM. Specimens were decalcified, embedded in Epon after determination of the tartrate-resistant acid phosphatase activity (TRAP), and TRAP-positive cells for investigation were serially sectioned by alternating semithin and ultrathin sections, and then examined by TEM. By SEM, many cellular contacts were seen between the cells cultured on the dentin, but by TEM there were few special structures on the cell membranes between osteoclasts and osteoblasts/stroma cells, or between osteoclasts and bone marrow cells. A special structure on the cell membranes of osteoclasts was observed between an osteoclast and a cytoplasmic process of osteoblast/stroma cells, and this cell membrane was coated with electron dense or bristle-like structures. These bristle-like structures were very similar to those of coated pits. The present results show that the coated pit-like structure plays an important role in cell-cell interaction between osteoclasts and osteoblasts/stroma cells in vitro, and suggest that macromolecules binding to the osteoclast-surface receptor via ligands, accumulate in the coated pits, and enter the osteoclast as receptor-macromolecule complexes in endocytic vesicles.     

Inhibitory effect of Zn2+ in zinc-containing beta-tricalcium phosphate on resorbing activity of mature osteoclasts

Long term effect of the growing instability of the bone-implant interface due to bone resorption at the interface is a problem for the implants, including bioactive ceramics. Zn2+ -containing tricalcium phosphate (ZnTCP) is a material which may overcome this problem. The present study aims to clarify whether Zn2+ -containing tricalcium phosphate (ZnTCP) ceramics with a Zn2+ content of 0.316 (ZnTCP316) and 0.633 (ZnTCP633) wt % suppress resorption by mature osteoclasts in vitro. Suppression would be due to an increase in the number of apoptotic osteoclasts and the inhibition of the resorbing activity of osteoclasts, the latter being the major mechanism of the suppression. The number of apoptotic osteoclasts was significantly 6.3 times higher with ZnTCP633 than with tricalcium phosphate ceramic (TCP) after 24-h culture. The net contribution to resorption of this change in apoptotic cell numbers is much smaller than that of the change in resorbing activity. The osteoclasts cultured on ZnTCP formed fewer actin rings than those cultured on the TCP. The mRNA expression of CAII and cathepsin K/OC2 in the osteoclasts on ZnTCP633 was downregulated 0.5-fold and 0.6-fold, respectively, compared with that on the TCP. The volume of resorption pits was downregulated 0.4-fold in the ZnTCP633 than that in TCP. These results suggest that ZnTCPs suppressed the resorbing activity of mature osteoclasts probably through a local increase in the level of Zn2+. Bone substitutes or coating layers containing ZnTCP would be promising biomaterials from the viewpoint of counteracting osteoclastic bone resorption at the bone-implant interface.     

Osteoclast adhesion and activity on synthetic hydroxyapatite, carbonated hydroxyapatite, and natural calcium carbonate: relationship to surface energies.

This study investigates the adhesion, cytoskeletal changes, and resorptive activity of disaggregated rat osteoclasts cultured on polished slices of three biomaterials: crystalline synthetic hydroxyapatite (HA), carbonated hydroxyapatite (C-HA), and natural calcium carbonate (C). The surface chemistry of each substrate was defined by X-ray diffraction and IR spectroscopy, surface wettability by the dispersive, and the polar components of the surface energies. Osteoclast adhesion was modulated by the polar component of the surface energy: fewer (p < 0.01) osteoclasts adhered to C-HA (97 +/- 20/slice, surface energy 9 +/- 5 mJ/m2) than to HA (234 +/- 16/slice, surface energy 44 +/- 2 mJ/m2) or to C (268 +/- 37/slice, surface energy 58 +/- 0.5 mJ/m2). Actin rings, which are the cytoskeletal structure essential for resorption, developed on all three materials. The area of the actin ring, which is resorbed by local acidification, and the osteoclast area, which reflects osteoclast spreading, were both greater in osteoclasts cultured on HA and C-HA than in those cultured on C. C was resorbed, but HA and C-HA were not. Thus, the surface energy plays an essential role in osteoclast adhesion, whereas osteoclast spreading may depend on the surface chemistry, especially on protein adsorption and/or on newly formed apatite layers. Resorption may be limited to the solubility of the biomaterial.     

