钛-6铝-4钒颗粒对破骨细胞形态和功能的影响

目的在体外条件下观察钛-6铝-4钒（Ti-6AL-4V）颗粒对破骨细胞形态和功能的影响。方法取新西兰大耳兔破骨细胞培养在玻璃盖玻片和牛皮质骨片上，实验组用0．1mg／ml颗粒刺激，经不同时段培养后行抗酒石酸酸性磷酸酶（TRAP）染色，观察颗粒对破骨细胞形态的影响。对骨片上形成的骨吸收陷窝进行甲苯胺蓝染色观察，并用计算机图像分析软件评估吸收面积的大小。结果破骨细胞可以吞噬颗粒，形态变得不规则，特征性TRAP染色加深，较早出现凋亡征象。经颗粒刺激后在骨片上形成的骨吸收陷窝面积较大、数量较多。结论破骨细胞有吞噬功能，吞噬Ti-6AL-4V颗粒后发生形态和功能的变化，骨吸收功能增强。     

γ射线对人外周血单核细胞向破骨样细胞诱导分化的影响

目的 探讨电离辐射对人外周血单核细胞来源的破骨细胞体外诱导分化的影响。方法 将人外周血采用密度梯度离心法获得单核细胞,经核因子κB受体活化因子配体(RANKL)和巨噬细胞集落刺激因子(M-CSF)诱导培养24 h,分别给予0、0.75和2 Gy的¹³⁷Cs γ射线照射,第7天行TRAP染色后计数TRAP阳性多核破骨细胞,第10天取象牙骨片基质行甲苯胺蓝染色观察骨吸收陷窝形成;qRT-PCR检测破骨细胞特征标志物组织蛋白酶K和integrin β3的mRNA表达水平;ELISA法检测培养上清液中TRAcP-5b含量。结果 0.75 Gy照射组TRAP染色阳性多核破骨细胞计数显著多于对照组(0 Gy)(t=3.451,P<0.05),组织蛋白酶K和integrin β3的mRNA表达水平明显上调(t=2.343、2.728,P<0.05),且培养液中TRAcP-5b浓度明显增加(t=3.631,P<0.05)。而2 Gy照射组的TRAP染色阳性多核破骨细胞计数少于对照组,并伴有组织蛋白酶K和integrin β3的mRNA表达水平下调、培养上清液中TRAcP-5b浓度下降,但差异无统计学意义。结论 电离辐射可改变人外周血单核细胞破骨样细胞分化趋势,小剂量照射时可促进破骨细胞分化和骨吸收活性,而较大剂量照射后破骨细胞分化和骨吸收活性均降低。     

