HtrA1丝氨酸蛋白酶在原发性肝癌中的表达及其临床意义

目的 探讨原发性肝细胞癌(HCC)中HtrA1丝氨酸蛋白酶(HtrA1 serine protease)的表达水平及其与临床病理特征之间的关系.方法 采用实时荧光定量逆转录-聚合酶链反应(Realtime-PCR)检测手术切除的肝细胞癌患者的肝癌组织标本及癌旁肝组织标本中HtrA1 mRNA的表达.结果 肝癌组织中HtrA1 mRNA值为1.2729±0.9911,癌旁组织中的HtrA1 mRNA值3.8667±2.8099,肝癌组织中 HtrA1 mRNA明显低于相应的癌旁肝组织中的表达(P=0.000).多因素Logistic回归分析显示HtrA1 mRNA表达,肝内外转移及包膜是影响肝癌静脉浸润的独立因素.结论 HtrA1在肝癌中的表达可望作为判断肝癌预后或转移复发的指标之一.     

Cartilage synthesizes the serine protease inhibitor PAI-1: support for the involvement of serine proteases in cartilage remodeling

The work described here demonstrates the synthesis by human articular cartilage of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the serine protease tissue plasminogen activator (tPA). We also present data demonstrating an increase in PAI-1 messenger ribonucleic acid (mRNA) in chondrocytes exposed to the cytokine interleukin-1 (IL-1). Interestingly, this elevation of steady-state mRNA levels does not appear to result in an increase in synthesis of PAI-1 protein. Northern blot analysis reveals that of the two mRNA species (3.4 kb, 2.4 kb) previously reported for PAI-1, only the larger species (3.4 kb) appears to be synthesized by chondrocytes. Our data demonstrate the IL-1-stimulated production by cartilage of tissue plasminogen activator. We also show evidence for the presence of plasminogen in cartilage. A scheme is presented indicating the probable importance of the serine proteases (tPA and plasminogen) and PAI-1 in cartilage degradation.     

Expression of mouse osteoclast K-Cl Co-transporter-1 and its role during bone resorption.

To assess the role of Cl- transport during osteoclastic bone resorption, we studied the expression and function of K+/Cl- co-transporters (KCCs). KCC1 and chloride channel-7 were found to be expressed in mouse osteoclasts. The KCC inhibitor, R(+)-butylindazone (DIOA), KCC1 antisense oligo-nucleotides, and siRNA suppressed osteoclastic pit formation. DIOA also decreased Cl- extrusion and reduced H+ extrusion activity. These results show that KCC1 provides a Cl- extrusion mechanism accompanying the H+ extrusion during bone resorption. INTRODUCTION: Mice with deficient chloride (Cl-) channels, ClC7, show severe osteopetrosis, resulting from impairment of Cl- extrusion during osteoclastic bone resorption. However, the expression and functional role of Cl- transporters other than ClC7 in mammalian osteoclasts is unknown. The aim of this study was to determine expression of K+/Cl- co-transporters (KCCs) and their functional role for bone resorption in mouse osteoclasts. MATERIALS AND METHODS: Mouse osteoclasts were derived from cultured bone marrow cells with macrophage-colony stimulating factor (M-CSF) and RANKL or from co-culture of bone marrow cells and primary osteoblasts. We examined the expression of Cl- transporters using RT-PCR, immunochemical, and Western blot methods. The effects of Cl- transport inhibitors on H+ and Cl- extrusion were assessed by measuring intracellular H+ ([H+]i) and Cl- ([Cl-]i). The effects of inhibitors, antisense oligo-nucleotides, and siRNA for Cl- transporters on bone resorption activities were evaluated using a pit formation assay. RESULTS AND CONCLUSIONS: Mouse osteoclasts express not only ClC7 but also K+/Cl- co-transporter mRNA. The existence of KCC1 in the cell membrane of mouse osteoclasts was confirmed by immunochemical staining and Western blot analysis. KCC inhibitors and Cl- channels blockers increased [Cl-]i and [H+]i in resorbing osteoclasts, suggesting that the suppression of Cl- extrusion through KCC and Cl- channels leads to reduced H+ extrusion activity. The combination of both inhibitors greatly suppressed these extrusion activities. KCC inhibitors and Cl- channel blockers also decreased osteoclastic bone resorption in our pit area essay. Furthermore, KCC1 antisense oligo-nucleotides and siRNA suppressed osteoclastic pit formation as well as treatment of ClC7 inhibitors. These results indicate that K+/Cl- co-transporter-1 expressed in mouse osteoclasts acts as a Cl- extruder and plays an important role for H+ extrusion during bone resorption.     

