EphB/EphrinB信号通路对成骨分化能力的影响

目的探讨EphB/EphrinB信号通路对去势小鼠骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSCs)的成骨分化能力和绝经后骨质疏松动物模型骨量的影响。方法将24只8周龄健康雌性BALB/c小鼠随机分为去势组(OVX组)和假手术组(Sham组),检测小鼠BMSCs中EphB/EphrinB信号通路表达水平。(2)将去势小鼠分为4组,分别进行腹腔注射相应Eph受体激动剂:EfnB1-Fc及EfnB2-Fc,Eph受体抑制剂EphB2-Fc,对照组腹腔注射human IgG-Fc。各组分别于术后10周将小鼠脱颈处死,在Micro-CT分析下比较股骨骨质情况。去势组和假手术组小鼠取股骨与胫骨骨髓,经密度梯度离心法分离培养并鉴定其骨髓间充质干细胞至P3用于实验,各组BMSCs在成骨诱导条件下7 d后,通过Realtime PCR检测成骨相关基因(Runx2、ALP、Osterix)及破骨细胞分化相关基因(OPG、RANKL)的表达水平,成骨分化14、21 d后,采用ALP染色和茜素红染色观察成骨分化能力。结果与假手术组(Sham组)相比较,去势组(OVX组)中EfnA2、EphB4、EfnB2、EfnB1及EphA4表达显著增高(P0. 01); Sham组中EphA2及EfnB2表达显著降低(P0. 01)。10周后Micro-CT结果显示,与去势对照Fc组比较,Sham组、EfnB1-Fc、EfnB2-Fc及EphB2-Fc去势组骨小梁结构均较完整、骨小梁密度较好(P0. 01);与EfnB1-Fc、EfnB2-Fc去势组相比较,Sham组和EphB2-Fc去势组的骨密度及骨体积分数均明显增高(P0. 01)。Realtime PCR检测成骨相关基因提示,与去势对照Fc组比较,EfnB1-Fc、EfnB2-Fc和EphB2-Fc去势组中ALP与Osterix的表达明显升高(P0. 01); EfnB1-Fc和EphB2-Fc可显著提高BMSCs成骨分化中ALP的活性和骨基质矿化能力,以EphB2-Fc效果最为显著(P0. 01)。与去势对照Fc组比较,EfnB1-Fc、EfnB2-Fc和EphB2-Fc去势组中RANKL的表达未见明显差异(P0. 05),但OPG的表达明显升高(P0. 01),其中,EfnB1-Fc去势组与EphB2-Fc去势组中OPG/RANKL的比率升高最为明显(P0. 01)。结论 EphB2/EfnB1双向信号通路可逆转去势所导致的骨量减少,上调ALP与Osterix的表达,促进BMSCs的成骨分化能力,并可通过调节OPG/RNAKL比率影响去势骨髓间充质干细胞的功能,从而间接影响破骨细胞系的分化。     

铁蓄积小鼠去势前后骨吸收活性的差异

目的了解小鼠在去势前与去势后,铁对骨吸收影响的差异。方法建立高铁小鼠模型,将小鼠随机分为假手术组(SHAM组)、高铁去势组(F+OVX组)、去势组(OVX组)。铁剂干预两周后去势。在去势前(雌激素存在)与去势7周后(雌激素缺乏)检测小鼠体重,血清铁蛋白;反转录聚合酶链反应(RT-PCR)检测胫骨内抗酒石酸酸性磷酸酶(TRAP)、降钙素受体(CTR)、基质金属蛋白酶9(MMP9)、组织蛋白酶K(cathepsin K)、雌激素受体α(ERα)、雌激素受体β(ERβ)表达情况,股骨远端Micro-CT三维分析,提取各组小鼠股骨骨髓细胞进行破骨细胞培养,抗酒石酸酸性磷酸酶(TRAP)染色观察破骨细胞分化情况。结果各组小鼠体重无明显差异。F+OVX组铁蛋白含量明显升高(P<0.05)。Micro-CT分析表明:雌激素存在时,正常组与铁干预组骨密度及相关指标无明显差异(P>0.05);雌激素缺乏时,与OVX组比较,F+OVX组骨量下降更为严重(P<0.05)。半定量PCR结果显示:雌激素存在时,两组TRAP、CTR、MMP9、cathepsin K基因表达无统计学差异;雌激素缺乏时,F+OVX组各基因表达水平较OVX组显著升高(P<0.05)。去势后ERβ表达下降(P<0.05),ERα无明显差异。TRAP染色显示去势前各组小鼠破骨细胞数无明显差异(P>0.05);而去势后,F+OVX组破骨细胞数明显高于OVX组(P<0.05)。结论未去势时(有雌激素),FAC对骨吸收影响甚微;去势后(无雌激素),FAC能显著增强破骨活性。     