唑来膦酸钠对假膜组织细胞介导的骨溶解的抑制作用观察

目的 检验松动假体周围假膜组织中的细胞是否能够分化为破骨细胞并观察唑来膦酸钠(ZOL)对这一过程的影响.方法 采用酶消化法从假膜组织中分离细胞,应用免疫磁珠法将细胞分离成CD14+和CD14-细胞并在各种条件下分别培养.将CD14+细胞分为5组,其中A组培养液中加入巨噬细胞集落刺激因子(M-CSF),B组培养液中加入NF-κB受体活化因子配体(RANKL),C组培养液中加入M-CSF+RANKL,D组培养液中加入M-CSF+RANKL+ZOL(于培养开始时加入),E组培养液中加入M-CSF+RANKL+ZOL(于培养第10天时加入).CD14-细胞分为3组,其中F组培养液中加入M-CSF,G组培养液中加入RANKL,H组培养液中加入M-CSF+RANKL.CD14+和CD14-混合细胞分为I-M共5组,其培养液中加入的成分分别同A-E组.细胞分别接种于玻璃盖玻片(培养10d)及皮质骨磨片(培养14d)上,培养结束后检测抗酒石酸磷酸酶(TRAP)的表达及骨吸收陷窝的形成,以骨吸收陷窝面积为指标比较各组细胞的骨吸收活性.结果 CD14+细胞在缺乏RANKL(A组)或M-CSF(B组)时不能形成TRAP阳性多核细胞及骨吸收陷窝,在M-CSF和RANKL均存在的条件下(C组)可形成TRAP阳性多核细胞,并在皮质骨磨片上形成骨吸收陷窝(面积为8.10%±1.58%);于培养开始时加入ZOL(D组),骨吸收陷窝的面积(6.28%±1.40%)减少,与C组比较差异有统计学意义(P=0.043);于培养第10天加入ZOL,骨吸收陷窝面积(7.92%±1.64%)与C组比较差异无统计学意义(P=0.224).CD14-细胞在各种条件下培养均未观察到TRAP阳性多核细胞或骨吸收陷窝的形成.混合细胞在M-CSF和RANKL均存在的条件下(K组)可形成TRAP阳性多核细胞,并在皮质骨磨片上形成骨吸收陷窝(面积为3.30%±1.21%);于培养开始及第10天时加入ZOL(L组及M组),骨吸收陷窝的面积(分别为1.67%±0.46%、2.02%±0.45%)均减少,与K组比较差异有统计学意义(P=0.006,P=0.023).结论 假膜组织中的CD14+巨噬细胞能够分化成为具有骨吸收活性的破骨细胞;ZOL可抑制假膜组织介导的溶骨作用.     

低氧对破骨细胞活性的影响及其机制

目的:观察低氧对乳兔破骨细胞分化、活性的影响,并研究其可能的机制.方法:培养乳兔破骨细胞、成骨细胞,TRAP染色鉴定破骨细胞,比较破骨细胞在常氧(含氧量20％)及低氧(含氧量3％)条件下骨吸收陷窝面积.破骨细胞加入到第3代成骨细胞中分别于常氧和低氧条件下进行共培养,第24、48、72、96 h检测RANK、OPG、RANKL、TRAP及CtsK mRNA的表达.结果:各时间点低氧组RANK、RANKL、CtsK、TRAP mRNA表达均高于常氧组;OPG mRNA的表达低于常氧组组,具有统计学差异(P＜0.05或P＜0.01).结论:低氧可激活破骨细胞TRAP和CtsK基因的上游信号,增强破骨细胞活性.     

人破骨细胞的体外培养及鉴定

 目的:探索人破骨细胞的体外培养及鉴定方法,为各类试验研究提供大量高活性破骨细胞.方法:分离骨髓血得到单个核细胞,用不同培养液、诱导剂在不同浓度下培养破骨细胞,通过TRAP染色鉴定破骨细胞形态,ELISA法检测特异性蛋白表达水平,扫描电镜分析骨片骨陷窝面积.结果:用α-MEM培养液,10(-7)mmol/L浓度的1,25(OH)2VD3或50ng/ml浓度的RANKL和30ng/ml的M-CSF作为诱导剂,所培养的破骨细胞多核、特异性蛋白表达水平高、蚀骨能力强.结论:用DMEM培养液和α-MEM培养液培养均是一种能在体外诱导生成大量人破骨细胞的方法.同时,建议采用1,25(OH)2VD3作为诱导剂.     

