胸椎黄韧带骨化患者黄韧带细胞的体外培养及初步鉴定

目的探讨胸椎黄韧带骨化的发病机理。方法2004年3月-2004年12月对8例行后路减压的下胸椎黄韧带骨化患者（男性5例，女性3例，平均年龄55岁）进行术中取材，采用组织块培养法，体外培养下胸椎黄韧带骨化患者非骨化区域的黄韧带细胞，并进行细胞化学，免疫细胞化学等方面研究；同时对7例急性外伤性下胸椎压缩骨折行胸椎后路减压术的患者（男性5例，女性2例，平均年龄28岁）进行术中取材，体外培养青壮年患者的下胸椎黄韧带细胞作为正常对照。结果体外成功地培养出黄韧带细胞15株，其中骨化患者黄韧带细胞8株（OLF1～OLF8），正常黄韧带细胞7株（NLF1～NLF7），黄韧带细胞可以在体外增殖和传代，通常正常黄韧带生长较慢，而骨化患者的黄韧带细胞则生长较快，并且可形成典型的钙结节样结构；80％以上的细胞呈碱性磷酸酶（ALP）强阳性反应；细胞内的ALP活性及其合成的骨钙素（BGP）含量均较正常对照组明显升高；细胞浆有BMP-2与TGF—betal的阳性表达，表明体外培养的下胸椎黄韧带骨化患者非骨化区域的黄韧带细胞呈现典型的成骨细胞表型特征：而正常黄韧带细胞主要为成纤维细胞表型。结论胸椎黄韧带骨化患者的非骨化区域中存在大量具备典型的成骨细胞表型特征的细胞，可能被骨形成蛋白等骨生长因子所调控。     

骨桥蛋白及其受体在人黄韧带骨化标本中的表达及意义

目的探讨骨桥蛋白(OPN)及其受体CD44、整合素在人黄韧带骨化(OLF)标本中的表达及意义。方法2014年6月—2016年6月,收集8例在本院接受胸椎后路椎板切除的OLF患者的骨化黄韧带标本以及8例非黄韧带骨化患者的正常黄韧带标本。对所得标本进行脱钙、切片、免疫组织化学染色以及细胞体外培养和蛋白质印迹检测,观察黄韧带标本中OPN及其受体CD44、整合素的表达变化。结果 OLF标本中的弹性纤维排列杂乱无章,同时在骨化层可见大量软骨细胞;正常黄韧带标本中则无上述表现。OLF标本中OPN及其受体CD44呈高表达,且多在骨化区域的软骨细胞周围,而整合素β1的表达较弱;正常黄韧带标本中OPN及其受体呈低表达或不表达状态。通过对体外培养的细胞进行蛋白质印迹检测发现骨化黄韧带细胞OPN的表达较正常黄韧带细胞增加。免疫细胞化学染色发现骨化黄韧带细胞整合素β3染色呈阳性,而正常黄韧带细胞整合素β3的表达呈阴性或弱阳性;蛋白质印迹检测发现骨化黄韧带细胞整合素β3的表达明显高于正常黄韧带细胞。结论 OPN及其受体可能在OLF的发生过程中起关键作用。     

机械应力因素在脊柱韧带骨化发生机制中的作用研究进展

正脊柱韧带骨化是一种以脊柱正常韧带发生异位骨化为特点的慢性、退行性疾病。主要包括后纵韧带骨化(OPLL)、黄韧带骨化(OLF)及弥漫性特发性骨肥大症(DISH)~([1])。后纵韧带和黄韧带解剖位置特殊,直接参与椎管结构的组成,因此,OPLL和OLF的发生常常导致脊髓和神经根受压,引起严     