成骨与破骨细胞促红细胞生成素肝细胞受体B4/肝配蛋白B2双向信号通路

成骨细胞与破骨细胞以直接接触的方式共同调控骨重建平衡,这也决定了两者的不可分割性。最新研究表明前破骨细胞肝配蛋白(Ephrin)B2与成骨细胞膜上促红细胞生成素肝细胞受体(Eph)B4受体的直接接触来调控骨稳态平衡。具有EphrinB2配体的前破骨细胞可通过直接接触具有EphB4受体的前成骨细胞从而触发各自相应的下游信号转导分子。通过激活成骨细胞膜表面Eph受体而起正向作用,进一步去抑制下游信号转导分子RhoA活性促进前体细胞分化成熟。反之,EphrinB2配体的激活起到反向作用,抑制破骨相关转录因子的C-fos/NFATc1转录级联反应来抑制前破骨细胞的分化,磨损颗粒导致的骨溶解会使破骨细胞上EphrinB2配体表达明显升高,并且会促进NFATc1的高表达,可以通过这种双向信号的机制来减弱甚至是抑制磨损颗粒导致的破骨细胞的进一步分化。     

复合振动抑制RAW264.7细胞分化成熟

目的研究复合振动对核因子-κB受体活化因子配体(RANKL)诱导的RAW264.7细胞分化的影响,探讨复合振动对破骨细胞分化的影响及机制。方法 RAW264.7细胞RANKL诱导培养3或4d并施加复合振动干预,通过抗酒石酸酸性磷酸酶(TRAP)染色观察TRAP阳性多核细胞形成的变化,real-time RT-PCR分析破骨细胞特异性基因组织蛋白酶K(cathepsin K),金属蛋白酶-9(MMP-9)和TRAP表达的变化。结果复合振动能抑制RANKL诱导破骨细胞形成,下调破骨细胞特异基因cathepsin K,MMP-9和TRAP的表达。结论 RANKL促进RAW264.7细胞向破骨细胞分化,并增加特异基因的表达,但RANKL的促进作用受复合振动抑制。这进一步的阐释复合振动抗骨质疏松的作用机制。     

胃癌组织中 EphB4受体及其配体 EphrinB2的表达与血管生成及肿瘤转移的关系

目的探讨EphB4受体与其配体EphrinB2在胃癌组织中的表达及其与血管生成和肿瘤转移的关系。方法应用免疫组织化学(免疫组化)SABC法检测93例胃癌和30例癌旁组织中EphB4受体与其配体EphrinB2的表达及间质微血管密度(MVD)。结果胃癌组织中肿瘤细胞、间质新生血管的EphB4与EphrinB2高度表达。癌组织中EphB4与EphrinB2的阳性表达(49.5%和50.5%)明显高于癌旁组织(20.0%和23.3%,P=0.005和P=0.009);EphB4与EphrinB2阳性表达组的平均MVD值(56.21±15.3和62.41±16.9)明显高于EphB4与EphrinB2阴性表达组(32.22±12.9和34.12±14.7),两组比较,分别为P=0.018和P=0.004。此外,EphB4与EphrinB2表达程度还与胃癌的转移和癌浸润程度密切相关。结论EphB4与EphrinB2可以促进肿瘤间质的血管生成,加速肿瘤浸润和转移。     