模拟失重条件下小鼠骨组织尿卟啉原Ⅲ合酶通过增加破骨细胞活性促进骨量流失

目的 探究尿卟啉原Ⅲ合酶（UROS）对失重性骨丢失的影响。方法 将8只C57BL/6J雄性小鼠（9周龄分为对照组和尾吊组（HLU）,每组4只。小鼠尾吊28 d后,取小鼠后肢分别进行Micro CT和力学实验检测。利用旋转式细胞培养系统（RCCS）建立失重的破骨细胞模型,通过实时定量PCR(qPCR)和抗酒石酸酸性磷酸酶（TRAP）染色明确失重导致破骨细胞活性的变化,qPCR和Western印迹检测UROS表达水平变化;构建UROS敲除RAW264.7稳定株,通过qPCR和TRAP染色进一步明确UROS缺失后破骨细胞活性的变化。结果 小鼠尾吊后骨组织骨量流失明显,UROS的蛋白表达水平显著上调;破骨细胞失重处理后,UROS的蛋白表达水平上调,并且破骨分化基质金属蛋白酶9、抗酒石酸酸性磷酸酶等表达水平上调。敲低UROS后,破骨细胞分化相关标志物表达水平显著下调。结论 失重应激条件下,破骨细胞中UROS表达水平上调,进而导致破骨细胞活性增强,骨吸收大于骨形成,从而导致骨量流失。     

力学拉伸强度对破骨细胞形成和分化的影响

目的 探讨早期力学拉伸强度对破骨细胞形成和分化的影响. 方法 对采用巨噬细胞集落刺激因子和破骨细胞分化因子诱导的RAW 264.7细胞分别施加0,1 000,1 500,2 000,2 500和5 000με的力学拉伸3 d.于培养第7天观察各组破骨细胞形态、计数及检测前体细胞增殖,并于培养第4,7天检测各组细胞培养液中抗酒石酸酸性磷酸酶(TRAP)活性. 结果 2 500με组的破骨细胞形成数量明显减少,5 000με组的破骨细胞数量显著增加;2 000,2 500 με组前体细胞增殖明显,5 000με组前体细胞增殖显著降低;1 000,1 500με组与静态组在破骨细胞形成数量和前体细胞增殖方面差异无统计学意义.与静态组相比,应变组第4,7天培养液的破骨细胞TRAP活性均降低. 结论 早期力学因素可直接影响破骨细胞的形成和分化,较高强度的生理载荷可抑制破骨细胞形成,病理过载促进破骨细胞的形成,而低强度载荷几乎不影响破骨细胞形成;早期力学载荷可抑制破骨细胞分化.     

运动调节破骨细胞活性改善溶骨性疾病研究进展

破骨细胞是来源于骨髓造血干细胞的骨吸收细胞,其职责在于清除老化受损的骨基质,通过与成骨细胞的紧密耦合来维持骨重塑平衡。然而,由破骨细胞过度形成与活化引起的溶骨性疾病已成为全球性的公共卫生问题,如骨质疏松症、骨关节炎、类风湿性关节炎、骨肉瘤骨病和佩吉特式骨病等。在此背景下,运动被视为一种调节骨骼细胞命运的重要手段,通过多种微环境信号来协调骨骼的合成与分解代谢平衡,缓解因过度骨吸收而导致的骨破坏。本文以运动引起的机械与分子信号为线索,系统总结了运动调控破骨细胞产生与骨吸收活性的分子机制,并总结了运动在改善溶骨性疾病中的作用,为通过规律运动防治骨流失相关疾病提供理论依据。     

上、下坡跑台运动对去卵巢小鼠破骨细胞分化NF-κB信号通路的影响

目的:通过去卵巢手术制备小鼠骨质疏松模型,对比研究上、下坡跑台运动对去卵巢小鼠破骨细胞分化的影响,以及NF-κB信号通路在介导运动对破骨细胞分化影响过程中的作用。方法:32只8周龄C57 BL/6雌性小鼠被随机分为4组:假手术安静组(S组)、去卵巢安静组(O组)、去卵巢上坡跑组(U组)和去卵巢下坡跑组(D组)。U组和D组小鼠于摘除卵巢一周后分别进行为期8周的上坡跑和下坡跑训练,每周训练5次,每次训练40 min,跑台坡度分别为±9°,跑速均为0.8 km/h。S组和O组在笼中正常饲养,不进行任何训练。8周后对股骨组织进行抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色检测,并对其骨髓进行破骨细胞原代培养,检测破骨细胞分化过程中NF-κB信号通路相关蛋白的表达。结果:各组小鼠股骨TRAP阳性染色区域的平均光密度值和平均阳性染色面积百分比存在显著差异,表现为O组显著高于S组,两运动组显著低于O组,且D组显著低于U组。各组小鼠破骨细胞前体IκBα和p65蛋白相对表达量无显著性差异;而p-IκBα和p-p65蛋白相对表达量组间差异显著,表现为O组显著高于S组,两运动组显著低于O组,且D组显著低于U组。结论:小鼠去卵巢后骨组织破骨细胞特异性酶TRAP大量生成,显示去卵巢手术可引起小鼠骨组织破骨细胞的大量分化和活化,而跑台运动可通过抑制破骨细胞分化过程中p65和IκBα蛋白的磷酸化而有效抑制破骨细胞分化NF-κB信号通路,从而有效抑制骨吸收,且下坡跑效果优于上坡跑。     

