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…     

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.     

槲皮苷抑制RANKL诱导的破骨细胞形成及骨吸收

[目的]探讨槲皮苷对核因子κB受体激动剂配体(receptor activator of nuclear factor kappa B ligand,RANKL)诱导的破骨细胞形成及骨吸收功能的影响。[方法]通过CCK-8法观察不同浓度槲皮苷(0~800μmol/L)干预不同时间(48 h、96 h)对RAW 264.7细胞的生存影响,确定合适的体外用药浓度;利用体外RANKL诱导RAW 264.7细胞形成破骨细胞体系,通过抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色计数评价槲皮苷(200、400μmol/L)对破骨细胞形成和生存的影响;通过骨片吸收实验对骨凹陷和骨吸收面积统计分析评价槲皮苷(200、400μmol/L)3 d内对成熟破骨细胞骨吸收功能的影响;釆用实时定量(Real-Time)PCR技术,检测槲皮苷(200、400μmol/L)对RANKL诱导的破骨细胞特异性基因NFATc1、TRAP和c-fos表达水平的影响。[结果]细胞生存实验发现槲皮苷干预96 h后,槲皮苷(0~800μmol/L)对RAW 264.7细胞4 d内生存未发现显著影响;通过TRAP染色发现200、400μmol/L槲皮苷能显著抑制体外RANKL诱导的破骨细胞形成;通过骨片吸收实验发现200、400μmol/L槲皮苷3d内能显著降低骨吸收面积,提示其抑制成熟破骨细胞骨吸收功能;同时,槲皮苷能呈剂量依赖性抑制RANKL诱导活化T细胞核因子(nuclear factor of activated T cells,NFAT)c1、TRAP和c-fos基因表达。[结论]槲皮苷通过抑制NFATc1,TRAP和c-fos的表达,来抑制体外RANKL诱导的破骨细胞形成和骨吸收功能,是一种潜在治疗骨质疏松药物。     

破骨细胞体外培养研究进展

破骨细胞是骨吸收的主要功能细胞,体外培养破骨细胞是骨吸收研究和抑制骨吸收药物开发研究的基础.该文综述破骨细胞体外培养常用的5种方法及近年研究进展,并对各种方法进行比较.成熟破骨细胞分离培养法仍是目前获得成熟破骨细胞的最佳途径;骨髓诱导破骨细胞培养法是目前最常用的破骨细胞培养法;脾干细胞诱导培养法和外周血单核细胞诱导培养法是近年新发展起来的培养方法,能排除骨髓基质细胞及其他造血干细胞干扰,更具有应用价值;骨巨细胞瘤破骨细胞样细胞分离培养法是其他破骨细胞培养方法的一个重要补充.     

M-CSF和RANKL体外诱导培养小鼠破骨样细胞及其骨吸收功能的动态观察

目的以巨噬细胞集落刺激因子(Macrophage colony-stimulating factor,M-CSF)和核因子κB受体激动剂配体(Ligand of receptor activator of NF-κB,RANKL)联合体外诱导小鼠骨髓干细胞分化为破骨样细胞,并对其骨吸收功能进行动态观察。方法分离小鼠四肢骨获取骨髓干细胞,以M-CSF和RANKL诱导培养,将盖玻片及骨磨片置入培养基内,在诱导培养的第3,6,9天分别对盖玻片行抗酒石酸酸性磷酸酶(The tartrate-resistant acid phosphatase TRAP)染色,观察细胞形态和染色情况,并计算破骨样细胞数量;同时对骨磨片进行骨吸收陷窝的观察。结果诱导培养3 d后出现含TRAP(+)颗粒的细胞,可见淡染单核和双核;诱导培养6 d后可见TRAP(+)细胞较诱导培养3 d时增多,仍以双核为主;诱导培养9 d后出现多核巨型TRAP(+)细胞,细胞核达到3个以上。并且随着诱导培养的时间延长,破骨样细胞数量逐渐增长。诱导培养第3,6天骨磨片上均未发现骨吸收陷窝,第九天出现不同形态的呈蓝紫色的吸收陷窝。结论 M-CSF和RANKL联合体外诱导小鼠骨髓干细胞形成破骨样细胞是一种有效的诱导培养方法,本实验在诱导培养第九天发现破骨样细胞具有骨吸收功能。     

