脂多糖、前列腺素E_2在骨再生中的作用
摘要
研究背景：骨再生是由成骨细胞、破骨细胞和其他多种细胞及细胞因子参与的复杂的修复过程,其核心是破骨细胞介导的骨吸收和成骨细胞介导的骨形成的一个动态平衡过程。骨折后的愈合和贯穿人一生的骨代谢都是靠骨再生过程来完成的。然而,中年后随着年龄增长由于骨吸收速度大于骨形成速度所造成的骨质疏松、以及由于骨形成停滞造成的骨折后延迟愈合甚至骨不连等疾病一直是困扰医务工作者的难题。近年来,参与及调控骨再生过程中的细胞因子已成为人们研究的热点,而作为激发骨折愈合起始阶段并起着重要作用的促炎症因子在骨再生中的作用机理还未被很好地阐明。脂多糖(lipopolysaccharide, LPS),前列腺素E2 (prostaglandin E2, PGE2)都是骨再生过程中重要的促炎症因子。LPS,可诱导多种炎症因子的释放。LPS对细胞及组织的作用被喻为“双刃剑”。早期的研究表明LPS能直接导致细胞及组织的凋亡或坏死,而最新的研究发现低浓度的LPS又可促进细胞或组织的增殖。而低浓度的LPS是否能促使成骨细胞增殖还鲜有报道。PGE2,一直是研究骨再生过程中的热点,内源性和外源性的PGE2、体外和体内PGE2在骨再生过程中的作用各不相同。PGE2促进骨吸收的作用研究已相对成熟,而其在成骨方面的作用机制还未被阐明。NF-κB,是广泛存在于胞浆中的一种快反应转录因子,通常以p65-p50二聚体的形式存在,在LPS等外界因素刺激成骨细胞分泌PGE2的同时,往往伴随着NF-κB的活化,而抑制其活化可阻碍由于雌激素缺乏造成的骨丢失。本课题将研究LPS、PGE2和NF-κB在成骨细胞增殖和分化以及在体内骨折愈合中的相互作用。
目的：探讨LPS和PGE2两种促炎症因子在骨再生过程中的作用及信号途径,试图找到一个关键性的靶点,为以后基因治疗骨不连、骨延迟愈合等疾病提供基础实验支持。
方法：
1.采用500 ng/mL的LPS、500ng/mL的LPS和COX-2抑制剂sc791、500ng/mL的LPS和NF-κB抑制剂BAY11-7082分别处理MC3T3-E1成骨细胞1h、6h和12h。采用流式细胞仪检测各组细胞的细胞周期,利用RT-PCR和westernblot法检测和比较LPS对成骨细胞NF-κB mRNA和蛋白水平的变化,采用western blot法检测各组细胞NF-κB、COX-2蛋白表达的变化,利用免疫荧光和EMSA法检测各组细胞NF-κB蛋白核转移情况和活力的变化情况。
2.采用10μmol/L PGE2、5μmol/L BAY 11-7082和10μmol/L PGE2分别处理MC3T3-E1成骨细胞30 min,2 h和4 h。通过MTT法检测细胞增殖活力,利用流式细胞检测法检测各组的细胞周期和凋亡率变化,通过检测细胞ALP活力了解细胞的分化情况,采用western blot法检测各组细胞NF-κB、BMP-7和Id2蛋白的表达、利用免疫荧光和EMSA法检测各组细胞NF-κB蛋白的核转移和活力变化。
3.采用wistar大鼠造桡骨骨折模型,用BAY11-7082进行局部骨折部位注射干预,分别于3 d,7 d,14 d和28 d取出骨痂组织,检测骨痂组织ALP活力了解其成骨情况,采用elisa法检测骨痂组织PGE2含量,利用western blot法检测骨痂组织中NF-κB、BMP-7和Id2蛋白的表达情况,利用组织切片HE染色法观察14 d和28 d时处理组和非处理组骨痂组织结构。
4.10μmol/L PGE2处理细胞30 min后,收集正常对照组和处理组的细胞,用TRIZOL冰上悬浮后立即转交至上海生物芯片公司做RNA的提取、RNA的检测和基因芯片的杂交。本课题采用的基因芯片为美国西北大学基因芯片中心实验室制备的人类全基因组芯片,芯片包含34995个基因探针点。芯片结果采用Agilent扫描仪进行扫描,Feature extraction软件读取数据后进行Normalize处理分析。将Ratio≥2或≤0.5, p Value Log Ratio<0.01作为表达差异显著的基因。
结果：
1.500 ng/mL LPS处理细胞6 h、12 h后,细胞的增殖指数有显著性增高(P<0.01), COX-2抑制剂sc791不能抑制由LPS诱导的成骨细胞增殖,NF-κB抑制剂BAY 11-7082可显著抑制由LPS诱导的成骨细胞增殖。LPS处理成骨细胞6 h后,NF-κB的mRNA表达与对照组比较无显著差异(P>0.05),蛋白表达水平与对照组相比有显著的增高(P<0.01),说明LPS作用在成骨细胞NF-κB的蛋白水平,而不是在mRNA水平。LPS处理成骨细胞后NF-κB发生核易位和活力的增强,而COX-2蛋白的表达与对照组比较无显著变化(P>0.05), sc791并不能抑制由LPS诱导的NF-κB活力增强,说明低浓度的LPS是通过非COX-2依赖的NF-κB途径诱导成骨细胞增殖的。
2.10μmol/L PGE2处理成骨细胞2 h和4 h时,细胞增殖活力与对照组比较有显著下降(P<0.01),而细胞的ALP活力较正常组显著增加(P<0.01),说明PGE2可抑制成骨细胞增殖,促进其分化。PGE2处理成骨细胞30 min时细胞的NF-κB和BMP-7蛋白表达与对照组比较即显著增加(P<0.01),Id2蛋白表达与对照组比较显著下降(P<0.01),细胞内NF-κB蛋白出现核转移和活力增加,说明PGE2可诱导成骨细胞内NF-κB的活化,BMP-7蛋白的表达增强,Id2蛋白表达下降。PGE2单独处理成骨细胞时细胞的凋亡率与正常组比较无显著差异(P>0.05),BAY11-7082和PGE2共同处理细胞后,细胞的早期凋亡率和晚期凋亡率与正常对照组比较都有显著增高(P<0.01),且NF-κB、BMP-7蛋白表达与对照组比较显著下降(P<0.01),Id2蛋白表达与对照组比较显著增加(P<0.01),ALP活力较对照组显著下降(P<0.01),说明NF-κB抑制剂可显著抑制由PGE2诱导的细胞分化,与PGE2共同作用可引起细胞凋亡,并可显著抑制由PGE2诱导的成骨细胞内BMP-7和Id2蛋白表达的变化。
3.体内实验中骨折未处理组3 d和7 d时骨痂组织的PGE2含量和ALP活力都较对照组显著增加(P<0.01), NF-κB、BMP-7蛋白表达较对照组显著增加(P<0.01),Id2蛋白表达较对照组显著下降(P<0.01),说明在正常骨折愈合过程中,PGE2含量和ALP活力增高,NF-κB、BMP-7蛋白表达升高,Id2蛋白表达下降。用BAY11-7082干预后,骨痂的PGE2含量和ALP活力都较对照组显著下降(P<0.01)。NF-κB、BMP-7蛋白表达较对照组显著下降(P<0.01),Id2蛋白表达较对照组显著增加(P<0.01)。组织学切片HE染色可见BAY11-7082处理组术后14 d时骨痂组织为软骨性骨痂,而骨折未处理组术后14d时骨痂组织的边缘处已经形成骨性骨痂,BAY11-7082处理组28 d时骨痂组织中开始出现骨性骨痂,而骨折未处理组28 d时骨痂组织中均为骨性骨痂。表明BAY11-7082可抑制体内骨折愈合过程中PGE2含量和ALP的活力,最终导致骨折延迟愈合。
4.PGE2处理30 min后,成骨细胞基因表达谱中有276个基因表达上调,168个基因表达下调。与炎症、骨再生有关的PGE2诱导的上调基因有：monocyte to macrophage differentiation-associated (MMD)基因,即单核细胞向巨噬细胞转化基因；nuclear receptor subfamily 4, group A, member 2基因,即NR4A2, NF-κB的活化是和NR4A2的启动子密切相联的,且NF-κB和NR4A2密切相连的位点正是p65-p50异源二聚体或p50同源二聚体；catenin(cadherin-associated protein),即钙粘蛋白,Wnt/β-catenin通路通常被称为Wnt通路,在骨再生过程中起重要作用；osteoblast specific factor (POSTN),即成骨细胞特异性因子；bone morphogenetic protein 7 (osteogenic protein 1),即BMP-7。与炎症、骨再生有关的PGE2诱导的上调基因有：Id1、Id2、Id3。
结论：
1.低浓度的LPS可诱导成骨细胞增殖。LPS诱导的成骨细胞增殖作用是通过NF-κB信号途径,而与COX-2的蛋白表达无关。我们推测在骨再生炎症反应的初始阶段,NF-κB信号途径起核心作用。
2.PGE2可通过NF-κB信号途径来诱导BMP-7蛋白的高表达和Id2蛋白表达下降,从而引起成骨细胞分化,促进骨形成。BAY11-7082可抑制PGE2对成骨细胞的分化作用,共同作用于成骨细胞可引起细胞凋亡。NF-κB可调节骨折愈合过程中PGE2的产量。BAY11-7082可抑制骨折愈合过程中PGE2产量的增加,降低ALP活力,最终导致骨折延迟愈合。PGE2-NF-κB的相互调节作用在骨再生过程中是非常重要的信号传导途径。
3.应用基因芯片技术研究PGE2对成骨细胞基因表达谱的影响,研究发现PGE2处理后成骨细胞促成骨的基因和核受体基因上调,Id蛋白家族表达下调,基本支持我们前两章的结论。因此,我们可以把NF-κB作为骨创伤修复过程中的一个重要靶点,开发其特异性干预剂,用于骨不连或骨延迟愈合的治疗中。
Background:The process of bone remodeling is complicated and many cells and cytokines inculding osteoblasts, osteoclasts participate in the process. The central of which is a dynamic balance precess result from the coupled activity of these two cell populations—the osteoblasts which mediate the bone formation process and the osteoclasts which mediate the bone resorbing process. However, the osteoporosis due to the bone resorbing rate exceed the bone formation rate and the non-union of bone owing to the stopping of the bone formation process are always the medical problems. Inflammation is a likely feature of bone repair processes, and several cytokines such as prostaglandins, leukotrienes, cytokines, and growth factors have been reported to be involved in bone metabolism. LPS (lipopolysaccharide, LPS) and PGE2 (prostaglandin E2, PGE2) are both important proinflammatory cytokines in bone regeneration.
The mechanism of LPS on bone remodelling, however, is unclear as it has a biphasic response on cells and organs. PGE2 is the most potent stimulator during bone regeneration. The mechanism of PGE2 on bone formation has not been clarified. The activation of the fast response transcription factor nuclear factorκB (NF-κB),which exists normally as an complex composed of p65 and p50 had been reported to be involved in the regulation of the LPS-induced production of PGE2. The inhibition of NF-κB activity could block estrogen-deficiency induced bone loss. In this study, we focus on the relationship of LPS、PGE2 and NF-κB in bone regeneration in vitro and in vivo.
Objective:To investigate the role of LPS and PGE2 in the bone regeneration process and the mechanism of signal conduction. To find the central factor for the therapy of nonunion or delayed union fractures providing a laboratory evidence.
Methods:
1 MC3T3-E1 osteoblasts were challenged with 500 ng/mL LPS,500 ng/mL LPS plus 50 nmol/L sc791 (selective COX-2 inhibitor), and 500 ng/mL LPS plus 5μmol/L BAY 11-7082(NF-κB inhibitor) respectively for Flow cytometry analysis、RT-PCR、western blot analysis, emsa and Immunofluorescence Analysis.
2 Mouse MC3T3-E1 osteoblasts were challenged with 10μmol/L PGE2、5μmol/L BAY 11-7082 for 30 min,2 h and 4 h respectively. MTT method for Assessment of cell viability, cell differentiation was analyzed using ALP activity. Western blot analysis for NF-κB、BMP-7 and Id2 protein expression. Flow cytometry analysis for cell cycle and apoptosis rate. EMSA and Immunofluorescence analysis for NF-κB translocation and activation.
3 Rats were randomly divided into three groups, including the controls, the radius fracture group-1 locally injected with normal saline and the radius fracture group-2 locally injected with 50 umol/L BAY 11-7082. The callus tissue were harvested at 3 d,7 d,14 d and 28 d after being fracture for Western blot analysis, ALP assessment, PGE2 production assay and histological observation.
4 The cells were collected from the control group and the PGE2 treated group.The cell suspension with TRIZOL were immediately transmited to the "ShangHai biology gene chip company" for RAN extracted、measurement and hybridization with gene chip. By adopting software Feature extraction to analyze acquired genes of differential expression. Ratio≥2 or≤0.5, p Value Log Ratio<0.01 means the difference is significant.
Results:
1 500 ng/mL LPS could induce osteoblasts proliferation and increase the expression of NF-kB p65 in the nucleus and the specific NF-kB DNA binding activity, but had no influence on COX-2 expression at the same time. The cellular DPI value and NF-kB p65 protein expression had significant increase (P<0.01) at 6 h and 12 h after LPS treatment comparing to the control group. Whereas COX-2 expression had no significant difference (P>0.05) compared with untreated cells. NF-kB mRNA had no significant difference (P>0.05) but the protein expression had significant increase (P<0.01) comparing to the control group in the presence of LPS. BAY 11-7082 could inhibit the cell proliferation produced by 500 ng/mL LPS whereas sc791 could not do it. The results showed that low concentrations of LPS is beneficial for MC3T3-E1 osteoblast proliferation. LPS can induce proliferation of osteoblasts through NF-κB pathway and its induction is not correlated to COX-2.
2 10μmol/L PGE2 exposure obviously caused inhibition of cell proliferation (P<0.01) and significant increasing the activity of ALP (P<0.01) in MC3T3-E1 cells at the time course of 2 h and 4 h. Preincubation with 5μmol/L NF-κB inhibitor can inhibit the PGE2-medicated ALP activity. Our data implied that PGE2 can increase ALP production through NF-κB activity. Western blot analysis indicated that the expression of NF-κB/p65 and BMP-7 protein in the stimulated cells were significant higher (P<0.01) than that of the controls after 10μmol/L PGE2 treated,whereas the Id2 expression was decreased (P<0.01) under the same conditions. NF-κB inhibitor BAY 11-7082 can inhibit the inductuion of PGE2. This means that PGE2 can inhibit the expresson of Id2 factors and increase the expression of NFKB/p65 and BMP-7 through NF-κB pathway. BAY 11-7082 plus PGE2 can induce osteoblast apoptosis but PGE2 alone can not do it.
3 A significant increase of PGE2 production (P<0.01) was gained in the fracture groups compared with the controls after being fractured for 3 d and 7 d, while the production of PGE2 was substantially inhibited by the injection of specific inhibitors of NF-κB. In the same time, the ALP activity of callus was also increased and can be inhibited by the injection of NF-κB inhibitor BAY 11-7082. Just as the in vitro data, accompanying with the inhibition of NF-κB activation, the expression of BMP-7 was decreased, while the expression of Id2 was increased. These results implied that PGE2 production can be blocked through inhibition of the NF-κB pathway in vivo. Histological analysis results suggested that local injection of NF-κB inhibitor BAY 11-7082 at the fracture sites could result in delaying the union of fractures.
