关键词： 椎间盘退变   基因治疗   端粒酶逆转录酶   蛋白多糖   腺病毒  

摘要： 椎间盘退变(intervertebral disc degeneration, IDD)是引起椎间盘突出症(lumbar intervertebral disc protrusion, LIDP)等椎间盘退行性疾病(discdegenerated disease, DDD)的主要原因,其发病机制较为复杂。目前认为IDD主要是细胞因子、营养代谢、遗传、生物力学等因素共同作用的结果。在退变早期,髓核(nucleus pulposus, NP)和纤维环(anulus fibrosus, AF)分界逐渐模糊,纤维环细胞密度有所增加,髓核内细胞聚集,椎间盘内细胞增殖、群集和死亡增多；软骨终板裂缝、变薄、软骨下骨微骨折和骨硬化。随着终板的硬化,软骨终板的血运通道、纤维环的血管逐渐减少,直接影响通过终板的营养途径。营养供给的减少限制了椎间盘髓核细胞合成新的基质,髓核组织内氧供的减少使髓核细胞进行无氧代谢、产生乳酸产物,而且代谢产物难以从无血管的椎间盘内排出,乳酸聚集使细胞外环境变为酸性,这是适宜于蛋白酶作用的环境。蛋白酶是参与老化和退行性变的重要因素,基质金属蛋白酶(matrix metalloproteinases, MMPs)引起细胞外基质降解。胶原酶(CollagenasesMMP1,8,13)、白明胶酶(gelatinases MMP2,9)和(stromelysin, MMP3)降解椎间盘大多数胶原质、蛋白多糖(proteoglycan, PG)和连接蛋白(connexin, CN)。随着退变的进展,髓核逐渐脱水化,高度降低,细胞数量减少,蛋白含量明显降低。纤维环逐渐失去正常板层排列,板层数目减少。在退变晚期阶段,椎间盘变薄、弹性降低,髓核和纤维环的边界不清。髓核膨胀压降低,纤维环硬度增加,分散负荷的能力降低,使转移到骨的应力增加。纤维环裂隙和撕裂的数量和程度增加,纤维环在应力下不能包含限制髓核,致使椎间盘疝出,肉芽组织和神经血管从纤维环外层向内长入。 IDD主要生化改变为蛋白多糖和水分减少、髓核细胞凋亡、纤维环老化变性、细胞外基质(extracellular matrix, ECM)降解和纤维化、加速退变的细胞因子出现等一系列级联反应(cascade reaction)。正常椎间盘细胞通过各种途径调节代谢活动,其中影响椎间盘退变主要包括四个因素：①促进基质分解的因素如炎症因子、基质金属蛋白酶(matrix metalloproteinases, MMPs)等,如一氧化氮(nitrogen monoxidum, NO)-.白细胞介素(interleukin, IL)-1,6、肿瘤坏死因子a (tumour necrosis factor,TNF-α)、前列腺素E2、MMP3、环氧化酶2、蛋白聚糖酶(ADAMTS-4,属于MMPs家族)等；②抑制基质分解的因素如基质金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinase, TIMP);③促进细胞分裂的因素如血小板源性生长因子(platelet-derived growth factor, PDGF),细胞生长因子；④促进软骨形成因素如转化生长因子(transforming growth factor, TGF)、骨形态发生蛋白(bone morphogenetic protein, BMP)、成骨蛋白-1(OP-1)等；分子调节因子如Sox-9等。ECM分解代谢的增加促进了椎间盘细胞的加速衰老(aging)和凋亡(apoptosis),从而加速了IDD进程。椎间盘退变过程中最显著的特征是髓核细胞凋亡、蛋白多糖及Ⅱ型胶原(collagen typeⅡ, Col.Ⅱ)减少、Ⅰ型胶原(collagen typeⅠ, Col.Ⅰ)增多。 如何抑制和延缓椎间盘细胞凋亡、增加退变椎间盘ECM中的蛋白多糖以及Ⅱ型胶原含量,达到抑制、延缓或者治疗椎间盘退变是临床共同关注的问题。研究报道直接向椎间盘内注射细胞因子、椎间盘细胞、间充质干细胞(mesenchyme stem cell, MSC)等能够延缓或逆转椎间盘细胞的退变。有学者进行了将外源性生长因子的编码基因转入动物椎间盘的一系列体外和体内实验研究。Nishida等应用腺病毒载体将人TGF-β1基因转导入新西兰兔椎间盘中,发现TGF-β1浓度比对照组高出约5倍,合成的蛋白多糖量也较对照组高出1倍。Yoon也发现TGF-β1基因腺病毒可显著提高Ⅱ型胶原(collagen typeⅡ, Col.Ⅱ)及蛋白聚糖(proteoglycan, PG)的含量。因为椎间盘细胞为非分裂细胞,故实验中应用介导目的基因转入的载体为质粒型病毒载体如腺病毒。目的基因的选择：主要为具有促进蛋白多糖和Ⅱ型胶原合成的作用的转