The glycosaminoglycans of dog compact bone and epiphyseal cartilage in the normal state and in experimental hyperparathyroidism

In normal dogs and in dogs treated with 20 or 30 U.S.P. parathyroid extract for 5 and 3–4 days, respectively, the glycosaminoglycans of compact bone tissue were identified using the cetylpyridinium chloride precipitation method, and the concentrations of total hexosamines and the hexosamines corresponding to cetylpyridinium chloride precipitable acid glycosaminoglycans were determined. Further, the glycosaminoglycan pattern of the epiphyseal plate and the incorporation of³⁵S-sulphate into the glycosaminoglycans of bone tissue and epiphyseal cartilage after administration of³⁵S-sulphatein vivo was studied.In compact bone tissue, the hexosamines corresponding to acid glycosaminoglycans constuted approximately one third of the total hexosamine concentration and approximately 0.05–0.06% of the total dry weight. The main component of the acid glycosaminoglycans in bone was chondroitin-4-sulphate. This was sulphated to a higher degree and also of a higher molecular weight than thechondroitin sulphate of the epiphyseal cartilage, which in accordance with earlier investigations was found to have infrared characteristics of both chondroitin-4-sulphate and chondroitin-6-sulphate, with the former dominating. The molecular weights of the main part of bone chondroitin sulphate ranged from approximately 45,000 to 56,000. A small component of the bone glycosaminoglycans was hyaluronic acid.Large regularly recurring differences in the specific activity of fractions with differences in molecular weight in the condroitin sulphate of bone tissue and epiphyseal cartilage were noted.Treatment of the dogs with parathyroid extract gave no effect on the molecular weights of the chondroitin sulphate of the bone matrix or of the epiphyseal cartilage. Nor was there any unequivocal effect on the concentrations of total hexosamines or on the acid glycosaminoglycans. No evident stimulatory or depressant effect on the incorporation of³⁵S-sulphate into the chondroitin sulphate or in the molecular distribution of newly sulphated and/or synthesized molecules of the condroitin sulphate within these tissues occurd.

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Zusammenfassung An normale Hunden und an Hunden die unter 5 respektive 3–4 Tagen 20 oder 30 U.S.P./kg Parathyreoidea-Extract erhalten hatten, wurden im kompakten Knochen die Glykosaminoglykane unter Anwendung der Cetylpyridiniumchlorid-Fällungsmethode identifiziert und die Konzentration des Total-Hexosamins und des Hexosamins, entsprechend den Cetylpyridiniumchlorid fällbaren sauren Glykosaminoglykanen, wurde bestimmt. Außerdem wurden das Glykosaminoglykanmuster der Epiphysenplatte und der Einbau von³⁵S-Sulfat in die Glykosaminoglykane des Knochengewebes und des Epiphysenknorpels nach Zufuhr von³⁵S-Sulfatin vivo studiert.Im kompakten Knochengewebe macht des Hexosamin, entsprechend den sauren Glykosaminoglykanen, ungefähr ein Drittel der totalen Hexosaminkonzentration und ungefähr 0,05–0,06% des totalen Trockengewichtes aus. Der Hauptanteil der sauren Glykosaminoglykane im Knochen war Chondroitin-4-Sulfat. Dieses war in höherem Grad sulfatiert und hatte ein höheres Molekülgewicht als das Chondroitinsulfat der Epiphysenplatte, welches, in Übereinstimmung mit früheren Untersuchungen, Infrarot spektra kennzeichnend für sowohl Chondroitin-4-sulfat als auch für Chondroitin-6-sulfat, das Erstere überwiegend, hatte. Das Molekülgewicht des Hauptanteiles des Knochen-Chondroitinsulfates lag zwischen ungefähr 45000–56000. Ein kleiner Teil der Knochen-Glykosaminoglykane war Hyaluronsäure.Sowohl im Knochengewebe als auch im Epiphysenknorpel wurde in verschiedenen Fraktionen des Chondroitinsulfates, mit unterschiedlichem Molekülgewicht, grße und regelmäßig reproduzierbare Unterschiede in der spezifischen Aktivität gefunden.Behandlung der Hunde mit Parathyreoideaextrakt gab keinen Ausschlag in den Molekülgewichten des Chondroitinsulfates, weder des Knochens noch des Epiphysenknorpels. Ebenso wurde kein eindeutiger Effekt auf die Konzentration des totalen Hexosamins oder der sauren Glykosaminoglykane gefunden. Kein offenbarer, weder anregender noch senkender, Effekt auf den Einbau von³⁵S-Sulfat in das Chondroitinsulfat oder in der molekularen Verteilung der neulich sulfatierten und/oder synthetisierten Moleküle des Chondroitinsulfates dieser Gewebe wurde gefunden.

