破骨细胞相关受体研究进展

人破骨细胞相关受体是一种细胞表面分子,属白细胞受体复合物编码的家族成员,广泛表达于破骨细胞、单核细胞、巨噬细胞、单核源树突状细胞等髓系来源细胞,与破骨细胞分化过程中的重要调控因子相互作用,参与破骨细胞分化,在骨代谢中发挥重要作用,受多种因素调节。同时,破骨细胞相关受体与Fc受体γ链特异性结合,调节免疫反应。     

大麻素受体CB2在机械牵张力介导的人牙周膜细胞成骨分化中的作用

目的:研究大麻素Ⅱ型受体(cannabinoi dreceptor Ⅱ,CB2)在机械牵张力介导的人牙周膜细胞中的表达以及成骨分化中的作用。方法:体外培养人牙周膜细胞,构建细胞-机械牵张力加载模型,施加不同大小的机械牵张力,采用Real-timePCR和细胞免疫荧光化学技术检测CB2在人牙周膜细胞中mRNA和蛋白的表达。用碱性磷酸酶(ALP)试剂盒检测机械牵张力介导的细胞ALP活性。结果:对人牙周膜细胞施加不同大小的机械牵张力24h,CB2 mRNA的表达随机械牵张力的力值增大而显著性增加(P<0.05),在18%拉伸应变率作用下表达量最高(P<0.05),此时CB2蛋白的表达显著增加。加入CB2激动剂HU-308后,施加18%拉伸应变率的机械牵张力作用于人牙周膜细胞24h,ALP活性显著性增加(P<0.05)。结论:CB2在人牙周膜细胞中的表达与机械牵张力的力值具有相关性。在机械牵张力作用下,大麻素受体CB2与其配体结合能够促进人牙周膜细胞的成骨分化,从而在正畸牙槽骨改建中发挥重要作用。     

培养基对小鼠单核巨噬细胞RAW264.7诱导分化破骨细胞的影响

目的 探讨培养基对小鼠单核巨噬细胞RAW264.7向破骨细胞分化的影响。方法 实验分为3组：高糖DMEM培养基组（DMEM组）;高糖DMEM/α-MEM培养基组（DMEM/α-MEM组）;α-MEM培养基组（α-MEM组）。按常规方法采用上述3种培养基进行破骨细胞培养,培养5 d后,采用抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase, TRAP）染色观察各组破骨细胞的形成情况,并采用实时荧光定量PCR方法观察破骨细胞分化相关标志物NFATc-1、c-Fos和TRAF-6 mRNA的表达;培养11 d后采用甲苯胺蓝染色进行骨陷窝面积分析,观察破骨细胞骨吸收功能情况。结果 3组均可以观察到典型的TRAP+破骨细胞。与DMEM组相比,DMEM/α-MEM组、α-MEM组TRAP+破骨细胞数量明显增加（P<0.01）,但各组形态略有不同。在骨吸收功能上,与DMEM组和DMEM/α-MEM组相比,α-MEM组骨陷窝面积明显增加（P<0.01）。在破骨细胞分化相关调控因子表达上,与DMEM组相比,α-MEM组NFATc-1、c-Fos 和TRAF-6 mRNA表达显著增加（P<0.01,P<0.05）;DMEM/α-MEM组NFATc-1 mRNA表达显著增加（P<0.01）,c-Fos 和TRAF-6 mRNA表达有增加的趋势,但差异无统计学意义;α-MEM组与DMEM/α-MEM组比较差异无统计学意义。结论 高糖DMEM培养基、高糖DMEM/α-MEM培养基、α-MEM培养基均可用于小鼠单核巨噬细胞RAW264.7诱导分化破骨细胞的实验;从破骨细胞数量、状态及功能来看,α-MEM培养基更适合做为破骨细胞分化培养基。     