关键词： 腰椎间盘退化   亚洲人群   易感基因   骨关节炎   免疫组织化学方法   骨细胞外基质   免疫组织化学定位   基因表达模式  

摘要： Asporin也被称为牙周韧带相关蛋白-1(PLAP-1),属于富含亮氨酸重复序列的小分子蛋白多糖(SLRP)家族,它存在于软骨细胞外基质(ECM),与骨关节炎有相关性。最近发现亚洲人群的腰椎间盘退化和它的D14等位基因有相关性。但是,这种基因的正常免疫组织化学定位在人类椎间盘,在椎间盘退化的高加索人群中的表达水平还没有研究。方法本研究是由人类研究审查委员会通过批准,用免疫组织化学方法研究asporin基因在人类和沙鼠椎间盘的表达(一种与年龄相关的、自发的、椎间盘退变的小型啮齿类动物),并用Affymetrix公司的微点阵系统分析(基因芯片技术)asporin基因在体内和体外的表达。结果免疫组织化学方法研究人类的椎间盘发现,asporin基因不仅在外环的部分细胞中,而且在内环的少量细胞中均表达,但是几乎不能在髓核中表达。在沙鼠的腰椎间盘中也发现了类似的asporin基因表达模式,在椎间盘组织和立体培养的细胞中也发现了大量的asporin相关基因的表达。椎间盘退化严重组(汤普森Ⅳ级)与椎间盘退化轻微组(汤普森Ⅰ、Ⅱ、Ⅲ级)比较,有显著的差异(P=0.004),asporin基因显著高表达。结论高加索受试者和沙鼠的椎间盘在免疫组化水平上asporin阳性表达在外环比内环多,而且几乎没有在髓核表达。基因表达研究也证实了asporin基因在椎间盘退化严重的人群体内表达最为明显。

关键词： 椎间盘   Sox9基因   基因治疗  

摘要： 目的 观察腺病毒介导的Sox9基因（AdSox9）对兔腰椎间盘退变模型的疗效.方法 25只新西兰大白兔用针刺法制作腰椎间盘退变模型,24周后,将AdSox9注射到退变椎间盘组织中,6周后用MRI观察椎间盘变化,免疫组织化学法观察椎间盘组织Ⅱ型胶原（Col2al）的表达,逆转录-聚合酶链反应（RT-PCR）观察Col2al mRNA的变化.结果 MRI示：治疗后L2～3、L3～4、L4～5的椎间盘信号值分别为：580.43±76.43、612.74±56.73、605.87±76.53,模型对照组为：238.43±43.65、217.65±38.95、236.98±76.53 Col2al表达的标记指数：治疗后L2～3、L3～4、L4～5的椎间盘分别为：42.48±5.53、45.34±15.43、42.47±25.45,模型对照组为：11.68±10.08、12.68±13.75、13.38±26.53 Col2al mRNA的表达：治疗后L2～3、L3～4、L4～5的椎间盘分别为6570.43±174.53、7653.75±158.93、6905.83±119.23,模型对照组为：1256.76±89.76、1217.65±18.65、1132.97±47.65.3种方法均示,治疗组与模型对照组之间比较差异有统计学意义（P〈0.01）.结论 AdSox9对退变椎间盘动物模型有治疗作用.

关键词： Asporin   Transforming growth factor-beta   Type II collagen   Protein interaction   Leucine-rich repeat  

摘要： Asporin is an extracellular matrix (ECM) protein that regulates cartilage matrix gene expression and cartilage formation by modulating the transforming growth factor-beta (TGF-beta) signaling pathway. Our previous studies have indicated that asporin binds to TGF-beta 1 directly and inhibits TGF-beta 1-mediated expression of cartilage matrix genes. However, it is still unknown how asporin interacts with TGF-beta 1 and influences its activity. Using competition assays, we determined that amino acids 159-205 of asporin mediate its interaction with TGF-beta 1 and effectively repress TGF-beta 1-induced cartilage matrix gene expression. Asporin also has a binding ability to type II collagen in vitro, but its binding pattern is different from that of TGF-beta 1. In contrast with previous in vivo findings, asporin did not affect the interaction between TGF-beta 1 and the TGF-beta type II receptor (T beta RII) by itself or in the presence of type II collagen in vitro. However, in the presence of heparin/heparan sulfate, asporin inhibits the interaction between TGF-beta and T beta RII in vitro. These findings suggest that asporin is one of the important cartilage matrix proteins that binds to the ECM and TGF-beta 1 and thereby modulates interactions between TGF-beta and its signaling receptors.