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Résumé Les glycosaminoglycanes de l'os compact ont été identifiés, par la méthode de précipitation au chlorure de cetylpyridinium, chez le chien normal et des chiens soumis à 20 ou 30 U.S.P. d'extrait parathyroïdien pendant 5 et 3–4 jours. Les concentrations d'héxosamines totales ainsi que les héxosamines, en rapport avec les glycoaminoglycanes acides, précipités par chlorure de cetylpyridinium, sont déterminées. En outre, la répartition du glycosaminoglycane de la zone d'ossification épiphysaire ainsi que l'incorporation du³⁵S-sulfate dans les glycosaminoglycanes du tissu osseux et du cartilage épiphysaire, après administration de³⁵S-sulfatein vivo, ont été étudiées.Dans l'os compact, les héxosamines, en rapport avec les glycosaminoglycances acides, constitutent environ un tiers de la concentration totale en héxosamine et environ 0,05–0,06% du poids sec total. Le constituant principal des glycosaminoglycances acides osseux est formé par le chondroitine-4-sulfate. Ce dernier est plus riche en sulfate et présente un poids moléculaire plus élevé que le chondroitine sulfate du cartilage épiphysaire, qui, selon des travaux antérieurs, présente des caractéristiques infra-rouges du chondroitine-4-sulfate et du chondroitine-6-sulfate, avec prédominance du premier. Les poids moléculaires du chondroitine sulfate osseux varient surtout d'environ 45000 et 56000. L'acide hyaluronique constitute une faible fraction des glycosaminoglycanes osseux.Des différences marquées de l'activité spécifique des fractions de cohondroitine sulfate de l'os et du cartilage épiphysaire, à poids moléculaires variables, on tété notées de façon répétée. L'administration d'extrait parathyroïdien à des chiens n'a pas d'effet sur les poids moléculaires en chondroitine sulfate de l'os ou du cartilage épiphysaire. Elle n'influence pas non plus les concentrations en héxosamines totales ou en glycosaminoglycances acides. Dans ces tissus, il ne se produit pas d'effect de stimulation ou de dépression concernant l'incorporation de³⁵S-sulfate dans le chondroitine sulfate our sur la séparation moléculaire de molécules transformées en sulfates et/ou de molécules synthétiques de sulfate chondroitine.

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The nomenclature ofBalasz andJeanloz (1965) has been followed.This work was reported, in part, at the meeting of Svenska Patologföreningen in Stockholm, Sweden, December 26th–27th, 1965; see Nordisk Medicin 75, 434–435 (1965); and at the Fourth European Symposium on Calcified Tissues, Leiden, Noordwijk aan Zee, The Netherlands, March 28th–April 1st. 1966; see Exerpta Medica Foundation, International Congress Series No 120, 1966, p. 55–56.     

Ultrastructural study of articular cartilage in experimental pyogenic arthritis

Ultrastructural changes of the articular cartilage at the early stage of experimental pyogenic arthritis were studied by electron microscopy. The superficial zone of the cartilage at the site of the cartilage-synovium junction demonstrated far more changes than those at the site of the free surface of the articular cartilage. Changes were observed at the cartilage-synovium junction as early as four hours after the intraarticular injection of the infecting organism. The most interesting finding was migration of erythrocytes and polymorphonuclear leucocytes into the superficial zone of the cartilage at the early stage. The pericellular matrix in the middle zone became so loose in some areas that collagen fibrils were partially exposed after three days. The naked collagen fibrils were tapered, nicked and irregular in size after seven days.     

