人正常软骨和软骨肉瘤蛋白多糖的比较研究

取三例一般型一级的软骨肉瘤及不同年令正常人股骨头关节软骨,经4mol/L盐酸胍提取和氯化铯平衡等密度梯度离心,分析蛋白多糖组份的化学组成,并用Sepharose CL-2B层析分析蛋白多糖分子大小。初步结果,两例软骨肉瘤蛋白多糖的化学组成和分子大小与新生儿接近,另一例软骨肉瘤蛋白多糖的化学组成和分子大小与成年人相似。此结果说明软骨肉瘤生物学行为的多变性及其蛋白多糖的异质性。     

Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species.

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.     

Establishment of a reliable method for direct proteome characterization of human articular cartilage

Articular cartilage consists mainly of extracellular matrix, mostly made of collagens and proteoglycans. These macromolecules have so far impaired the detailed two-dimensional electrophoresis-based proteomic analysis of articular cartilage. Here we describe a method for selective protein extraction from cartilage, which excludes proteoglycans and collagen species, thus allowing direct profiling of the protein content of cartilage by two-dimensional electrophoresis. Consistent electrophoretic patterns of more than 600 protein states were reproducibly obtained after silver staining from 500 mg of human articular cartilage from joints with diverse pathologies. The extraction yield increased when the method was applied to a chondrosarcoma sample, consistent with selective extraction of cellular components. Nearly 200 of the most intensely stained protein spots were analyzed by MALDI-TOF mass spectrometry after trypsin digestion. They represented 127 different proteins with diverse functions. Our method provides a rapid, efficient, and pertinent alternative to previously proposed approaches for proteomic characterization of cartilage phenotypes. It will be useful for detecting protein expression patterns that relate pathophysiological processes of cartilaginous tissues such as osteoarthritis and chondrosarcoma.     

Doublecortin is expressed in articular chondrocytes

Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.     

Pharmacological actions of 17 beta-oestradiol on articular cartilage chondrocytes and chondrosarcoma chondrocytes in the absence of oestrogen receptors

(1) Pharmacological concentrations (greater than 10(-5) M) of 17 beta-oestradiol inhibited 35S-labelled proteoglycan synthesis in bovine articular cartilage explant cultures. They also inhibited 35S-labelled proteoglycan synthesis and 3H-labelled protein synthesis in cell cultures of chondrocytes from bovine articular cartilage and Swarm rat chondrosarcoma. Maximal inhibition was about 30-50%. Physiological concentrations (10(-9)-10(-8) M) of oestradiol had no effect on the synthesis of either protein or proteoglycan. (2) The inhibitory action of high concentrations of oestradiol on these biosynthetic pathways is not common to all steroids since 10(-4) M cortisol had no effect on articular chondrocyte cell cultures. 10(-4) M testosterone had a similar action to oestradiol. (3) Neither physiological nor pharmacological concentrations of 17 beta-oestradiol had any effect on 35S-labelled proteoglycan turnover in the cartilage explant system. (4) 10(-5) M oestradiol inhibited cell division in cultures of articular chondrocytes which had entered the log growth phase. 10(-7) M oestradiol had no effect on articular chondrocyte growth. (5) In male rats implanted with silastic capsules releasing 17 beta-oestradiol, increase in body weight was retarded by about 25% over a period of 6 weeks, compared to control rats. Rat chondrosarcoma grew to the same size in oestrogen-treated rats as it did in controls. (6) Oestrogen receptors could not be detected in freshly isolated bovine articular chondrocytes or in rat chondrosarcoma. (7) In conclusion, neither the mitotic rate of articular chondrocytes nor their proteoglycan metabolism is under the direct physiological control of oestradiol. Growth and biosynthetic activity of the rat chondrosarcoma chondrocytes are independent of either direct control by the hormone or control effected by oestradiol regulation of a second hormone or growth factor.     

The N-terminal sequence of the large proteoglycan of articular cartilage

A peptide with hyaluronic acid-binding properties was isolated from trypsin digests of bovine articular cartilage proteoglycan aggregate. This peptide originated from the N-terminus of the proteoglycan core protein, retained its function of forming complexes with hyaluronate and link protein and contained at least one keratan sulfate chain. Amino acid sequence data demonstrated that the first six amino acid residues of the N-terminus of bovine articular cartilage proteoglycan core protein differed from the same region from the rat chondrosarcoma proteoglycan. Further sequence data indicate areas of considerable sequence homology in the hyaluronic acid-binding regions of proteoglycans from the two species.     

Localization of type IX collagen in chondrons isolated from porcine articular cartilage and rat chondrosarcoma

Summary Chondrocytes, each with their pericellular matrix bounded by a fibrous capsule, can be extracted singly or in groups from both mature pig articular cartilage and chondrosarcoma tissue. These structures, termed chondrons, are thought to anchor the chondrocytes in the matrix and protect them from the compressive forces experienced when articular cartilage is under load. The capsule of these chondrons contains both type II and type IX collagens and is composed of fine fibrillar material, unlike the large banded fibres of type II collagen found in the rest of the matrix. This suggests a rote for type IX collagen in regulating the diameter of type II fibres to produce the fine fibrillar structure of the chondron capsules.     

Insulin stimulates secretion of a collagenase inhibitor by Swarm rat chondrosarcoma chondrocytes

Swarm rat chondrosarcoma chondrocytes produce an inhibitor of collagenase similar to that found in bovine articular chondrocytes and extracts of bovine scapular cartilage. These cells synthesize normal levels of cartilage type proteoglycans when cultured in serum free medium with insulin. Collagen synthesis is also increased when insulin is added to chondrosarcoma chondrocytes. We have demonstrated that insulin stimulates collagenase inhibitor production by these chondrocytes. Enhancement of inhibitory activity occurs over the range of 10 to 1000 ng/ml. A 3.2 fold stimulation was observed at a concentration of 1 microgram/ml. There was a lag period of 24 to 48 hours before the insulin effect became evident. Latent or active collagenase was not detectable under these conditions. These results suggest that the hormone insulin controls the levels of collagen in this tumor by stimulating synthesis of collagen and inhibitors of collagenase.     

Isolation of proteoglycan-specific T lymphocytes from patients with ankylosing spondylitis

Three T-cell lines and clones of the OKT4 phenotype have been isolated from the peripheral blood of three patients with ankylosing spondylitis. Antigen specificities of T cells were determined with purified protein derivative-(PPD) and cartilage-derived antigens, namely proteoglycans from human articular cartilage and intervertebral disc, bovine nasal cartilage, and rat chondrosarcoma and human type II collagen from cartilage. A cell line from one patient reacted with proteoglycans from human articular cartilage and human intervertebral disc, but the other two cell lines (each from a different patient) and four clones from one of the latter two lines proved to be highly specific for the human articular cartilage proteoglycan. From a study of four proteoglycan specific clones isolated from one patient, it is clear that removal of chondroitin sulfate had no effect on immunoreactivity but digestion of proteoglycan with pronase or alkali/sodium borohydride treatment abolished all reactivity. A OKT4-positive T-cell clone isolated from a healthy adult which was reactive to PPD was used to compare the antigen specificity of cells: this clone showed no reactivity to any of the other putative antigens listed above.     

Interleukin-1β and cyclic AMP mediate the invasion of sheared chondrosarcoma cells via a matrix metalloproteinase-1-dependent mechanism

Matrix metalloproteinase-1 (MMP-1) is a potential biomarker for chondrosarcoma that is overexpressed at the invading edges of articular cartilage, and its expression correlates with poor survival rates. However, the molecular mechanisms of MMP-1 regulation and its potential contribution to chondrosarcoma cell invasion have yet to be elucidated, especially in shear-activated cells. Using molecular biology tools and an in vitro fluid shear model, we report that shear stress upregulates cyclic AMP (cAMP) and interleukin-1β (IL-1β) release, which in turn promotes the invasion of chondrosarcoma cells via the induction of MMP-1 in a phosphoinositide 3-kinase (PI3-K)- and ERK1/2-dependent manner. Activated PI3-K and ERK1/2 signaling pathways phosphorylate c-Jun, which in turn transactivates MMP-1 in human chondrosarcoma cells. Collectively, fluid shear stress upregulates matrix MMP-1 expression, which is responsible for the enhanced invasion of human chondrosarcoma cells.     