骨桥蛋白对RANKL诱导破骨细胞分化的影响

 目的 初步探讨骨桥蛋白对破骨细胞体外分化的影响。方法 体外培养小鼠破骨前体RAW264.7细胞,RANKL诱导刺激其分化为破骨细胞,并加入骨桥蛋白进行干预。MTT法及TRAP染色分别检测破骨细胞细胞增殖及前体破骨细胞分化情况,RT-PCR检测RANK的mRNA表达水平。结果 骨桥蛋白可明显促进RAW264.7细胞向破骨细胞分化、增加细胞增殖,与对照组相比差异具有统计学意义(P＜0.05),并使破骨细胞特征性基因RANK的mRNA表达上调约2.67倍。结论 骨桥蛋白可能通过影响RANK/RANKL信号通路促进RAW264.7细胞分化为破骨细胞,有望成为防治骨质疏松等骨相关疾病的新靶点。
     

Physiopathological basis of bone turnover.

Bone remodeling involves the continuous removal of bone (bone resorption) followed by synthesis of new bone matrix and subsequent mineralization (bone formation). The principal cells that mediate the boneforming processes of the skeleton are osteoblast cells. They are responsible for the production of the matrix constituents and the differentiation of osteoblasts from stromal cell precursors is stimulated by several hormonal and non-hormonal molecules. On the other hand, the osteoclasts are giant multinucleated cells responsible of bone resorption. They are formed in the bone marrow and mature cells are stimulated by PTH and locally acting agents such as transforming growth factor alpha (TGFalpha), tumor necrosis factor (TNF) interleukin 1 (IL-1) and interleukin 6 (IL-6). The first events during bone remodeling is osteoclast activation, followed by osteoclast formation, polarization constitution of the ruffled border, resorption and ultimately apoptosis. Osteoclast apoptosis is followed by a series of sequential changes in cells in the osteoblast lineage, including osteoblast chemotaxis, proliferation and differentiation, which in turn is followed by formation of mineralized bone and cessation of osteoblast activity. The final phase of the formation process is cessation of osteoblast activity. The resorption lacunae are usually repaired either completely or almost completely. Understanding the sequence of cellular events may be important to better know the mechanisms responsible for bone loss that occurs in age and in several pathological conditions.     