Expression of Noggin and Gremlin1 and its implications in fine‐tuning BMP activities in mouse cartilage tissues

Increasing evidence supports the idea that bone morphogenetic proteins (BMPs) regulate cartilage maintenance in the adult skeleton. The aim of this study is to obtain insight into the regulation of BMP activities in the adult skeletal system. We analyzed expression of Noggin and Gremlin1 , BMP antagonists that are known to regulate embryonic skeletal development, in the adult skeletal system by Noggin‐LacZ and Gremlin1‐LacZ knockin reporter mouse lines. Both reporters are expressed in the adult skeleton in a largely overlapping manner with some distinct patterns. Both are detected in the articular cartilage, pubic symphysis, facet joint in the vertebrae, and intervertebral disk, suggesting that they regulate BMP activities in these tissues. In a surgically induced knee osteoarthritis model in mice, expression of Noggin mRNA was lost from the articular cartilage, which correlated with loss of BMP2/4 and pSMAD1/5/8, an indicator of active BMP signaling. Both reporters are also expressed in the sterna and rib cartilage, suggesting an extensive role of BMP antagonism in adult cartilage tissue. Moreover, Noggin‐LacZ was detected in sutures in the skull and broadly in the nasal cartilage, while Gremlin1‐LacZ exhibits a weaker and more restricted expression domain in the nasal cartilage. These results suggest broad regulation of BMP activities by Noggin and Gremlin1 in cartilage tissues in the adult skeleton, and that BMP signaling and its antagonism by NOGGIN play a role in osteoarthritis development. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1671–1682, 2017.

     

Collagen type VI content in healthy and arthritis knee joint cartilage

AIM OF THE STUDY: Since collagen type VI seems to play an important role in cartilage metabolism and is increased in osteoarthritis, the aim of this study was to investigate whether different stages of osteoarthritis can be characterised by the content of collagen type VI in human knee cartilage. MATERIAL AND METHODS: Collagen type VI was investigated in 148 histologically normal and 117 osteoarthritic cartilage samples from 18 different localisations of human knee joints. It was quantified in cartilage extracts using an inhibition-ELISA. RESULTS: In normal cartilage the average content of collagen type VI was 0.48 per cent of total collagens. Interestingly, there was a high variability not only in osteoarthritic, but also in normal cartilage. The statistical analysis showed significant differences between normal femoral, tibial or retropatellar cartilage samples. Therefore, normal and osteoarthritic samples from different localizations had to be compared separately. A significant increase of collagen type VI was already found in early osteoarthritic lesions. CONCLUSIONS: Inspite of a statistically significant increase of collagen type VI in osteoarthritic cartilage, the range of concentrations found in normal and osteoarthritic samples overlap. In view of the high interindividual variability, collagen type VI is not very precise in the diagnosis of early osteoarthritic lesions if used as the only marker.     

小鼠胆囊酪氨酸激酶受体的表达及其意义

目的研究酪氨酸激酶受体（c-kit）在CD1小鼠胆囊的表达及其意义。方法采用免疫组化方法，应用激光扫描共聚焦显微镜检测CD1小鼠胆囊组织切片和急性分离单细胞的c-kit免疫反应阳性细胞的表达；RT-PCR法检测胆囊c-kit mRNA的表达；Western blot检测胆囊c-kit蛋白的表达。结果免疫荧光激光扫描共聚焦显微镜检测结果显示CD1小鼠胆囊从黏膜层到肌层均有c-kit免疫反应阳性细胞存在，其呈类似网络状分布和点和（或）丝状分布。急性分离胆囊单细胞检测结果显示c-kit免疫反应阳性细胞大致可区分为两种类型，一种为分支状，大多数细胞呈现该形态；一种为类梭形，少见分支，类似平滑肌细胞，部分细胞呈现此形态。而典型呈梭形的平滑肌细胞则无荧光显现，呈c-kit免疫反应阴性。RT-PCR法检测结果表明胆囊c-kit mRNA表达阳性。Western印迹显示c-kit蛋白表达阳性。结论c-kit在CD1小鼠胆囊表达阳性，即CD1小鼠胆囊存在Cajal间质细胞（ICC）。ICC可能与胆囊运动有关，有可能为胆囊起搏细胞。     

Three-dimensional visualization and pathologic characteristics of cartilage and subchondral bone changes in the lumbar facet joint of an ovariectomized mouse model

Background Context

Low back pain (LBP) is more prevalent among postmenopausal women than men. Ovariectomy (OVX) is an established animal model that mimics the estrogen deficiency of postmenopausal women. Little is known about the three-dimensional (3D) morphologic properties of cartilage and subchondral bone changes in the lumbar facet joint (LFJ) of an OVX mouse model.