过量氟化物导致大鼠腰椎黄韧带退变与骨化

[目的]研究氟化物在黄韧带骨化中可能的作用机理。[方法]36只雄性SD大鼠，实验组饮用含NaF（质量分数为1014）蒸馏水，分别于3个月及6个月时，检测骨密度，血清、骨组织标本中Ca、P^3＋、Mg、Zn、Cu、Fe、F^-含量和血清碱性磷酸酶（ALP）活性；行大鼠腰段标本X线检查后组织病理观察。[结果]3个月实验组10只大鼠中6只出现氟斑牙：6个月实验组均出现氟斑牙，且大鼠中轴骨骨密度显著增加（P〈0．05）。X线检查，6个月实验组中有4只可见黄韧带嵴状骨化影。病理观察，3个月实验组大鼠黄韧带呈退行性改变；6个月实验组部分黄韧带骨化，骨化类型以膜内骨化为主。[结论]过量氟化物可造成SD大鼠腰椎黄韧带的退变、骨化，在黄韧带的骨化中可能起重要作用。     

胸椎黄韧带骨化易感基因及蛋白质组学研究进展

正黄韧带骨化(ossification of the ligamentum flavum,OLF)系脊柱韧带病理性异位骨化性疾病之一,全脊柱颈椎、胸椎和腰椎黄韧带附着部位均可发生,其中以胸椎尤其是下胸段最为常见~([1])。脊柱OLF及后纵韧带骨化(ossification of the posterior longitudinal ligament,OPLL)是导致脊柱椎管狭窄、脊髓受压的常见原因,严重危害患者运动神经功能~([2])。脊柱韧带的解剖特征、局部生物应力刺激、微循环炎症免疫反应、内分泌与遗传等因素均与脊柱黄韧带骨化的     

氟与黄韧带骨化

目的：探讨氟与黄韧带骨化的关系。方法：采用氟离子电极等方法测定１１ 例胸椎黄韧带骨化症（ 包括５ 例氟骨症） 患者血清及黄韧带标本中氟、钙含量,分别选取胸腰椎急性外伤性截瘫及腰椎管狭窄患者为正常及退变对照。结果：氟骨症骨化患者与非氟骨症骨化患者黄韧带中氟、钙含量均显著增高（ Ｐ＜ ０－０１） 。结论：氟在黄韧带骨化中起重要作用,可能是诱导退变黄韧带进一步骨化的重要诱因。     

颈椎后纵韧带骨化患者韧带成纤维细胞中Cx43的表达及其在成骨化中的作用

目的:观察Connexin43(Cx43)在颈椎后纵韧带骨化患者韧带成纤维细胞中的表达,探讨其在成骨化过程中的作用.方法:选择2013年1月～12月期间在我科行颈前路手术治疗的15例颈椎后纵韧带骨化症患者(骨化组)与15例不伴后纵韧带骨化的颈椎疾病患者(非骨化组).术中切取两组患者韧带标本并采用组织块培养法进行细胞体外培养,应用免疫细胞化学及免疫荧光技术检测胞浆内波形蛋白进行细胞鉴定.采用RT-PCR方法检测细胞内成骨特异指标骨钙素(OCN)、碱性磷酸酶(ALP)及Ⅰ型胶原(COL Ⅰ)mRNA表达,Western blot技术检测两组细胞Cx43蛋白表达,评估其成骨活性.采用siRNA技术对骨化组第3代细胞之Cx43抑制72h,检测抑制组与非抑制组OCN、ALP及COL Ⅰ的表达变化.结果:经鉴定培养出的细胞为成纤维细胞.韧带骨化组细胞OCN、ALP与COL Ⅰ的mRNA表达量分别为1.36±0.21、0.53±0.18、1.64±0.37,明显高于非骨化组(0.78±0.21、0.29±0.13、1.01±0.26);骨化组Cx43蛋白表达量(1.00±0.30)亦明显高于非骨化组(0.48±0.18).对骨化组细胞Cx43抑制72h后,其蛋白表达下调69％(P＜0.01);非抑制组OCN、ALP及COL Ⅰ的mRNA表达量为1.54±0.33、1.32±0.36、1.86±0.44,抑制组分别为0.91±0.32、0.47±0.21、0.88±0.29,两组比较差异具有统计学意义(P＜0.05).结论:颈椎后纵韧带骨化症患者韧带成纤维细胞中存在较高水平的Cx43,并在其成骨化过程中发挥着积极作用.     