CTGF对破骨前体细胞RAW264.7增殖及碳酸酐酶Ⅱ分化的影响

目的研究结缔组织生长因子(CTGF)对体外培养的破骨细胞前体细胞RAW264.7增殖及对核因子Kappa B配体受体(RANKL)诱导体外培养的破骨细胞前体细胞RAW264.7分化为成熟多核破骨细胞的影响。方法使用200 ng/mLCTGF干预培养的破骨细胞前体细胞RAW264.7,采用3H-TdR掺入法检测RAW264.7细胞增殖率;使用200 ng/mL CTGF与RANKL单独或共同处理RAW264.7细胞,抗酒石酸酸性磷酸酶(TRAP)染色观察TRAP阳性多核细胞,Western blot检测碳酐酶Ⅱ蛋白的表达。结果 CTGF可显著促进RAW264.7细胞增殖;200 ng/mLCTGF与RANKL共同处理RAW264.7细胞可促进RAW264.7细胞分化为成熟多核破骨细胞;200 ng/mL CTGF与RANKL共同处理RAW264.7细胞可促进RAW264.7细胞碳酐酶Ⅱ蛋白的表达。结论 CTGF促进体外培养的破骨细胞前体细胞RAW264.7增殖,促进RANKL诱导的破骨细胞前体细胞RAW264.7分化为成熟多核破骨细胞。     

骨保护素及其配体对破骨细胞功能的调控作用

骨保护素及其配体的发现是近年来骨代谢基础研究方面的重要进展，它从分子水平阐明了破骨细胞的分化和激活过程以及骨髓基质微环境在其中所起的重要作用，作为破骨细胞的终末调控因子，骨保护素及其配体也是其它细胞因子调控骨代谢的关键作用环节，具有很高的临床应用价值。本文对此做一简要综述。     

骨细胞对破骨细胞形成和功能的影响

破骨细胞的骨吸收作用和成骨细胞骨形成作用的交替进行维持了骨量的平衡。破骨细胞可以选择性吸收损伤部位的骨质,其激活和定位机制目前还未阐明。近年来的研究认为骨细胞是感知骨环境的基本单位,而且骨细胞还可以将所感知的信号传递给其它骨细胞,骨衬细胞,成骨细胞及破骨细胞等。对骨细胞和破骨细胞的研究中发现骨细胞可能在破骨细胞的激活和定位中起到了重要的作用,但是具体机制还有待研究。     

细胞因子RANKL对破骨细胞的分化调节作用

骨是一个代谢活跃的器官 ,在整个生命周期中都在进行不断的更新与改建。其形态发生和重建由两个对立统一的过程所控制 :破骨细胞 (ｏｓｔｃｏｃｌａｓｔ,ＯＣ)介导的骨吸收过程和成骨细胞 (ｏｓｔｅｏｂｌａｓｔ,ＯＢ)介导的骨基质合成过程。ＯＣ、ＯＢ之间功能的失调将导致骨骼形态结构的异常 ,如骨质疏松时骨量减少 ,而骨硬化症时骨量增多。Ｕｄａｇａｗａ等[1 ] 首次发现 ,许多激素、细胞因子影响ＯＣ的分化、激活 ,而这种作用是由ＯＢ/ＳＴ介导的 ,并且是通过细胞膜与细胞膜的直接接触或旁分泌机制而实现的[2 ,3 ] ,但其分子学基础一直…     

RANKL诱导小鼠单核细胞RAW264.7分化成成熟破骨细胞

目的观察小鼠的单核／巨噬细胞RAW264．7的一般生物学特征及在RANKL诱导下形成成熟破骨细胞的特征。方法RANKI，诱导RAW264．7细胞6d后，用抗酒石酸酸性磷酸酶(TRAP)染色法观察TRAP阳性多核细胞，吖啶橙染色激光共聚焦显微镜(LCSM)观察多核细胞形态；诱导RAW264．7细胞9d后，RT、PCR检测RAW264．7细胞的破骨细胞表型和功能基因表达及其RANKL诱导后变化；诱导RAW264．7细胞12d后，钙磷覆盖的破骨细胞活性分析板观察破骨细胞的骨吸收功能。结果RAW264．7细胞TRAP染色阴性，单核或2个核，能表达破骨细胞表型和功能基因，无骨吸收功能。RANKL可诱导RAW264．7细胞形成TRAP阳性成熟的多核破骨细胞，上调CathepsinK、CAⅡ、integrinβ3等基因mRNA的表达。结论RAW264．7具有破骨细胞特征性基因表达谱，是一种较好的破骨前体细胞模型。RANKL可诱导RAW264．7细胞形成成熟破骨细胞。     

The immunosuppressant rapamycin,alone or with transforming growth factor-beta,enhances osteoclast differentiation of RAW264.7 monocyte-macrophage cells in the presence of RANK-ligand