Monoclonal antibodies directed against epitopes within the core protein structure of the large aggregating proteoglycan (aggrecan) from the swarm rat chondrosarcoma.

The core protein of the large hyaline cartilage proteoglycan, aggrecan, is composed of six distinct domains: globular 1 (G1), interglobular, globular 2 (G2), keratan sulfate attachment, chondroitin sulfate (CS) attachment, and globular 3 (G3). Monoclonal antibodies that recognize epitopes in these domains were raised against Swarm rat chondrosarcoma aggrecan that was either denatured through reduction and alkylation or partially deglycosylated through chondroitinase ABC digestion or alkali elimination, the latter with or without sulfite addition. Monoclonal antibodies were further characterized for reactivity to purified aggrecan substructures including rat chondrosarcoma G1 and CS attachment domains, a recombinant rat chondrosarcoma G3 domain fusion protein, bovine articular cartilage G2 domain, and rat chondrosarcoma link protein (LP). Biochemical characterization of the specificities of these monoclonal antibodies indicated that one (1C6) recognized an epitope shared by both the G1 and the G2 domains; one (5C4) recognized an epitope shared by both LP and the G1 domain; one (7D1) recognized an epitope shared by both the G1 and the CS attachment domains; two (14A1 and 15B2) recognized epitopes in the CS attachment domain; one (14B4) recognized an epitope in the G3 domain; and one (13D1) recognized a ubiquitous epitope shared by the G1, G2, G3, and CS attachment domains of aggrecan and also LP. Collectively the specificities of these antibodies confirm the occurrence of multiple repeated epitopes (both carbohydrate and protein in nature) throughout the different domain structures of aggrecan. These antibodies have been proven to be useful for identifying aggrecan-like molecules in several connective tissues other than cartilage.     

Molecular structure and tissue distribution of matrilin-3, a filament-forming extracellular matrix protein expressed during skeletal development

Matrilin-3 is a recently identified member of the superfamily of proteins containing von Willebrand factor A-like domains and is able to form hetero-oligomers with matrilin-1 (cartilage matrix protein) via a C-terminal coiled-coil domain. Full-length matrilin-3 and a fragment lacking the assembly domain were expressed in 293-EBNA cells, purified, and subjected to biochemical characterization. Recombinantly expressed full-length matrilin-3 occurs as monomers, dimers, trimers, and tetramers, as detected by electron microscopy and SDS-polyacrylamide gel electrophoresis, whereas matrilin-3, purified from fetal calf cartilage, forms homotetramers as well as hetero-oligomers of variable stoichiometry with matrilin-1. In the matrix formed by cultured chondrosarcoma cells, matrilin-3 is found in a filamentous, collagen-dependent network connecting cells and in a collagen-independent pericellular network. Affinity-purified antibodies detect matrilin-3 expression in a variety of mouse cartilaginous tissues, such as sternum, articular, and epiphyseal cartilage, and in the cartilage anlage of developing bones. It is found both inside the lacunae and in the interterritorial matrix of the resting, proliferating, hypertrophic, and calcified cartilage zones, whereas the expression is lower in the superficial articular cartilage. In trachea and in costal cartilage of adult mice, an expression was seen in the perichondrium. Furthermore, matrilin-3 is found in bone, and its expression is, therefore, not restricted to chondroblasts and chondrocytes.     