颈椎后纵韧带骨化症合并黄韧带骨化的诊断和治疗

目的　深化对颈椎后纵韧带骨化 (OPLL)合并黄韧带骨化 (OLF)的认识。方法　复习并分析 1987年 10月至 1997年10月证实为颈椎OPLL合并OLF ,并经过治疗的 9例影像学及病理学资料。结果　 9例OPLL合并OLF均采用后路椎板成形术治疗并获得显著神经功能恢复。结论　颈椎OPLL合并OLF主要依靠影像学检查进行诊断 ,后路椎板成形手术为治疗此种病症的有效方法。     

脊柱后纵韧带骨化及黄韧带骨化的易感基因研究进展

脊柱后纵韧带骨化(ossification of posterior longitudi-nal ligamentum,OPLL)及黄韧带骨化(ossification of theligamentum flavum,OLF)均为发生于脊柱韧带的疾病,是导致颈胸部椎管狭窄、脊髓压迫的常见原因。外界因素及易感基因是影响OPLL与OLF发病率的重要原因。外界致病因子如慢性退行性变,内分泌和代谢性疾病,身高/体     

腰椎退变黄韧带的生物力学测定

目的:测定人腰椎黄韧带的生物力学指标,为研究黄韧带退变的力学机制提供依据.方法:30例腰椎退变患者为退变组,同时期的15例年轻腰椎外伤患者为对照组,黄韧带标本均取自两组患者L4/5间黄韧带.标本清洗、修剪后测量初始长度和横截面积.将标本固定在力学试验机上的夹具后,调零处理,对标本施加拉伸载荷,直至标本断裂,计算机自动输出实验数据,根据直接获得的位移、载荷数据.通过计算得出应变、应力以及弹性模量的数值,并对两组数据进行统计学分析.结果:对照组所能承受的最大拉力载荷为65.98±6.58N,退变组为81.39±8.32N;对照组位移为3.74±0.37mm,退变组为3.08±0.29mm:对照组应力为32.99±5.48MPa.退变组为41.59±3.72MPa;对照组应变为0.209±0.09,退变组为0.183±0.02;对照组弹性模量为157.79±5.76MPa,退变组为229.32±21.95MPa.各指标两组相比均有统计学意义(P＜0.05).结论:腰椎退变黄韧带与正常黄韧带相比,应力增加,应变降低,弹性模量增加.说明退变黄韧带抗拉伸作用增强,但弹性却降低.这可能与腰椎退变导致黄韧带受力改变有关.     

Characterization of cultured human ligamentum flavum cells in lumbar spine stenosis.

To investigate the pathogenesis of the degenerative changes of the ligamentum flavum occurring in lumbar spine stenosis, yellow ligament cells from patients with lumbar spine stenosis were cultured for the first time and subjected to biochemical, histochemical and immunohistochemical study. Stenotic ligamentum flavum (SLF) cells were seen to express high levels of alkaline phosphatase (ALP) activity and to produce a matrix rich in type I and III collagen, fibronectin and osteonectin. The matrix mineralized only following beta-glycerophosphate (betaGP) and ascorbic acid supplementation. Stimulation with human parathyroid hormone (PTH) increased intracellular cAMP concentration. These findings indicate that there was significant evidence of osteoblast-like activity in these cells. SLF cells also stained for S100 protein, type II and type X collagen, and co-localized type II collagen and ALP labelling, reflecting the presence of hypertrophic chondrocyte-like cells. Cultures from control patients showed neither osteoblastic nor chondrocytic features: they expressed type I and type III collagen and fibronectin, but did not stain for osteonectin, nor were bone-like calcifications observed in presence or absence of betaGP. Normal ligamentum flavum (NLF) cells did not synthesized S100 protein or type II or type X collagen, and showed a weaker response to PTH stimulation. Our data demonstrated the presence of hypertrophic chondrocytes with an osteoblast-like activity in the ligamentum flavum of patients with spinal stenosis suggesting that they could have a role in the pathophysiology of the heterotopic ossification of ligamentum flavum (OLF) in lumbar spine stenosis.     