4 Analysis on hybridezed chip sifts suggested that 276 significant up-regulated genes and 168 significant down-regulated genes after PGE2 treatment.The up-regulated genes induced by PGE2 related to inflammation and bone regeneration are:monocyte to macrophage differentiation-associated (MMD) gene. nuclear receptor subfamily 4, group A, member 2 gene, NR4A2, which is connected tightly with the p65-p50 heterodimer or homodimer of NF-κB. catenin (cadherin-associated protein). osteoblast specific factor (POSTN). bone morphogenetic protein 7 (osteogenic protein 1), BMP-7. The down-regulated genes induced by PGE2 related to inflammation and bone regeneration are:Id1, Id2 and Id3.
Conclusion:
1 Low concentration of lipopolysaccharide can induce directly proliferation of MC3T3-E1 osteoblasts mediated by COX-2-independent NF-κB pathway, and These studies provide insight into a potential mechanism by which NF-κB signal pathway has a central role in the initial phase of inflammation of the bone regeneratrion.
2 PGE2 can induce osteoblast differentiation and promote bone formation by increasing BMP-7 protein expression and decreasing Id2 protein expression through NF-κB signal pathway. BAY 11-7082 can inhibit the osteoblast differentiation induced by PGE2 and induce the cell apoptosis with the role of PGE2. NF-κB can regulate the PGE2 production in vivo. BAY11-7082 can inhibit the PGE2 production, decrease the ALP activity and finally result in delaying the union of fractures. Cooperation between PGE2 production and NF-κB activation is the most primary step during the bone remodeling in vitro and in vivo.
3 The up-regulated genes of promoting bone formation and nuclear receptor and the down-regulated genes of Id family found in expression of osteoblasts gene profile induced by PGE2 support our study results. Therefore, NF-κB could be taken as an important targeted gene in the bone healing process_and has a potential therapy role for nonunion or delayed union fractures.
目的：探讨LPS和PGE2两种促炎症因子在骨再生过程中的作用及信号途径,试图找到一个关键性的靶点,为以后基因治疗骨不连、骨延迟愈合等疾病提供基础实验支持。
方法：
1.采用500 ng/mL的LPS、500ng/mL的LPS和COX-2抑制剂sc791、500ng/mL的LPS和NF-κB抑制剂BAY11-7082分别处理MC3T3-E1成骨细胞1h、6h和12h。采用流式细胞仪检测各组细胞的细胞周期,利用RT-PCR和westernblot法检测和比较LPS对成骨细胞NF-κB mRNA和蛋白水平的变化,采用western blot法检测各组细胞NF-κB、COX-2蛋白表达的变化,利用免疫荧光和EMSA法检测各组细胞NF-κB蛋白核转移情况和活力的变化情况。
2.采用10μmol/L PGE2、5μmol/L BAY 11-7082和10μmol/L PGE2分别处理MC3T3-E1成骨细胞30 min,2 h和4 h。通过MTT法检测细胞增殖活力,利用流式细胞检测法检测各组的细胞周期和凋亡率变化,通过检测细胞ALP活力了解细胞的分化情况,采用western blot法检测各组细胞NF-κB、BMP-7和Id2蛋白的表达、利用免疫荧光和EMSA法检测各组细胞NF-κB蛋白的核转移和活力变化。
3.采用wistar大鼠造桡骨骨折模型,用BAY11-7082进行局部骨折部位注射干预,分别于3 d,7 d,14 d和28 d取出骨痂组织,检测骨痂组织ALP活力了解其成骨情况,采用elisa法检测骨痂组织PGE2含量,利用western blot法检测骨痂组织中NF-κB、BMP-7和Id2蛋白的表达情况,利用组织切片HE染色法观察14 d和28 d时处理组和非处理组骨痂组织结构。
4.10μmol/L PGE2处理细胞30 min后,收集正常对照组和处理组的细胞,用TRIZOL冰上悬浮后立即转交至上海生物芯片公司做RNA的提取、RNA的检测和基因芯片的杂交。本课题采用的基因芯片为美国西北大学基因芯片中心实验室制备的人类全基因组芯片,芯片包含34995个基因探针点。芯片结果采用Agilent扫描仪进行扫描,Feature extraction软件读取数据后进行Normalize处理分析。将Ratio≥2或≤0.5, p Value Log Ratio<0.01作为表达差异显著的基因。
结果：
1.500 ng/mL LPS处理细胞6 h、12 h后,细胞的增殖指数有显著性增高(P<0.01), COX-2抑制剂sc791不能抑制由LPS诱导的成骨细胞增殖,NF-κB抑制剂BAY 11-7082可显著抑制由LPS诱导的成骨细胞增殖。LPS处理成骨细胞6 h后,NF-κB的mRNA表达与对照组比较无显著差异(P>0.05),蛋白表达水平与对照组相比有显著的增高(P<0.01),说明LPS作用在成骨细胞NF-κB的蛋白水平,而不是在mRNA水平。LPS处理成骨细胞后NF-κB发生核易位和活力的增强,而COX-2蛋白的表达与对照组比较无显著变化(P>0.05), sc791并不能抑制由LPS诱导的NF-κB活力增强,说明低浓度的LPS是通过非COX-2依赖的NF-κB途径诱导成骨细胞增殖的。
2.10μmol/L PGE2处理成骨细胞2 h和4 h时,细胞增殖活力与对照组比较有显著下降(P<0.01),而细胞的ALP活力较正常组显著增加(P<0.01),说明PGE2可抑制成骨细胞增殖,促进其分化。PGE2处理成骨细胞30 min时细胞的NF-κB和BMP-7蛋白表达与对照组比较即显著增加(P<0.01),Id2蛋白表达与对照组比较显著下降(P<0.01),细胞内NF-κB蛋白出现核转移和活力增加,说明PGE2可诱导成骨细胞内NF-κB的活化,BMP-7蛋白的表达增强,Id2蛋白表达下降。PGE2单独处理成骨细胞时细胞的凋亡率与正常组比较无显著差异(P>0.05),BAY11-7082和PGE2共同处理细胞后,细胞的早期凋亡率和晚期凋亡率与正常对照组比较都有显著增高(P<0.01),且NF-κB、BMP-7蛋白表达与对照组比较显著下降(P<0.01),Id2蛋白表达与对照组比较显著增加(P<0.01),ALP活力较对照组显著下降(P<0.01),说明NF-κB抑制剂可显著抑制由PGE2诱导的细胞分化,与PGE2共同作用可引起细胞凋亡,并可显著抑制由PGE2诱导的成骨细胞内BMP-7和Id2蛋白表达的变化。
3.体内实验中骨折未处理组3 d和7 d时骨痂组织的PGE2含量和ALP活力都较对照组显著增加(P<0.01), NF-κB、BMP-7蛋白表达较对照组显著增加(P<0.01),Id2蛋白表达较对照组显著下降(P<0.01),说明在正常骨折愈合过程中,PGE2含量和ALP活力增高,NF-κB、BMP-7蛋白表达升高,Id2蛋白表达下降。用BAY11-7082干预后,骨痂的PGE2含量和ALP活力都较对照组显著下降(P<0.01)。NF-κB、BMP-7蛋白表达较对照组显著下降(P<0.01),Id2蛋白表达较对照组显著增加(P<0.01)。组织学切片HE染色可见BAY11-7082处理组术后14 d时骨痂组织为软骨性骨痂,而骨折未处理组术后14d时骨痂组织的边缘处已经形成骨性骨痂,BAY11-7082处理组28 d时骨痂组织中开始出现骨性骨痂,而骨折未处理组28 d时骨痂组织中均为骨性骨痂。表明BAY11-7082可抑制体内骨折愈合过程中PGE2含量和ALP的活力,最终导致骨折延迟愈合。
4.PGE2处理30 min后,成骨细胞基因表达谱中有276个基因表达上调,168个基因表达下调。与炎症、骨再生有关的PGE2诱导的上调基因有：monocyte to macrophage differentiation-associated (MMD)基因,即单核细胞向巨噬细胞转化基因；nuclear receptor subfamily 4, group A, member 2基因,即NR4A2, NF-κB的活化是和NR4A2的启动子密切相联的,且NF-κB和NR4A2密切相连的位点正是p65-p50异源二聚体或p50同源二聚体；catenin(cadherin-associated protein),即钙粘蛋白,Wnt/β-catenin通路通常被称为Wnt通路,在骨再生过程中起重要作用；osteoblast specific factor (POSTN),即成骨细胞特异性因子；bone morphogenetic protein 7 (osteogenic protein 1),即BMP-7。与炎症、骨再生有关的PGE2诱导的上调基因有：Id1、Id2、Id3。
结论：
1.低浓度的LPS可诱导成骨细胞增殖。LPS诱导的成骨细胞增殖作用是通过NF-κB信号途径,而与COX-2的蛋白表达无关。我们推测在骨再生炎症反应的初始阶段,NF-κB信号途径起核心作用。
2.PGE2可通过NF-κB信号途径来诱导BMP-7蛋白的高表达和Id2蛋白表达下降,从而引起成骨细胞分化,促进骨形成。BAY11-7082可抑制PGE2对成骨细胞的分化作用,共同作用于成骨细胞可引起细胞凋亡。NF-κB可调节骨折愈合过程中PGE2的产量。BAY11-7082可抑制骨折愈合过程中PGE2产量的增加,降低ALP活力,最终导致骨折延迟愈合。PGE2-NF-κB的相互调节作用在骨再生过程中是非常重要的信号传导途径。
3.应用基因芯片技术研究PGE2对成骨细胞基因表达谱的影响,研究发现PGE2处理后成骨细胞促成骨的基因和核受体基因上调,Id蛋白家族表达下调,基本支持我们前两章的结论。因此,我们可以把NF-κB作为骨创伤修复过程中的一个重要靶点,开发其特异性干预剂,用于骨不连或骨延迟愈合的治疗中。
Background:The process of bone remodeling is complicated and many cells and cytokines inculding osteoblasts, osteoclasts participate in the process. The central of which is a dynamic balance precess result from the coupled activity of these two cell populations—the osteoblasts which mediate the bone formation process and the osteoclasts which mediate the bone resorbing process. However, the osteoporosis due to the bone resorbing rate exceed the bone formation rate and the non-union of bone owing to the stopping of the bone formation process are always the medical problems. Inflammation is a likely feature of bone repair processes, and several cytokines such as prostaglandins, leukotrienes, cytokines, and growth factors have been reported to be involved in bone metabolism. LPS (lipopolysaccharide, LPS) and PGE2 (prostaglandin E2, PGE2) are both important proinflammatory cytokines in bone regeneration.
The mechanism of LPS on bone remodelling, however, is unclear as it has a biphasic response on cells and organs. PGE2 is the most potent stimulator during bone regeneration. The mechanism of PGE2 on bone formation has not been clarified. The activation of the fast response transcription factor nuclear factorκB (NF-κB),which exists normally as an complex composed of p65 and p50 had been reported to be involved in the regulation of the LPS-induced production of PGE2. The inhibition of NF-κB activity could block estrogen-deficiency induced bone loss. In this study, we focus on the relationship of LPS、PGE2 and NF-κB in bone regeneration in vitro and in vivo.
Objective:To investigate the role of LPS and PGE2 in the bone regeneration process and the mechanism of signal conduction. To find the central factor for the therapy of nonunion or delayed union fractures providing a laboratory evidence.
Methods:
1 MC3T3-E1 osteoblasts were challenged with 500 ng/mL LPS,500 ng/mL LPS plus 50 nmol/L sc791 (selective COX-2 inhibitor), and 500 ng/mL LPS plus 5μmol/L BAY 11-7082(NF-κB inhibitor) respectively for Flow cytometry analysis、RT-PCR、western blot analysis, emsa and Immunofluorescence Analysis.
2 Mouse MC3T3-E1 osteoblasts were challenged with 10μmol/L PGE2、5μmol/L BAY 11-7082 for 30 min,2 h and 4 h respectively. MTT method for Assessment of cell viability, cell differentiation was analyzed using ALP activity. Western blot analysis for NF-κB、BMP-7 and Id2 protein expression. Flow cytometry analysis for cell cycle and apoptosis rate. EMSA and Immunofluorescence analysis for NF-κB translocation and activation.
3 Rats were randomly divided into three groups, including the controls, the radius fracture group-1 locally injected with normal saline and the radius fracture group-2 locally injected with 50 umol/L BAY 11-7082. The callus tissue were harvested at 3 d,7 d,14 d and 28 d after being fracture for Western blot analysis, ALP assessment, PGE2 production assay and histological observation.
4 The cells were collected from the control group and the PGE2 treated group.The cell suspension with TRIZOL were immediately transmited to the "ShangHai biology gene chip company" for RAN extracted、measurement and hybridization with gene chip. By adopting software Feature extraction to analyze acquired genes of differential expression. Ratio≥2 or≤0.5, p Value Log Ratio<0.01 means the difference is significant.
Results:
1 500 ng/mL LPS could induce osteoblasts proliferation and increase the expression of NF-kB p65 in the nucleus and the specific NF-kB DNA binding activity, but had no influence on COX-2 expression at the same time. The cellular DPI value and NF-kB p65 protein expression had significant increase (P<0.01) at 6 h and 12 h after LPS treatment comparing to the control group. Whereas COX-2 expression had no significant difference (P>0.05) compared with untreated cells. NF-kB mRNA had no significant difference (P>0.05) but the protein expression had significant increase (P<0.01) comparing to the control group in the presence of LPS. BAY 11-7082 could inhibit the cell proliferation produced by 500 ng/mL LPS whereas sc791 could not do it. The results showed that low concentrations of LPS is beneficial for MC3T3-E1 osteoblast proliferation. LPS can induce proliferation of osteoblasts through NF-κB pathway and its induction is not correlated to COX-2.
2 10μmol/L PGE2 exposure obviously caused inhibition of cell proliferation (P<0.01) and significant increasing the activity of ALP (P<0.01) in MC3T3-E1 cells at the time course of 2 h and 4 h. Preincubation with 5μmol/L NF-κB inhibitor can inhibit the PGE2-medicated ALP activity. Our data implied that PGE2 can increase ALP production through NF-κB activity. Western blot analysis indicated that the expression of NF-κB/p65 and BMP-7 protein in the stimulated cells were significant higher (P<0.01) than that of the controls after 10μmol/L PGE2 treated,whereas the Id2 expression was decreased (P<0.01) under the same conditions. NF-κB inhibitor BAY 11-7082 can inhibit the inductuion of PGE2. This means that PGE2 can inhibit the expresson of Id2 factors and increase the expression of NFKB/p65 and BMP-7 through NF-κB pathway. BAY 11-7082 plus PGE2 can induce osteoblast apoptosis but PGE2 alone can not do it.
3 A significant increase of PGE2 production (P<0.01) was gained in the fracture groups compared with the controls after being fractured for 3 d and 7 d, while the production of PGE2 was substantially inhibited by the injection of specific inhibitors of NF-κB. In the same time, the ALP activity of callus was also increased and can be inhibited by the injection of NF-κB inhibitor BAY 11-7082. Just as the in vitro data, accompanying with the inhibition of NF-κB activation, the expression of BMP-7 was decreased, while the expression of Id2 was increased. These results implied that PGE2 production can be blocked through inhibition of the NF-κB pathway in vivo. Histological analysis results suggested that local injection of NF-κB inhibitor BAY 11-7082 at the fracture sites could result in delaying the union of fractures.