Immunosuppressant therapy is known to cause bone loss. Since this may partly result from direct effects on osteoclast development, we investigated whether cyclosporin A (CsA), rapamycin, or FK506 affect osteoclastic differentiation of RAW264.7 monocytic cells induced by RANK-ligand (RANKL). Furthermore, since the rapamycin receptor protein binds transforming growth factor beta (TGF-beta) receptors, and TGF-beta enhances osteoclastogenesis induced by RANKL, we also examined potential synergistic effects of rapamycin and TGF-beta1. Rapamycin inhibited cell proliferation and stimulated tartrate-resistant acid phosphatase (TRAP) activity of RAW cells in a dose-dependent manner. At the optimal concentration of 10 ng/ml, it increased the number of TRAP+ multinucleated cells (MNC) more than 20-fold and enhanced the expression of TRAP and calcitonin receptor (CTR) mRNAs 2.1- and 10-fold, respectively. CsA, at 125-2000 ng/ml, similarly inhibited proliferation, but at high doses (1000-2000 ng/ml) it decreased TRAP activity, TRAP+MNC formation, and the expression of TRAP and CTR mRNAs. FK506 had no effect on cell proliferation or TRAP activity at concentrations up to 2000 ng/ml; however, like CsA, 1000 ng/ml FK506 inhibited TRAP+MNC formation and the expression of TRAP and CTR mRNAs. The combination of rapamycin (10 ng/ml) and TGF-beta1 (1 ng/ml) increased TRAP+MNC 3.1- and 6.9-fold as compared with rapamycin or TGF-beta1 alone, respectively, and enhanced CTR mRNA expression induced by TGF-beta1 by 1.9-fold. Rapamycin also increased osteoclastic resorption activity by 6.5-fold compared with control, and this was enhanced further by the addition of TGF-beta by 3-fold, compared with rapamycin alone. These data thus indicate that rapamycin, alone or in synergy with TGF-beta, directly enhances osteoclastogenesis and may affect bone metabolism in vivo after long-term use.     

组蛋白修饰在破骨细胞分化中的作用

破骨细胞(osteoclast,OC)是来源于造血干细胞的多核巨细胞,是机体内唯一具有骨吸收功能的细胞.OC功能失衡与多种骨代谢疾病的发生发展密切相关,如骨质疏松症、骨关节炎和Paget's病等,常作为骨代谢疾病临床治疗和药物研发的靶细胞.组蛋白(histone,H)是细胞核内序列高度保守的蛋白质,组蛋白修饰是指组蛋白...     

The role of osteoclast differentiation in aseptic loosening.

The major cause of orthopaedic implant loosening is thought to be accelerated osteoclastic bone resorption due to the action of cytokines produced in response to phagocytosis of implant-derived wear particles. This accelerated osteoclastic bone resorption could be due to increases in any of the following processes: recruitment of osteoclast precursors to the local microenvironment, differentiation of precursors into mature multinucleated osteoclasts. activation of mature osteoclasts, and/or survival of osteoclasts. Our studies have focused on differentiation and survival to complement work by others who have focused on recruitment of precursors and activation. Taken together, our studies and those of other investigators provide strong evidence that increased recruitment of osteoclast precursors and their subsequent differentiation play major roles in wear particle-induced osteolysis. In contrast, increased osteoclast activation and survival appear to play minor roles. These studies suggest that development of therapeutic interventions that reduce either recruitment or differentiation of osteoclast precursors would improve the performance of orthopaedic implants.     

小鼠单核巨噬细胞白血病细胞（RAW264.7）的培养及其在诱导破骨细胞中的应用

小鼠单核巨噬细胞白血病细胞(RAW264.7)是通过Abelson鼠白血病病毒诱导BALB/c小鼠产生肿瘤后得到的细胞株。RAW264.7细胞在医学研究中应用十分普遍:它是许多白血病相关研究模型的常用细胞株;在微生物学、免疫学等研究中也十分常用;同时也是小鼠源性破骨前体细胞,可通过特定细胞因子的诱导在体外获得成熟破骨细胞,因此也成为许多骨骼疾病研究的体外模型。但RAW264.7细胞形态和分化状态多变,在实际培养中较难把握,给科研工作带来了一定困难。科研工作者在RAW264.7细胞的培养方法、细胞的形态及分化状态等方面始终观点不一,对于用RAW264.7细胞在体外诱导获得破骨细胞的方法也有许多差别。本文结合文献资料及实践,探索了RAW264.7细胞的培养条件,冻存、复苏、传代方法,以及用核因子κB受体活化因子配基(receptor activator for nuclear factor-κB ligand,RANKL)诱导其成为成熟破骨细胞的技术关键;旨在总结RAW264.7细胞的培养及诱导其分化为破骨细胞的经验教训,探讨RAW264.7细胞的培养和在体外用RANKL诱导RAW264.7细胞分化为破骨细胞的方法、技巧,供广大科研工作者借鉴。     

Effects of selective prostaglandin EP4 receptor antagonist on osteoclast formation and bone resorption in vitro.