MR水激励序列显示膝关节软骨的临床应用研究

目的：探讨三维快速梯度回波水激励膝关节软骨成像序列（3D-FFE-WATS）相对于三维快速梯度回波预饱和反转恢复法脂肪抑制序列（3D-FFE-SPIR）在显示膝关节软骨方面的优势,选择显示膝关节软骨的最佳序列。方法：应用3D-FFE-WATS及3D-FFE-SPIR序列组合对20名志愿者及30例疑诊关节软骨损伤的单膝关节进行检查,获得膝关节各软骨的3D图像,并利用3D最大密度投影法（MIP）进行横断面和冠状面3D重建。分析上述2种序列对软骨病变的显示及检出能力,计算其对关节软骨的信噪比（SNR）和对比噪声比（CNR）,并进行统计学分析。结果：两序列在显示膝关节软骨SNR方面,无统计学意义（P〉0.05）;两种序列在显示软骨与关节液的CNR、软骨与骨皮质的CNR、软骨与骨髓的CNR、软骨与肌肉的CNR差异方面t值分别为（-30.619;2.348;-2.408;2.216）,有统计学意义（P〈0.05）。结论：3D-FFE-WATS序列可作为膝关节软骨成像的首选序列。     

Biosynthesis and cell-free translation of Swarm rat chondrosarcoma and bovine cartilage link proteins

In cartilage, link protein(s) (LP) stabilize proteoglycan aggregates via their specific association with hyaluronic acid and proteoglycan monomers. Two major link glycoproteins are produced in bovine articular cartilage, designated LP1 (49.5 kDa) and LP2 (44.0 kDa), whereas rat chondrosarcoma produces a single link protein species similar in size to bovine LP2. Although multiple link proteins differ to a significant degree in carbohydrate content, it is not known whether they arise from variable glycosylation of a single common protein core or from complete glycosylation of different protein cores. Biosynthesis of these molecules has been studied under conditions where differences generated by N-linked glycosylation would not be evident. Link proteins were immunoprecipitated 1) from cell-free translation products of total cellular and size fractionated RNA and 2) from cell lysates and medium of cultured chondrocytes using short term radioactive labeling of the protein in the presence and absence of tunicamycin. A 42-kDa link protein precursor is synthesized by cell-free translation of either rat chondrosarcoma or bovine chondrocyte mRNa. An apparently single 41.5-kDa link protein is synthesized with inhibition of N-linked glycosylation by tunicamycin, whereas LP1 and LP2 are the mature products of cultured bovine chondrocytes. The size range of translatable rat chondrosarcoma LP mRNA is 4.0-5.5 kilobase pairs and bovine LP mRNA is 3.0-4.5 kilobase pairs, both much larger than required to encode the link protein molecule. These results suggest that a single link protein precursor gives rise to multiple fully glycosylated forms and that link protein is not synthesized as a significantly larger "pro" form.     

Notch通路与基质金属蛋白酶在软骨肉瘤中的表达及意义

目的 研究软骨肉瘤组织中Notch通路、p38丝裂原活化蛋白激酶(MAPK)及基质金属蛋白酶(MMPs)的表达情况,探讨它们在软骨肉瘤间质浸润中的作用机制.方法 收集正常软骨组织标本10例、内生性软骨瘤标本23例和软骨肉瘤标本32例,分别用免疫组化、Western blot和real-time PCR检测Notch1、Jagged1、MMP-1、MMP-13、p38 MAPK及p-p38 MAPK的表达情况.结果 与正常软骨组织相比,Notch1、Jagged1、MMP-1、MMP-13及p-p38 MAPK在内生性软骨瘤表达部分升高,在软骨肉瘤表达均明显增加(P＜0.01).p38 MAPK在正常软骨组织、内生性软骨瘤及软骨肉瘤组织中表达无明显差异(P＞0.05).结论 Notch通路和p38 MAPK通过调节基质金属蛋白酶在软骨肉瘤中的表达,来增加软骨肉瘤的浸润转移能力.     

Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues

Introduction

The small leucine-rich proteoglycans (SLRPs) modulate tissue organization, cellular proliferation, matrix adhesion, growth factor and cytokine responses, and sterically protect the surface of collagen type I and II fibrils from proteolysis. Catabolism of SLRPs has important consequences for the integrity of articular cartilage and meniscus by interfering with their tissue homeostatic functions.