破骨细胞及其骨吸收调控研究进展

一、概述破骨细胞是一个高度分化的多核巨细胞,直接参与骨吸收,是骨组织吸收的主要功能细胞。破骨细胞的来源:由于长期以来对于破骨细胞(Osteoclast,OC)的来源问题一直不清楚,给研究工作带来许多困难,因而对临床各种骨疾患的诊断及其防治水平,也不可能进一步深入和提高。对于破骨细胞来源的认识,在本世纪40～70年代,普遍应用的经典理论为多潜能的骨源细胞学说,认为破骨细胞是由骨源细胞融合而成。直至70年代中期还认为OC与成骨细胞(OB)为共同的祖代来源。这种观点多年来一直是疑问,不能被证实,现已被否定。自80年代初开始,提出了OC来源…     

应用体外分离培养破骨细胞的技术评价人工骨

本文介绍如何应用分离培养破骨细胞的技术评价人工骨。家兔４８只,随机分成Ａ、Ｂ、Ｃ、Ｄ四组,每组１２只,人工造成家兔同侧桡骨１０ｍｍ缺损。分别植入不同材料的骨移植物,Ａ组为珊瑚羟基磷灰石,Ｂ组为人工合成羟基磷灰石,Ｃ组为人工多聚体,Ｄ组为自体骨。移植术后６、８、１２、１６周,分期处死动物,取出植入动物体内骨移植物并制成骨磨片,与体外分离的破骨细胞共同培养１周,扫描电镜发现,破骨细胞能够在植入动物体内     

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。     

体外培养破骨细胞的功能观察

观察破骨细胞在离体状态下的骨吸收功能。新鲜牛皮质骨经锯式切片机横切成５０μｍ厚０．５×０．５ｃｍ大小骨片。参照已建立的破骨细胞分离培养方法，从新生Ｗｉｓｔａｒ大鼠四肢长骨分离破骨细胞，培养于２．５ｃｍ细胞培养皿内或上述骨片上，相差倒置显微镜或扫描电镜观察，可见培养的细胞具有典型破骨细胞的形态特点，接种子骨片上的破骨细胞分别培养１、３、５、７天，扫描电镜观察，可见破骨细胞能在骨片表面形成吸收陷窝，其形态多样，深浅不一，边界清晰，底面粗糙，而且随培养时间延长，陷窝扩大，数量增多。结论，破骨细胞在体外培养条件下具有良好的骨吸收功能，可进行药物干预的研究。     

骨保护蛋白及配体与破骨细胞

破骨细胞对骨量的维持起重要作用,它的发育、成熟信号是由成骨细胞传递的。当受到骨吸收因子作用时,成骨细胞表达骨保护蛋白配体分子,与破骨前体细胞膜上的核因子κB受体激活子结合,使之分化、成熟为破骨细胞。而成骨细胞旁分泌的骨保护蛋白分子,则作为伪受体与核因子κB受体激活子竞争结合骨保护蛋白配体,从而抑制破骨细胞的生成。骨保护蛋白配体-核因子κB受体激活子-骨保护蛋白组成了破骨细胞分化的信号传导通路,对它的认识有助于临床治疗代谢性骨病。     