共轭亚油酸c9,t11-CLA及t10,c12-CLA对大鼠骨髓细胞骨代谢相关基因表达的影响

目的 观察c9,t11-CLA及t10,c12-CLA干预后,大鼠骨髓细胞过氧化物酶体增殖物激活受体2(PPARγ2)及核因子(NF)-κB活化受体配体(RANKL)、碱性磷酸酶(ALP)、骨保护素(OPG)、NF-κB活化受体(RANK)、抗酒石酸酸性磷酸酶(TRAP) mRNA表达的变化,探讨其对骨代谢的影响.方法 体外培养大鼠骨髓细胞,分别加入c9,t11-CLA及t10,c12-CLA干预24h,逆转录-聚合酶链反应(RT-PCR)检测PPARγ2、RANKL、ALP、OPG、RANK、TRAP mRNA表达水平,比较其对骨髓细胞上述基因表达的影响.结果 (1)c9,t11-CLA呈剂量依赖性下调RANK、TRAP mRNA表达,组间比较差异有统计学意义(P＜0.05,P＜0.01),而其对RANKL、ALP、OPG及PPARγ2 mRNA 表达的影响不明显.(2) t10,c12-CLA呈剂量依赖性上调RANKL、OPG mRNA的表达,同时下调 RANK、TRAP及PPARγ2 mRNA的表达,组间比较差异有统计学意义(P＜0.05,P＜0.01),但对ALP mRNA的表达无明显影响.结论 c9,t11-CLA可能通过抑制破骨细胞标记物基因表达阻断骨髓细胞向破骨细胞分化,而t10,c12-CLA在抑制破骨细胞标记物基因表达的同时也可促进成骨细胞标记物基因表达,两者均有利于骨形成.     

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。     

应用颅骨-破骨细胞联合培养体系研究先天性成骨不全破骨细胞骨吸收活性

目的 先天性成骨不全(OI)的主要临床表现为骨矿化过程不良,骨量丢失,骨骼畸形和骨折.但是其发病机理,尤其在其骨再建过程中成骨细胞(OB)及破骨细胞(OC)的功能改变尚不清楚.本实验以先天性成骨不全小鼠模型,oim/oim为基础,应用破骨细胞-颅骨联合培养体系研究OB和OC两种细胞在骨再建过程中的功能改变和相互作用.方法 本实验采用小鼠颅骨(CAL)组织培养模型.本模型采用颅骨组织培养,利用颅骨中成骨细胞可以从颅骨片游离出到培养皿及颅骨表面,从而支持培养皿及颅骨表面前体破骨细胞分化成为成熟破骨细胞,并吸收颅骨产生吸收陷窝.本实验中,共2组颅骨-破骨细胞联合培养体系:(1) 对照组(WT)颅骨与对照破骨细胞(WTCAL-WTOC);(2) OI颅骨与OI破骨细胞(OICAL-OIOC).联合培养颅骨及骨髓组织14日后,以TRAP免疫组化染色方法识别破骨细胞,ALP免疫组化染色方法识别成骨细胞,计算OC/OB.破骨细胞骨吸收活性以颅骨表面骨吸收陷窝占颅骨表面百分比并除以培养系统中的破骨细胞数表达.结果 第14日,OICAL-OIOC组的破骨细胞数低于WTCAL-WTOC组(92.50+23.18/mm2 对比 379.00+ 136.53/mm2,P<0.01); OICAL-OIOC组的OC/OB明显低于WTCAL-WTOC组(0.68+0.57对比1.65+0.67,P<0.01);OICAL-OIOC组OI破骨细胞的吸收能力高于WTCAL-WTOC组(27.76+22.81对比7.32+5.09,P<0.001).结论 oim/oim小鼠破骨细胞-颅骨培养体系中破骨细胞的数目明显减少,成骨细胞支持破骨细胞分化能力减低;但其破骨细胞骨吸收活性明显增强,以代偿成骨细胞功能,维持骨再建过程中成骨过程及骨吸收过程的平衡.     