破骨细胞在强直性脊柱炎滑膜组织中的分化及其在外周关节骨质破坏病理机制中的作用

目的检测破骨细胞在强直性脊柱炎（AS）外周关节滑膜组织中的分化情况,了解破骨细胞的分化与AS患者外周关节骨质破坏病理改变的相关性。方法应用单克隆抗体,通过免疫组织化学及酶组织化学染色的方法检测13例AS、16例类风湿关节炎（RA）、17例骨关节炎（OA）及6例健康对照关节滑膜组织中CD68蛋白表达及TRAP染色阳性滑膜细胞的分布状况,并通过计算机辅助图像分析系统和半定量分析方法确定CD68分子表达水平及TRAP染色阳性细胞在各研究组滑膜组织中的差异性。结果滑膜组织中均有CD68阳性细胞,AS和RA组CD68表达水平较OA组和健康对照组显著增高（P〈0．05）,其阳性细胞主要分布于滑膜衬里层,衬里下层也见少量表达;OA和健康者滑膜中仅有少量CD68阳性细胞分布。TRAP染色阳性细胞位于滑膜组织衬里层、淋巴细胞聚集区及滑膜软骨交界区,其中AS组阳性细胞百分数显著低于RA组,OA及正常对照组阳性细胞少见。RA组阳性细胞百分数与RANKL表达水平呈正相关（r=0．442,P=0．043）。结论炎症关节滑膜组织中表达CD68分子及TRAP染色阳性滑膜细胞数量的增加为破骨细胞的分化、成熟提供了充足的细胞数量基础,破骨细胞数量和活性上调是造成强直性脊柱炎外周关节骨质破坏的重要前提。     

运动性骨疲劳的模拟实验研究

目的 :探讨运动性骨疲劳发生发展的机理。方法 :实验雄性成年兔 2 5只 ,运用高电压、低电流动物刺激仪 ,刺激实验兔进行主动跑跳训练 ,成功地模拟出运动性骨疲劳的动物模型。结果 :2周训练后 ,X线及核素骨显像均呈阴性 ,血甲状旁腺素 (PTH)和血骨钙素 (BGP)显著高于正常组 ,血睾酮 (T)显著低于正常组 ,胫骨皮质骨吸收腔显著增加 ,组织学表现为破骨细胞增加 ;电镜观察骨细胞呈吸收相。继续跑跳训练( 3 -4周 )可使疲劳向损伤转化 ,X线出现轻微骨膜反应 ,核素骨显像呈现阳性 ,血生化及组织学出现应力性损伤的变化。结论 :运动性骨疲劳的发生是一个由骨细胞直接参与的破骨细胞性重吸收大于成骨细胞性骨形成的生理过程 ;高血PTH、高血BGP和低血T是其发生发展过程中的血生化基础     

女性体力活动水平和骨矿物质含量及骨代谢关系的研究

目的 :探讨成年女性体力活动水平与骨矿含量和反映骨代谢有关生化指标的关系。方法 :以江门市 2 2 0位健康成年女性作为研究对象 ,采用MOSPA体力活动问卷评价其体力活动水平 ,并测量骨矿含量以及血清钙、磷、碱性磷酸酶、酸性磷酸酶、非前列腺酸性磷酸酶及尿钙和尿肌酐比值等反映骨代谢的生化指标的水平。结果 :校正有关混杂因素后 ,与低体力活动组相比 ,高体力活动组女性骨矿含量和骨皮质系数增加 ,血清碱性磷酸酶含量增加 ,酸性磷酸酶和非前列腺酸性磷酸酶含量下降。结论 :与低体力活动组比较 ,高体力活动组女性骨矿含量增加 ,骨形成较活跃 ,而骨吸收减少。     

He-Ne激光对正畸牙根吸收影响的组织形态学研究

 目的通过组织形态学观察He-Ne激光照射对实验性牙移动根吸收的影响与作用。方法选用Wistar大鼠建立根吸收的动物模型。利用HE和TRAP酶染色定量根吸收面积并进行破牙骨质细胞计数,对照射组与对照组大鼠牙移动过程中根吸收的严重程度进行分析比较。结果根吸收最早出现在加力4 d组,7 d时破牙骨质细胞开始增多,照射组细胞数低于对照组（P<0.05）;14 d时根表面破牙骨质细胞数达高峰,照射组细胞数明显低于对照组（P<0.01）;21 d时根吸收基本停止,照射组根吸收面积明显低于对照组（P<0.01）,并在照射组观察到根吸收的修复现象。结论He-Ne激光的局部照射可有效地减少实验性牙移动根吸收的程度。     

Clinical utility of biochemical marker of bone remodelling in patients with bone metastases of solid tumors.