二甲双胍抑制破骨分化改善糖尿病模型鼠骨量的研究

目的 探讨二甲双胍对骨髓源性巨噬细胞(BMDM)破骨分化的影响,并观察二甲双胍对糖尿病模型鼠骨量的改善作用。方法 在体外实验中,通过梯度浓度的二甲双胍干预RANKL诱导的大鼠BMDM破骨分化,并检测破骨分化标志基因Nfatc1、Ctsk、C-fos、Traf6及相应标志蛋白的表达;在体内实验中,将24只8周龄大鼠随机分为对照组、糖尿病模型组(模型组)、糖尿病模型组+二甲双胍干预组(治疗组),每组各8只,其中对照组与模型组行生理盐水灌胃,治疗组使用二甲双胍灌胃,治疗时间为3个月,检测大鼠体重、空腹血糖,并检测各组大鼠骨量的变化。结果 在体外实验中,二甲双胍显著抑制大鼠BMDM破骨分化,并呈浓度依赖性,显著下调破骨分化标志基因Nfatc1、Ctsk、C-fos、Traf6的表达(P<0.05),显著下调破骨分化标志蛋白C-FOS、NFATC1、TRAF6、CTSK的表达;在体内实验中,模型组相比较于对照组,体重减轻、空腹血糖上升、骨量降低;治疗组相比较于模型组,体重增加、空腹血糖降低、骨量增加。结论 二甲双胍能够抑制破骨分化,在增加体重、降低空腹血糖的同时,可以减少骨表面的破骨细胞数...     

Suppressive effects of <Emphasis Type="Italic">Anoectochilus formosanus</Emphasis> extract on osteoclast formation in vitro and bone resorption in vivo

Anoectochilus formosanus, a plant native to Taiwan, is used as a folk medicine. It was found that oral administration of A. formosanus extract (AFE) (500 mg/kg) for 4 weeks suppressed bone weight loss and trabecular bone loss in ovariectomized mice, an experimental model of osteoporosis. Although AFE at 12.5 and 25 μg/ml inhibited osteoclast formation in co-culture of osteoblasts and bone marrow cells, AFE did not inhibit the formation of osteoclast progenitor cells and preosteoclast cells in bone marrow cells and RAW264 cells. However, AFE (at 12.5 and 25 μg/ml) decreased RANKL expression. These results suggested that AFE might suppress the bone loss caused by estrogen deficiency through suppression of RANKL expression required for osteoclast formation.     

经皮微创锁定加压钢板内固定治疗老年股骨粗隆间骨折

[目的]探讨经皮微创钢板接骨术(MIPPO)结合锁定加压钢板(LCP)内固定治疗老年股骨粗隆间骨折的手术方法及临床效果.[方法]对2008年4月～2009年10月本院收治的20例股骨粗隆间骨折患者采用MIPPO技术结合LCP进行内固定治疗.其中男9例,女11例,年龄72～92岁,平均81岁.Tronzo-Evans分型:Ⅰ型2例,Ⅱ型3例,Ⅲ型10例,Ⅳ型3例,Ⅴ型2例.[结果]20例均获随访,随访时间12～30个月,平均18个月.愈合时间11～14周,仅1例不愈合.无感染、深静脉血栓、骨折不愈合、内固定松动、断钉并发症.术后功能按Hams评分:优15例、良4例、可1例,优良率95%.[结论]MIPPO技术结合LCP内固定治疗老年股骨粗隆间骨折,手术时间短,术中创伤小,固定简约可靠,术后骨折愈合率高,并发症少,功能良好,是治疗老年股骨粗隆间骨折的良好方法.     

Does use of a quadrilateral surface plate improve outcome in elderly acetabular fractures?

