Bid 蛋白在骨关节病关节软骨细胞中的表达及意义

目的研究促凋亡蛋白Bid在正常关节软骨细胞和骨关节病时关节软骨细胞中的表达,探索Bid蛋白在骨关节病发病中的作用。方法取20例髋关节骨关节病行关节置换的关节软骨标本和10例正常关节软骨标本,应用免疫组织化学方法检测Bid蛋白在软骨细胞中的表达。结果Bid蛋白表达阳性率在骨关节病关节软骨细胞中较正常关节软骨细胞高,两者有显著性差异。结论Bid蛋白可能通过参与调控关节软骨细胞凋亡,在骨关节病发病中起作用。     

一氧化氮对软骨代谢影响的研究进展

人和动物的炎症关节中一氧化氮（ＮＯ）含量增高,关节中ＮＯ产生主要来源于关节软骨细胞和滑膜细胞。增高的ＮＯ可引起软骨代谢紊乱;抑制软骨细胞增殖,促使软骨细胞凋亡;抑制软骨细胞蛋白多糖（ＰＧ）、胶原合成,促进软骨细胞ＰＧ、胶原分解;促进软骨细胞糖酵解,从而导致关节软骨修复能力降低、软骨破坏增加。ＮＯ对软骨代谢的影响与类风湿性关节炎（ＲＡ）、骨关节病（ＯＡ）的发生、发展关系密切。抑制ｉＮＯＳ的表达、ＮＯ的产生,有可能成为治疗ＲＡ、ＯＡ的新途径     

Ⅳ型胶原在颞下颌关节紊乱病中的表达

目的 探讨颞下颌关节紊乱病病变与Ⅳ型胶原之间的关系.方法 应用链菌素亲生物素-过氧化酶连结法检测19例颞下颌关节病变关节盘和盘后组织中Ⅳ型胶原的表达.结果 19例颞下颌关节紊乱病患者中,不可复性盘前移位3例,盘前移位伴关节盘穿孔5例,骨关节病伴关节盘穿孔11例.Ⅳ型胶原主要存在于病变关节盘和双板区的血管基底膜.其中骨关节病伴关节盘穿孔的阳性反应最为明显.6例在软骨细胞周围有弱阳性反应.结论 Ⅳ型胶原在病变颞下颌关节盘和盘后组织的血管基底膜以及软骨细胞周围可出现表达,Ⅳ型胶原免疫组织化学的染色强度与病变的严重程度有一定关系.     

骨关节炎软骨中Ⅰ型和Ⅱ型胶原的分布

目的：研究骨关节炎软骨中Ⅰ型和Ⅱ型胶原的分布。方法：从正常关节软骨和骨关节炎软骨上取样本做切片，所有样本行HE、蕃红0染色及Ⅰ型和Ⅱ型胶原免疫组化。结果：骨关节炎软骨中Ⅱ型胶原免疫组化染色不均匀。Ⅰ型胶原染色，在表层和中层的部分区域有不规则着色，纤维样组织中，Ⅰ型胶原免疫组化呈阳性，Ⅱ型胶原免疫组化不着色，结论：骨关节软骨基质中Ⅱ型胶原和蛋白聚糖的破坏增强与软骨细胞对其合成增强同时存在，软骨修复的过程中，部分软骨细胞发生去分化，而表达Ⅰ型胶原。     

大鼠不同部位软骨细胞的形态及表型特征比较研究

目的:比较研究大鼠椎间盘软骨终板和膝关节软骨的细胞表型特征的相关性.方法:大鼠的软骨终板和关节软骨细胞分别予以消化培养.进行光镜、电镜观察其形态.使用免疫组化技术分别检测不同部位细胞的Ⅱ型胶原表达.结果:大鼠椎间盘软骨终板和关节软骨细胞形状相似,并且均表达Ⅱ型胶原.结论:本研究提示软骨终板表达软骨细胞的特征性胶原,与关节软骨细胞相似.     