Methods

SLRPs were dissociatively extracted from articular cartilage from total knee and hip replacements, menisci from total knee replacements, macroscopically normal and fibrillated knee articular cartilage from mature age-matched donors, and normal young articular cartilage. The tissue extracts were digested with chondroitinase ABC and keratanase-I before identification of SLRP core protein species by Western blotting using antibodies to the carboxyl-termini of the SLRPs.

Results

Multiple core-protein species were detected for all of the SLRPs (except fibromodulin) in the degenerate osteoarthritic articular cartilage and menisci. Fibromodulin had markedly less fragments detected with the carboxyl-terminal antibody compared with other SLRPs. There were fewer SLRP catabolites in osteoarthritic hip than in knee articular cartilage. Fragmentation of all SLRPs in normal age-matched, nonfibrillated knee articular cartilage was less than in fibrillated articular cartilage from the same knee joint or total knee replacement articular cartilage specimens of similar age. There was little fragmentation of SLRPs in normal control knee articular cartilage. Only decorin exhibited a consistent increase in fragmentation in menisci in association with osteoarthritis. There were no fragments of decorin, biglycan, lumican, or keratocan that were unique to any tissue. A single fibromodulin fragment was detected in osteoarthritic articular cartilage but not meniscus. All SLRPs showed a modest age-related increase in fragmentation in knee articular and meniscal cartilage but not in other tissues.

Conclusion

Enhanced fragmentation of SLRPs is evident in degenerate articular cartilage and meniscus. Specific decorin and fibromodulin core protein fragments in degenerate meniscus and/or human articular cartilage may be of value as biomarkers of disease. Once the enzymes responsible for their generation have been identified, further research may identify them as therapeutic targets.     

LED照射对培养SW1353细胞MPP-3与MMP-13的影响

在骨关节疾病中,基质金属蛋白酶(matrix metalloproteinase,MMP)对关节软骨缺损的机理扮演着重要的角色。为了进一步明确低强度激光在关节软骨缺损中的治疗作用,应用细胞因子IL-1β与TNF-α刺激培养SW 1353细胞复制炎症细胞模型,观察发光二极管(LED)照射对培养细胞的生存率和死亡率的影响、细胞活性的影响、以及对MMP-3和MMP-13的调节作用。实验结果显示,10μg/L IL-1β与20μg/L TNF-α对培养细胞生存率无明显影响,而细胞活性明显下降(P<0.01),培养液中MMP-3和MMP-13含量明显上升(P<0.01,P<0.05);LED照射后可见与相应的模型组比较细胞死亡率下降(P<0.01)、培养液中MMP-3和MMP-13含量显著性降低在(P<0.01,P<0.05)。研究表明,LED照射对炎症因子刺激的SW 1 353细胞的MMP过多表达具有抑制性作用,可能对于关节软骨缺损性疾病的LED照射治疗起一定的指导意义。     

Hyaluronan regulates PPARγ and inflammatory responses in IL-1β-stimulated human chondrosarcoma cells, a model for osteoarthritis

The carbohydrate polymer, hyaluronan, is a major component of the extracellular matrix in animal tissues. Exogenous hyaluronan has been used to treat osteoarthritis (OA), a degenerative joint disease involving inflammatory changes. The underlying mechanisms of hyaluronan in OA are not fully understood. Pro-inflammatory interleukin (IL)-1β downregulates peroxisome proliferator-activated receptor gamma (PPARγ), and increases expression of matrix metalloproteinases (MMPs) which are responsible for the degeneration of articular cartilage. The effects of low- and high-molecular-weight hyaluronan (oligo-HA and HMW-HA) on the inflammatory genes were determined in human SW-1353 chondrosarcoma cells. HMW-HA antagonized the effects of IL-1β by increasing PPARγ and decreasing cyclooxygenase (COX)-2, MMP-1, and MMP-13 levels. It promoted Akt, but suppressed mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NFκB) signaling, indicating anti-inflammatory effects. In contrast, the cells had overall opposite responses to oligo-HA. In conclusion, HMW-HA and oligo-HA exerted differential inflammatory responses via PPARγ in IL-1β-treated chondrosarcoma cells.