4 Analysis on hybridezed chip sifts suggested that 276 significant up-regulated genes and 168 significant down-regulated genes after PGE2 treatment.The up-regulated genes induced by PGE2 related to inflammation and bone regeneration are:monocyte to macrophage differentiation-associated (MMD) gene. nuclear receptor subfamily 4, group A, member 2 gene, NR4A2, which is connected tightly with the p65-p50 heterodimer or homodimer of NF-κB. catenin (cadherin-associated protein). osteoblast specific factor (POSTN). bone morphogenetic protein 7 (osteogenic protein 1), BMP-7. The down-regulated genes induced by PGE2 related to inflammation and bone regeneration are:Id1, Id2 and Id3.
Conclusion:
1 Low concentration of lipopolysaccharide can induce directly proliferation of MC3T3-E1 osteoblasts mediated by COX-2-independent NF-κB pathway, and These studies provide insight into a potential mechanism by which NF-κB signal pathway has a central role in the initial phase of inflammation of the bone regeneratrion.
2 PGE2 can induce osteoblast differentiation and promote bone formation by increasing BMP-7 protein expression and decreasing Id2 protein expression through NF-κB signal pathway. BAY 11-7082 can inhibit the osteoblast differentiation induced by PGE2 and induce the cell apoptosis with the role of PGE2. NF-κB can regulate the PGE2 production in vivo. BAY11-7082 can inhibit the PGE2 production, decrease the ALP activity and finally result in delaying the union of fractures. Cooperation between PGE2 production and NF-κB activation is the most primary step during the bone remodeling in vitro and in vivo.
3 The up-regulated genes of promoting bone formation and nuclear receptor and the down-regulated genes of Id family found in expression of osteoblasts gene profile induced by PGE2 support our study results. Therefore, NF-κB could be taken as an important targeted gene in the bone healing process_and has a potential therapy role for nonunion or delayed union fractures.
引文
[1]刘和娣.细胞因子与骨代谢、骨质疏松[J].国外医学生理、病理科学与临床分册.1999：19(2)：150-153.
[2]赵定麟.现代骨科学[M].北京：科学出版社.2004：305.
[3]康献刚,王志强.骨折愈合过程中细胞因子的作用[J].中国修复重建外科杂志.2008,22(7)：877-879.
[4]马德媛,吕志伟.细胞因子在骨质疏松症发病机理中的作用及运动对其的影响[J].科技信息,2008,20：506.
[5]Frost HM. The biology of fracture healing. An overview for clinicians Part 1[J].Clin Orthop,1989,248:283-293.
[6]Hogevold HE, Grogaard B. Effects of short-time treatment with corticosteroids and indomethacin on bone healing:a mechanical study of osteotomies in rat[J]. Acad Orthop Scand,1992,63:607-611.
[7]Phillips JE, Gersbach CA, Garcia AJ. Virus-based gene therapy strategies for bone regeneration[J]. Biomaterials,2007,28(2):211-229.
[8]Fernandez-Tresguerres-Hernandez-Gil Ⅰ, Alobera-Gracia MA, del-Canto-Pingarron M,et al. Physiological bases of bone regeneration Ⅱ. The remodeling process[J]. Med Oral Patol Oral Cir Bucal,2006,11(2):E151-157.
[9]Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor[J]. Endocrin Rev,1997,18:4-25.
[10]Bancroft GN,Mikos AG. Bone tissue engineering by cell transplantation. In:Ikada Y, Oshima N (eds) Tissue engineering for therapeutic use 5. Elsevier,New York,2001:151.
[11]Yaszemski MJ, Payne RG, Hayes WC, et al. Evolution of bone transplantation:molecular, cellular and tissue strategies to engineer human bone[J]. Biomaterials,1996,17(2): 175-185.
[12]William M. Mihalko, A. US Bone and Joint Decade:The Burden of Musculoskeletal Diseases in the United States. Rosemont, IL. American Academy of Orthopaedic Surgeons. 2008:199-209.
[13]Praemer AFS, Rice D. Musculoskeletal Conditions in the United States. Chicago: American Academy of Orthopaedic Surgeons,1992:85-24.
[14]Sen MK, Miclau T. Autologous iliac crest bone graft:should it still be the gold standard for treating nonunions? [J]. Injury,2007,38(Suppl 1):S75-S80.
[15]Nauth A, Miclau T, Li R, et al. Gene therapy for fracture healing [J]. J Orthop Trauma,2010 Suppl 1:S17-24.
[16]Bishop GB, Einhorn TA. Current and future clinical applications of bone morphogenetic proteins in orthopaedic trauma surgery [J]. Int Orthop,2007 (6):721-727.
[17]Rundle CH, Wang H, Yu H, et al.Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair[J]. Bone,2006,38(4):521-529.
[18]Hecht J, Kuhl H, Haas SA, et al. Gene identification and analysis of transcripts differentially regulated in fracture healing by EST sequencing in the domestic sheep[J]. BMC Genomics,2006,7:172.
[19]Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing [J]. J Bone Miner Res,2002,17(3):513-520.
[20]Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular aspects of bone healing [J]. Injury,2005,36(12):1392-1404.
[21]Rundle CH, Wang H, Yu H, et al. Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair.[J]. Bone,2006,38(4):521-529.
[22]Barnes GL, Kostenuik PJ, Gerstenfeld LC, et al. Growth factor regulation of fracture repair [J].J Bone Miner Res,1999,14(11):1805-1815.
[23]Gerstenfeld LC, Cho TJ, Kon T, et al. Impaired fracture healing in the absence of TNF-alpha signaling:the role of TNF-alpha in endochondral cartilage resorption [J]. J Bone Miner Res,2003,18(9):1584-1592.
[24]Kon T, Cho TJ, Aizawa T, et al.Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing[J]. J Bone Miner Res,2001,16(6):1004-1014.
[25]Compston JE. Sex steroids and bone[J]. Physiol Rev,2001,81:419-447.
[26]Nair SP, Meghji S, Wilson M, et al.Bacterially induced bone destruction:mechanisms and misconceptions[J]. Infect Immun,1996,64(7):2371-2380.
[27]Loomer PM, Sigusch B, Sukhu B, et al. Direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis 2561 on osteogenesis in vitro[J]. Infect Immun,1994, 62:1289-1297.
[28]Nair SP, Meghji S, Wilson M, et al. Bacterially induced bone destruction:mechanisms and misconceptions[J]. Infect Immun,1996,64:2371-2380.
[29]Schwartz Z, Goultschin J, Dean DD, et al. Mechanisms of alveolar bone destruction in periodontitis[J]. Periodontol2000,1997,14:158-172.
[30]Shoji M, Tanabe N, Mitsui N, et al. Lipopolysaccharide stimulates the production of prostaglandin E2 and the receptor Ep4 in osteoblasts[J]. Life Sci,2006,78(17):2012-2018.
[31]Chung YH, Chang EJ, Kim SJ, et al. Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts[J]. J Periodontal Res,2006,41(4):288-296.
[32]Babu AN, Meng X, Zou N, et al. Lipopolysaccharide stimulation of human aortic valve interstitial cells activates inflammation and osteogenesis.[J]. Ann Thorac Surg,2008,86(1): 71-76.
[33]Dubios RN, Abramson SB, Crofford L, et al. Cyclooxygenase in biology and disease[J]. FASEB J,1998,12:1063-1073.
[34]Raisz LG, Pilbeam CC, Fall PM. Prostaglandins: mechanisms of action and regulation of production in bone[J]. Osteoporos Int,1993,3:136-140.
[35]Martin P, D'Souza D, Martin J, et al.Wound healing in the PU.1 null mouse--tissue repair is not dependent on inflammatory cells[J]. Curr Biol,2003,13(13):1122-1128.
[36]Mountziaris PM, Mikos AG. Modulation of the Inflammatory Response for Enhanced Bone Tissue Regeneration. Tissue Eng Part B Rev,2008,14(2):179-186.
[37]Aspenberg, P. Drugs and fracture repair. Acta Orthop,2005,76(6),741-748.
[38]Barrere F, van Blitterswijk CA, de Groot K. Bone regeneration:molecular and cellular interactions with calcium phosphate ceramics[J]. Int J Nanomedicine,2006,1(3):317-332.
[39]Annaz B, Hing KA, Kayser M, et al. Porosity variation in hydroxyapatite and osteoblast morphology:a scanning electron microscopy study[J]. J Microsc,2004,215(Pt 1):100-110.
[40]Zuscik MJ, Gunter TE, Rosier RN, et al.Activation of phosphoinositide metabolism by parathyroid hormone in growth plate chondrocytes[J]. Cell Calcium,1994,16(2):112-122.
[41]Blomberg P, Islam KB. Genes tame pro-inflammatory cytokines and promote bone healing[J]. Acta Orthop Scand,1999,70(5):415-418.
[42]Miagkov AV, Kovalenko DV, Brown CE, et al. NF-kappaB activation provides the potential link between inflammation and hyperplasia in the arthritic joint[J]. Proc Natl Acad Sci U S A,1998,95(23):13859-13864.
[43]Bondeson J, Foxwell B, Brennan F, et al. Defining therapeutic targets by using adenovirus: blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators[J]. Proc Natl Acad Sci U S A,1999,96(10):5668-5673.
[1]Klerekooper M, L Avioli. Evaluation and treatment of postmenopausal osteoporosis. In M. J. Favus (ed), Primer on the metabolic bone diseases and disorders of mineral metabolism,2nd ed. Raven Press, New York.1993,223-229.
[2]Mundy GR. Bone remodeling. In:Bone Remodeling and Its Disorders. London:Martin Dunitz Ltd,1995:1-11.
[3]McCarty, DG., Koopman WJ. Arthritis and allied conditions,12th ed. Lea and Febiger, Philadelphia.1993:1723-1734.
[4]Sen MK, Miclau T.Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions? [J]. Injury,2007,38(Suppl1):S75-80.
[5]Barnes GL, Kostenuik PJ, Gerstenfeld LC, et al. Growth factor regulation of fracture repair [J]. J Bone Miner Res,1999,14(11):1805-1815.
[6]Compston JE. Sex steroids and bone[J]. Physiol Rev,2001,81:419-447.
[7]Nair SP, Meghji S, Wilson M, et al. Bacterially induced bone destruction:mechanisms and misconceptions[J]. Infect Immun.,1996,64(7):2371-2380.
[8]Loomer PM, Sigusch B, Sukhu B, et al. Direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis 2561 on osteogenesis in vitro[J]. Infect Immun,1994,62:1289-1297.
[9]Nair SP, Meghji S, Wilson M, et al. Bacterially induced bone destruction:mechanisms and misconceptions[J]. Infect Immun,1996,64:2371-2380.
[10]Schwartz Z, Goultschin J, Dean DD, et al. Mechanisms of alveolar bone destruction in periodontitis[J]. Periodontol 2000,1997,14:158-172.
[11]Shoji M, Tanabe N, Mitsui N, et al. Lipopolysaccharide stimulates the production of prostaglandin E2 and the receptor Ep4 in osteoblasts[J]. Life Sci,2006,78(17):2012-2018.
[12]Chung YH, Chang EJ, Kim SJ, et al. Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts[J]. J Periodontal Res,2006,41(4):288-296.
[13]Retzlaff C, Yamamoto Y, Hoffman PS, et al. Bacterial heat shock proteins directly induce cytokine mRNA and interleukin-1 secretion in macrophage cultures[J]. Infect Immun, 1994,62(12):5689-5693.
[14]Islam S, Hassan F, Tumurkhuu G, et al.Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells[J]. Biochem Biophys Res Commun,2007,360 (2):346-351.
[15]Zhuang L, Jung JY, Wang EW, et al. Pseudomonas aeruginosa lipopolysaccharide induces osteoclastogenesis through a toll-like receptor 4 mediated pathway in vitro and in vivo[J]. Laryngoscope,2007,117(5):841-847.
[16]Doucet P, Lowenstein MOsteoclast activation by bacterial endotoxins during periodontal diseases[J].Med.Sci,2006,22:614-620.
[17]Suda K, Woo JT, Takami M, et al. Lipopolysaccharide supports survival and fusion of preosteoclasts independent of TNF-alpha, IL-1, and RANKL[J]. J Cell Physiol,2002,190 (1):101-108.
[18]Mormann M, Thederan M, Nackchbandi I, et al. Lipopolysaccharides (LPS) induce the differentiation of human monocytes to osteoclasts in a tumour necrosis factor (TNF) alpha-dependent manner:a link between infection and pathological bone resorption [J].Mol Immunol,2008,45(12):3330-3337.
[19]AN Babu, X Meng, N Zou et al. Lipopolysaccharide Stimulation of Human Aortic Valve Interstitial Cells Activates Inflammation and Osteogenesis[J]. Ann Thorac Surg,2008,86 (1):71-76.
[20]Thammasitboon K, Goldring SR, Boch JA.Role of macrophages in LPS-induced osteoblast and PDL cell apoptosis[J]. Bone,2006,38(6):845-852.
[21]Reikeras O, Shegarfi H, Wang JE, et al. Lipopolysaccharide impairs fracture healing:an experimental study in rats[J]. Acta Orthop,2005,76(6):749-753.
[22]Gamier Y, Kadyrov M, Gantert M, et al. Proliferative responses in the placenta after endotoxin exposure in preterm fetal sheep[J]. Eur J Obstet Gynecol Reprod Biol, 2008,138 (2):152-157.
[23]Li XZ, Zhou Y, Liu CF, et al. A dual effect on the growth of astrocytes of lipopolysaccharide[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2007,23(10):934-936.
[24]杨一新,李桂源.LPS所介导的信号转导通路研究进展[J].中南大学学报(医学版),2006,31(1)：141.
[25]Shishodia S, Koul D, Aggarwal BB. Cyclooxygenase (COX) -inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma:correlation with supp ression of COX-synthesis [J]. J Imm unol,2004,173 (3):2011-2022.
[26]Li Q, Verma IM. NF-kappaB regulation in the immune system[J]. Nat Rev Immunol, 2002,2 (10):725-734.
[27]Calzado MA, Bacher S, Schmitz ML.NF-kappaB inhibitors for the treatment of inflammatory diseases and cancer[J]. Curr Med Chem.2007,14(3):367-376.
[28]Ackerman WE 4th, Summerfield TL, Vandre DD, et al. Nuclear factor-kappa B regulates inducible prostaglandin E synthase expression in human amnion mesenchymal cells[J]. Biol Reprod,2008,78(1):68-76.
[29]Noguchi K, Ishikawa I.The roles of cyclooxygenase-2 and prostaglandin E2 in periodontal disease[J]. Periodontol 2000,2007,43:85-101.
[30]Miyauchi M, Hiraoka M, Oka H, et al. Immuno-localization of COX-1 and COX-2 in the rat molar periodontal tissue after topical application of lipopolysaccharide[J]. Arch Oral Biol,2004,49(9):739-746.
[31]Noguchi K, Yanai M, Shitashige M, et al. Cyclooxygenase-2-dependent prostaglandin production by peripheral blood monocytes stimulated with lipopolysa- ccharides isolated from periodontopathogenic bacteria[J]. J Periodontol,2000,71(10):1575-1582.
[32]Anderson DM, Maraskovsky E, Billingsley WL, et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function[J]. Nature,1997 13,390(6656):175-179.
[33]Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation[J]. Cell,1998,93(2):165-176.
[34]Suda K, Udagawa N, Sato N, et al. Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation[J]. J Immunol,2004,172(4):2504-2510.
