关键词： Asporin   Single nucleotide polymorphism   Knee osteoarthritis  

摘要： The aim of this study was to investigate associations between single nucleotide polymorphisms rs13301537 in asporin (ASPN) and rs373444 in the bone morphogenetic protein 5 (BMP5) gene with knee osteoarthritis (OA) susceptibility in a Chinese Han population. ASPN rs13301537 and BMP5 rs373444 polymorphisms were genotyped in patients with knee OA and age- and sex-matched OA-free controls from a Chinese Han population. A total of 510 patients with knee OA and 520 controls were enrolled in the study. CT and CC genotypes of rs13301537, and variant C, were associated with a significantly increased risk of knee OA. On stratification analysis, the association between the risk of OA and rs13301537 CT heterozygotes compared with TT homozygotes was stronger in females and those aged > 65 years. In contrast, the CT and CC genotypes of rs373444 in BMP5 were not significantly associated with the risk of knee OA, even after further stratification analysis according to age or sex. Our results showed that ASPN rs13301537 T to C change and variant C genotype may contribute to knee OA risk in a Chinese Han population.

关键词： 腰椎   椎间盘退行性变   基因疗法   综述  

摘要： 下腰痛是一种常见病、多发病，它影响人们的生活质量和精神状态，成为困扰人类的疾病之一［1-3］。很多因素都会导致下腰痛，其中椎间盘退变是最重要的因素之一［4］。目前临床上针对椎间盘退变引起相关疾病的治疗策略是解除椎间盘退变相关的脊髓、神经和血管刺激或压迫，并非针对椎间盘退变的病理过程，因而临床症状可反复发作，而且手术后病变节段的相邻节段椎间盘退变加速［5-6］。随着科学技术的发展，特别是分子生物学技术不断进步，延缓甚至逆转椎间盘退变正成为可能。目前治疗椎间盘退变的分子生物学技术主要包括三大类：细胞治疗、组织工程治疗及基因治疗。本综述主要着重基因治疗。

关键词： 椎间盘退变   Asporin   TGF-β1   L929  

摘要： 目的:构建高表达asporin基因L929细胞株,研究其对子小鼠正常成纤维细胞L929 TGF-β1及Col2α1表达的影响。方法:运用RT-PCR技术从小鼠下颌骨组织中扩增asporin cDNA,连接酶连接至真核表达载体pcDNA3.1(-),所得的重组质粒pcDNA3.1(-)-ASPNⅢ用脂质体法转染小鼠成纤维细胞L929,经过G418筛选,使用RT-PCR以及Western blot技术筛选asporin基因高表达的细胞株。并使用RT-PCR以及Western blot技术检测asporin基因高表达对细胞TGF-β1及Col2α1表达的影响。结果 :成功获得asporin高表达的L929细胞株。RT-PCR以及Western blot技术检测高表达asporin对TGF-β1及Col2α1的表达有明显的抑制作用。结论:asporin基因在L929细胞中高表达能抑制细胞TGF-β1及Col2α1的表达。

关键词： ASPORIN   MINERALIZATION   ENAMEL   DENTIN   FLUOROSIS  

摘要： Microarray analysis of odontoblastic cells treated with sodium fluoride has identified the asporin gene as a fluoride target. Asporin is a member of the small leucine-rich repeat proteoglycan/protein (SLRP) family that is believed to be important in the mineralization process. In this study, asporin expression and distribution were investigated by systematic analysis of dentin and enamel, with and without fluoride treatment. Specific attention was focused on a major difference between the two mineralized tissues: the presence of a collagenous scaffold in dentin, and its absence in enamel. Normal and fluorotic, continually growing incisors from Wistar rats treated with 2.5 to 7.5mM sodium fluoride (NaF) were studied by immunochemistry, in situ hybridization, Western blotting, and RT-qPCR. Asporin was continuously expressed in odontoblasts throughout dentin formation as expected. Asporin was also found, for the first time, in dental epithelial cells, particularly in maturation-stage ameloblasts. NaF decreased asporin expression in odontoblasts and enhanced it in ameloblasts, both in vivo and in vitro. The inverse response in the two cell types suggests that the effector, fluoride, is a trigger that elicits a cell-type-specific reaction. Confocal and ultrastructural immunohistochemistry evidenced an association between asporin and type 1 collagen in the pericellular nonmineralized compartments of both bone and dentin. In addition, transmission electron microscopy revealed asporin in the microenvironment of all cells observed. Thus, asporin is produced by collagen-matrix-forming and non-collagen-matrix-forming cells but may have different effects on the mineralization process. A model is proposed that predicts impaired mineral formation associated with the deficiency and excess of asporin. (c) 2014 American Society for Bone and Mineral Research.