骨形态发生蛋白对体外培养胎儿关节软骨细胞胶原基因表达…

为了解骨形态发生蛋白对关节软骨细胞的作用特点及意义，本研究用部分纯化的牛骨形态发生蛋白对胎儿关节软骨细胞进行诱导，并用人Ⅰ、Ⅱ、Ⅲ、Ⅳ型胶原的前胶原ｃＤＮＡ探针对被诱导关节软骨细胞进行原位杂交。结果发现，ｂＢＭＰ可明显增加被诱导软骨细胞中Ⅰ型胶原的表达，中断Ⅱ型胶原的表达，并轻度增加软骨细胞中Ⅲ型胶原的表达，但对Ⅳ型胶原的表达影响不大。本研究认为，ｂＢＭＰ诱导的软骨细胞胶原基因表达的这种改变是软骨     

正常关节软骨的胶原表型与骨关节炎关节软骨的表型异常

正常关节软骨细胞特异性表达及形成Ⅱ、Ⅸ、和Ⅺ型胶原,关节软骨胶原的正常表型和含量是关节软骨力学特征的基础。业已证明,关节软骨细胞表型表达改变所致胶原代谢及成分的异常,是骨关节炎发生、发展的主要因素之一。     

正常关节软骨的胶原表型与骨关节炎关节软骨的表型…

正常关节软骨细胞特异性表达及形成Ⅱ，Ⅸ，和Ⅺ型胶原，关节软骨胶原的正常表型和含量是关节软骨力学特征的基础。业已证明，关节软骨细胞表型表达改变所致胶原代谢及成分的异常，是骨关节炎发生，发展的主要因素之一。     

中药对软骨细胞代谢作用的研究现状及思路探讨

近年来对关节软骨细胞代谢的研究逐渐深入广泛 ,发现关节软骨细胞的代谢异常致变性、退化、凋亡 ,继而发生软骨的破坏、骨质和滑膜改变与骨关节病 (Ｏｓｔｅｏａｒｔｈｒｉｔｉｓ ,ＯＡ)的发生关系密切。目前ＯＡ的治疗药物以非甾体类药物为主 ,现正逐步转向软骨保护剂。祖国医学中并无骨关节病的病名记载 ,而将其纳入“痹症”范畴 ,并从“肾主骨”理论出发 ,使用补肾、柔肝、活血及行气消肿类中药治疗该病 ,取得较好的疗效。目前国内有关中药对软骨细胞代谢影响的报道较少 ,现将近年来中药与软骨细胞代谢方面的相关研究概况介绍如下 :1　软…     

不同浓度hTNFa对软骨细胞及其基质相关基因表达 的影响

目的探讨软骨细胞在肿瘤坏死因子(TNFa)作用下对其分泌基质蛋白及相关基因mRNA表达的影响。方法体外培 养的小鼠软骨细胞用25,50,100 ng/ml的hTNFa干预,不加人TNFa作为对照,通过RT-qPCR检测hTNFa对软骨细胞分泌基质 蛋白及相关基因mRNA表达的影响。结果hTNFa能降低软骨细胞分泌的n型胶原及刺激促n型胶原分泌的SOX9基因的表达; 对基质可提高基质酶MMP~9,MMP43,ADAMTS-4和ADAMTS-5基因的表达,提高炎性因子IL4a,TGFp,并增加凋亡基因BAX, caspase3的表达。结论hTNFa通过不同途径影响软骨细胞及基质相关基因的表达,从而促进了关节软骨的降解。     

Metabolic activation of chondrocytes in human osteoarthritis. Expression of type II collagen]

OBJECTIVE/METHODS: Type II collagen is the dominating collagen in articular cartilage. It is essential for the structural integrity and the biomechanical properties of cartilage. Using immunohistology and in situ hybridization we systematically analyzed the protein and mRNA-expression of type II collagen in cartilage/bone sections without any signs of osteoarthritis and osteoarthritic samples with various degrees of osteoarthritis. RESULTS: In normal articular cartilage without any histologic signs of osteoarthritis type II collagen was distributed homogeneously. An expression of the type II collagen-mRNA was not detectable in any of these samples. In cartilage sections with a roughening of the cartilage surface and a superficial loss of the safranin O staining as early histologic signs of osteoarthritis the immunohistologic staining for type II collagen was reduced in the deep cartilage zone. An expression of the type II collagen-mRNA was found in 19 of 35 preparations. This expression, however, was restricted to the middle and deep zone of cartilage. A good and reproducible correlation of the specific gene and protein expression was found in samples with more severe osteoarthritic lesions. CONCLUSIONS: Detailed information on metabolic changes and the activation of chondrocytes in osteoarthritic cartilage are important to characterize certain stages of osteoarthritis and thus identify new prognostic factors. Increasing knowledge of the factors regulating the matrix synthesis and degradation in cartilage will provide the basis for new disease modifying therapies in osteoarthritis.     