ACEI对去卵巢骨质疏松大鼠激肽释放酶-激肽系统及骨丢失的影响

目的探究血管紧张素转换酶抑制剂(angiotensin-converting enzyme inhibitor,ACEI)卡托普利对去卵巢(OVX)骨质疏松大鼠骨丢失及激肽释放酶-激肽(kallikrein-kinin system,KKS)系统的影响。方法去卵巢法制备绝经骨质疏松大鼠模型,分为OVX组、ACEI组[6 mg/(kg·d)卡托普利]、阳性对照雌激素组[0.05 mg/(kg·d)己烯雌酚],另不去卵巢为Sham组。给药8周后,全自动生化分析仪检测血清钙(Ca)、Ⅰ型前胶原N-端肽(PINP)、碱性磷酸酶(ALP)、骨钙素(OCN)、抗酒石酸酸性磷酸酶(TRAP)及Ⅰ型胶原交联C端肽β序列(β-CTX)水平,微型计算机断层摄影术(micro CT)法检测骨密度及微结构,TRAP法观察骨组织破骨细胞数量,Western blot法检测骨组织中骨形态发生蛋白2(BMP2)、Runt相关转录因子2(Runx2)、组织激肽释放酶(KLK)、缓激肽受体1(B1R)及缓激肽(BK)蛋白表达。结果相较于Sham组,OVX组大鼠骨密度、骨小梁数量及厚度、骨体积分数、SMI、PINP、ALP、OCN水平、BMP2、Runx2蛋白表达均降低(P均0.05),骨小梁分离度、TRAP、β-CTX水平、破骨细胞数量、KLK、B1R及BK蛋白水平均增加(P均0.05)。相较于OVX组,ACEI组、阳性组大鼠骨密度、骨小梁数量及厚度、骨体积分数、SMI、PINP、ALP、OCN水平、BMP2、Runx2蛋白表达均增加,骨小梁分离度、TRAP、β-CTX水平、破骨细胞数量、KLK、B1R及BK蛋白水平降低(P0.05)。结论 ACEI可抑制KKS系统,降低破骨细胞活性减少骨吸收,增强成骨细胞活性增加骨形成,提高骨密度,改善骨微结构,进而改善OVX大鼠骨质疏松症状。     

柚皮苷对共培养体系中成骨细胞活性及破骨细胞分化的影响

目的研究在成骨细胞-破骨细胞共培养体系中,柚皮苷对成骨细胞活性和破骨细胞分化的影响。方法将成骨细胞(MC3T3-E1细胞株)和破骨细胞(RAW264.7细胞株)以2∶1的比例分别培养至Transwell小室的上室和下室。根据培养基是否含有柚皮苷分为对照组和柚皮苷(2ng/ml组、20 ng/ml组、200 ng/ml组),培养7 d后对下室破骨细胞进行TRAP染色和骨吸收陷窝鉴定;荧光定量PCR分析成骨细胞骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子配体(receptor activator for nuclear factor-κB ligand,RANKL)基因和破骨细胞分化基因活化T细胞核因子-1(NFATc-1)、活化T细胞核因子-2(NFATc-2)和核因子κB受体激活蛋白(receptor activator of NF-κB,RANK)的相对表达量。结果与对照组相比,柚皮苷可以促进成骨细胞OPG、RANKL的表达量且可提高OPG/RANKL的比值,差异有统计学意义(P0.05);20 ng/ml柚皮苷TRAP(+)细胞数(5.82±3.37)明显少于对照组(20.56±7.69),差异有统计学意义(P0.05);柚皮苷可抑制破骨细胞NFATc-1、NFATc-2的表达,促进RANK的表达,差异有统计学意义(P0.05)。结论柚皮苷可促进共培养条件体系中成骨细胞OPG和RANKL的表达以及抑制破骨细胞的分化,与上调OPG/RANKL的比值有关。     