关键词： Osteoarthritis   Asporin   Polymorphism   Meta-analysis  

摘要： Our aim was to determine whether asporin (ASPN) D-repeat polymorphisms are associated with susceptibility to osteoarthritis (OA). A meta-analysis was conducted to examine the association between the ASPN D14, D13, and D15 alleles and OA of the knee and hip in each ethnic group. In total, 9 studies from eight articles involving 4,417 OA patients and 3,403 controls were considered in the meta-analysis. Meta-analysis showed no association between OA and the ASPN allele coding for 14 D-repeats (D14) in the overall population (OR 1.161, 95 % CI 0.934-1.444, p = 0.178). Stratification by ethnicity identified no association between the ASPN D14 allele and OA in Europeans or Asians (OR 1.035, 95 % CI 0.914-1.173, p = 0.589;OR 1.537, 95 % CI 0.899-2.626, p = 0.116), respectively. However, high heterogeneity was found in Asians (I (2) = 81.2, p = 0.001). Meta-analysis of OA by site showed no association between knee and hip OA and the ASPN D14 allele (OR 1.240, 95 % CI 0.946-1.627, p = 0.119;OR 1.130, 95 % CI 0.767-1.665, p = 0.537). Meta-analysis of D14 versus D13 allele showed the same pattern of OA association as the D14 allele. No association was found between the ASPN D13 and D15 alleles and risk of developing OA by meta-analysis (OR 0.942, 95 % CI 0.840-1.056, p = 0.304;OR 1.050, 95 % CI 0.956-1.154, p = 0.306), respectively. This meta-analysis shows that the ASPN D14, D13, and D15 alleles are not associated with the development of OA in Europeans and Asians. Thus, further study of this relationship is required in homogenous populations because of the heterogeneity of the ASPN D14 allele observed in Asians.

关键词： 牙周膜   基因表达   离心法  

摘要： 目的通过对体外培养的人牙周膜细胞加载相对离心力,观察asporin、骨形成蛋白2（bone morphogenetic protein-2,BMP-2）、碱性磷酸酶（alkaline phosphatase,ALP）基因表达的变化,探讨相对离心力对牙周膜细胞asporin基因表达的影响及asporin的作用机制,为进一步探究asporin对根-骨粘连的预防作用提供依据。方法加力组采用离心机对体外培养的人牙周膜细胞加载0、1、2、6、10 h的30＆#215; g相对离心力,以相同培养条件和培养时间的未加力细胞作为对照组,实时定量PCR（ real time-PCR）检测asporin基因、BMP-2和ALP的表达。结果各时间点对照组及加力组细胞形态均正常。对照组各时间点asporin、BMP-2、ALP基因相对表达量的总体差异均无统计学意义（ P〉0．05）。加力组加力1 h后asporin、BMP-2基因相对表达量分别为0．50±0．05、0．40±0．13,加力2 h后分别为0．42±0．09、0．58±0．19,均较0 h（均为1）显著降低（P〈0．05）；加力6 h后两种基因相对表达量均较加力1、2 h显著升高（P〈0．05）,加力10 h后asporin表达降至0 h水平（P〉0．05）,BMP-2表达量也迅速降低,且显著低于0 h（P〈0．05）。加力组细胞asporin和BMP-2的表达呈正相关（r=0．995, P〈0．05）；asporin与ALP表达无相关性（r=-0．221,P〉0．10）。结论 asporin基因在牙周膜细胞中表达稳定,轻力刺激能促进asporin表达迅速升高,使同时升高的BMP-2最后降低至低于正常水平,提示asporin可在预防BMP-2诱导的细胞分化中起一定作用。