Expression of type X collagen in young and old C57Bl/6 and Balb/c mice. Relation with articular cartilage degeneration

OBJECTIVE: To investigate whether the development of osteoarthritic lesions in the knee joints of mice is associated with increased immunostaining of type X collagen. METHODS: Sections of total knee joints in combination with immunohistochemistry were used to study the distribution of type X collagen in the cartilage of young and old mice of two mouse strains, Balb/c and C57Bl/6, known to develop osteoarthritic lesions at different locations. Expression of type X collagen and PTH/PTHrP-receptor mRNA were studied by RT-PCR. RESULTS: Young adult Balb/c and C57Bl/6 mice both expressed type X collagen in the non-calcified cartilage of the tibia-femoral joint. Old mice of both strains had a strongly increased deposition of type X collagen in the patella-femoral but not in the tibia-femoral joint. The locations in the murine knee joints prone to develop osteoarthritis (OA) did not preferentially express increased amounts of type X collagen. Thus, whereas increased type X was observed in both strains in the patella-femoral joints, only Balb/c mice preferentially developed osteoarthritic lesions in these joints. Also cartilage degeneration was usually seen only in the lateral compartment of the knee joints of C57Bl/6 mice but this was not accompanied by increased type X collagen immunostaining. Increased deposition of type X collagen was not associated with elevated levels of type X collagen mRNA or with decreased levels of PTH/PTHrP-receptor mRNA. CONCLUSION: Type X collagen expression and spontaneous OA in mice are not necessarily related since OA prone locations in the murine knee joint do not preferentially express type X collagen.     

Expression of type I,type II,and type X collagen genes during altered endochondral ossification in the femoral epiphysis of osteosclerotic (oc/oc) mice

The osteosclerotic (oc/oc) mouse, a genetically distinct murine mutation that has a functional defect in its osteoclasts, also has rickets and shows an altered endochondral ossification in the epiphyseal growth plate. The disorder is morphologically characterized by an abnormal extension of hypertrophic cartilage at 10 days after birth, which is later (21 days after birth) incorporated into the metaphyseal woven bone without breakdown of the cartilage matrix following vascular invasion of chondrocyte lacunae. In situ hybridization revealed that the extending hypertrophic chondrocytes expressed type I and type II collagen mRNA, as well as that of type X collagen and that the osteoblasts in the metaphysis expressed type II and type X collagen mRNA, in addition to type I collagen mRNA. The topographic distribution of the signals suggests a possible co-expression of each collagen gene in the individual cells. Immunohistochemically, an overlapping deposition of type I, type II, and type X collagen was observed in both the extending cartilage and metaphyseal bony trabeculae. Such aberrant gene expression and synthesis of collagen indicate that pathologic ossification takes place in the epiphyseal/metaphyseal junction of oc/oc mouse femur in different way than in normal endochondral ossification. This abnormality is probably not due to a developmental disorder in the epiphyseal plate but to the failure in conversion of cartilage into bone, since the epiphyseal plate otherwise appeared normal, showing orderly stratified zones with a proper expression of cartilage-specific genes.     

Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic cartilage

OBJECTIVE: To test the hypothesis that terminal differentiation of chondrocytes in human osteoarthritic cartilage might lead to the failure of repair mechanisms and might cause progressive loss of structure and function of articular cartilage. DESIGN: Markers for terminally differentiated chondrocytes, such as alkaline phosphatase, annexin II, annexin V and type X collagen, were detected by immunohistochemical analysis of human normal and osteoarthritic knee cartilage from medial and lateral femoral condyles. Apoptosis in these specimens was detected using the TUNEL labeling. Mineralization and matrix vesicles were detected by alizarin red S staining and electron microscopic analysis. RESULTS: Alkaline phosphatase, annexin II, annexin V and type X collagen were expressed by chondrocytes in the upper zone of early stage and late stage human osteoarthritic cartilage. However, these proteins, which are typically expressed in hypertrophic and calcifying growth plate cartilage, were not detectable in the upper, middle and deep zones of healthy human articular cartilage. TUNEL labeling of normal and osteoarthritic human cartilage sections provided evidence that chondrocytes in the upper zone of late stage osteoarthritic cartilage undergo apoptotic changes. In addition, mineral deposits were detected in the upper zone of late stage osteoarthritic cartilage. Needle-like mineral crystals were often associated with matrix vesicles in these areas, as seen in calcifying growth plate cartilage. CONCLUSION: Human osteoarthritic chondrocytes adjacent to the joint space undergo terminal differentiation, release alkaline phosphatase-, annexin II- and annexin V-containing matrix vesicles, which initiate mineral formation, and eventually die by apoptosis. Thus, these cells resume phenotypic changes similar to terminal differentiation of chondrocytes in growth plate cartilage culminating in the destruction of articular cartilage in osteoarthritis.     

Differential expression of type X collagen in a mechanically active 3-D chondrocyte culture system: a quantitative study

Objective  

Mechanical loading of cartilage influences chondrocyte metabolism and gene expression. The gene encoding type X collagen is expressed specifically by hypertrophic chondrocytes and up regulated during osteoarthritis. In this study we tested the hypothesis that the mechanical microenvironment resulting from higher levels of local strain in a three dimensional cell culture construct would lead to an increase in the expression of type X collagen mRNA by chondrocytes in those areas.     

Molecular principles of induction and progression of arthrosis

From a morphological point of view, osteoarthritis is characterized by continuous loss of the cartilage matrix, an increased density of the subchondral bone, and partial involvement of the synovial compartment. Research activities are focussing on gene expression and gene regulation in normal and osteoarthritic cartilage to develop prognostic markers and new therapeutic strategies. In general, chondrocytes from normal adult articular cartilage show low metabolic activity. However in osteoarthritis, activation and differentiation of chondrocytes occur. Activation involves anabolic pathways such as an enhanced expression of type II collagen as well as catabolic patterns such as the increased expression of matrix metalloproteinases. These metabolic pathways are unbalanced, leading to insufficient cartilage architecture, unable to meet the requirements for mechanical stability and load compensation. In osteoarthritis, chondrocyte differentiation is characterized by the expression of type X collagen. Further differentiation stages have been observed as shown for the expression of osteocalcin, osteopontin, or alkaline phosphatase in articular cartilage. This altered expression pattern of chondrocytes is likely to influence the biochemical and biomechanical properties of the cartilage matrix. In conclusion, new analytic and comparative methods to analyze gene and protein expression offer powerful tools to elucidate candidate genes in osteoarthritis. Detailed information on the regulatory pathways will be the basis for modulation of chondrocyte behavior and, therefore, may lead to new therapeutic approaches in the treatment of osteoarthritis.     

Immunohistochemical analysis of type-X-collagen expression in osteoarthritis of the hip joint.