胸椎黄韧带骨化与腰椎黄韧带退变的病理及基本代谢元素的比较研究

目的：探讨胸椎黄韧带骨化的病因。方法：对１４例胸椎黄韧带骨化（包括５例氟骨症）及１４例腰椎管狭窄症患者手术切除的黄韧带标本作病理研究；对患者血清及黄韧带采用雾化原子吸收法等方法测定钙、磷、镁、锌、铜、锰、钼、氟含量，取急性外伤性截瘫患者为对照。结果：（１）骨化黄韧带初期的病理改变与黄韧带退变性质类似；（２）除氟元素外，７种基本代谢元素在骨化与退变患者血清及黄韧带中含量均呈基本一致的变化规律；（３）非氟骨症骨化患者黄韧带中氟含量显著增高（Ｐ＜０．０１）。结论：本文证实胸椎黄韧带骨化发生于黄韧带退变的基础之上，但退变不直接导致骨化，元素氟是诱导退变黄韧带进一步骨化的重要诱因。     

Sodium fluoride modulates caprine osteoblast proliferation and differentiation

The cellular and molecular pathways of fluoride toxicity in osteoblasts are not very well understood. Therefore, the objective of the present study was to evaluate the effects of sodium fluoride (NaF) on caprine osteoblasts cultured in vitro. Caprine osteoblasts at 2.0 x 10(-4) cells/ml were incubated in vitro with NaF at 0, 10(-8), 10(-7), 10(-6), 10(-5), 10(-4), 5.0 x 10(-4), and 10(-3) M, and then proliferation, differentiation, apoptosis, calcification, and alkaline phosphatase activity were examined. Also, the effect of NaF on osteoblastic cell viability and the molecular events leading to apoptosis were determined. Electron microscopy revealed cytoplasmic and nuclear alterations in the ultrastructure of osteoblasts exposed to various NaF concentrations. A cell-based quantitative evaluation of the MTT assay showed that NaF at concentrations of 10(-8) to 10(-5) M promoted cell proliferation, whereas at 10(-4) to 10(-3) M it suppressed cell proliferation and induced apoptosis. Alkaline phosphatase (ALP) activity and mineralization ability increased in cells treated at 10(-8) to 10(-5) M with sodium versus the controls, but decreased at 5.0 x 10(-4) to 10(-3) M dosage. The highest incidence of early apoptotic cells and late apoptotic cells was reached (3.33% and 2.92%, respectively) under NaF concentration of 10(-4) M. In conclusion, results of this study indicated that NaF modulates osteoblast proliferation and differentiation in a dose-dependent manner and modified osteoblast metabolism bidirectionally, suggesting NaF may play a significant role in osteoblast physiology.     

氟化钠及其拮抗剂硫酸锌对成骨细胞的影响

目的观察不同剂量氟化钠（ＮａＦ）对成骨细胞的影响及硫酸锌（ＺｎＳＯ４）的拮抗作用,为临床治疗提供实验依据。方法取新生２４ｈＳＤ鼠头盖骨分离成骨细胞,在不同浓度的ＮａＦ和／或ＺｎＳＯ４（１０＾－５ｍｏｌ／Ｌ）中培养。经ＭＴＴ法测定细胞增殖,细胞分化由碱性磷酸酶（ＡＬＰ）和骨钙素水平测定得到。结果ＮａＦ对成骨细胞增殖及分化呈双向调节,主要表现为大剂量抑制,小剂量促进。当ＮａＦ浓度为１０＾－６ｍｏｌ／Ｌ     