EphB4-EphrinB2双向信号传导在骨重建中的作用

Eph-Ephrin双向信号传导机制是近年来细胞间的通讯机制的研究热点,在神经系统、血管内皮系统及肿瘤形成和转移方面发挥着重要的作用。研究发现该信号传导系统在骨重建中发挥重要作用。破骨细胞上存在EphrinB2配体,成骨细胞前体上存在EphB4受体,以细胞直接接触的方式,通过胞内酪氨酸激酶信号系统,在成骨细胞和破骨细胞内分别产生正向和反向信号,促进成骨细胞前体分化成熟,同时抑制破骨细胞功能,并促进其凋亡。     

Down-regulation of osteoclast differentiation by daidzein via caspase 3.

Phytoestrogens are plant-derived compounds with estrogen-like activity. Phytoestrogen-rich diets may prevent postmenopausal osteoporosis and these molecules maintain bone mass in ovariectomized animals. We compared the effects of the isoflavone daidzein, which has no action on tyrosine kinases, and 17beta-estradiol on the development and activity of osteoclasts in vitro. Nonadherent porcine bone marrow cells were cultured on dentine slices or on culture slides in the presence of 10-8 M of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], with or without 10(-8) M of daidzein, 10(-8) M of 17beta-estradiol for 9-11 days. Multinucleated tartrate-resistant acid phosphatase-positive (TRAP+) cells that resorbed bone (osteoclasts) developed in the presence of 1,25(OH)2D3. The number of osteoclasts formed in response to 1,25(OH)2D3 was reduced by 58 +/- 8% by daidzein and 52 +/- 5% by estrogen (p < 0.01); these effects were reversed by 10-6 M of ICI 182,780. The area resorbed by mature osteoclasts was reduced by 39 +/- 5% by daidzein and 42 +/- 6% by estradiol (p < 0.01). Both compounds also inhibited the 1,25(OH)2D3-induced differentiation of osteoclast progenitors (mononucleated TRAP+ cells), 53 +/- 8% by daidzein and 50 +/- 7% by estradiol (p < 0.05). Moreover, daidzein and estradiol promoted caspase-8 and caspase-3 cleavage and DNA fragmentation of monocytic bone marrow cells. Caspase-3 cleavage was reversed by 10-8 M of ICI 182,780. Both compounds up-regulated the expression of nuclear estrogen receptors ER-alpha and ER-beta. Thus, daidzein, at the same concentration as 17beta-estradiol, inhibits osteoclast differentiation and activity. This may be caused by, at least in part, greater apoptosis of osteoclast progenitors mediated by ERs.     

大鼠去卵巢后骨髓源性破骨细胞形成的动态变化及其与骨髓IL-6、IL-6受体表达的关系

目的 观察大鼠去卵巢后骨髓源性破骨细胞形成的动态变化及其与骨髓细胞IL-6、IL-6受体以及gpl30基因表达水平改变的关系。方法 健康3月龄雌性SD大鼠60只,30只作为假手术对照组,30只经腹手术去卵巢。分别于去卵巢后2,4,6,8,12周取去卵巢组和对照组各6只大鼠骨髓细胞作细胞培养和提取RNA。培养的第6天分别作瑞氏-吉姆萨染色和抗酒石酸酸性磷酸酶(TRAP)染色,以胞核3个或3个以上以及TRAP(+)为破骨细胞标志,计数细胞数。骨髓细胞提取总RNA进行逆转录PCR。结果 术后2周去卵巢组破骨细胞形成数即多于对照组(P<0.05);第4-6周,去卵巢组破骨细胞形成达高峰,明显多于2周(P<0.01);去卵巢后的8周,较峰值下降,直至12周去卵巢组破骨细胞形成数仍高于对照组(P<0.05)。与破骨细胞的变化相对应,术后2周,骨髓细胞IL-6及IL-6RmRNA表达均明显增高(分别为P<0.01,P<0.05);第4周时,IL-6表达水平达到高峰,保持至第6周,IL-6R表达则在第6周达最高;第8周,IL-6、IL-6R基因表达水平较峰值下降,但直至第12周仍高于对照组。去卵巢后全程未见gp130基因表达水平有明显变化。结论 大鼠去卵巢后骨髓干细胞分化形成破骨细胞明显增加,呈时间相关动态过程,第6周达高峰,这一过程与骨髓细胞IL-6、IL-6R基因表达的动态变化一致,提示去卵巢后骨髓细     