关键词： asporin   keloid   keratin 10   proteomic   skin  

摘要： Background Keloids are pathological scars and, despite numerous available treatment modalities, continue to plague physicians and patients. Objectives Identification of molecular mediators that contribute to this fibrotic phenotype. Methods Two-dimensional gel electrophoresis, MALDI-TOF, Mascot online database searching algorithm and Melanie 5 gel analysis software were employed for comparative proteomic analysis between normal skin (NS) and keloid scar (KS) tissue extracts. Results Seventy-nine protein spots corresponding to 23 and 32 differentially expressed proteins were identified in NS and KS, respectively. Isoforms of heat shock proteins, gelsolin, carbonic anhydrase and notably keratin 10 were strongly expressed in NS along with manganese superoxide dismutase, immune components, antitrypsin, prostatic binding protein and crystalline. Various classes of proteins were found either to be present or to be upregulated in keloid tissue: (i) inflammatory/differentiated keratinocyte markers: S100 proteins, peroxiredoxin I;(ii) wound healing proteins: gelsolin-like capping protein;(iii) fibrogenetic proteins: mast cell beta-tryptase, macrophage migration inhibitory factor (MIF);(iv) antifibrotic proteins: asporin;(v) tumour suppressor proteins: stratifin, galectin-1, maspin;and (vi) antiangiogenic proteins: pigment epithelium-derived factor. Significant increases in expression of asporin, stratifin, galectin-1 and MIF were observed by Western blot analysis in KS. Conclusions This work has identified differentially expressed proteins specific to KS tissue extracts which can potentially be used as specific targets for therapeutic intervention.

关键词： 椎间盘退变   载体   基因治疗   目的基因   病毒载体   基因调控  

摘要： 椎间盘退变是一种慢性疾病,是引起腰腿痛的重要病因,主要是由于椎间盘中各种细胞因子、生长因子等合成代谢和分解代谢失衡引起椎间盘组织内环境的不稳定,最终导致退变,基因治疗是将目的基因导入靶细胞内,成为宿主细胞遗传物质的一部分,目的基因表达产物对疾病起治疗作用,有效目的基因与载体的选择为基因治疗椎间盘退变提供了可能。

关键词： 椎间盘退变   基因治疗   疾病   病理生理改变   细胞功能   生物学治疗   生化因子   临床症状  

摘要： 椎间盘退变是受诸如年龄、机械物理、细胞、基因以及生化因子等多种因素影响的复杂的病理生理改变。目前各种手术及保守治疗手段仅仅是针对椎间盘退变引起的各种临床症状的治疗,而不是从恢复椎间盘细胞功能的角度上去逆转椎间盘的退变。基因治疗作为一种新的治疗手段,由于能够改变和持续影响细胞的生理功能,而成为生物学治疗研究领域中的一个新热点.

关键词： GENETIC polymorphisms   ANKYLOSING spondylitis   PATIENTS   SPINE -- Diseases   ASPARTIC acid   CHINESE   HEALTH   THERAPEUTIC use  

摘要： Objective: To investigate the role of a functional polymorphism consisting of an aspartic acid (D) repeat located in the asporin gene (ASPN) gene in the susceptibility to and clinical outcome of ankylosing spondylitis (AS). Methods: A total of 374 Chinese patients with ankylosing spondylitis and 421 controls of the same ethnic origin matched for age and sex were included in the study. The asporin D repeat polymorphism was genotyped by polymerase chain reaction with a fluorescent primer. Results: Significant differences between AS patients and controls were detected in the distribution of the 7 alleles found in our population. D14 and D16 alleles were significantly over-represented in AS patients (D14, P=0.001, odds ratio (OR)=1.857, 95% confidence interval(CI) 1.27-2.715;and D16, P<0.0001, OR=2.605, 95% CI 1.75-3.879). D16 over-representation was more common in early-onset patients than in late-onset patients, although the difference did not reach significance (P=0.071). Conclusion: The results support a role for an asporin D repeat polymorphism in the susceptibility to AS and an influence of this gene on the outcome of the disease. D14 and D16 allele variants of ASPN might be the susceptibility alleles for AS in the Han Chinese population, whereas the D13 allele variant may have a protective effect on the onset of AS.

关键词： TRANSFORMING growth factors-beta   GENE expression   BONE cells   TRANSCRIPTION factors   BONE spurs   BONES -- Metabolism   OSTEOARTHRITIS   PATIENTS   FRACTURES -- Diagnosis   FEMUR neck   WOUNDS & injuries  

摘要： To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta 1 (TGF-beta 1), TGF-beta 2, TGF-beta 3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. Osteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-beta 1, TGF-beta 2, TGF-beta 3, and Runx2 was analyzed using real-time RT-PCR. Expression of ASPN, TGF-beta 1, and TGF-beta 3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta 1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. The increased ratio of ASPN mRNA to TGF-beta 1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-beta 1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

关键词： asporin   biomineralization   calcium   collagen binding   decorin   small leucine-rich repeat proteoglycan/protein (SLRP)  

摘要： The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2 were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosettagami (TM)) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins).