近β钛合金对人成骨细胞骨保护素/核因子-κB受体活化因子配体mRNA表达影响的研究

[目的]探讨近B钛合金Ti-5Zr-3Sn-5Mo-15Nb(TLM)对人成骨样MG63细胞骨保护素(osteoprotegerin,OPG)及核因子-κB受体活化因子配体(receptor activator of nuclear factor kappa B ligand,RANKL)mRNA表达水平的影响.[方法]将人成骨样MG63细胞分别接种于纯钛钛片、TLM及微弧氧化处理的TLM表面,采用荧光实时定量PCR法检测OPG/RANKL mRNA表达水平.[结果]TLM组OPG mRNA表达水平略高于纯钛钛片组,但没有统计学意义(P>0.05),而微弧氧化处理组OPG mRNA水平明显升高,与纯钛钛片组及TLM组相比有显著性差异(P<0.05),三组之间的RANKL mRNA水平没有明显差异(P>0.05).[结论]经过微弧氧化处理的TLM可以上调成骨细胞OPG mRNA水平,从而影响OPG/RANKL mRNA的比值,调节成骨细胞与破骨细胞之间的平衡,进一步促进骨质重建.     

间歇性负压培养对人BMSCs骨保护素和骨保护素配体mRNA表达水平的影响

目的 探讨间歇性负压对体外培养的人BMSCs骨保护素(osteoprotegerin,OPG)mRNA和骨保护素配体(osteoprotegerin ligand,OPGL)mRNA表达水平的影响. 方法 2008年1月由2例髋关节骨性关节炎行人工关节置换患者自愿捐赠骨髓,分离并体外培养人BMSCs,取第3代细胞.实验组进行负压诱导,设置压力为50 kPa,30 min/次,2次/d,间歇性负压干预2周;对照组于普通CO2培养箱中常规培养.倒置相差显微镜下观察细胞形态,并通过实时定量PCR检测OPG mRNA和OPGL mRNA表达水平. 结果 实验组细胞增殖速度略低于对照组,细胞逐渐由梭形向多角形转变,有数个突起,数目和形态不定,10～14d细胞融合成单层,2周后逐渐形成多个散在致密岛状结构;对照组细胞大部分为梭形.间歇性负压培养2周后,与对照组比较,实验组细胞OPG mRNA表达水平显著提高,OPGL mRNA表达水平显著降低,OPGL mRNA与OPG mRNA比率显著下降,差异均有统计学意义(P＜0.01). 结论 间歇性负压促进入BMSCsOPG表达,同时抑制其OPGL表达.     

甲状旁腺激素对MG63细胞OPG、OPGL及其相关因子的调节

目的探讨甲状旁腺激素（PTH）调节骨吸收作用的途径和机理。方法用0．2-20μg PTH干预人成骨肉瘤MG63细胞2d，观察PTH对MG63细胞表达护骨素（OPG）、护骨素配体（OPGL）及其相关因子，如肿瘤坏死因子相关性凋亡诱导配体（TRAIL）、巨噬细胞集落刺激因子（M—CSF）的影响，用RT-PCR法检测OPG、OPGL、M—CSF和TRAIL mRNA表达情况，用Western blot分析法检测OPG和OPGL蛋白表达情况。结果PTH下调MG63细胞中OPG的表达，上调OPGL、M—CSF及TRAIL的表达。结论删能够减低成骨样细胞MG63中OPG／OPGL表达比率，增加M—CSF、TRAIL的表达。这可能有利于促进破骨细胞生成及活化，进而促进骨吸收。     

骨保护素和骨保护素配体在假体周围骨溶解中作用的实验研究

目的通过动物实验模拟聚乙烯颗粒诱导假体周围骨溶解,观察骨溶解的组织学反应和界膜内肿瘤坏死因子-α(TNF-α)、骨保护素(OPG)和骨保护素配体(OPGL)的变化,了解上述物质在磨损颗粒诱导假体周围骨溶解中的作用机理。方法实验选用SPF级雄性SD大鼠24只,经双侧膝关节向股骨远端植入钛合金棒,术后2、4、6、8、10周分别向左侧膝关节注入聚乙烯颗粒,右侧注射生理盐水作为对照,术后12周处死动物取材,观察界膜的组织学改变、TNF-α的浓度和OPG、OPGL的mRNA含量变化。所得数据选用配剥t检验进行统计学分析。结果注射颗粒侧钛合金棒周围有明显界膜形成,界膜中的TNF-α也有明显增高。RT-PCR半定量分析发现,注射颗粒侧界膜组织中OPG mRNA水平低于对照侧,而OPGL mRNA水平则高于对照侧(P＜0．05)。结论聚乙烯颗粒可引起钛合金棒周围多种细胞参与的异物肉芽肿反应,成骨细胞/骨髓基质细胞OPGL的合成增加而OPG的合成减少,此过程可能与TNF-α的升高有关。     