[35]Coon D, Gulati A, Cowan C, et al. The role of cyclooxygenase-2 (COX-2) in inflammatory bone resorption[J]. J Endod,2007,33(4):432-436.
[36]Lu K, Liang CL, Chen HJ, et al. Nuclear factor-kappaB-regulated cyclooxygenase-2 expression in surgery-associated paraspinal muscle injury in rats[J]. J Neurosurg, 2003,8(2 Suppl):181-187.
[37]Nakao S, Ogtata Y, Shimizu E, et al. Tumor necrosis factor alpha (TNF-alpha)-induced prostaglandin E2 release is mediated by the activation of cyclooxyge-nase-2 (COX2) transcription via NFkappaB in human gingival fibroblasts[J]. Mol Cell Biochem,2002, 238(1-2):11-18.
[38]Jung YJ, Isaacs JS, Lee S, et al. IL-1 beta-mediated up-regulation of HIF-lalpha via an NFkappaB/COX2 pathway identifies HIF-1 as a critical link between inflammation and oncogenesis[J]. FASEB J,2003,17(14):2115-2117.
[39]Thammasitboon K, Goldring SR, Boch JA. Role of macrophages in LPS-induced osteoblast and PDL cell apoptosis[J]. Bone,2006,38(6):845-852.
[40]Ren Y, Xie Y, Jiang GApoptotic cells protect mice against lipopolysaccharide-induced shock[J]. J Immunol,2008,180(7):4978-4985.
[41]Lakics V, Vogel SN. Lipopolysaccharide and ceramide use divergent signaling pathways to induce cell death in murine macrophages[J]. J Immunol,1998,161 (5):2490-2500.
[42]Yang H, Li F, Chai J. Influence of lipopolysaccharide on the biological characteristics of skin fibroblasts and its potential role in wound healing[J]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi,2006,20(9):873-876.
[43]Velickovic TC, Thunberg S, Polovic N, et al. Low levels of endotoxin enhance allergen-stimulated proliferation and reduce the threshold for activation in human peripheral blood cells. Int Arch Allergy Immunol[J],2008,146(1):1-10.
[44]Shankaran M, Marino ME, Busch R, et al. Measurement of brain microglial proliferation rates in vivo in response to neuroinflammatory stimuli:application to drug discovery[J]. J Neurosci Res,2007,85(11):2374-2384.
[45]Iino Y, Hopps RM. The bone-resorbing activities in tissue culture of lipopolysaccharides from the bacteria Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Capnocytophaga ochracea isolated from human mouths[J]. Arch Oral Biol,1984,29(1): 59-63.
[46]Socransky SS, Haffajee AD. The bacterial etiology of destructive periodontal disease: current concepts[J]. J Periodontol,1992,63(4 Suppl):322-331.
[47]Bom-van Noorloos, JWM van der Meer, JS van de Gevel, et al. Bacteroides gingivalis stimulates bone resorption via interleukin-1 production by mononuclear cells. The relative role for B. gingivalis endotoxin[J]. J Clin Periodontol,1990,17(7 Pt 1):409-413
[48]Wilson M. Biological activities of lipopolysaccharides from oral bacteria and their relevance to the pathogenesis of chronic periodontitis[J]. Sci Prog,1995,78 (Pt1):19-34.
[49]Hussain Mian A, Saito H, et al. Lipopolysaccharide- induced bone resorption is increased in TNF type 2 receptor-deficient mice in vivo[J]. J Bone Miner Metab,2008,26(5): 469-477.
[50]Roberts HC, Moseley R, Sloan AJ, et al. Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal disease[J]. Eur J Oral Sci.2008;116(3):207-216.
[51]Luyendyk JP, Schabbauer GA, Tencati M, et al. Genetic analysis of the role of the PI3K-Akt pathway in lipopolysaccharide-induced cytokine and tissue factor gene expression in monocytes/macrophages[J]. J Immunol.2008;180(6):4218-4226.
[52]Shoji M, Tanabe N, Mitsui N, et al. Lipopolysaccharide stimulates the production of prostaglandin E2 and the receptor Ep4 in osteoblasts. Life Sci.2006;78(17):2012-2018.
[53]Tanaka H, Tanabe N, Shoji M, et al. Nicotine and lipopolysaccharide stimulate the formation of osteoclast-like cells by increasing macrophage colony-stimulating factor and prostaglandin E2 production by osteoblasts. Life Sci.2006;78(15):1733-1740.
[54]Shoji M, Tanabe N, Mitsui N, et al. Lipopolysaccharide enhances the production of nicotine-induced prostaglandin E2 by an increase in cyclooxygenase-2 expression in osteoblasts[J]. Acta Biochim Biophys Sin (Shanghai),2007,39(3):163-172.
[55]Kwak HB, Sun HM, Ha H, et al. Tanshinone ⅡA suppresses inflammatory bone loss by inhibiting the synthesis of prostaglandin E2 in osteoblasts[J]. Eur J Pharmacol, 2008,601(1-3):30-37.
[56]Simon AM, Manigrasso MB, O'Connor JP. Cyclooxygenase 2 function is essential for bone fracture healing[J]. J Bone Miner Res,2002,17(6):963-976.
[57]Zhang X, Schwarz EM, Young DA, et al. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair[J]. J Clin Invest,2002,109(11):1405-1415.
[58]Zhang X, Morham SG, Langenbach R, et al. Evidence for a direct role of cyclo-oxygenase 2 in implant wear debris-induced osteolysis[J]. J Bone Miner Res,2001, 16(4):660-670.
[59]Igarashi K, Woo JT, Stern PH.Effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on bone resorption and osteoclastogenesis in vitro[J]. Biochem Pharmacol, 2002,63(3):523-532.
[60]Goodman S, Ma T, Trindade M, et al. COX-2 selective NSAID decreases bone ingrowth in vivo[J]. J Orthop Res,2002,20(6):1164-1169.
[61]Elder CL, Dahners LE, Weinhold PS. A cyclooxygenase-2 inhibitor impairs ligament healing in the rat[J]. Am J Sports Med,2001,29(6):801-805.
[62]Simon AM, Manigrasso MB, O'Connor JP. Cyclo-oxygenase 2 function is essential for bone fracture healing[J].J Bone Miner Res,2002,17(6):963-976.
[63]Forwood MR, Owan I, Takano Y, et al. Increased bone formation in rat tibiae after a single short period of dynamic loading in vivo[J]. Am J Physiol,1996,270(3 Pt 1):E419-423.
[64]Gerstenfeld LC, Cullinane DM, Barnes GL, et al. Fracture healing as a post-natal developmental process:molecular, spatial, and temporal aspects of its regulation[J]. J Cell Biochem,2003,88(5):873-884.
[65]Brown KM, Saunders MM, Kirsch T, et al. Effect of COX-2-specific inhibition on fracture-healing in the rat femur[J]. J Bone Joint Surg Am,2004,86-A(1):116-123.
[66]Ho ML, Chang JK, Wang GJ. Effects of ketorolac on bone repair:A radiographic study in modeled demineralized bone matrix grafted rabbits[J]. Pharmacology,1998,57(3):148-159.
[67]Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination:the control of NF-[kappa]B activity[J]. Annu Rev Immunol,2000,18:621-663.
[68]Baldwin AS Jr. The NF-kappa B and I kappa B proteins:new discoveries and insights[J]. Annu Rev Immunol,1996,14:649-683.
[69]Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation[J]. Cell,1998,93(2):165-176.
[70]Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis[J]. Nature,1999,397(6717): 315-323.
[71]Xu J, Tan JW, Huang L, et al. Cloning, sequencing, and functional characterization of the rat homologue of receptor activator of NF-kappaB ligand. J Bone Miner Res,2000, 15(11):2178-2186.
[72]Nakashima Y, Sun DH, Trindade MC, et al. Signaling pathways for tumor necrosis factor-alpha and interleukin-6 expression in human macrophages exposed to titanium-alloy particulate debris in vitro[J]. J Bone Joint Surg Am,1999,81(5):603-615.
[73]Roshak AK, Callahan JF, Blake SM. Small-molecule inhibitors of NF-kappaB for the treatment of inflammatory joint disease[J]. Curr Opin Pharmacol,2002,2(3):316-321.
[74]Handel ML, McMorrow LB, Gravallese EM. Nuclear factor-kappa B in rheumatoid synovium. Localization of p50 and p65[J]. Arthritis Rheum,1995,38(12):1762-1770.
[75]Fujisawa K, Aono H, Hasunuma T, et al. Activation of transcription factor NF-kappa B in human synovial cells in response to tumor necrosis factor alpha[J]. Arthritis Rheum, 1996,39(2):197-203.
[76]Karin M, Greten FR. NF-kappaB:linking inflammation and immunity to cancer development and progression[J]. Nat Rev Immunol,2005,5(10):749-759.
[77]Bharti AC, Takada Y, Aggarwal BB. Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis[J]. J Immunol,2004,172(10):5940-5947.
[78]Dai S, Hirayama T, Abbas S, et al. The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks osteoclastogenesis and bone erosion in inflammatory arthritis[J]. J Biol Chem,2004,279(36):37219-37222.
[79]Hong CY, Lin SK, Kok SH, et al. The role of lipopolysaccharide in infectious bone resorption of periapical lesion[J]. J Oral Pathol Med,2004,33(3):162-169.
[80]Simon AM, Manigrasso MB, O'Connor JP. Cyclo-oxygenase 2 function is essential for bone fracture healing[J]. J Bone Miner Res,2002,17(6):963-976.
[1]Chase LR, Aurbach GD. The effect of parathyroid hormone on the concentration of adenosine 3',5'-monophosphate in skeletal tissue in vitro.[J]. J Biol Chem,1970,245(7): 1520-1526.
[2]Ohya K, Yamada S, Felix R, et al. Effect of bisphosphonates on prostaglandin synthesis by rat bone cells and mouse calvaria in culture.[J]. Clin Sci (Lond),1985,69(4):403-411.
[3]Jr Tashjian AH, Hohmann EL, Antoniades HN, et al. Platelet-derived growth factor stimulates bone resorption via a prostaglandin-mediated mechanism.[J]. Endocrinology, 1982,111(1):118-124.
[4]Jr Tashjian AH, Voelkel EF, Lazzaro M, et al. Alpha and beta human transforming growth factors stimulate prostaglandin production and bone resorption in cultured mouse calvaria.[J]. Proc Natl Acad Sci U S A,1985,82(13):4535-4538.
[5]Tatakis DN, Schneeberger G, Dziak R. Recombinant interleukin-1 stimulates prostaglandin E2 production by osteoblastic cells:synergy with parathyroid hormone.[J]. Calcif Tissue Int,1988,42(6):358-362.
[6]Hurley MM, Fall P, Harrison JR, et al. Effects of transforming growth factor alpha and interleukin-1 on DNA synthesis, collagen synthesis, procollagen mRNA levels, and prostaglandin E2 production in cultured fetal rat calvaria.[J]. J Bone Miner Res,1989, 4(5):731-736.
[7]Raisz LG, Simmons HA, Sandberg AL, et al. Direct stimulation of bone resorption by epidermal growth factor.[J]. Endocrinology,1980,107(1):270-273.
[8]Akatsu T, Takahashi N, Debari K, et al. Prostaglandins promote osteoclastlike cell formation by a mechanism involving cyclic adenosine 3',5'-monophosphate in mouse bone marrow cell cultures.[J]. J Bone Miner Res,1989,4(1):29-35.
[9]Okuda A, Taylor LM, Heersche JN. Prostaglandin E2 initially inhibits and then stimulates bone resorption in isolated rabbit osteoclast cultures.[J]. Bone Miner,1989,7(3):255-266.
[10]Shinar DM, Rodan GA. Biphasic effects of transforming growth factor-beta on the production of osteoclast-like cells in mouse bone marrow cultures:the role of prostaglandins in the generation of these cells.[J]. Endocrinology,1990,126(6):3153-3158.
[11]Fuller K, Chambers TJ. Effect of arachidonic acid metabolites on bone resorption by isolated rat osteoclasts.[J]. J Bone Miner Res,1989,4(2):209-215.
[12]Conaway HH, Diez LF, Raisz LG. Effects of prostacyclin and prostaglandin El (PGE1) on bone resorption in the presence and absence of parathyroid hormone.[J]. Calcif Tissue Int,1986,38(3):130-134.
[13]Lerner UH, Ransjo M, Ljunggren O. Prostaglandin E2 causes a transient inhibition of mineral mobilization, matrix degradation, and lysosomal enzyme release from mouse calvarial bones in vitro.[J]. Calcif Tissue Int,1987,40(6):323-331.
[14]Boyce BF, Yates AJ, Mundy GR. Bolus injections of recombinant human interleukin-1 cause transient hypocalcemia in normal mice.[J]. Endocrinology,1989,125(5):2780-2783.
[15]Thompson DD, Rodan GA. Indomethacin inhibition of tenotomy-induced bone resorption in rats.[J]. J Bone Miner Res,1988,3(4):409-414.
[16]Blumenkrantz N, Sondergaard J. Effect of prostaglandins E1 and F1 on biosynthesis of collagen.[J]. Nat New Biol,1972,239(95):246.
[17]Raisz LG, Koolemans-Beynen AR. Inhibition of bone collagen synthesis by prostaglandin E2 in organ culture.[J]. Prostaglandins,1974,8(5):377-385.
[18]Fall PM, Breault DT, Raisz LG. Inhibition of collagen synthesis by prostaglandins in the immortalized rat osteoblastic cell line Pyla:structure-activity relations and signal transduction mechanisms.[J]. J Bone Miner Res,1994,9(12):1935-1943.
[19]Hakeda Y, Ikeda E, Kurihara N, et al. Induction of osteoblastic cell differentiation by forskolin. Stimulation of cyclic AMP production and alkaline phosphatase activity.[J]. Biochim Biophys Acta,1985,838(1):49-53.
[20]Kjaersgaard-Andersen P, Nafei A, Teichert G, et al. Indomethacin for prevention of heterotopic ossification. A randomized controlled study in 41 hip arthroplasties.[J]. Acta Orthop Scand,1993,64(6):639-642.
[21]Jee WS, Ueno K, Deng YP, et al. The effects of prostaglandin E2 in growing rats: increased metaphyseal hard tissue and cortico-endosteal bone formation.[J]. Calcif Tissue Int,1985,37(2):148-157.
[22]Garrett IR, Mundy GR. Relationship between interleukin-1 and prostaglandins in resorbing neonatal calvaria.[J]. J Bone Miner Res,1989,4(5):789-794.
[23]Sakurai T, Terashima S, Miyakoshi J. Enhanced secretion of prostaglandin E2 from osteoblasts by exposure to a strong static magnetic field.[J]. Bioelectromagnetics,2008, 29(4):277-283.
[24]Whiteman M, Spencer JP, Zhu YZ, et al. Peroxynitrite-modified collagen-Ⅱ induces p38/ERK and NF-kappaB-dependent synthesis of prostaglandin E2 and nitric oxide in chondrogenically differentiated mesenchymal progenitor cells. [J]. Osteoarthritis Cartilage,2006,14(5):460-470.
[25]Kim MM, Rajapakse N, Kim SK. Anti-inflammatory effect of Ishige okamurae ethanolic extract via inhibition of NF-kappaB transcription factor in RAW 264.7 cells.[J]. Phytother Res,2009,23(5):628-634.
[26]Chung JW, Noh EJ, Zhao HL, et al. Anti-inflammatory activity of prosapogenin methyl ester of platycodin D via nuclear factor-kappaB pathway inhibition.[J]. Biol Pharm Bull,2008,31(11):2114-2120.
[27]Owen M. Uptake of [3H] uridine into precursor pools and RNA in osteogenic cells.[J]. J Cell Sci,1967,2(1):39-56.
[28]Owen M. The origin of bone cells.[J]. Int Rev Cytol,1970,28:213-238.
[29]Urist MR. Bone:formation by autoinduction.1965.[J]. Clin Orthop Relat Res,2002 (395):4-10.
[30]Urist MR, Strates BS. Bone morphogenetic protein.[J]. J Dent Res,1971,50(6):1392-1406.
[31]Miyazono K, Maeda S, Imamura T. BMP receptor signaling:transcriptional targets, regulation of signals, and signaling cross-talk.[J]. Cytokine Growth Factor Rev,2005,16(3): 251-263.
[32]Chen TL, Bates RL, Dudley A, et al. Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells:comparison with TGF-beta 1.[J]. J Bone Miner Res,1991,6(12):1387-1393.
[33]Hughes FJ, Collyer J, Stanfield M, et al. The effects of bone morphogenetic protein-2,-4, and -6 on differentiation of rat osteoblast cells in vitro.[J]. Endocrinology,1995,136 (6):2671-2677.
[34]Franceschi RT, Wang D, Krebsbach PH, et al. Gene therapy for bone formation:in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7.[J]. J Cell Biochem, 2000,78(3):476-486.
[35]Reddi AH. Bone morphogenetic proteins:from basic science to clinical applications.[J]. J Bone Joint Surg Am,2001,83-A Suppl 1(Pt 1):S1-S6.
[36]Buitenhuis M, van Deutekom HW, Verhagen LP, et al. Differential regulation of granulopoiesis by the basic helix-loop-helix transcriptional inhibitors Idl and Id2.[J]. Blood,2005,105(11):4272-4281.
[37]李晓军,秦浚川.细胞分化抑制因子(Id)研究进展[J].生物化学与生物物理进展,2004(10)：865-867
[38]Dedhar S, Mitchell MD, Pierschbacher MD. The osteoblast-like differentiated phenotype of a variant of MG-63 osteosarcoma cell line correlated with altered adhesive properties. [J]. Connect Tissue Res,1989,20(1-4):49-61.
[39]Hanazawa S, Ohmori Y, Amano S, et al. Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells.[J]. FEBS Lett,1986,203(2):279-284.
[40]Ohmori Y, Hanazawa S, Amano S, et al. Effects of recombinant human interleukin 1 alpha and interleukin 1 beta on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1.[J]. Biochim Biophys Acta,1988,970(1):22-30.
[41]Stashenko P, Dewhirst FE, Rooney ML, et al. Interleukin-1 beta is a potent inhibitor of bone formation in vitro.[J]. J Bone Miner Res,1987,2(6):559-565.
[42]Hughes FJ, Buttery LD, Hukkanen MV, et al. Cytokine-induced prostaglandin E2 synthesis and cyclooxygenase-2 activity are regulated both by a nitric oxide-dependent and -independent mechanism in rat osteoblasts in vitro.[J]. J Biol Chem,1999,274 (3):1776-1782.
[43]Hukkanen M, Hughes FJ, Buttery LD, et al. Cytokine-stimulated expression of inducible nitric oxide synthase by mouse, rat, and human osteoblast-like cells and its functional role in osteoblast metabolic activity.[J]. Endocrinology,1995,136(12):5445-5453.
[44]Nanes MS, Mckoy WM, Marx SJ. Inhibitory effects of tumor necrosis factor-alpha and interferon-gamma on deoxyribonucleic acid and collagen synthesis by rat osteosarcoma cells (ROS 17/2.8).[J]. Endocrinology,1989,124(1):339-345.
[45]Gowen M, Macdonald BR, Russell RG. Actions of recombinant human gamma-interferon and tumor necrosis factor alpha on the proliferation and osteoblastic characteristics of human trabecular bone cells in vitro.[J]. Arthritis Rheum,1988,31(12):1500-1507.
[46]Iraburu MJ, Dominguez-Rosales JA, Fontana L, et al. Tumor necrosis factor alpha down-regulates expression of the alphal(Ⅰ) collagen gene in rat hepatic stellate cells through a p20C/EBPbeta- and C/EBPdelta-dependent mechanism.[J]. Hepatology,2000, 31(5):1086-1093.
[47]Kahari VM, Chen YQ, Su MW, et al. Tumor necrosis factor-alpha and interferon-gamma suppress the activation of human type I collagen gene expression by transforming growth factor-beta 1. Evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels.[J]. J Clin Invest,1990,86(5):1489-1495.
[48]Mada Y, Miyauchi M, Oka H, et al. Effects of endogenous and exogenous prostaglandin E2 on the proliferation and differentiation of a mouse cementoblast cell line (OCCM-30).[J]. J Periodontol,2006,77(12):2051-2058.
[49]Oka H, Miyauchi M, Sakamoto K, et al. Prostaglandin E2 inhibits mineralization and enhances matrix metalloproteinase-13 in mature cementoblasts mainly via the EP4 pathway.[J]. Arch Oral Biol,2008,53(3):243-249.
[50]Raisz LG. Physiology and pathophysiology of bone remodeling.[J]. Clin Chem,1999,45(8 Pt 2):1353-1358.
[51]Hatakeyama D, Kozawa O, Niwa M, et al. Upregulation by retinoic acid of transforming growth factor-beta-stimulated heat shock protein 27 induction in osteoblasts:involvement of mitogen-activated protein kinases.[J]. Biochim Biophys Acta,2002,1589(1):15-30.
[52]Tokuda H, Kozawa O, Niwa M, et al. Mechanism of prostaglandin E2-stimulated heat shock protein 27 induction in osteoblast-like MC3T3-E1 cells.[J]. J Endocrinol, 2002,172(2):271-281.
[53]Ghayor C, Rey A, Caverzasio J. Prostaglandin-dependent activation of ERK mediates cell proliferation induced by transforming growth factor beta in mouse osteoblastic cells.[J]. Bone,2005,36(1):93-100.
[54]Coetzee M, Haag M, Claassen N, et al. Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3-E1 osteoblast-like cells.[J]. Prostaglandins Leukot Essent Fatty Acids,2005,73 (6):423-430.
[55]Joubert AM, Panzer A, Joubert F, et al. Comparative study of the effects of polyunsaturated fatty acids and their metabolites on cell growth and tyrosine kinase activity in oesophageal carcinoma cells.[J]. Prostaglandins Leukot Essent Fatty Acids, 1999,61(3):171-182.
[56]Christy BA, Sanders LK, Lau LF, et al. An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene.[J]. Proc Nat1 Acad Sci U S A,1991,88(5):1815-1819.
[57]Scott T, Owens MD. Thrombocytes respond to lipopolysaccharide through Toll-like receptor-4, and MAP kinase and NF-kappaB pathways leading to expression of interleukin-6 and cyclooxygenase-2 with production of prostaglandin E2.[J]. Mol Immunol,2008,45(4):1001-1008.
[58]Yang C M, Lee I T, Lin CC, et al. Cigarette smoke extract induces COX-2 expression via a PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB pathway and p300 in tracheal smooth muscle cells.[J]. Am J Physiol Lung Cell Mol Physiol,2009,297(5):L892-L902.
[59]de Mejia EG, Dia VP. Lunasin and lunasin-like peptides inhibit inflammation through suppression of NF-kappaB pathway in the macrophage.[J]. Peptides,2009,30(12):2388-2398.
[60]Zhang X, LiH, Feng H, et al. Valnemulin downregulates nitric oxide, prostaglandin E2, and cytokine production via inhibition of NF-kappaB and MAPK activity.[J]. Int Immunopharmacol,2009,9(7-8):810-816.
[61]Barakat W, Herrmann O, Baumann B, et al. NF-kappaB induces PGE2-synthesizing enzymes in neurons.[J]. Naunyn Schmiedebergs Arch Pharmacol,2009,380(2):153-160.
[62]Kingsley DM. The TGF-beta superfamily:new members, new receptors, and new genetic tests of function in different organisms.[J]. Genes Dev,1994,8(2):133-146.
[63]Paralkar VM, Grasser WA, Mansolf AL, et al. Regulation of BMP-7 expression by retinoic acid and prostaglandin E(2).[J]. J Cell Physiol,2002,190(2):207-217.
[64]Im HJ, Pacione C, Chubinskaya S, et al. Inhibitory effects of insulin-like growth factor-1 and osteogenic protein-1 on fibronectin fragment- and interleukin-1beta-stimulated matrix metalloproteinase-13 expression in human chondrocytes.[J]. J Biol Chem,2003,278(28): 25386-25394.
[65]Matsuyama A, Iwata H, Okumura N, et al. Localization of basic fibroblast growth factor-like immunoreactivity in the rat brain.[J]. Brain Res,1992,587(1):49-65.
[66]Takegami K, Thonar EJ, An HS, et al. Osteogenic protein-1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin-1.[J]. Spine (Phila Pa 1976),2002,27(12):1318-1325.
[67]Li X, An H S, Ellman M, et al. Action of fibroblast growth factor-2 on the intervertebral disc.[J]. Arthritis Res Ther,2008,10(2):R48.
[68]Lee MJ, Yang CW, Jin DC, et al. Bone morphogenetic protein-7 inhibits constitutive and interleukin-1 beta-induced monocyte chemoattractant protein-1 expression in human mesangial cells:role for JNK/AP-1 pathway.[J]. J Immunol,2003,170(5):2557-2563.
[69]Izumi N, Mizuguchi S, Inagaki Y, et al. BMP-7 opposes TGF-betal-mediated collagen induction in mouse pulmonary myofibroblasts through Id2.[J]. Am J Physiol Lung Cell Mol Physiol,2006,290(1):L120-L126.
[70]Rundle CH, Strong DD, Chen ST, et al. Retroviral-based gene therapy with cyclooxygenase-2 promotes the union of bony callus tissues and accelerates fracture healing in the rat.[J]. J Gene Med,2008,10(3):229-241.
[71]Kanegae Y, Tavares AT, Izpisua BJ, et al. Role of Rel/NF-kappaB transcription factors during the outgrowth of the vertebrate limb.[J]. Nature,1998,392(6676):611-614.
[72]Janssen R, van Wengen A, Hoeve MA, et al. The same IkappaBalpha mutation in two related individuals leads to completely different clinical syndromes.[J]. J Exp Med, 2004,200(5):559-568.
[73]Wu S, Flint JK, Rezvani G, et al. Nuclear factor-kappaB p65 facilitates longitudinal bone growth by inducing growth plate chondrocyte proliferation and differentiation and by preventing apoptosis.[J]. J Biol Chem,2007,282(46):33698-33706.
[74]White DE, Burchill SA. BAY 11-7082 induces cell death through NF-kappaB-independent mechanisms in the Ewing's sarcoma family of tumours.[J]. Cancer Lett,2008,268(2):212-224.
[75]Zanotto-Filho A, Delgado-Canedo A, Schroder R, et al. The pharmacological NFkappaB inhibitors BAY117082 and MG132 induce cell arrest and apoptosis in leukemia cells through ROS-mitochondria pathway activation.[J]. Cancer Lett,2010,288(2):192-203.
[1]梁国栋.最新分子生物学实验技术[M].北京：科学出版社,2001,336-354.
[2]陈志,曾照芳.基因芯片技术的最新进展[J].国际检验医学杂志,2006,27(3)：249.
[3]Chang JC, Hilsenbeck SG, Fuqua SA. Genomic approaches in the management and treatment of breast cancer.[J]. Br J Cancer,2005,92(4):618-624.
[4]Zhang X, Feng J, Cheng Y, et al. Characterization of differentially expressed genes in ovarian cancer by cDNA microarrays.[J]. Int J Gynecol Cancer,2005,15(1):50-57.
[5]Song B, Tang JW, Wang B, et al. Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip.[J]. World J Gastroenterol,2005,11(10):1463-1472.
[6]Slater EP, Diehl SM, Langer P, et al. Analysis by cDNA microarrays of gene expression patterns of human adrenocortical tumors.[J]. Eur J Endocrinol,2006,154(4):587-598.
[7]Baelde HJ, Eikmans M, Doran PP, et al. Gene expression profiling in glomeruli from human kidneys with diabetic nephropathy.[J]. Am J Kidney Dis,2004,43(4):636-650.
[8]Ishii T, Onda H, Tanigawa A, et al. Isolation and expression profiling of genes upregulated in the peripheral blood cells of systemic lupus erythematosus patients.[J]. DNA Res,2005,12(6):429-439.
[9]van Furth R, Raeburn JA, van Zwet TL. Characteristics of human mononuclear phagocytes.[J].Blood,1979,54(2):485-500.
[10]Lawrence T, Willoughby DA, Gilroy DW. Anti-inflammatory lipid mediators and insights into the resolution of inflammation.[J]. Nat Rev Immunol,2002,2(10):787-795.
[11]Gilroy DW, Lawrence T, Perretti M, et al. Inflammatory resolution:new opportunities for drug discovery.[J]. Nat Rev Drug Discov,2004,3(5):401-416.
[12]Gordon S. The macrophage:past, present and future.[J]. Eur J Immunol,2007,37 Suppl 1:S9-S17.
[13]Nahrendorf M, Swirski FK, Aikawa E, et al. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.[J]. J Exp Med,2007,204(12):3037-3047.
[14]Aranda A, Pascual A. Nuclear hormone receptors and gene expression.[J]. Physiol Rev, 2001,81(3):1269-1304.
[15]A unified nomenclature system for the nuclear receptor superfamily.[J]. Cell,1999,97(2): 161-163.
[16]Mages HW, Rilke O, Bravo R, et al. NOT, a human immediate-early response gene closely related to the steroid/thyroid hormone receptor NAK1/TR3.[J]. Mol Endocrinol,1994,8 (11):1583-1591.
[17]Tetradis S, Bezouglaia O, Tsingotjidou A. Parathyroid hormone induces expression of the nuclear orphan receptor Nurrl in bone cells.[J]. Endocrinology,2001,142(2):663-670.
[18]Lammi J, Huppunen J, Aarnisalo P. Regulation of the osteopontin gene by the orphan nuclear receptor NURR1 in osteoblasts.[J]. Mol Endocrinol,2004,18(6):1546-1557.
[19]Murphy EP, Mcevoy A, Conneely OM, et al. Involvement of the nuclear orphan receptor NURR1 in the regulation of corticotropin-releasing hormone expression and actions in human inflammatory arthritis.[J]. Arthritis Rheum,2001,44(4):782-793.
[20]Mcevoy AN, Murphy EA, Ponnio T, et al. Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5'-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.[J]. J Immunol,2002,168 (6):2979-2987.
[21]Akiyama T. Wnt/beta-catenin signaling.[J]. Cytokine Growth Factor Rev,2000,11(4): 273-282.
[22]Yang Y. Wnts and wing:Wnt signaling in vertebrate limb development and musculoskeletal morphogenesis.[J]. Birth Defects Res C Embryo Today,2003,69(4): 305-317.
[23]Fischer L, Boland G, Tuan RS. Wnt-3A enhances bone morphogenetic protein-2-mediated chondrogenesis of murine C3H10T1/2 mesenchymal cells.[J]. J Biol Chem,2002,277 (34):30870-30878.
[24]French DM, Kaul RJ, D'Souza AL, et al. WISP-1 is an osteoblastic regulator expressed during skeletal development and fracture repair.[J]. Am J Pathol,2004,165(3):855-867.
[25]Kakar S, Einhorn TA, Vora S, et al. Enhanced chondrogenesis and Wnt signaling in PTH-treated fractures.[J]. J Bone Miner Res,2007,22(12):1903-1912.
[26]Chen Y, Whetstone HC, Lin AC, et al. Beta-catenin signaling plays a disparate role in different phases of fracture repair:implications for therapy to improve bone healing.[J]. PLoS Med,2007,4(7):e249.
[27]Franceschi RT. The developmental control of osteoblast-specific gene expression:role of specific transcription factors and the extracellular matrix environment.[J]. Crit Rev Oral Biol Med,1999,10(1):40-57.
[28]Katagiri T, Yamaguchi A, Komaki M, et al. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage.[J]. J Cell Biol,1994,127(6 Pt1):1755-1766.
[2]赵定麟.现代骨科学[M].北京：科学出版社.2004：305.
[3]康献刚,王志强.骨折愈合过程中细胞因子的作用[J].中国修复重建外科杂志.2008,22(7)：877-879.
[4]马德媛,吕志伟.细胞因子在骨质疏松症发病机理中的作用及运动对其的影响[J].科技信息,2008,20：506.
[5]Frost HM. The biology of fracture healing. An overview for clinicians Part 1[J].Clin Orthop,1989,248:283-293.
[6]Hogevold HE, Grogaard B. Effects of short-time treatment with corticosteroids and indomethacin on bone healing:a mechanical study of osteotomies in rat[J]. Acad Orthop Scand,1992,63:607-611.
[7]Phillips JE, Gersbach CA, Garcia AJ. Virus-based gene therapy strategies for bone regeneration[J]. Biomaterials,2007,28(2):211-229.
[8]Fernandez-Tresguerres-Hernandez-Gil Ⅰ, Alobera-Gracia MA, del-Canto-Pingarron M,et al. Physiological bases of bone regeneration Ⅱ. The remodeling process[J]. Med Oral Patol Oral Cir Bucal,2006,11(2):E151-157.
[9]Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor[J]. Endocrin Rev,1997,18:4-25.
[10]Bancroft GN,Mikos AG. Bone tissue engineering by cell transplantation. In:Ikada Y, Oshima N (eds) Tissue engineering for therapeutic use 5. Elsevier,New York,2001:151.
[11]Yaszemski MJ, Payne RG, Hayes WC, et al. Evolution of bone transplantation:molecular, cellular and tissue strategies to engineer human bone[J]. Biomaterials,1996,17(2): 175-185.
[12]William M. Mihalko, A. US Bone and Joint Decade:The Burden of Musculoskeletal Diseases in the United States. Rosemont, IL. American Academy of Orthopaedic Surgeons. 2008:199-209.
[13]Praemer AFS, Rice D. Musculoskeletal Conditions in the United States. Chicago: American Academy of Orthopaedic Surgeons,1992:85-24.
[14]Sen MK, Miclau T. Autologous iliac crest bone graft:should it still be the gold standard for treating nonunions? [J]. Injury,2007,38(Suppl 1):S75-S80.
[15]Nauth A, Miclau T, Li R, et al. Gene therapy for fracture healing [J]. J Orthop Trauma,2010 Suppl 1:S17-24.
[16]Bishop GB, Einhorn TA. Current and future clinical applications of bone morphogenetic proteins in orthopaedic trauma surgery [J]. Int Orthop,2007 (6):721-727.
[17]Rundle CH, Wang H, Yu H, et al.Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair[J]. Bone,2006,38(4):521-529.
[18]Hecht J, Kuhl H, Haas SA, et al. Gene identification and analysis of transcripts differentially regulated in fracture healing by EST sequencing in the domestic sheep[J]. BMC Genomics,2006,7:172.
[19]Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing [J]. J Bone Miner Res,2002,17(3):513-520.
[20]Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular aspects of bone healing [J]. Injury,2005,36(12):1392-1404.
[21]Rundle CH, Wang H, Yu H, et al. Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair.[J]. Bone,2006,38(4):521-529.
[22]Barnes GL, Kostenuik PJ, Gerstenfeld LC, et al. Growth factor regulation of fracture repair [J].J Bone Miner Res,1999,14(11):1805-1815.
[23]Gerstenfeld LC, Cho TJ, Kon T, et al. Impaired fracture healing in the absence of TNF-alpha signaling:the role of TNF-alpha in endochondral cartilage resorption [J]. J Bone Miner Res,2003,18(9):1584-1592.
[24]Kon T, Cho TJ, Aizawa T, et al.Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing[J]. J Bone Miner Res,2001,16(6):1004-1014.
[25]Compston JE. Sex steroids and bone[J]. Physiol Rev,2001,81:419-447.
[26]Nair SP, Meghji S, Wilson M, et al.Bacterially induced bone destruction:mechanisms and misconceptions[J]. Infect Immun,1996,64(7):2371-2380.
[27]Loomer PM, Sigusch B, Sukhu B, et al. Direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis 2561 on osteogenesis in vitro[J]. Infect Immun,1994, 62:1289-1297.
[28]Nair SP, Meghji S, Wilson M, et al. Bacterially induced bone destruction:mechanisms and misconceptions[J]. Infect Immun,1996,64:2371-2380.
[29]Schwartz Z, Goultschin J, Dean DD, et al. Mechanisms of alveolar bone destruction in periodontitis[J]. Periodontol2000,1997,14:158-172.
[30]Shoji M, Tanabe N, Mitsui N, et al. Lipopolysaccharide stimulates the production of prostaglandin E2 and the receptor Ep4 in osteoblasts[J]. Life Sci,2006,78(17):2012-2018.
[31]Chung YH, Chang EJ, Kim SJ, et al. Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts[J]. J Periodontal Res,2006,41(4):288-296.
[32]Babu AN, Meng X, Zou N, et al. Lipopolysaccharide stimulation of human aortic valve interstitial cells activates inflammation and osteogenesis.[J]. Ann Thorac Surg,2008,86(1): 71-76.
[33]Dubios RN, Abramson SB, Crofford L, et al. Cyclooxygenase in biology and disease[J]. FASEB J,1998,12:1063-1073.
[34]Raisz LG, Pilbeam CC, Fall PM. Prostaglandins: mechanisms of action and regulation of production in bone[J]. Osteoporos Int,1993,3:136-140.
[35]Martin P, D'Souza D, Martin J, et al.Wound healing in the PU.1 null mouse--tissue repair is not dependent on inflammatory cells[J]. Curr Biol,2003,13(13):1122-1128.
[36]Mountziaris PM, Mikos AG. Modulation of the Inflammatory Response for Enhanced Bone Tissue Regeneration. Tissue Eng Part B Rev,2008,14(2):179-186.
[37]Aspenberg, P. Drugs and fracture repair. Acta Orthop,2005,76(6),741-748.
[38]Barrere F, van Blitterswijk CA, de Groot K. Bone regeneration:molecular and cellular interactions with calcium phosphate ceramics[J]. Int J Nanomedicine,2006,1(3):317-332.
[39]Annaz B, Hing KA, Kayser M, et al. Porosity variation in hydroxyapatite and osteoblast morphology:a scanning electron microscopy study[J]. J Microsc,2004,215(Pt 1):100-110.
[40]Zuscik MJ, Gunter TE, Rosier RN, et al.Activation of phosphoinositide metabolism by parathyroid hormone in growth plate chondrocytes[J]. Cell Calcium,1994,16(2):112-122.
[41]Blomberg P, Islam KB. Genes tame pro-inflammatory cytokines and promote bone healing[J]. Acta Orthop Scand,1999,70(5):415-418.
[42]Miagkov AV, Kovalenko DV, Brown CE, et al. NF-kappaB activation provides the potential link between inflammation and hyperplasia in the arthritic joint[J]. Proc Natl Acad Sci U S A,1998,95(23):13859-13864.
[43]Bondeson J, Foxwell B, Brennan F, et al. Defining therapeutic targets by using adenovirus: blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators[J]. Proc Natl Acad Sci U S A,1999,96(10):5668-5673.
[1]Klerekooper M, L Avioli. Evaluation and treatment of postmenopausal osteoporosis. In M. J. Favus (ed), Primer on the metabolic bone diseases and disorders of mineral metabolism,2nd ed. Raven Press, New York.1993,223-229.
[2]Mundy GR. Bone remodeling. In:Bone Remodeling and Its Disorders. London:Martin Dunitz Ltd,1995:1-11.
[3]McCarty, DG., Koopman WJ. Arthritis and allied conditions,12th ed. Lea and Febiger, Philadelphia.1993:1723-1734.
[4]Sen MK, Miclau T.Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions? [J]. Injury,2007,38(Suppl1):S75-80.
[5]Barnes GL, Kostenuik PJ, Gerstenfeld LC, et al. Growth factor regulation of fracture repair [J]. J Bone Miner Res,1999,14(11):1805-1815.
[6]Compston JE. Sex steroids and bone[J]. Physiol Rev,2001,81:419-447.
[7]Nair SP, Meghji S, Wilson M, et al. Bacterially induced bone destruction:mechanisms and misconceptions[J]. Infect Immun.,1996,64(7):2371-2380.
[8]Loomer PM, Sigusch B, Sukhu B, et al. Direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis 2561 on osteogenesis in vitro[J]. Infect Immun,1994,62:1289-1297.
[9]Nair SP, Meghji S, Wilson M, et al. Bacterially induced bone destruction:mechanisms and misconceptions[J]. Infect Immun,1996,64:2371-2380.
[10]Schwartz Z, Goultschin J, Dean DD, et al. Mechanisms of alveolar bone destruction in periodontitis[J]. Periodontol 2000,1997,14:158-172.
[11]Shoji M, Tanabe N, Mitsui N, et al. Lipopolysaccharide stimulates the production of prostaglandin E2 and the receptor Ep4 in osteoblasts[J]. Life Sci,2006,78(17):2012-2018.
[12]Chung YH, Chang EJ, Kim SJ, et al. Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts[J]. J Periodontal Res,2006,41(4):288-296.
[13]Retzlaff C, Yamamoto Y, Hoffman PS, et al. Bacterial heat shock proteins directly induce cytokine mRNA and interleukin-1 secretion in macrophage cultures[J]. Infect Immun, 1994,62(12):5689-5693.
[14]Islam S, Hassan F, Tumurkhuu G, et al.Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells[J]. Biochem Biophys Res Commun,2007,360 (2):346-351.
[15]Zhuang L, Jung JY, Wang EW, et al. Pseudomonas aeruginosa lipopolysaccharide induces osteoclastogenesis through a toll-like receptor 4 mediated pathway in vitro and in vivo[J]. Laryngoscope,2007,117(5):841-847.
[16]Doucet P, Lowenstein MOsteoclast activation by bacterial endotoxins during periodontal diseases[J].Med.Sci,2006,22:614-620.
[17]Suda K, Woo JT, Takami M, et al. Lipopolysaccharide supports survival and fusion of preosteoclasts independent of TNF-alpha, IL-1, and RANKL[J]. J Cell Physiol,2002,190 (1):101-108.
[18]Mormann M, Thederan M, Nackchbandi I, et al. Lipopolysaccharides (LPS) induce the differentiation of human monocytes to osteoclasts in a tumour necrosis factor (TNF) alpha-dependent manner:a link between infection and pathological bone resorption [J].Mol Immunol,2008,45(12):3330-3337.
[19]AN Babu, X Meng, N Zou et al. Lipopolysaccharide Stimulation of Human Aortic Valve Interstitial Cells Activates Inflammation and Osteogenesis[J]. Ann Thorac Surg,2008,86 (1):71-76.
[20]Thammasitboon K, Goldring SR, Boch JA.Role of macrophages in LPS-induced osteoblast and PDL cell apoptosis[J]. Bone,2006,38(6):845-852.
[21]Reikeras O, Shegarfi H, Wang JE, et al. Lipopolysaccharide impairs fracture healing:an experimental study in rats[J]. Acta Orthop,2005,76(6):749-753.
[22]Gamier Y, Kadyrov M, Gantert M, et al. Proliferative responses in the placenta after endotoxin exposure in preterm fetal sheep[J]. Eur J Obstet Gynecol Reprod Biol, 2008,138 (2):152-157.
[23]Li XZ, Zhou Y, Liu CF, et al. A dual effect on the growth of astrocytes of lipopolysaccharide[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2007,23(10):934-936.
[24]杨一新,李桂源.LPS所介导的信号转导通路研究进展[J].中南大学学报(医学版),2006,31(1)：141.
[25]Shishodia S, Koul D, Aggarwal BB. Cyclooxygenase (COX) -inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma:correlation with supp ression of COX-synthesis [J]. J Imm unol,2004,173 (3):2011-2022.
[26]Li Q, Verma IM. NF-kappaB regulation in the immune system[J]. Nat Rev Immunol, 2002,2 (10):725-734.
[27]Calzado MA, Bacher S, Schmitz ML.NF-kappaB inhibitors for the treatment of inflammatory diseases and cancer[J]. Curr Med Chem.2007,14(3):367-376.
[28]Ackerman WE 4th, Summerfield TL, Vandre DD, et al. Nuclear factor-kappa B regulates inducible prostaglandin E synthase expression in human amnion mesenchymal cells[J]. Biol Reprod,2008,78(1):68-76.
[29]Noguchi K, Ishikawa I.The roles of cyclooxygenase-2 and prostaglandin E2 in periodontal disease[J]. Periodontol 2000,2007,43:85-101.
[30]Miyauchi M, Hiraoka M, Oka H, et al. Immuno-localization of COX-1 and COX-2 in the rat molar periodontal tissue after topical application of lipopolysaccharide[J]. Arch Oral Biol,2004,49(9):739-746.
[31]Noguchi K, Yanai M, Shitashige M, et al. Cyclooxygenase-2-dependent prostaglandin production by peripheral blood monocytes stimulated with lipopolysa- ccharides isolated from periodontopathogenic bacteria[J]. J Periodontol,2000,71(10):1575-1582.
[32]Anderson DM, Maraskovsky E, Billingsley WL, et al. A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function[J]. Nature,1997 13,390(6656):175-179.
[33]Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation[J]. Cell,1998,93(2):165-176.
[34]Suda K, Udagawa N, Sato N, et al. Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation[J]. J Immunol,2004,172(4):2504-2510.
[35]Coon D, Gulati A, Cowan C, et al. The role of cyclooxygenase-2 (COX-2) in inflammatory bone resorption[J]. J Endod,2007,33(4):432-436.
[36]Lu K, Liang CL, Chen HJ, et al. Nuclear factor-kappaB-regulated cyclooxygenase-2 expression in surgery-associated paraspinal muscle injury in rats[J]. J Neurosurg, 2003,8(2 Suppl):181-187.
[37]Nakao S, Ogtata Y, Shimizu E, et al. Tumor necrosis factor alpha (TNF-alpha)-induced prostaglandin E2 release is mediated by the activation of cyclooxyge-nase-2 (COX2) transcription via NFkappaB in human gingival fibroblasts[J]. Mol Cell Biochem,2002, 238(1-2):11-18.
[38]Jung YJ, Isaacs JS, Lee S, et al. IL-1 beta-mediated up-regulation of HIF-lalpha via an NFkappaB/COX2 pathway identifies HIF-1 as a critical link between inflammation and oncogenesis[J]. FASEB J,2003,17(14):2115-2117.
[39]Thammasitboon K, Goldring SR, Boch JA. Role of macrophages in LPS-induced osteoblast and PDL cell apoptosis[J]. Bone,2006,38(6):845-852.
[40]Ren Y, Xie Y, Jiang GApoptotic cells protect mice against lipopolysaccharide-induced shock[J]. J Immunol,2008,180(7):4978-4985.
[41]Lakics V, Vogel SN. Lipopolysaccharide and ceramide use divergent signaling pathways to induce cell death in murine macrophages[J]. J Immunol,1998,161 (5):2490-2500.
[42]Yang H, Li F, Chai J. Influence of lipopolysaccharide on the biological characteristics of skin fibroblasts and its potential role in wound healing[J]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi,2006,20(9):873-876.
[43]Velickovic TC, Thunberg S, Polovic N, et al. Low levels of endotoxin enhance allergen-stimulated proliferation and reduce the threshold for activation in human peripheral blood cells. Int Arch Allergy Immunol[J],2008,146(1):1-10.
[44]Shankaran M, Marino ME, Busch R, et al. Measurement of brain microglial proliferation rates in vivo in response to neuroinflammatory stimuli:application to drug discovery[J]. J Neurosci Res,2007,85(11):2374-2384.
[45]Iino Y, Hopps RM. The bone-resorbing activities in tissue culture of lipopolysaccharides from the bacteria Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Capnocytophaga ochracea isolated from human mouths[J]. Arch Oral Biol,1984,29(1): 59-63.
[46]Socransky SS, Haffajee AD. The bacterial etiology of destructive periodontal disease: current concepts[J]. J Periodontol,1992,63(4 Suppl):322-331.
[47]Bom-van Noorloos, JWM van der Meer, JS van de Gevel, et al. Bacteroides gingivalis stimulates bone resorption via interleukin-1 production by mononuclear cells. The relative role for B. gingivalis endotoxin[J]. J Clin Periodontol,1990,17(7 Pt 1):409-413
[48]Wilson M. Biological activities of lipopolysaccharides from oral bacteria and their relevance to the pathogenesis of chronic periodontitis[J]. Sci Prog,1995,78 (Pt1):19-34.
[49]Hussain Mian A, Saito H, et al. Lipopolysaccharide- induced bone resorption is increased in TNF type 2 receptor-deficient mice in vivo[J]. J Bone Miner Metab,2008,26(5): 469-477.
[50]Roberts HC, Moseley R, Sloan AJ, et al. Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal disease[J]. Eur J Oral Sci.2008;116(3):207-216.
[51]Luyendyk JP, Schabbauer GA, Tencati M, et al. Genetic analysis of the role of the PI3K-Akt pathway in lipopolysaccharide-induced cytokine and tissue factor gene expression in monocytes/macrophages[J]. J Immunol.2008;180(6):4218-4226.
[52]Shoji M, Tanabe N, Mitsui N, et al. Lipopolysaccharide stimulates the production of prostaglandin E2 and the receptor Ep4 in osteoblasts. Life Sci.2006;78(17):2012-2018.
[53]Tanaka H, Tanabe N, Shoji M, et al. Nicotine and lipopolysaccharide stimulate the formation of osteoclast-like cells by increasing macrophage colony-stimulating factor and prostaglandin E2 production by osteoblasts. Life Sci.2006;78(15):1733-1740.
[54]Shoji M, Tanabe N, Mitsui N, et al. Lipopolysaccharide enhances the production of nicotine-induced prostaglandin E2 by an increase in cyclooxygenase-2 expression in osteoblasts[J]. Acta Biochim Biophys Sin (Shanghai),2007,39(3):163-172.
[55]Kwak HB, Sun HM, Ha H, et al. Tanshinone ⅡA suppresses inflammatory bone loss by inhibiting the synthesis of prostaglandin E2 in osteoblasts[J]. Eur J Pharmacol, 2008,601(1-3):30-37.
[56]Simon AM, Manigrasso MB, O'Connor JP. Cyclooxygenase 2 function is essential for bone fracture healing[J]. J Bone Miner Res,2002,17(6):963-976.
[57]Zhang X, Schwarz EM, Young DA, et al. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair[J]. J Clin Invest,2002,109(11):1405-1415.
[58]Zhang X, Morham SG, Langenbach R, et al. Evidence for a direct role of cyclo-oxygenase 2 in implant wear debris-induced osteolysis[J]. J Bone Miner Res,2001, 16(4):660-670.
[59]Igarashi K, Woo JT, Stern PH.Effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on bone resorption and osteoclastogenesis in vitro[J]. Biochem Pharmacol, 2002,63(3):523-532.
[60]Goodman S, Ma T, Trindade M, et al. COX-2 selective NSAID decreases bone ingrowth in vivo[J]. J Orthop Res,2002,20(6):1164-1169.
[61]Elder CL, Dahners LE, Weinhold PS. A cyclooxygenase-2 inhibitor impairs ligament healing in the rat[J]. Am J Sports Med,2001,29(6):801-805.
[62]Simon AM, Manigrasso MB, O'Connor JP. Cyclo-oxygenase 2 function is essential for bone fracture healing[J].J Bone Miner Res,2002,17(6):963-976.
[63]Forwood MR, Owan I, Takano Y, et al. Increased bone formation in rat tibiae after a single short period of dynamic loading in vivo[J]. Am J Physiol,1996,270(3 Pt 1):E419-423.
[64]Gerstenfeld LC, Cullinane DM, Barnes GL, et al. Fracture healing as a post-natal developmental process:molecular, spatial, and temporal aspects of its regulation[J]. J Cell Biochem,2003,88(5):873-884.
[65]Brown KM, Saunders MM, Kirsch T, et al. Effect of COX-2-specific inhibition on fracture-healing in the rat femur[J]. J Bone Joint Surg Am,2004,86-A(1):116-123.
[66]Ho ML, Chang JK, Wang GJ. Effects of ketorolac on bone repair:A radiographic study in modeled demineralized bone matrix grafted rabbits[J]. Pharmacology,1998,57(3):148-159.
[67]Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination:the control of NF-[kappa]B activity[J]. Annu Rev Immunol,2000,18:621-663.
[68]Baldwin AS Jr. The NF-kappa B and I kappa B proteins:new discoveries and insights[J]. Annu Rev Immunol,1996,14:649-683.
[69]Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation[J]. Cell,1998,93(2):165-176.
[70]Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis[J]. Nature,1999,397(6717): 315-323.
[71]Xu J, Tan JW, Huang L, et al. Cloning, sequencing, and functional characterization of the rat homologue of receptor activator of NF-kappaB ligand. J Bone Miner Res,2000, 15(11):2178-2186.
[72]Nakashima Y, Sun DH, Trindade MC, et al. Signaling pathways for tumor necrosis factor-alpha and interleukin-6 expression in human macrophages exposed to titanium-alloy particulate debris in vitro[J]. J Bone Joint Surg Am,1999,81(5):603-615.
[73]Roshak AK, Callahan JF, Blake SM. Small-molecule inhibitors of NF-kappaB for the treatment of inflammatory joint disease[J]. Curr Opin Pharmacol,2002,2(3):316-321.
[74]Handel ML, McMorrow LB, Gravallese EM. Nuclear factor-kappa B in rheumatoid synovium. Localization of p50 and p65[J]. Arthritis Rheum,1995,38(12):1762-1770.
[75]Fujisawa K, Aono H, Hasunuma T, et al. Activation of transcription factor NF-kappa B in human synovial cells in response to tumor necrosis factor alpha[J]. Arthritis Rheum, 1996,39(2):197-203.
[76]Karin M, Greten FR. NF-kappaB:linking inflammation and immunity to cancer development and progression[J]. Nat Rev Immunol,2005,5(10):749-759.
[77]Bharti AC, Takada Y, Aggarwal BB. Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis[J]. J Immunol,2004,172(10):5940-5947.
[78]Dai S, Hirayama T, Abbas S, et al. The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks osteoclastogenesis and bone erosion in inflammatory arthritis[J]. J Biol Chem,2004,279(36):37219-37222.
[79]Hong CY, Lin SK, Kok SH, et al. The role of lipopolysaccharide in infectious bone resorption of periapical lesion[J]. J Oral Pathol Med,2004,33(3):162-169.
[80]Simon AM, Manigrasso MB, O'Connor JP. Cyclo-oxygenase 2 function is essential for bone fracture healing[J]. J Bone Miner Res,2002,17(6):963-976.
[1]Chase LR, Aurbach GD. The effect of parathyroid hormone on the concentration of adenosine 3',5'-monophosphate in skeletal tissue in vitro.[J]. J Biol Chem,1970,245(7): 1520-1526.
[2]Ohya K, Yamada S, Felix R, et al. Effect of bisphosphonates on prostaglandin synthesis by rat bone cells and mouse calvaria in culture.[J]. Clin Sci (Lond),1985,69(4):403-411.
[3]Jr Tashjian AH, Hohmann EL, Antoniades HN, et al. Platelet-derived growth factor stimulates bone resorption via a prostaglandin-mediated mechanism.[J]. Endocrinology, 1982,111(1):118-124.
[4]Jr Tashjian AH, Voelkel EF, Lazzaro M, et al. Alpha and beta human transforming growth factors stimulate prostaglandin production and bone resorption in cultured mouse calvaria.[J]. Proc Natl Acad Sci U S A,1985,82(13):4535-4538.
[5]Tatakis DN, Schneeberger G, Dziak R. Recombinant interleukin-1 stimulates prostaglandin E2 production by osteoblastic cells:synergy with parathyroid hormone.[J]. Calcif Tissue Int,1988,42(6):358-362.
[6]Hurley MM, Fall P, Harrison JR, et al. Effects of transforming growth factor alpha and interleukin-1 on DNA synthesis, collagen synthesis, procollagen mRNA levels, and prostaglandin E2 production in cultured fetal rat calvaria.[J]. J Bone Miner Res,1989, 4(5):731-736.
[7]Raisz LG, Simmons HA, Sandberg AL, et al. Direct stimulation of bone resorption by epidermal growth factor.[J]. Endocrinology,1980,107(1):270-273.
[8]Akatsu T, Takahashi N, Debari K, et al. Prostaglandins promote osteoclastlike cell formation by a mechanism involving cyclic adenosine 3',5'-monophosphate in mouse bone marrow cell cultures.[J]. J Bone Miner Res,1989,4(1):29-35.
[9]Okuda A, Taylor LM, Heersche JN. Prostaglandin E2 initially inhibits and then stimulates bone resorption in isolated rabbit osteoclast cultures.[J]. Bone Miner,1989,7(3):255-266.
[10]Shinar DM, Rodan GA. Biphasic effects of transforming growth factor-beta on the production of osteoclast-like cells in mouse bone marrow cultures:the role of prostaglandins in the generation of these cells.[J]. Endocrinology,1990,126(6):3153-3158.
[11]Fuller K, Chambers TJ. Effect of arachidonic acid metabolites on bone resorption by isolated rat osteoclasts.[J]. J Bone Miner Res,1989,4(2):209-215.
[12]Conaway HH, Diez LF, Raisz LG. Effects of prostacyclin and prostaglandin El (PGE1) on bone resorption in the presence and absence of parathyroid hormone.[J]. Calcif Tissue Int,1986,38(3):130-134.
[13]Lerner UH, Ransjo M, Ljunggren O. Prostaglandin E2 causes a transient inhibition of mineral mobilization, matrix degradation, and lysosomal enzyme release from mouse calvarial bones in vitro.[J]. Calcif Tissue Int,1987,40(6):323-331.
[14]Boyce BF, Yates AJ, Mundy GR. Bolus injections of recombinant human interleukin-1 cause transient hypocalcemia in normal mice.[J]. Endocrinology,1989,125(5):2780-2783.
[15]Thompson DD, Rodan GA. Indomethacin inhibition of tenotomy-induced bone resorption in rats.[J]. J Bone Miner Res,1988,3(4):409-414.
[16]Blumenkrantz N, Sondergaard J. Effect of prostaglandins E1 and F1 on biosynthesis of collagen.[J]. Nat New Biol,1972,239(95):246.
[17]Raisz LG, Koolemans-Beynen AR. Inhibition of bone collagen synthesis by prostaglandin E2 in organ culture.[J]. Prostaglandins,1974,8(5):377-385.
[18]Fall PM, Breault DT, Raisz LG. Inhibition of collagen synthesis by prostaglandins in the immortalized rat osteoblastic cell line Pyla:structure-activity relations and signal transduction mechanisms.[J]. J Bone Miner Res,1994,9(12):1935-1943.
[19]Hakeda Y, Ikeda E, Kurihara N, et al. Induction of osteoblastic cell differentiation by forskolin. Stimulation of cyclic AMP production and alkaline phosphatase activity.[J]. Biochim Biophys Acta,1985,838(1):49-53.
[20]Kjaersgaard-Andersen P, Nafei A, Teichert G, et al. Indomethacin for prevention of heterotopic ossification. A randomized controlled study in 41 hip arthroplasties.[J]. Acta Orthop Scand,1993,64(6):639-642.
[21]Jee WS, Ueno K, Deng YP, et al. The effects of prostaglandin E2 in growing rats: increased metaphyseal hard tissue and cortico-endosteal bone formation.[J]. Calcif Tissue Int,1985,37(2):148-157.
[22]Garrett IR, Mundy GR. Relationship between interleukin-1 and prostaglandins in resorbing neonatal calvaria.[J]. J Bone Miner Res,1989,4(5):789-794.
[23]Sakurai T, Terashima S, Miyakoshi J. Enhanced secretion of prostaglandin E2 from osteoblasts by exposure to a strong static magnetic field.[J]. Bioelectromagnetics,2008, 29(4):277-283.
[24]Whiteman M, Spencer JP, Zhu YZ, et al. Peroxynitrite-modified collagen-Ⅱ induces p38/ERK and NF-kappaB-dependent synthesis of prostaglandin E2 and nitric oxide in chondrogenically differentiated mesenchymal progenitor cells. [J]. Osteoarthritis Cartilage,2006,14(5):460-470.
[25]Kim MM, Rajapakse N, Kim SK. Anti-inflammatory effect of Ishige okamurae ethanolic extract via inhibition of NF-kappaB transcription factor in RAW 264.7 cells.[J]. Phytother Res,2009,23(5):628-634.
[26]Chung JW, Noh EJ, Zhao HL, et al. Anti-inflammatory activity of prosapogenin methyl ester of platycodin D via nuclear factor-kappaB pathway inhibition.[J]. Biol Pharm Bull,2008,31(11):2114-2120.
[27]Owen M. Uptake of [3H] uridine into precursor pools and RNA in osteogenic cells.[J]. J Cell Sci,1967,2(1):39-56.
[28]Owen M. The origin of bone cells.[J]. Int Rev Cytol,1970,28:213-238.
[29]Urist MR. Bone:formation by autoinduction.1965.[J]. Clin Orthop Relat Res,2002 (395):4-10.
[30]Urist MR, Strates BS. Bone morphogenetic protein.[J]. J Dent Res,1971,50(6):1392-1406.
[31]Miyazono K, Maeda S, Imamura T. BMP receptor signaling:transcriptional targets, regulation of signals, and signaling cross-talk.[J]. Cytokine Growth Factor Rev,2005,16(3): 251-263.
[32]Chen TL, Bates RL, Dudley A, et al. Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells:comparison with TGF-beta 1.[J]. J Bone Miner Res,1991,6(12):1387-1393.
[33]Hughes FJ, Collyer J, Stanfield M, et al. The effects of bone morphogenetic protein-2,-4, and -6 on differentiation of rat osteoblast cells in vitro.[J]. Endocrinology,1995,136 (6):2671-2677.