Ephrin B2/EphB4 Mediates the Actions of IGF‐I Signaling in Regulating Endochondral Bone Formation

Ephrin B2/EphB4 mediates interactions among osteoblasts (OBs), osteoclasts (OCLs), and chondrocytes to regulate their differentiation. We investigated the role of ephrin B2/EphB4 signaling in mediating the anabolic effects of insulin‐like growth factor‐I (IGF‐I) and parathyroid hormone (PTH) on those cells and overall endochondral bone formation. Immunohistochemistry demonstrated that the expression of ephrin B2 in OBs, OCLs, and osteocytes, and the expression of EphB4 in OBs and osteocytes was dramatically decreased in global IGF‐I knockout mice. Inactivation of EphB4 by EphB4 small, interfering RNA (siRNA) in cultured bone marrow stromal cells significantly decreased the mRNA levels of OB differentiation markers and abolished the stimulatory effects of IGF‐I on these markers. Blocking the interaction of EphB4 and ephrin B2 in the OB‐OCL cocultures with the EphB4 specific peptide TNYL‐RAW or deletion of ephrin B2 in OCL prior to coculture led to fewer and smaller tartrate‐resistant acid phosphatase (TRAP)‐positive cells, decreased expression of OB differentiation markers, and blunted response to IGF‐I for both OCL and OB differentiation. In the growth plate, both ephrin B2 and EphB4 are expressed in late stage proliferating and prehypertrophic chondrocytes, and their expression was decreased in mice lacking the IGF‐I receptor specifically in chondrocytes. In vitro, blocking the interaction of EphB4 and ephrin B2 in chondrogenic ATDC5 cells with TNYL‐RAW significantly decreased both basal and IGF1‐induced expression of type II and type X collagen. In the cocultures of ATDC5 cells and spleen cells (osteoclast precursors), TNYL‐RAW decreased the numbers of TRAP‐positive cells and the expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and receptor activator of NF‐κB (RANK), and blocked their stimulation by IGF‐I. Our data indicate that IGF‐I/IGF‐IR signaling promotes OB, OCL, and chondrocyte differentiation via ephrin B2/EphB4 mediated cell‐cell communication. © 2014 American Society for Bone and Mineral Research.     

体外研究橙皮苷抑制钛颗粒介导的破骨细胞分化

目的研究橙皮苷对钛颗粒介导前破骨细胞分化及成熟的影响。 方法骨髓巨噬细胞在巨噬细胞集落刺激因子(M-CSF)30 ng/ml及核因子κB受体活化因子配体(Rankl)50 ng/ml刺激下,诱导分化为破骨细胞。扫描电镜观察钛磨损颗粒形貌结构。对不同浓度下橙皮苷对巨噬细胞增殖的影响进行t检验分析得出最低有效浓度;对抗酒石酸酸性磷酸酶(TRAP)阳性细胞数及骨吸收陷窝面积判断橙皮苷对破骨细胞分化及成熟的影响。最后通过实时定量PCR(RT-PCR)验证橙皮苷对钛颗粒介导的破骨基因,包括活化T细胞核因子(NFATc1)、组织蛋白酶K (CTSK)、TRAP的影响。TRAP阳性细胞数及骨吸收陷窝面积、RT-PCR结果数据均采用t检验分析。 结果扫描电镜显示钛磨损颗粒大小在1～3 μm。CCK-8实验结果显示橙皮苷对巨噬细胞增殖有促进作用,浓度超过40 μmol/L后,会对巨噬细胞产生抑制作用(F＝40.1, P<0.01),所以选择40 μmol/L作为对巨噬细胞分化的影响。在抗酒石酸酸性磷酸酶染色中发现40 μmol/L的橙皮苷会明显抑制前破骨细胞的分化,TRAP阳性细胞数目及破骨细胞的面积明显减少(t＝5.5,P<0.05)。与对照组相比,扫描电镜观察钛颗粒介导骨吸收陷窝面积明显增多,但加入40 μmol/L的橙皮苷后,这种吸收效果或明显减少(t＝6.1,P<0.05)。最后,通过RT-PCR实验得出,40 μmol/L的橙皮苷会明显抑制破骨细胞分化相关NFATc1, CTSK,TRAP基因(t＝7.1、4.8、9.1,均为P<0.05)。 结论橙皮苷抑制钛颗粒介导的破骨细胞分化及成熟。     

Expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals

Metatarsal bones of 15-day-old mouse embryos contain proliferative tartrate-resistant acid phosphatase (TRAP) negative (-) osteoclast progenitors that progressively differentiate into multinucleated TRAP positive (+) osteoclasts. Using histochemical and autoradiographic techniques, we have examined the expression of calcitonin receptors during osteoclast differentiation in mouse metatarsals. Fresh mouse metatarsals from embryos aged 14-17 days and metatarsals from 15-day-old embryos cultured for 1, 2, 3, and 6 days were stained for TRAP. Calcitonin binding to osteoclasts and their precursors was studied by incubating metatarsals with [125I]salmon calcitonin (sCT) and quantitating grain counts from autoradiographs of tissue sections. Calcitonin receptors first appear on nonproliferating osteoclast precursors, most often just after or simultaneously with the development of TRAP activity. The effect of sCT on the development of TRAP+ mononuclear preosteoclasts was examined by culturing 15-day-old metatarsals in the continuous presence of 5 mU sCT for periods of up to 3 days and quantitating the number of TRAP+ mononuclear preosteoclasts that develop. Calcitonin did not affect the differentiation of osteoclasts up to the stage of the TRAP+ mononuclear preosteoclast.     

A Novel,Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice

Most US Food and Drug Administration (FDA)‐approved treatments for osteoporosis target osteoclastic bone resorption. Only PTH derivatives improve bone formation, but they have drawbacks, and novel bone‐anabolic agents are needed. Nitrates, which generate NO, improved BMD in estrogen‐deficient rats and may improve bone formation markers and BMD in postmenopausal women. However, nitrates are limited by induction of oxidative stress and development of tolerance, and may increase cardiovascular mortality after long‐term use. Here we studied nitrosyl‐cobinamide (NO‐Cbi), a novel, direct NO‐releasing agent, in a mouse model of estrogen deficiency–induced osteoporosis. In murine primary osteoblasts, NO‐Cbi increased intracellular cGMP, Wnt/β‐catenin signaling, proliferation, and osteoblastic gene expression, and protected cells from apoptosis. Correspondingly, in intact and ovariectomized (OVX) female C57Bl/6 mice, NO‐Cbi increased serum cGMP concentrations, bone formation, and osteoblastic gene expression, and in OVX mice, it prevented osteocyte apoptosis. NO‐Cbi reduced osteoclasts in intact mice and prevented the known increase in osteoclasts in OVX mice, partially through a reduction in the RANKL/osteoprotegerin gene expression ratio, which regulates osteoclast differentiation, and partially through direct inhibition of osteoclast differentiation, observed in vitro in the presence of excess RANKL. The positive NO effects in osteoblasts were mediated by cGMP/protein kinase G (PKG), but some of the osteoclast‐inhibitory effects appeared to be cGMP‐independent. NO‐Cbi increased trabecular bone mass in both intact and OVX mice, consistent with its in vitro effects on osteoblasts and osteoclasts. NO‐Cbi is a novel direct NO‐releasing agent that, in contrast to nitrates, does not generate oxygen radicals, and combines anabolic and antiresorptive effects in bone, making it an excellent candidate for treating osteoporosis. © 2016 American Society for Bone and Mineral Research.