骨细胞分离培养及其与成骨细胞鉴别比较的实验研究

目的:建立比较稳定的骨细胞实验室分离培养方法,并将其部分生物学特性与实验室培养的成骨细胞进行比较研究,明确两者区别.方法:采用序列酶消化法,分别从3只3dSD乳鼠骨骼中分离培养骨细胞和成骨细胞,培养24 h后进行细胞形态学观察.第1代细胞用碱性磷酸酶试剂盒采用重氮盐法(改良Kaplow氏法)染色,采用免疫细胞化学法对细胞的骨钙素(BGP)染色,测定碱性磷酸酶并计算其活性.结果:骨细胞多呈星状或树枝状,且有很多的突触;成骨细胞呈长梭形,有少量的突触.骨细胞碱性磷酸酶染色,胞浆内cAKP颗粒不明显;成骨细胞碱性磷酸酶染色,胞浆内可见众多cAKP颗粒.骨细胞BGP染色阳性明显,成骨细胞BGP染色阳性不如骨细胞明显.ALP在骨细胞中分泌较成骨细胞低,且有统计学差异.结论:实验室条件下能培养出骨细胞,该类细胞和成骨细胞有明显区别.     

骨保护蛋白基因转染成骨细胞后的表达及对其生物学行为的影响

目的 将人骨保护蛋白(OPG)基因转入体外培养的人成骨细胞,研究OPG基因在转染成骨细胞中的表达,并分析OPG基因转染对成骨细胞生物学行为的影响.方法 首先行人成骨细胞体外培养,然后用质粒peDNA3.1-hOPG转染成骨细胞,应用逆转录-聚合酶链反应(RT-PCR)和Western blot检测转染细胞OPG mRNA和蛋白质的表达.观察OPG基因转染后成骨细胞的增殖情况,对成骨细胞表达的碱性磷酸酶(ALP)和骨钙素(BGP)的含量进行检测,观察转基因细胞的生物学特性.结果 RT-PCR和Western blot检测结果表明在OPG基因转染后,成骨细胞表达的OPGmRNA和蛋白质含量均较未转染组增加.在OPG基因转染后,可明显促进成骨细胞的增殖,成骨细胞表达的ALP和BGP的含量均显著上升,而且在量效图中可以观察到随着OPG基因转染剂量增加,其增加程度也增加.结论 成骨细胞可作为转基因的受体细胞,成功表达目的 基因.转染OPG基因的成骨细胞可稳定、高效的表达OPG.OPG基因转染成骨细胞生物学特性稳定,而且可明显促进转染成骨细胞的成骨特性的表达.     

Suggestion of a deficient osteoblastic function in diabetes mellitus: The possible cause of osteopenia in diabetics

Summary The mechanism underlying diabetic osteopenia is still unclear and may involve osteoblastic activity and/or the deficit of insulin's anabolic action. Bone gla protein (BGP) is synthesized by the osteoblast and its synthesis increases with 1,25(OH)₂D₃ and fluoride. Because 1,25(OH)₂D₃ also stimulates insulin secretion, sodium fluoride administration can be used to investigate deficient osteoblastic activity in diabetics, as reflected by BGP levels. BGP was determined before and after administering sodium fluoride at a dosage of 50 mg/day/15 days to three groups: 14 patients with insulin-dependent diabetes, 16 diabetics on oral antidiabetic treatment, and 25 controls, all of similar age, sex, and characteristics. Basal BGP values (mean±SD) were low in diabetics on insulin treatment (4.3±1.1 ng/ml) and in diabetics on oral antidiabetics (5.8±1.2 ng/ml) as compared with controls (6.5±0.7 ng/ml) (P<0.001 and <0.05, respectively). After giving fluoride, BGP values did not change in the two diabetic groups but did vary in controls (8.1±0.6 ng/ml,P<0.001). These results suggest that deficient osteoblast function could be responsible for osteopenia in diabetics.     