骨保护素和骨保护素配体在人骨髓基质细胞向成骨细胞分化过程中的表达

目的研究骨保护素和骨保护素配体在人骨髓基质细胞向成骨细胞诱导分化过程中的表达情况，探讨其在骨重建过程中的调节作用。方法实验中采用梯度离心法和酶消化法分别获得人骨髓基质细胞和成骨细胞，并将骨髓基质细胞向成骨细胞方向诱导分化。通过形态学观察、生化指标检测、细胞染色和矿化结节测定等方法，确定骨髓基质细胞的功能状态和分化程度。采用RT-PCR和Westem blot方法，检测骨髓基质细胞向成骨细胞分化过程中骨保护素和骨保护素配体的表达情况。结果获得的骨髓基质细胞和成骨细胞生长状态良好，生化指标稳定。骨髓基质细胞分化后，碱性磷酸酶分泌明显增加，可以产生大量的矿化结节，具有成熟成骨细胞的表型特征。RT-PCR和Western blot检测，在骨髓基质细胞向成骨细胞分化过程中，骨保护素在mRNA和蛋白质水平的表达明显升高，而骨保护素配体的表达则逐渐下降。细胞中OPG mRNA表达在第21天时达到最大，约为未分化时水平的2．5倍。而OPGLmRNA表达减少为未分化时1／2；细胞中OPG的蛋白质表达水平提高约为未分化细胞的6倍。统计学分析，P〈0．01，差异有显著性。结论在人骨髓基质细胞向成骨细胞分化过程中，骨保护素表达逐渐升高而骨保护素配体表达显著降低，两者比值的逐渐增大，从而发挥促进骨形成，抑制骨吸收的作用，这可能是协调骨重建周期有序进行的重要机制之一。     

不同浓度钛微粒对护骨素/护骨素配体基因表达影响的体外研究

目的: 探讨磨损微粒引起假体周围骨溶解的机理, 为人工关节假体松动的预防研究打下基础。方法: 采用不同浓度钛微粒 (粒径 <3μm) 对成骨细胞MG 63细胞进行 24h干预, 半定量RT PCR观察其对护骨素 (OPG) 及护骨素配体 (OPG- L) 基因表达的影响。结果:钛微粒能上调MG 63细胞OPG及OPG- L基因表达, 且对后者作用强于前者。结论: OPG- L/OPG比率随着钛微粒浓度的加大而增高, 引起骨溶解大于骨形成, 这可能是磨损微粒引起人工关节假体松动的重要原因, 这表明降低OPG- L/OPG比率有可能预防或逆转此病理过程。     

骨保护素/骨保护素配体在大鼠创伤性股骨头坏死中的表达及意义

[目的]探讨大鼠创伤性股骨头坏死模型中OPG/OPGL蛋白表达及意义。[方法]6个月龄SD大鼠32只,雌雄不限。分组:实验组按术后1、2、4、6周4个时间点将动物随机分成4组,每组8只。对照组采用自身非实验侧股骨头做正常对照。采用圆韧带离断方法制备大鼠创伤性股骨头坏死动物模型;分别于术后1,2,4,6周处死动物。光镜下观察股骨头坏死病理形态学改变;采用CM IAS真彩色医学图像分析系统检测骨髓腔内脂肪组织与造血组织比值并计数骨组织内空骨陷窝百分率;免疫组化技术检测OPG/OPGL蛋白在股骨头坏死组织中的表达。[结果]成功的复制出大鼠股骨头坏死进展期模型,呈现出典型股骨头坏死不同时期病理改变;股骨头坏死不同时期实验组空骨陷窝百分率高于对照组(P0.05);实验组骨髓腔内脂肪组织与造血组织的面积比值高于对照组(P0.05);免疫组化检测结果显示实验组OPG蛋白在股骨头坏死不同时期表达的光密度值(OD值)均明显低于对照组(P0.05);OPGL蛋白表达高于对照组(P0.05)。[结论](1)本实验成功复制了大鼠股骨头坏死模型并在一定程度上反映了股骨头坏死的病理变化过程;(2)股骨头坏死局部OPG、OPGL表达水平与病变严重程度密切相关,OPG表达随病程的延长逐渐降低;而OPGL表达水平则相反。提示OPG的过度表达可抑制破骨细胞功能,减少骨吸收;反之则促进骨吸收。如能提高坏死局部OPG浓度可以抑制骨丢失,将有助于股骨头修复,可能为股骨头坏死的早期治疗提供新方法。     