关键词： 椎间盘退变   基因治疗   蛋白多糖   II型胶原  

摘要： 椎间盘退变作为一种与年龄相关的退变性疾病,在脊柱外科最为常见。目前临床治疗仍然致力于退变终末期(例如椎间盘突出症、椎管狭窄症、椎体滑脱症等),不能延缓椎间盘退变的进展。近年来,随着人们对椎间盘退变研究的不断深入,对椎间盘退变的治疗也有了新的发现。基因治疗为延缓椎间盘退变提供了新方法,现就目前的研究状况做一综述。

关键词： cytodifferentiation   BMP-2   extracellular matrix   asporin   tissue homeostasis   gene polymorphism  

摘要： PLAP-1/asporin is an extracellular matrix protein that is predominantly expressed in the human periodontal ligament (PDL) and has an aspartic acid (D) repeat polymorphism in its N-terminal region. In this study, we hypothesized that the D repeat polymorphism of PLAP-1/asporin may affect the physiological functions of periodontal ligaments. We established periodontal ligament cell lines transfected with the D13- or D14-PLAP-1 gene. Alkaline phosphatase staining and alizarin red staining revealed that the cytodifferentiation of the D14-PLAP-1-expressing PDL cells was more repressed compared with that of the D13-PLAP-1-expressing cells. Furthermore, the D14-PLAP-1-expressing cells inhibited BMP-2-induced cytodifferentiation more strongly than did the D13-PLAP-1-expressing cells. Western blotting analysis and luciferase assay revealed that D14-PLAP-1 suppressed BMP-2 signal transduction more efficiently than did D13-PLAP-1, and co-immunoprecipitation demonstrated the stronger affinity of the D14-PLAP-1 protein to BMP-2 compared with the D13-PLAP-1 protein. Analysis of these data suggests that the D repeat polymorphism of PLAP-1/asporin has a significant influence on the functions of PDL cells.

关键词： TNFα   牙周膜相关蛋白1   人牙周膜细胞  

摘要： 目的:检测TNF-α对人牙周膜细胞Asporin表达的影响,初步探讨Asporin在牙周炎致病过程中扮演的角色,为牙周炎的发病机制研究提供线索。方法:改良酶消化法体外分离培养人牙周膜细胞,qRT-PCR检测在不同浓度TNF-α(0.5、1、2、5、10 ng/mL)作用下牙周膜细胞Asporin的表达及使用1 ng/mL的TNF-α作用于牙周膜细胞3、6、24和48 h后,牙周膜细胞中Asporin的表达;应用免疫细胞化学检测Asporin在牙周膜细胞中表达定位和在TNF-α作用下的表达变化,其结果用Image-Pro Plus(IPP)软件分析。结果:qRT-PCR结果显示,1ng/mL TNF-α能最大程度降低人牙周膜细胞中Asporin的表达(P<0.05),TNF-α作用后3 h开始,Asporin表达下降,作用24 h,48 h后,其表达降低最为明显(P<0.05)。免疫细胞化学的检测结果与qRT-PCR一致。结论:TNF-α能显著下调人牙周膜细胞Asporin的表达,推测Asporin可能参与了牙周炎的发生发展过程。