Bone turnover is characterized both by the formation of new bone by the osteoblasts and the resorption of old tissue by the osteoclast. This process takes place only on the surface of bone and can be described in terms of spatio-temporal events that are the bone metabolic unit and the bone remodelling cycle. The former consists of a discrete group of cells (osteoblasts and osteoclasts) involved in a particular remodelling event while the latter represents the succession of resorption and formation. In a typical remodelling cycle, resorption takes 7-10 days, whereas formation requires 2-3 months. Remodelling is regulated either by local or systemic factors, including electrical and mechanical forces, hormones (e.g. parathyroid hormone, sexual steroids, calcitriol, cortisol, thyroid hormones, calcitonin), growth factors and cytokines. Recently different circulating biochemical markers have been proposed for the investigation of bone turnover. In addition to classical parameters such as serum alkaline phosphatase and urinary calcium and hydroxyproline, new markers have gained clinical attention because of their accuracy in assessing the dynamic changes in bone remodelling (bone alkaline phosphatase, osteocalcin, propeptides PICP and PINP, tartrate-resistant acid phosphatase, deoxypyridinoline, pyridinoline, telopeptide CTx and NTx). The aim of this review is to present the recent advances in this field and the clinical application of markers of bone turnover in patients with bone metastases from solid tumors. Also the cellular and molecular bases of bone remodelling are reported with details.     

Large gradient high magnetic field affects FLG29.1 cells differentiation to form osteoclast-like cells

Abstract

Purpose: We aimed to investigate the effects of different apparent gravities (μ g, 1 g and 2 g) produced by large gradient high magnetic field (LGHMF) on human preosteoclast FLG29.1 cells.

Materials and methods: FLG29.1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. Cells were exposed to LGHMF for 72 h. On culture day 1, 2, 3, cell proliferation was detected by 3-(4,5)-dimethylthiahi-azo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. On day 3, cell apoptosis and necrosis were assayed by Hoechst and propidium iodide (PI) staining. After cells were exposed to LGHMF for 72 h with the induction of 12-o-tetradecanoylphorbol 13-acetate (TPA), Tartrate-Resistant Acid Phosphatase (TRAP) positive cells and nitric oxide (NO) release were detected by TRAP staining and Griess method, respectively. Intracellular TRAP activity was measured using nitrophenylphosphate (pNPP) as the substrate.

Results: MTT detection revealed that compared to control, FLG 29.1 cell proliferation in the μ g and 2 g groups were promoted. However, there is no obvious difference between the 1 g and control groups. Hoechst-PI staining showed that LGHMF promoted cell apoptosis and necrosis, especially in the 2 g group. Exposure to LGHMF inhibited the NO concentration of supernatant. Both the TRAP activity and the number of TRAP positive cells were higher in cells of μ g group than those in 2 g group. In the 1 g group, they were decreased significantly compared to control.

Conclusions: These findings indicate that LGHMF could directly affect human preosteoclast FLG29.1 cells survival and differentiation. High magnetic flux inhibited osteoclasts formation and differentiation while reduced apparent gravity enhanced osteoclastogenesis.     

Histomorphologic study of the bones of monkeys after 14-day anti-orthostatic hypokinesia

Using a semi-automatic image analyzer MOP Videoplan, a quantitative histomorphometric investigation of sponge bone of tibial metaphyses, iliac crests and lumbar vertebrae of monkeys kept for 14 days in the head-down position (at -10 degrees) was carried out. This exposure was found to cause spongy bone reduction in all the above bones. The lowered number and activity of osteoblasts as well as the reduced amount of osteoids during the exposure give evidence that the inhibition of bone neoformation makes a significant contribution to osteopenia. The involvement of osteoclast resorption in the spongiosa reduction during head-down tilt needs further study because in our experiments the osteoclast number or acid phosphatase activity did not grow significantly.