葛根素对人牙周膜干细胞成骨分化的作用

目的:探讨葛根素对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)成骨分化的影响。方法:向体外培养的hPDLSCs中分别加入0.01mmol/L、0.1mmol/L和1mmol/L葛根素作为实验组,另设阳性对照组和空白组。MTT法检测葛根素对hPDLSCs活性影响,采用免疫荧光法、碱性磷酸酶(alkaline phosphotase,ALP)试剂盒、茜素红染色及qRT-PCR对葛根素作用后hPDLSCs生物学特性作初步观察。结果:0.01mmol/L葛根素可明显促进hPDLSCs增殖,免疫荧光染色显示实验组I型胶原蛋白(collagen-I,COL-I)和骨桥蛋白(osteopontin,OPN)均阳性表达;与空白组对比,实验组诱导7天后ALP活性升高,14天后茜素红染色见矿化结节形成,qRT-PCR检测ALP和骨钙蛋白(osteocalcin,OCN)分别在加药后7天和14天表达上调。结论:葛根素能够促进牙周膜干细胞向成骨细胞分化。     

Phenytoin and fluoride act in concert to stimulate bone formation and to increase bone volume in adult male rats

We have recently demonstrated that phenytoin is an osteogenic agent at low doses. The present paper describes observations that a mitogenic dose (i.e., 20 M in BGJ_b medium) of fluoride significantly augments the phenytoin-dependent stimulation of normal human bone cell proliferation and alkaline phosphatase (ALP) activity in cell culture. Additionally, the present study was designed to investigate whether fluoride and phenytoin would interact to increase bone formation in rats in vivo. Four groups of weight-matched adult male rats received daily I.P. injection of (1) vehicle (10% DMSO), (2) 5 mg/kg/day phenytoin, (3) 5 mg/kg/day phenytoin and 50 ppm NaF, and (4) 50 ppm NaF and vehicle, respectively, for 36 days. Sodium fluoride (NaF) was delivered in drinking water. Blood samples were drawn weekly and analyzed for serum osteocalcin, ALP, calcium, phosphorus, and 25 (OH)D₃. Rats were labeled with fetracycline at day 21 and 30 and histomorphometric analysis was carried out on the tibia at the end of the experiment. Neither agent by itself or together affected the serum calcium, phosphorus, or 25 (OH)D₃ levels. All measures of bone formation, i.e., serum osteocalcin level and ALP activity, bone ALP specific activity, mineral apposition rate, bone formation rate, and % bone formation surface, were increased by each agent. Fluoride and phenytoin together produced bigger increases in each parameter than did each agent alone. Trabecular bone volume was increased in the bibial metaphysis by fluoride or phenytoin alone; and when administered together, the two agents produced a greater increase. The combined effect of fluoride and phenytoin on each serum and bone formation parameter appeared to be less than additive. Phenytoin or fluoride alone did not significantly reduce the metaphyseal % resorptive surface. However, treatments with both agents together caused a highly significant reduction in the metaphyseal % resorptive surface. Phenytoin and fluoride together also significantly reduced (by 36%) the mineralization lag time, indicating that these agents did not promote osteomalacia. In summary, fluoride and phenytoin act in concert to stimulate bone formation and increase trabecular bone volume without causing mineralization defects in vivo and thus, may be a potential combination therapy for low bone mass in osteoporosis.     

Experimental studies on the ossification of the posterior longitudinal ligament of the cervical spine

Fluor, which is a natural substance contained in rice, vegetables, marine products and some seasonings, is assimilated into the body through ingestion. Approximately 60% of the total amount of this fluor intake would be based on rice. A histological study of cervical spine, knee ligaments, Achilles tendon and viscera of rabbits (approx. 12-16 weeks old) was made after injection with sodium fluoride in this study. The rabbits were divided into three groups: Group A (administered NaF 86.2 mg/kg, 5.7 mg/ml, 7 rabbits); Group B (administered NaF 31.5 mg/kg, 2.85 mg/ml, 6 rabbits) and a Control Group of 3 rabbits. Five rabbits in Group A (71.4%) and all six rabbits in Group B (100%) developed ossification of the posterior longitudinal ligament. Ossification of the yellow ligament was also found in two rabbits in Group A and one in Group B. No ossification was found in the Control Group. Both enchondral and intramembranous ossification were found in ossification of posterior longitudinal ossification in the rabbits.