T3对MG63细胞株增殖及TRAIL，OPG，OPGL表达的影响

目的 观察在不同浓度三碘甲状腺原氨酸环境下人成骨肉瘤MG63细胞株增殖和肿瘤坏死因子相关凋亡诱导配体(TRAIL)及其护骨素(OPG)、护骨素配体(OPGL)的表达,探讨甲亢性骨质疏松症的发病机制.方法 用不同浓度T3(0、1.0×10-12、1.0×10-10、1.0×10-8 mol/L)分别刺激培养的MG63细胞24 h,MTT比色分析法测定细胞增殖,流式细胞仪检测细胞周期,逆转录PCR(RT-PCR)法检测基因表达情况,免疫细胞化学法检测MG63细胞中TRAIL的表达及分布.结果 T3以浓度依赖方式抑制MG63细胞的增殖,并将细胞阻滞于G1期.T3能下调MG63细胞中OPG的表达,上调OPGL和TRAIL的表达.TRAIL免疫反应阳性物质密度高浓度T3组明显高于对照组.结论 T3可抑制成骨细胞的增殖,导致成骨细胞中TRAIL和OPGL表达增多,OPG的表达减少.这可能是甲亢性骨质疏松症的重要发病机制之一.     

雄激素对体外成骨细胞QPG及其配体基因表达的影响

目的检测小鼠胎鼠成骨细胞体外培养并将不同浓度雄激素干预后,OPG和OPGLmRNA表达的变化,探讨雄激素在成骨细胞介导破骨细胞分化、活化过程中的调控机制。方法分离得到小鼠胎鼠颅盖骨成骨细胞,培养传代并选择第二代细胞用于实验;对第二代成骨细胞实施含10-10mol/L、10-9mol/L、10-8mol/L3种浓度雄激素的培养液干预;抽提细胞RNA,采用RT-PCR方法半定量观察成骨细胞中OPG和OPGL基因mRNA表达的变化。结果实验选用的各浓度组均未出现细胞毒性反应,雄激素干预使成骨细胞中OPG基因表达上调,而OPGL与OPG比率随时间呈递减趋势。结论雄激素可以特异性地在转录水平调节成骨细胞中OPG和OPGL基因的表达。     

LC调节人成骨样MG-63细胞表达OPG、RANKL分子机制的研究

目的 探讨大黄酸哌嗪雌酚酮（rhein-piperizinyl-estrone,命名为 LC）调节人成骨样MG-63细胞表达骨保护素(osteoprotegerin, OPG)、NF-κB激活受体配体(receptor activator of NF-κB ligand, RANKL)的分子机制。方法 在原工作基础上,选择兼有两种雌激素受体(estrogen receptor, ER)亚型表达的人成骨样MG-63细胞为研究模型,采用RT-PCR、免疫印迹及小RNA干扰等技术,探讨LC对人成骨细胞产生的骨吸收调节因子OPG、RANKL表达的作用及作用机制。结果 LC可上调MG-63细胞OPG表达及下调RANKL表达,该作用可被纯ER阻断剂ICI 182,780完全阻断,应用小RNA干扰技术进一步证实LC对成骨细胞OPG、RANKL表达的调节作用主要是由ERα介导的。结论 LC调节成骨细胞表达OPG、RANKL是经ER途径、主要是由ERα介导的。     

Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing.