流体剪应力对大鼠破骨细胞骨吸收活性的影响

探讨流体剪应力对大鼠破骨细胞骨吸收活性的影响。我们采用低温离心法获取6月龄健康雌性SD大鼠椎骨骨髓细胞,以1.6×106细胞密度种植于血盖片上,采用1,25-(OH)2维生素D3体外诱导获取破骨细胞。于破骨细胞诱导的第7d取出细胞爬片,置于流体剪应力装置中,分别加载5.97、11.36、16.08、20.54dyne/cm2大小的流体剪应力,持续30min,以未加载流体剪应力的破骨细胞为对照组。实验结束时,以2.5%戊二醛固定细胞爬片,置于0.25mol/L氢氧化铵液超声处理10min,清除细胞爬片上的破骨细胞,经1%硪酸固定,梯度酒精脱水,醋酸异戊酯置换酒精,CO2临界点干燥,喷金后扫描电镜观察骨吸收陷窝并计数,图像分析法测定骨吸收陷窝的面积;同时分别收集每次灌流液10ml,冻干,用1ml复溶后,紫外分光光度仪检测抗酒石酸酸性磷酸酶(Tartrate-resistant acid phosphatase,TRAP)活性的变化。结果显示:在本实验中所选用的流体剪应力可增强破骨细胞TRAP活性,而骨吸收陷窝的数量和面积也增加,尤其是剪应力在16.08dyne/cm2时,破骨细胞TRAP活性增强以及骨吸收陷窝的数量和面积增高最为明显。结果表明,一定范围内的流体剪应力可以增强破骨细胞的骨吸收活性。     

Glutamate suppresses osteoclastogenesis through the cystine/glutamate antiporter

Previous studies have demonstrated functional expression of different glutamate receptor subtypes (GluRs) in both osteoblasts and osteoclasts. In the present study, we investigated the possible functional expression by osteoclasts of different glutamatergic signaling machineries including GluRs. In disagreement with the aforementioned prevailing view, no mRNA expression was found for all GluRs examined in primary cultured mouse osteoclasts differentiated from bone marrow precursors. Constitutive expression of mRNA was seen with glutamate transporters, such as excitatory amino acid transporters and cystine/glutamate antiporter, in primary osteoclasts. Glutamate significantly inhibited osteoclastogenesis at a concentration over 500 mumol/L in both primary osteoclasts and preosteoclastic RAW264.7 cells without affecting the cell viability in a manner sensitive to the antiporter inhibitor. In RAW264.7 cells stably overexpressing the cystine/glutamate antiporter, the inhibition by glutamate was more conspicuous than in cells transfected with empty vector alone. The systemic administration of glutamate significantly prevented the decreased bone mineral density in both femur and tibia in addition to increased osteoclastic indices in ovariectomized mice in vivo. These results suggest that glutamate may play a pivotal role in mechanisms associated with osteoclastogenesis through the cystine/glutamate antiporter functionally expressed by osteoclasts devoid of any GluRs cloned to date.     

白细胞介素1β对破骨细胞空泡型质子泵活性与胞内酸化的影响

目的:通过体外实验研究白细胞介素1β(IL-1β)对破骨细胞空泡型质子泵与细胞内pH值的影响,以探讨IL-1β促进破骨细胞骨吸收的机制。方法:骨髓单核巨噬细胞经过诱导成为破骨细胞后,予以0.1、0.3和0.5μg/L的IL-1β干预48 h,检测破骨细胞空泡型质子泵mRNA和蛋白的表达以及IL-1β对破骨细胞胞内酸化的影响,观察空泡型质子泵活性的改变与破骨细胞骨吸收功能的变化。结果:研究结果提示破骨细胞经IL-1β干预48 h后,空泡型质子泵的表达与活性均显著增加,并且细胞内pH值下降。破骨细胞与含有IL-1β的细胞培养液共同孵育后,其骨吸收能力增强,培养液中IL-1β的浓度越高,该作用越趋明显。结论:IL-1β可能是通过上调空泡型质子泵的表达与酶的活性,促进氢离子的产生和(或)转运,引发破骨细胞骨吸收活动增加,最终导致骨质破坏,由此参与骨关节炎症性疾病的发生。     

Suppressive effect of genistein on rat bone osteoclasts: involvement of protein kinase inhibition and protein tyrosine phosphatase activation

The suppressive effect of genistein on osteoclast-like multinucleated cells from rat femoral tissues was investigated. The bone cells isolated from rat femoral tissues were cultured for 48 h in an alpha-minimal essential medium (5% fetal bovine serum) containing either vehicle or genistein (10(-7)-10(-5) M). Osteoclasts were estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of genistein caused a significant decrease in the number of osteoclasts. Such a decrease was also seen in the presence of calcium choride (10(-5) M). Magnesium chloride (10(-5)-10(-3) M), a blocker of Ca2+ channels, had no effect on the number of osteoclasts. The effect of genistein (10(-5) M) or calcium (10(-3) M) in decreasing osteoclasts was significantly prevented by the presence of magnesium (10-3 M). Vanadate (10(-6)-10(-4) M), an inhibitor of protein tyrosine phosphatase activity, did not have an effect on the number of osteoclasts. The genistein's effect was not altered by vanadate. When isolated osteoclasts were cultured for 24 h in the presence of genistein (10(-7)-10(-5) M), protein kinase activity in the 5500 g supernatant of homogenate of the cells was significantly decreased, while protein tyrosine phosphatase activity was significantly elevated. Such an effect was also seen by the addition of genistein (10(-7)-10(-5) in the enzyme reaction mixture in vitro. The present study suggests that the suppressive effect of genistein on rat bone osteoclasts is partly involved in the inhibition of protein kinase and the activation of protein tyrosine phosphatase in osteoclasts.