BackgroundAcetabular fractures in the elderly frequently involve segmental quadrilateral plate injury, yet no consensus exists on how to best control the femoral head medial displacement. Quadrilateral surface plates (QSP) were developed to help buttress these challenging fractures. The study aims to 1) Determine the prevalence of segmental quadrilateral plate fractures (SQPF) in elderly patients; and 2) Assess if utilization of a QSP is associated with improved acetabulum fracture reduction and outcome.MethodsThis was a retrospective study conducted at a level-1 trauma centre. . All patients over 60-years that sustained an acetabular fracture between 2007 and 2019 were reviewed. Pre-operative pelvic radiographs and CT imaging were reviewed for 96 patients, to assess for SQPF. From the 96 patients reviewed, over one third of patients (n = 40, 41.6%) sustained a SQPF. Patients that had an acute-THA (n = 7) were excluded as were patients that underwent an ORIF but did not have a QSP or an anterior column buttress plate (n = 3). The remaining 30 formed the study’s cohort. We assessed the ability to achieve and maintain reduction in this elderly population, and compared outcomes using traditional anterior column buttress plates (ilioingual or intra-pelvic approach) versus an intra-pelvic pre-contoured buttress suprapectineal plate (QSP). Outcome measures included: fracture reduction using the Matta classification (desirable: anatomical/imperfect and poor), re-operations, conversion to THA and Oxford Hip Score (OHS) (for the preserved hips).ResultsTen patients had an ORIF with utilization of a QSP (QSP-group), and 20 had an ORIF but did not have the QSP (non-QSP-group). There was no difference in patient demographics between groups. Fracture patterns were also similar (p = 0.6). Postoperative fracture reduction was desirable (anatomical/imperfect) in 17 patients and poor in 13. Improved ability to achieve a desirable reduction was seen in the QSP-group (p = 0.02). Conversion to THA was significantly lower in patients that had a desirable fracture reduction (appropriate: 3/17; poor: 7/13). No patients in the QSP-group have required a THA to-date, compared to 10/20 patients in the non-QSP-group (p = 0.01). The mean time to THA was 1.6 ± 2.1 year. There was no difference in OHS between the two groups (34.4 ± 10.3).ConclusionElderly acetabulum fractures have a high incidence (approaching 40%) of segmental QPF. Desirable (anatomical/imperfect) fracture reduction was associated with improved outcome. The use of a QSP was associated with improved ability to achieve an appropriate reduction. A QSP should be considered as they are both reliable and reproducible with a significantly improved fracture reduction and lower conversion to THA.     

Does use of a quadrilateral surface plate improve outcome in elderly acetabular fractures?

BackgroundAcetabular fractures in the elderly frequently involve segmental quadrilateral plate injury, yet no consensus exists on how to best control the femoral head medial displacement. Quadrilateral surface plates (QSP) were developed to help buttress these challenging fractures. The study aims to 1) Determine the prevalence of segmental quadrilateral plate fractures (SQPF) in elderly patients; and 2) Assess if utilization of a QSP is associated with improved acetabulum fracture reduction and outcome.MethodsThis was a retrospective study conducted at a level-1 trauma centre. . All patients over 60-years that sustained an acetabular fracture between 2007 and 2019 were reviewed. Pre-operative pelvic radiographs and CT imaging were reviewed for 96 patients, to assess for SQPF. From the 96 patients reviewed, over one third of patients (n = 40, 41.6%) sustained a SQPF. Patients that had an acute-THA (n = 7) were excluded as were patients that underwent an ORIF but did not have a QSP or an anterior column buttress plate (n = 3). The remaining 30 formed the study’s cohort. We assessed the ability to achieve and maintain reduction in this elderly population, and compared outcomes using traditional anterior column buttress plates (ilioingual or intra-pelvic approach) versus an intra-pelvic pre-contoured buttress suprapectineal plate (QSP). Outcome measures included: fracture reduction using the Matta classification (desirable: anatomical/imperfect and poor), re-operations, conversion to THA and Oxford Hip Score (OHS) (for the preserved hips).ResultsTen patients had an ORIF with utilization of a QSP (QSP-group), and 20 had an ORIF but did not have the QSP (non-QSP-group). There was no difference in patient demographics between groups. Fracture patterns were also similar (p = 0.6). Postoperative fracture reduction was desirable (anatomical/imperfect) in 17 patients and poor in 13. Improved ability to achieve a desirable reduction was seen in the QSP-group (p = 0.02). Conversion to THA was significantly lower in patients that had a desirable fracture reduction (appropriate: 3/17; poor: 7/13). No patients in the QSP-group have required a THA to-date, compared to 10/20 patients in the non-QSP-group (p = 0.01). The mean time to THA was 1.6 ± 2.1 year. There was no difference in OHS between the two groups (34.4 ± 10.3).ConclusionElderly acetabulum fractures have a high incidence (approaching 40%) of segmental QPF. Desirable (anatomical/imperfect) fracture reduction was associated with improved outcome. The use of a QSP was associated with improved ability to achieve an appropriate reduction. A QSP should be considered as they are both reliable and reproducible with a significantly improved fracture reduction and lower conversion to THA.     