Prostaglandin estradiol (PGE(2)) stimulates bone resorption by a cyclic AMP (cAMP)-dependent mechanism that involves prostaglandin E receptors of the EP2 and EP4 subtypes. We tested a potent selective EP4 antagonist (EP4RA), which blocks PGE(2) binding to EP4 receptors. We examined the effects of EP4RA on osteoclastogenesis in murine marrow cultures, on cAMP production in primary osteoblastic (POb) cell cultures, and on bone resorption in organ cultures. EP4RA (1 micromol/L) decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+) MNC) by 46%-48% in cultures treated with 0.1-1.0 micromol/L PGE(2) and by 96% in cultures treated with 0.01 micromol/L PGE(2). EP4RA also decreased TRAP(+) MNC formation by 60% in 1,25-dihydroxyvitamin D (1,25D)-treated cultures and by 62% in parathyroid hormone (PTH)-treated cultures. A chemically related analog of EP4RA that lacks antagonist activity did not inhibit TRAP(+) MNC formation. EP4RA decreased cAMP production in PGE(2)-treated POb by 44% but did not block cAMP response to PTH. EP4RA inhibited the increase in receptor activator of NF-kappaB ligand (RANKL) mRNA levels produced by PGE(2). In fetal rat long bone cultures, EP4RA decreased 45Ca release from control, unstimulated cultures by 12%-25% and from PGE(2)-stimulated cultures by 22%-37%. Because EP4RA partially inhibited osteoclastogenesis not only in response to PGE(2) but also in response to 1,25D and PTH, these results suggest that activation of the EP4 receptor may play a general role in osteoclastic bone resorption. EP4RA showed partial inhibition of PGE(2)-stimulated osteoclastogenesis at 1 micromol/L, but almost complete inhibition at 0.01 micromol/L PGE(2). This could be due to the limited efficacy of the antagonist at high concentrations of PGE(2), or an alternative pathway, such as activation of the EP2 receptor.     

Treatment with hydrogen molecules prevents RANKL-induced osteoclast differentiation associated with inhibition of ROS formation and inactivation of MAPK,AKT and NF-kappa B pathways in murine RAW264.7 cells

The bone protective effects of the hydrogen molecule (H₂) have been demonstrated in several osteoporosis models while the underlying molecular mechanism has remained unclear. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. In this work, we evaluated the effects of incubation with H₂ on receptor activator of NFκB ligand (RANKL)-induced osteoclast differentiation. We found that treatment with H₂ prevented RANKL-induced osteoclast differentiation in RAW264.7 cells and BMMs. Treatment with H₂ inhibits the ability to form resorption pits of BMMs stimulated by RANKL. Treatment with H₂ reduced mRNA levels of osteoclast-specific markers including tartrate resistant acid phosphatase, calcitonin receptor, cathepsin K, metalloproteinase-9, carbonic anhydrase typeII, and vacuolar-type H⁺-ATPase. Treatment with H₂ decreased intracellular reactive oxygen species (ROS) formation, suppressed NADPH oxidase activity, down-regulated Rac1 activity and Nox1 expression, reduced mitochondrial ROS formation, and enhanced nuclear factor E2-related factor 2 nuclear translocation and heme oxygenase-1 activity. In addition, treatment with H₂ suppressed RANKL-induced expression of nuclear factor of activated T cells c1 and c-Fos. Furthermore, treatment with H₂ suppressed NF-κB activation and reduced phosphorylation of p38, extracellular signal-regulated kinase, c-Jun-N-terminal kinase, and protein kinases B (AKT) stimulated with RANKL. In conclusion, hydrogen molecules prevented RANKL-induced osteoclast differentiation associated with inhibition of reactive oxygen species formation and inactivation of NF-κB, mitogen-activated protein kinase and AKT pathways.