关键词： 牙周膜成纤维细胞   Asporin表达   炎性因子   牙周炎   致病机制  

摘要： 研究背景：\n 牙周炎是一种侵犯牙周支持组织的炎症性和破坏性疾病，其主要特征为牙龈及牙周膜胶原纤维溶解破坏，牙周附着丧失，牙槽骨吸收，最终导致牙齿松动脱落，跟癌症和心血管疾病一样严重威胁人类健康，因此明确各种宿主因素在牙周炎发生发展过程中所扮演的角色，对了解牙周炎病因及病理生理机制就显得尤为重要。\n 牙周炎的发病机制错综复杂，是近年来学者深入研究探讨的课题之一，细菌是牙周炎的始动因素，除全身易感因素外，细胞因子的作用不容忽视。学者认为牙周炎主要是通过致病因子引发的宿主免疫和炎症反应失调所造成的间接损害。多种炎性因子形成细胞因子网络，大多参与牙槽骨破骨与成骨、牙周组织破坏与修复过程。其中在破骨前体细胞分化成成熟破骨细胞过程中，促进破骨细胞分化的有白细胞介素1β(Interleukin-1β，IL-1β)、肿瘤坏死因子α(Tumornecrosis factor-α，TNF-α)、IL-17和抑制破骨细胞分化促进成骨的IL-10、转化生长因子β(Transforming growth factor，TGF-β)等。\n 白细胞介素1(IL-1)主要由单核-吞噬细胞、中性粒细胞和血管内皮细胞等诱导产生，具有促炎功能，可分解、代谢、促进前列腺素E2合成，激活破骨细胞，促进破骨细胞合成导致骨吸收。并可促进体质内的间质细胞诱导产生金属蛋白酶，导致基质中的胶原降解、破坏。有研究表明，在炎症部位的龈沟液中，IL-1活性明显高于健康部位，IL-1的量也与牙周袋的深度呈正相关关系。其中龈沟液中IL-1β的水平与慢性牙周炎的严重程度密切相关。IL-1β是一种多效性细胞因子，具有广泛的细胞免疫调节作用，包括促进胸腺细胞和T细胞增殖和分化，刺激破骨细胞和胶原酶，促进破骨细胞形成和骨吸收，最终导致骨和牙周结缔组织破坏，牙周组织修复能力丧失等。\n TNF-α主要由被G-细菌脂多糖(Lipopolysaccharide，LPS)激活的巨噬细胞产生，对白细胞、血管内皮细胞及结缔组织中多种细胞具有广泛的生物学效应，并可活化前破骨细胞使之分化成熟为破骨细胞，促进骨吸收，还可增强血管通透性，具有促炎作用。\n IL-8是中性粒白细胞强有力的趋化因子，可聚集并激活中性白细胞，使其释放溶菌酶，Gamonal等研究发现，IL-8在正常牙龈组织和牙周炎患者牙龈组织中均有表达，但在病变牙龈组织中明显高于正常牙龈组织，且经治疗后显著下降。并有研究发现IL-8可以刺激RANKL mRNA表达和破骨细胞生成，促进骨吸收。\n IL-17作为一种促炎症性细胞因子，可以与受体特异性结合，有促进炎症反应、免疫应答、造血等多种作用。并可刺激成纤维细胞释放IL-6、IL-8、前列素E2(Prostaglandin E2，PGE2)等，诱导巨噬细胞产生IL-1β、TNF-α等，抑制IL-10的生成，促进骨吸收。\n 转化生长因子β(TGF-β)是一种多功能的细胞活性调节因子，具有调节机体的免疫应答、炎症反应功能，与组织修复密切相关。在慢性牙周炎发生发展过程中，TGF-β参与了牙周组织炎症的免疫反应和软硬组织的再生修复，影响着牙周疾病的发展速度和愈合过程。\n Asporin(ASPN)也叫牙周膜相关蛋白1(periodontal ligament associated protein1，PLAP1)，是小富亮氨酸重复序列蛋白多糖(small leucine-rich proteoglycans，SLRPs)家族中的一员，此家族成员还有二聚糖(Biglycan)，核心聚糖(Decorin)，fibromodulin，lumican等。SLRPs可以和许多不同的细胞因子、表面受体、生长因子结合达到调节细胞的功能。如Toll样受体、TGF-β、骨形成蛋白(Bonemorphogenetic protein-4，BMP-4)等。现有的研究表明SLRPs在调节胶原纤维的形成，组装和降解过程中扮演着重要角色。SLRPs与胶原纤维和弹性纤维结合，可以增强纤维的稳定性，保护纤维不被各种胶原酶降解。\n Asporin是2001年发现的SLRPs家族新成员，与其他成员一样它具有亮氨酸重复序列，不同的是Asporin不是严格意义的蛋白多糖，其氨基末端有个独特的天门冬氨酸残基重复区域，Loughlin J发现Asporin天门冬氨酸残基量D14等位基因与骨关节炎易感性显著相关。Sebastian K等对细胞外基质的相互作用研究结果显示Asporin与Decorin相互竞争，直接影响成骨细胞胶原矿化。Kei等报道Asporin在骨关节炎发病机制中抑制TGF-β成软骨和骨重构功能，从而抑制成骨。Yamada等报道Asporin对牙周组织中的胶原纤维同样发挥着调节