Conflicting data have been reported on the spatial distribution of type X-collagen expression in osteoarthritis, and no concise data exist on a possible correlation between type X-collagen expression and clinical and radiological alterations. Well defined clinical and radiological data were compared with histopathological and immunohistochemical findings to investigate the expression of type-X collagen in osteoarthritis of the hip joint. Femoral heads were obtained in toto from 11 patients undergoing routine hip arthroplasty for femoral neck fractures (n = 3) or osteoarthritis (n = 8) and from 13 patients (age: 12 days to 69 years) without any evidence of hip-joint pathology. Whole coronal sections from the femoral head were decalcified for routine histology and immunohistochemical analysis with use of type-specific monoclonal antibodies to type-X collagen. Our results demonstrate that type-X collagen is consistently found in osteoarthritic cartilage and is absent from normal adult cartilage (including the region of calcified cartilage). Except for the occurrence of type-X collagen in the middle zone of articular cartilage in advanced stages of osteoarthritis, there is no specific change in the staining pattern or intensity for the collagen during osteoarthritis, particularly when the staining is related to clinical and radiological parameters. Hardly more than 20% of the extracellular matrix stained for type-X collagen; therefore, we suggest that, in most cases, this type of collagen may not play a direct biomechanical role in the weakening of osteoarthritic cartilage but rather may contribute indirectly to a disturbance of the disc biomechanics by altering matrix-molecule interaction. However, expression of type-X collagen may indicate a change in chondrocyte phenotype that consistently coincides with the formation of chondrocyte clusters, one of the first alterations in osteoarthritis visible on histologic examination.     

Localization of type X collagen in canine growth plate and adult canine articular cartilage

Type X collagen was extracted from ends of canine growth plates by pepsin digestion after 4 M guanidine hydrochloride extraction, purified by stepwise salt precipitation (2.0 M NaCl in 0.5 M acetic acid), and chromatographed on a Bio-Gel A1.5 M column in 1.0 M CaCl2. Without reduction on sodium dodecyl sulfate (SDS) polyacrylamide gels, the preparation yielded a single, high-molecular-weight (mol wt) band; after reduction, a single band of relative mol wt 5.0 x 10(4) was found. Polyclonal sera were raised against the purified collagen and used in the immunolocalization of canine type X collagen. As expected, indirect immunoperoxidase (IP) or indirect immunofluorescent staining with the polyclonal sera demonstrated that most of the immunoreactivity was localized in the zone of provisional calcification of the growth plate and in cartilage remnants in the metaphyseal region of the physis. A progressive decrease in staining toward the diaphysis of the fetal canine long bone was apparent as the trabecular structures were remodeled to bone. Unexpectedly, type X collagen was also detected in the zone of calcified, mature articular cartilage. It was concentrated in the pericellular matrix of the chondrocytes, appeared at or just above the tidemark, and was expressed immediately before mineralization. Identification of type X collagen in both the canine growth plate and the zone of calcified articular cartilage suggests that cells in the deep layer of cartilage and in the zone of calcified cartilage in the adult animal retain some characteristics of a growth plate and may be involved in regulation of mineralization at this critical interface. The expression of growth plate-like properties would allow the deep chondrocytes of mature articular cartilage to play a role in remodeling of the joint with age and in the pathogenesis of osteoarthritis.     

Type II collagen synthesis in the articular cartilage of a rabbit model of osteoarthritis: expression of type II collagen C-propeptide and mRNA especially during early-stage osteoarthritis

Background The aim of this study was to observe time course changes in type II collagen synthesis in various regions of articular cartilage affected with osteoarthritis (OA) by examining the expression of type II collagen C-propeptide (pCOL II-C) and mRNA in a rabbit OA model. Methods Osteoarthritis was experimentally induced by partial lateral meniscectomy in the knees of Japanese white rabbits. The cartilage of the animals was then examined histologically over time. The degenerative area of articular cartilage was divided into three areas, according to the degree of degeneration. The ability to synthesize type II collagen was estimated by the immunohistological staining of pCOL II-C and the in situ hybridization of mRNA in type II collagen. Results The positive rate of pCOL II-C immunostaining in chondrocytes was highest in the central-degenerative region 1 week after surgery, and the highest rate in the para-degenerative region was observed 2 and 4 weeks after surgery. The percentage of pCOL II-C positive cells increased as the histological degeneration score increased to moderate degeneration and then decreased with further progression of the severity of cartilage degeneration. Examination by in situ hybridization revealed that the regions marked by strong pCOL II-C mRNA expression were similar to those indicated by the immunohistology results. Conclusions These results suggest that the type II collagen-synthesizing potential of chondrocytes is highest in moderately degenerated areas of OA articular cartilage. Cartilage repair continues to be seen even as OA advances, although the reaction varies depending on the stage of OA.