Fracture healing is a unique biological process regulated by a complex array of signaling molecules and proinflammatory cytokines. Recent evidence for the role of tumor necrosis family members in the coupling of cellular functions during skeletal homeostasis suggests that they also may be involved in the regulation of skeletal repair. The expression of a number of cytokines and receptors that are of functional importance to bone remodeling (osteoprotegerin [OPG], macrophage colony-stimulating factor [M-CSF], and osteoprotegerin ligand [receptor activator of NF-kappaB ligand (RANKL)]), as well as inflammation (tumor necrosis factor alpha [TNF-alpha] and its receptors, and interleukin-1alpha [IL-1alpha] and -beta and their receptors) were analyzed over a 28-day period after the generation of simple transverse fractures in mouse tibias. OPG was expressed constitutively in unfractured bones and elevated levels of expression were detected throughout the repair process. It showed two distinct peaks of expression: the first occurring within 24 h after fracture and the second at the time of peak cartilage formation on day 7. In contrast, the expression of RANKL was nearly undetectable in unfractured bones but strongly induced throughout the period of fracture healing. The peak in expression of RANKL did not correlate with that of OPG, because maximal levels of expression were seen on day 3 and day 14, when OPG levels were decreasing. M-CSF expression followed the temporal profile of RANKL but was expressed at relatively high basal levels in unfractured bones. TNF-alpha, lymphotoxin-beta (LT-beta), IL-1alpha, and IL-1beta showed peaks in expression within the first 24 h after fracture, depressed levels during the period of cartilage formation, and increased levels of expression on day 21 and day 28 when bone remodeling was initiated. Both TNF-alpha receptors (p55 and p75) and the IL-1RII receptor showed identical patterns of expression to their ligands, while the IL-1R1 was expressed only during the initial period of inflammation on day 1 and day 3 postfracture. Both TNF-alpha and IL-1alpha expression were localized primarily in macrophages and inflammatory cells during the early periods of inflammation and seen in mesenchymal and osteoblastic cells later during healing. TNF-alpha expression also was detected at very high levels in hypertrophic chondrocytes. These data imply that the expression profiles for OPG, RANKL, and M-CSF are tightly coupled during fracture healing and involved in the regulation of both endochondral resorption and bone remodeling. TNF-alpha and IL-1 are expressed at both very early and late phases in the repair process, which suggests that these cytokines are important in the initiation of the repair process and play important functional roles in intramembraneous bone formation and trabecular bone remodeling.     

Prostate-specific antigen stimulates osteoprotegerin production and inhibits receptor activator of nuclear factor-kappaB ligand expression by human osteoblasts

BACKGROUND: Prostate cancer cells produce a large amount of prostate-specific antigen (PSA), which is widely used as a marker for this cancer. Even though it is widely used in the diagnosis of prostate cancer, many aspects of the pathophysiologic role of PSA in bone metastasis remain obscure. The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the activation of osteoclasts, while osteoprotegerin (OPG) neutralizes the action of RANKL. Various substances that act on bone have been shown to modulate the production of RANKL and OPG by osteoblasts. METHODS: In this study, we investigated the effect of PSA on the expression of OPG and RANKL mRNA and on protein production in human osteoblast-like cells. RESULTS: After addition of PSA and culture for 72 h, OPG mRNA expression and protein secretion by MG-63 and SaOS-2 cells showed a concentration-dependent increase. When osteoblasts were incubated with PSA (100 ng/ml), OPG mRNA expression and protein secretion increased with the passage of time. alpha1 -antichymotrypsin (ACT), which inactivates the serine protease activity of PSA, inhibited the increase of OPG mRNA expression and protein production in response to PSA, and this effect of PSA was also inhibited by anti-transforming growth factor-beta antibody. CONCLUSIONS: Based on our findings, PSA acts on human osteoblast-like cells via its own serine protease activity and promotes osteoblast differentiation. In addition, PSA stimulates OPG production and inhibits RANKL expression of osteoblasts, and inhibits bone resorption by osteoclasts, suggesting that it contributes to the characteristic osteoblastic features of bone metastases of prostate cancer.     

痩素对成骨细胞骨保护蛋白mRNA表达的影响初探

目的 初步探讨瘦素(Leptin)对成骨细胞骨保护蛋白(OPG)mRNA表达的影响。方法 用SYBR GreenⅠ染料实时监测定量RT-PCR的方法测定骨保护蛋白(OPG)mRNA表达。结果 瘦素(Leptin)使成骨细胞骨保护蛋白OPG mRNA表达呈剂量和时间依赖性降低。结论 瘦素(Leptin)作为一种内分泌因子，可直接与成骨细胞上leptin受体特异结合，通过受体后信号转导下调节成骨细胞OPG mRNA表达，使OPG mRNA表达降低，和其它因子共同参与骨再建调节。     

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.     

Serum osteoprotegerin and renal osteodystrophy.