螺钉结合重建钢板治疗Hoffa骨折

正2007年1月~2015年1月,我科采用螺钉结合重建钢板治疗23例Hoffa骨折患者,疗效满意,报道如下。1材料与方法1.1病例资料本组23例,男17例,女6例,年龄24~63岁。均为新鲜闭合骨折,外侧髁骨折16例,内侧髁骨折7例。骨折Letenneur分型:Ⅰ型12例,Ⅱ型5例,Ⅲ型6例。受伤至手术时间4~     

解剖锁定接骨板结合改良加垫式植骨治疗锁骨骨不连疗效分析

背景：近年来,锁骨骨折接受手术治疗患者越来越多,因内固定失效致骨不连患者也渐多。国内外报道多针对锁骨骨不连的治疗方法,但在植骨方式的选择上尚存争议。目的：探讨解剖锁定接骨板结合自体骨改良加垫式植骨治疗锁骨骨不连的临床效果。方法：2006年3月至2014年8月采用切开复位解剖锁定接骨板内固定结合自体骨髂骨加垫式植骨术治疗9例锁骨中段骨不连,记录手术时间,术后随访行X线片检查观察骨折愈合情况,并采用简短肩臂手残障功能评分系统（quick-DASH）评估其功能情况,末次随访胸部正位X线片,通过对比患侧与健侧的锁骨长度评价锁骨骨不连愈合后的短缩情况。结果：9例患者的平均手术时间为（112.22±19.22）min;均获得骨性愈合,无感染、接骨板断裂及再骨折发生,平均愈合时间为（10.67±2.83）周;术后quick-DASH为（14.33±1.66）分;末次随访患侧锁骨长度较健侧平均短缩（0.27±0.09）cm,两侧锁骨长度对比无显著性差异（P〉0.05）。结论：解剖锁定接骨板结合自体骨改良加垫式植骨是治疗锁骨骨不连的一种有效方法,能有效防止锁骨短缩,恢复患肢的良好功能。     

酶联免疫斑点法在快速诊断活动性肺结核中的应用

目的探讨酶联免疫斑点法（ELISPOT）在临床快速诊断活动性肺结核病中的应用价值。方法：采用T—SPOT．TB试剂盒对36例明确诊断为活动性肺结核的初治患者、30例健康体检者的外周血中结核分枝杆菌特异性T淋巴细胞进行检测,同时对26例活动性肺结核患者做结核菌素（PPD）试验。结果在36例活动性肺结核初治患者和30例健康对照者中,T-SPOT检测的阳性率分别为80．6％与6．7％,此技术用于诊断初治活动性肺结核患者的敏感性为80．6％,特异性为93．3％,阳性预测值为93．5％,阴性预测值为80．0％。在26例同时做PPD试验的活动性肺结核患者中,T-SPOT检测的阳性率略高于PPD试验（80．6％vs61．5％）,但差异无明显统计学意义（P〉0．05）。结论酶联免疫斑点法是一种具有较高敏感性和特异性的检测结核感染的技术,在活动性肺结核病的快速诊断中有较大应用价值。     

In vitro soft agar assay by use of multiplate for screening of tumor sensitivity to anticancer drugs

For use in routine clinical studies, modifications in Hamburger's stem cell assay were made. A multiplate with 24 wells made feasible, handling of a large number of samples. The addition of anticancer drugs to the bottom layer of agar led to avoidance of exposure to drugs before cell plating and evaluation of the effect of long-acting drugs such as 5-fluorouracil. Storage of the test plate including anticancer drugs in a freezer at −70°C showed no loss of the colony forming activity. This method is simple, reliable and readily facilitated.     

LISS钢板治疗股骨远端C型骨折

目的 探讨LISS钢板固定治疗股骨远端C型骨折的临床疗效及手术要点.方法对22例股骨远端C型骨折患者采用LISS钢板固定,骨缺损者一期植骨.结果 22例均获随访,时间12~24个月,骨折均愈合.膝关节功能按Kolmert评分标准：优8例,良11例,可3例.结论 对股骨远端C型骨折采用LISS钢板固定,既能使骨折达到关节面解剖复位,又提供有效稳定的固定,是治疗股骨远端C型骨折的良好方法.