[34]Franceschi RT, Wang D, Krebsbach PH, et al. Gene therapy for bone formation:in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7.[J]. J Cell Biochem, 2000,78(3):476-486.
[35]Reddi AH. Bone morphogenetic proteins:from basic science to clinical applications.[J]. J Bone Joint Surg Am,2001,83-A Suppl 1(Pt 1):S1-S6.
[36]Buitenhuis M, van Deutekom HW, Verhagen LP, et al. Differential regulation of granulopoiesis by the basic helix-loop-helix transcriptional inhibitors Idl and Id2.[J]. Blood,2005,105(11):4272-4281.
[37]李晓军,秦浚川.细胞分化抑制因子(Id)研究进展[J].生物化学与生物物理进展,2004(10)：865-867
[38]Dedhar S, Mitchell MD, Pierschbacher MD. The osteoblast-like differentiated phenotype of a variant of MG-63 osteosarcoma cell line correlated with altered adhesive properties. [J]. Connect Tissue Res,1989,20(1-4):49-61.
[39]Hanazawa S, Ohmori Y, Amano S, et al. Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells.[J]. FEBS Lett,1986,203(2):279-284.
[40]Ohmori Y, Hanazawa S, Amano S, et al. Effects of recombinant human interleukin 1 alpha and interleukin 1 beta on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1.[J]. Biochim Biophys Acta,1988,970(1):22-30.
[41]Stashenko P, Dewhirst FE, Rooney ML, et al. Interleukin-1 beta is a potent inhibitor of bone formation in vitro.[J]. J Bone Miner Res,1987,2(6):559-565.
[42]Hughes FJ, Buttery LD, Hukkanen MV, et al. Cytokine-induced prostaglandin E2 synthesis and cyclooxygenase-2 activity are regulated both by a nitric oxide-dependent and -independent mechanism in rat osteoblasts in vitro.[J]. J Biol Chem,1999,274 (3):1776-1782.
[43]Hukkanen M, Hughes FJ, Buttery LD, et al. Cytokine-stimulated expression of inducible nitric oxide synthase by mouse, rat, and human osteoblast-like cells and its functional role in osteoblast metabolic activity.[J]. Endocrinology,1995,136(12):5445-5453.
[44]Nanes MS, Mckoy WM, Marx SJ. Inhibitory effects of tumor necrosis factor-alpha and interferon-gamma on deoxyribonucleic acid and collagen synthesis by rat osteosarcoma cells (ROS 17/2.8).[J]. Endocrinology,1989,124(1):339-345.
[45]Gowen M, Macdonald BR, Russell RG. Actions of recombinant human gamma-interferon and tumor necrosis factor alpha on the proliferation and osteoblastic characteristics of human trabecular bone cells in vitro.[J]. Arthritis Rheum,1988,31(12):1500-1507.
[46]Iraburu MJ, Dominguez-Rosales JA, Fontana L, et al. Tumor necrosis factor alpha down-regulates expression of the alphal(Ⅰ) collagen gene in rat hepatic stellate cells through a p20C/EBPbeta- and C/EBPdelta-dependent mechanism.[J]. Hepatology,2000, 31(5):1086-1093.
[47]Kahari VM, Chen YQ, Su MW, et al. Tumor necrosis factor-alpha and interferon-gamma suppress the activation of human type I collagen gene expression by transforming growth factor-beta 1. Evidence for two distinct mechanisms of inhibition at the transcriptional and posttranscriptional levels.[J]. J Clin Invest,1990,86(5):1489-1495.
[48]Mada Y, Miyauchi M, Oka H, et al. Effects of endogenous and exogenous prostaglandin E2 on the proliferation and differentiation of a mouse cementoblast cell line (OCCM-30).[J]. J Periodontol,2006,77(12):2051-2058.
[49]Oka H, Miyauchi M, Sakamoto K, et al. Prostaglandin E2 inhibits mineralization and enhances matrix metalloproteinase-13 in mature cementoblasts mainly via the EP4 pathway.[J]. Arch Oral Biol,2008,53(3):243-249.
[50]Raisz LG. Physiology and pathophysiology of bone remodeling.[J]. Clin Chem,1999,45(8 Pt 2):1353-1358.
[51]Hatakeyama D, Kozawa O, Niwa M, et al. Upregulation by retinoic acid of transforming growth factor-beta-stimulated heat shock protein 27 induction in osteoblasts:involvement of mitogen-activated protein kinases.[J]. Biochim Biophys Acta,2002,1589(1):15-30.
[52]Tokuda H, Kozawa O, Niwa M, et al. Mechanism of prostaglandin E2-stimulated heat shock protein 27 induction in osteoblast-like MC3T3-E1 cells.[J]. J Endocrinol, 2002,172(2):271-281.
[53]Ghayor C, Rey A, Caverzasio J. Prostaglandin-dependent activation of ERK mediates cell proliferation induced by transforming growth factor beta in mouse osteoblastic cells.[J]. Bone,2005,36(1):93-100.
[54]Coetzee M, Haag M, Claassen N, et al. Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3-E1 osteoblast-like cells.[J]. Prostaglandins Leukot Essent Fatty Acids,2005,73 (6):423-430.
[55]Joubert AM, Panzer A, Joubert F, et al. Comparative study of the effects of polyunsaturated fatty acids and their metabolites on cell growth and tyrosine kinase activity in oesophageal carcinoma cells.[J]. Prostaglandins Leukot Essent Fatty Acids, 1999,61(3):171-182.
[56]Christy BA, Sanders LK, Lau LF, et al. An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene.[J]. Proc Nat1 Acad Sci U S A,1991,88(5):1815-1819.
[57]Scott T, Owens MD. Thrombocytes respond to lipopolysaccharide through Toll-like receptor-4, and MAP kinase and NF-kappaB pathways leading to expression of interleukin-6 and cyclooxygenase-2 with production of prostaglandin E2.[J]. Mol Immunol,2008,45(4):1001-1008.
[58]Yang C M, Lee I T, Lin CC, et al. Cigarette smoke extract induces COX-2 expression via a PKCalpha/c-Src/EGFR, PDGFR/PI3K/Akt/NF-kappaB pathway and p300 in tracheal smooth muscle cells.[J]. Am J Physiol Lung Cell Mol Physiol,2009,297(5):L892-L902.
[59]de Mejia EG, Dia VP. Lunasin and lunasin-like peptides inhibit inflammation through suppression of NF-kappaB pathway in the macrophage.[J]. Peptides,2009,30(12):2388-2398.
[60]Zhang X, LiH, Feng H, et al. Valnemulin downregulates nitric oxide, prostaglandin E2, and cytokine production via inhibition of NF-kappaB and MAPK activity.[J]. Int Immunopharmacol,2009,9(7-8):810-816.
[61]Barakat W, Herrmann O, Baumann B, et al. NF-kappaB induces PGE2-synthesizing enzymes in neurons.[J]. Naunyn Schmiedebergs Arch Pharmacol,2009,380(2):153-160.
[62]Kingsley DM. The TGF-beta superfamily:new members, new receptors, and new genetic tests of function in different organisms.[J]. Genes Dev,1994,8(2):133-146.
[63]Paralkar VM, Grasser WA, Mansolf AL, et al. Regulation of BMP-7 expression by retinoic acid and prostaglandin E(2).[J]. J Cell Physiol,2002,190(2):207-217.
[64]Im HJ, Pacione C, Chubinskaya S, et al. Inhibitory effects of insulin-like growth factor-1 and osteogenic protein-1 on fibronectin fragment- and interleukin-1beta-stimulated matrix metalloproteinase-13 expression in human chondrocytes.[J]. J Biol Chem,2003,278(28): 25386-25394.
[65]Matsuyama A, Iwata H, Okumura N, et al. Localization of basic fibroblast growth factor-like immunoreactivity in the rat brain.[J]. Brain Res,1992,587(1):49-65.
[66]Takegami K, Thonar EJ, An HS, et al. Osteogenic protein-1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin-1.[J]. Spine (Phila Pa 1976),2002,27(12):1318-1325.
[67]Li X, An H S, Ellman M, et al. Action of fibroblast growth factor-2 on the intervertebral disc.[J]. Arthritis Res Ther,2008,10(2):R48.
[68]Lee MJ, Yang CW, Jin DC, et al. Bone morphogenetic protein-7 inhibits constitutive and interleukin-1 beta-induced monocyte chemoattractant protein-1 expression in human mesangial cells:role for JNK/AP-1 pathway.[J]. J Immunol,2003,170(5):2557-2563.
[69]Izumi N, Mizuguchi S, Inagaki Y, et al. BMP-7 opposes TGF-betal-mediated collagen induction in mouse pulmonary myofibroblasts through Id2.[J]. Am J Physiol Lung Cell Mol Physiol,2006,290(1):L120-L126.
[70]Rundle CH, Strong DD, Chen ST, et al. Retroviral-based gene therapy with cyclooxygenase-2 promotes the union of bony callus tissues and accelerates fracture healing in the rat.[J]. J Gene Med,2008,10(3):229-241.
[71]Kanegae Y, Tavares AT, Izpisua BJ, et al. Role of Rel/NF-kappaB transcription factors during the outgrowth of the vertebrate limb.[J]. Nature,1998,392(6676):611-614.
[72]Janssen R, van Wengen A, Hoeve MA, et al. The same IkappaBalpha mutation in two related individuals leads to completely different clinical syndromes.[J]. J Exp Med, 2004,200(5):559-568.
[73]Wu S, Flint JK, Rezvani G, et al. Nuclear factor-kappaB p65 facilitates longitudinal bone growth by inducing growth plate chondrocyte proliferation and differentiation and by preventing apoptosis.[J]. J Biol Chem,2007,282(46):33698-33706.
[74]White DE, Burchill SA. BAY 11-7082 induces cell death through NF-kappaB-independent mechanisms in the Ewing's sarcoma family of tumours.[J]. Cancer Lett,2008,268(2):212-224.
[75]Zanotto-Filho A, Delgado-Canedo A, Schroder R, et al. The pharmacological NFkappaB inhibitors BAY117082 and MG132 induce cell arrest and apoptosis in leukemia cells through ROS-mitochondria pathway activation.[J]. Cancer Lett,2010,288(2):192-203.
[1]梁国栋.最新分子生物学实验技术[M].北京：科学出版社,2001,336-354.
[2]陈志,曾照芳.基因芯片技术的最新进展[J].国际检验医学杂志,2006,27(3)：249.
[3]Chang JC, Hilsenbeck SG, Fuqua SA. Genomic approaches in the management and treatment of breast cancer.[J]. Br J Cancer,2005,92(4):618-624.
[4]Zhang X, Feng J, Cheng Y, et al. Characterization of differentially expressed genes in ovarian cancer by cDNA microarrays.[J]. Int J Gynecol Cancer,2005,15(1):50-57.
[5]Song B, Tang JW, Wang B, et al. Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip.[J]. World J Gastroenterol,2005,11(10):1463-1472.
[6]Slater EP, Diehl SM, Langer P, et al. Analysis by cDNA microarrays of gene expression patterns of human adrenocortical tumors.[J]. Eur J Endocrinol,2006,154(4):587-598.
[7]Baelde HJ, Eikmans M, Doran PP, et al. Gene expression profiling in glomeruli from human kidneys with diabetic nephropathy.[J]. Am J Kidney Dis,2004,43(4):636-650.
[8]Ishii T, Onda H, Tanigawa A, et al. Isolation and expression profiling of genes upregulated in the peripheral blood cells of systemic lupus erythematosus patients.[J]. DNA Res,2005,12(6):429-439.
[9]van Furth R, Raeburn JA, van Zwet TL. Characteristics of human mononuclear phagocytes.[J].Blood,1979,54(2):485-500.
[10]Lawrence T, Willoughby DA, Gilroy DW. Anti-inflammatory lipid mediators and insights into the resolution of inflammation.[J]. Nat Rev Immunol,2002,2(10):787-795.
[11]Gilroy DW, Lawrence T, Perretti M, et al. Inflammatory resolution:new opportunities for drug discovery.[J]. Nat Rev Drug Discov,2004,3(5):401-416.
[12]Gordon S. The macrophage:past, present and future.[J]. Eur J Immunol,2007,37 Suppl 1:S9-S17.
[13]Nahrendorf M, Swirski FK, Aikawa E, et al. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.[J]. J Exp Med,2007,204(12):3037-3047.
[14]Aranda A, Pascual A. Nuclear hormone receptors and gene expression.[J]. Physiol Rev, 2001,81(3):1269-1304.
[15]A unified nomenclature system for the nuclear receptor superfamily.[J]. Cell,1999,97(2): 161-163.
[16]Mages HW, Rilke O, Bravo R, et al. NOT, a human immediate-early response gene closely related to the steroid/thyroid hormone receptor NAK1/TR3.[J]. Mol Endocrinol,1994,8 (11):1583-1591.
[17]Tetradis S, Bezouglaia O, Tsingotjidou A. Parathyroid hormone induces expression of the nuclear orphan receptor Nurrl in bone cells.[J]. Endocrinology,2001,142(2):663-670.
[18]Lammi J, Huppunen J, Aarnisalo P. Regulation of the osteopontin gene by the orphan nuclear receptor NURR1 in osteoblasts.[J]. Mol Endocrinol,2004,18(6):1546-1557.
[19]Murphy EP, Mcevoy A, Conneely OM, et al. Involvement of the nuclear orphan receptor NURR1 in the regulation of corticotropin-releasing hormone expression and actions in human inflammatory arthritis.[J]. Arthritis Rheum,2001,44(4):782-793.
[20]Mcevoy AN, Murphy EA, Ponnio T, et al. Activation of nuclear orphan receptor NURR1 transcription by NF-kappa B and cyclic adenosine 5'-monophosphate response element-binding protein in rheumatoid arthritis synovial tissue.[J]. J Immunol,2002,168 (6):2979-2987.
[21]Akiyama T. Wnt/beta-catenin signaling.[J]. Cytokine Growth Factor Rev,2000,11(4): 273-282.
[22]Yang Y. Wnts and wing:Wnt signaling in vertebrate limb development and musculoskeletal morphogenesis.[J]. Birth Defects Res C Embryo Today,2003,69(4): 305-317.
[23]Fischer L, Boland G, Tuan RS. Wnt-3A enhances bone morphogenetic protein-2-mediated chondrogenesis of murine C3H10T1/2 mesenchymal cells.[J]. J Biol Chem,2002,277 (34):30870-30878.
[24]French DM, Kaul RJ, D'Souza AL, et al. WISP-1 is an osteoblastic regulator expressed during skeletal development and fracture repair.[J]. Am J Pathol,2004,165(3):855-867.
[25]Kakar S, Einhorn TA, Vora S, et al. Enhanced chondrogenesis and Wnt signaling in PTH-treated fractures.[J]. J Bone Miner Res,2007,22(12):1903-1912.
[26]Chen Y, Whetstone HC, Lin AC, et al. Beta-catenin signaling plays a disparate role in different phases of fracture repair:implications for therapy to improve bone healing.[J]. PLoS Med,2007,4(7):e249.
[27]Franceschi RT. The developmental control of osteoblast-specific gene expression:role of specific transcription factors and the extracellular matrix environment.[J]. Crit Rev Oral Biol Med,1999,10(1):40-57.
[28]Katagiri T, Yamaguchi A, Komaki M, et al. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage.[J]. J Cell Biol,1994,127(6 Pt1):1755-1766.