BACKGROUND: Numerous growth factors and cytokines are known to modulate bone turnover. An important, recently discovered complex involved in osteoclastogenesis is the osteoprotegerin/osteoprotegerin-ligand (OPG/OPGL) cytokine complex, which is produced by osteoblasts. Many factors, including parathyroid hormone (PTH), appear to affect bone turnover through this pathway. In this disorder, the role of the OPG/OPGL system in the pathogenesis of renal osteodystrophy, a disease with either low or high bone turnover, has not been investigated so far. METHODS: Thirty-nine chronic haemodialysis patients had bone biopsies, including histomorphometric and histodynamic examinations. In addition, the following serum biochemistry parameters were measured: serum OPG, intact PTH, PTH 1-84, total PTH, osteocalcin, total and bone alkaline phosphatases, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. RESULTS: On average, serum OPG levels were above the normal range. They were lower in adynamic bone disease (ABD) patients, than in patients with predominant hyperparathyroidism (HP) or mixed osteodystrophy (MO). Significant negative correlations were found between serum OPG and PTH levels, and between serum OPG and parameters of bone resorption (ES/BS) and bone formation (ObS/BS and BFR/BS) in HP and MO patients with PTH values < or =1000 pg/ml. For intact PTH levels < or =300 pg/ml, serum OPG was significantly lower in the group with ABD than in those with HP or MO (P<0.05). CONCLUSION: In renal osteodystrophy the OPG/OPGL system is involved in the regulation of bone turnover induced by PTH. The determination of serum OPG levels could be of use in the diagnosis of low turnover bone disease, at least in association with PTH levels < or =300 pg/ml.     

人破骨细胞生成抑制因子编码区基因的克隆及在成肌细胞中的表达

目的：克隆人破骨细胞生成抑制因子（OPG／OCIF）编码区基因并在真核细胞中的表达。方法：以人骨肉瘤细胞系MG63的总RNA为模板，采用RT－PCR法得到OPG／OCIF的编码区cDNA，构建真核表达载体pIRES2-OPG-EGFP，在脂质体介导下转染小鼠成肌细胞系C2C12，经G418压力筛选建立稳定转染人OPG／OCIF的细胞系，共聚焦荧光显微镜及核酸杂交方法检测OPG／OCIF在细胞中的表达，结果：获得的人OPG／OCIF编码区全长cDNA序列与文献报道的核苷酸序列一致，核酸杂交证实稳定转染人OPG／OCIF编码区cDNA的小鼠成肌细胞系中有OPG／OCIF，mRNA的表达。结论：获得人破骨细胞生成抑制因子编码区全长cDNA并证实在真核细胞中稳定表达。     

前列腺癌、前列腺增生症患者血清骨保护素测定的临床价值

目的探讨前列腺癌（PCa）患者和前列腺增生（BPH）症患者血清中骨保护素（OPG）浓度的差异以及前列腺癌患者骨保护素浓度与血清前列腺特异性抗原（PSA）水平、前列腺体积是否具有相关性。方法采用双抗体夹心酶免法（ELISA）测定40例前列腺癌患者及40例前列腺增生患者血清OPG浓度,同时采集PCa患者的前列腺体积及PSA值。比较PCa患者及BPH患者血清OPG浓度的差异以及前列腺癌患者中OPG浓度与PSA、前列腺体积之间有无相关性。结果 PCa患者的血清OPG浓度平均值水平〔（14 900.19±5 168.65）pg/mL〕显著高于BPH组〔（10 457.87±4 786.29）pg/mL〕,差异有显著性意义（P〈0.01）。PCa患者血清OPG浓度与PSA值及前列腺体积之间均无明显相关性（r分别为=0.221、0.138,P均〉0.1）。结论血清OPG浓度对鉴别PCa和BPH有重要临床价值,PCa患者血清OPG浓度与血清PSA值及前列腺体积无明显相关性。     

不同性别及年龄因素对原发性骨质疏松症骨代谢指标、血清骨保护素及骨密度影响的研究

目的 研究不同性别及年龄因素对原发性骨质疏松症骨代谢指标、血清骨保护素及骨密度的影响。方法 选择92例原发性骨质疏松症患者为研究对象,根据性别的不同,将本组92例患者分为男性组36例和女性组56例;根据年龄的不同,将92例患者按10岁为一年龄段分组,分为40~50岁组38例,50~60岁组27例,60~70岁组17例,＞70岁组10例。分别对不同性别、不同年龄组患者的骨代谢指标（BGP、ALP）、血清骨保护素（OPG）及骨密度（BMD）进行检测,统计分析不同性别、年龄阶段患者各指标水平的变化趋势。结果 男性组患者平均BGP、ALP、OPG指标水平均低于女性组（P均＜0.05）,平均腰椎正位、右股骨颈BMD均高于女性组（P均＜0.05）。随着患者年龄的增加, BGP、ALP、OPG指标水平逐渐升高（P＜0.05）;同时,随着患者年龄的增加,腰椎正位、右股骨颈BMD指标值逐渐下降（P＜0.05）。结论 性别及年龄是影响原发性骨质疏松症患者的骨代谢指标、血清骨保护素及骨密度指标水平的重要因素,通过其影响机制的分析可为该病的防控提供参考依据。