Centrifugal and biochemical comparison of proteoglycan aggregates from articular cartilage in experimental joint disuse and joint instability

Two models involving altered joint loading were compared with regard to their effects on the biochemical composition and proteoglycan aggregate structure of articular cartilage. Disuse atrophy was created in greyhound dogs by nonrigid immobilization of the right knee in 90° of flexion, and joint instability was created by transection of the anterior cruciate ligament. Similarities and differences between the two experimental groups at two different time periods were examined to investigate why joint instability induces progressive and irreversible changes to the articular cartilage, whereas joint disuse induces changes that may be reversible when the joint is remobilized. The following studies were performed on the cartilage from all experimental and control groups: (a) compositional analyses to determine water, uronate, and hydroxyproline contents; (b) high performance liquid chromatography for detection of hyaluronan and chondroitin sulfates; and (c) centrifugation analyses of nondissociatively extracted and purified proteoglycans to isolate and quantify the populations of monomers and slow and fast-sedimenting families of aggregates. In general, all cartilage was found to have a decreased ratio of proteoglycan to collagen after 4 weeks of disuse, and this ratio returned to control values at 8 weeks. In contrast, cartilage had an elevated ratio of proteoglycan to collagen as well as increased hydration at 12 weeks after transection of the anterior cruciate ligament. The most striking contrast between the two models was the finding of an approximately 80% decrease in the content of hyaluronan at both time periods after transection of the anterior cruciate ligament, with no evidence of a change after disuse. The results of centrifugation analyses indicated a significant decrease in the quantity of proteoglycan aggregates in both models. However, this decrease was associated primarily with a loss of slow-sedimenting aggregates after disuse and a loss of both slow and fast-sedimenting aggregates after transection of the anterior cruciate ligament. Furthermore, the population of fast-sedimenting aggregates. The preservation of fast-sedimenting aggregates as well as hyaluronan after periods of joint disuse but not joint instability suggests a possible mechanism for the reversibility of cartilage changes. Although the proteoglycan aggregates were depleted after disuse atrophy, it is possible that an aggregate-depleted matrix could recover when normal proteoglycan synthesis is resumed. In contrast, although synthesis may be maintained or elevated after transection of the anterior cruciate ligament, the matrix may not be repopulated with aggregates because there is an insufficient amount of hyaluronan.     

Expression of type VI collagen in normal and osteoarthritic human cartilage

OBJECTIVE: This study was undertaken in order to study the expression of type VI collagen in normal and osteoarthritic human knee cartilage. METHODS: Seventy-two osteoarthritic cartilage/bone samples were obtained form 29 patients with primary OA undergoing surgery for a total knee replacement. Normal cartilage was collected from five human knees at the time of autopsy. Type VI collagen protein was localized using a polyclonal anti human type VI collagen antibody, the corresponding mRNA was detected with an 310 base antisense probe, specific for the alpha2(VI) collagen chain. RESULTS: In normal cartilage, type VI collagen protein is concentrated pericellularly around the chondrocytes of all cartilage zones. In the middle and deep zones, type VI collagen was also found in the interterritorial matrix. Type VI collagen mRNA expression was detected in chondrocytes of all cartilage zones. In moderately affected osteoarthritic cartilage, type VI collagen expression was increased. An intensive immunohistological interterritorial staining for type VI collagen was observed in the middle and deep cartilage zones. Specific mRNA signals were also increased especially in the middle and deep cartilage zone. In the superficial zone and calcified cartilage of these samples, type VI collagen mRNA expression was restricted to focal areas. In severe osteoarthritic cartilage, an intensive staining for type VI collagen mRNA was found in clusters of proliferating chondrocytes and in the deep cartilage zone. Type VI collagen was localized pericellularly and in the matrix of chondrocyte clusters. Furthermore, chondrocytes from the deep zone showed a territorial distribution of type VI collagen. CONCLUSIONS: These results demonstrate that in normal and osteoarthritic cartilage, type VI collagen is expressed in a zone specific pattern. The observed increase of type VI collagen expression in osteoarthritis suggests a potential role in the disease process.     

脂肪因子Vaspin在骨代谢中的研究进展

内脏脂肪组织来源特异性丝氨酸蛋白酶抑制剂(Vaspin)是一种主要由脂肪细胞分泌的脂肪因子,具有提高胰岛素敏感性、改善糖耐量、抑制血管炎症反应等作用,并可影响成骨细胞和破骨细胞的增殖与分化,对骨代谢有着重要调节作用。本文就Vaspin对正常骨代谢调节及病理情况下与骨疾病发生发展的关系作一综述。     

Osteoarthritis development in novel experimental mouse models induced by knee joint instability

OBJECTIVE: Although osteoarthritis (OA) is induced by accumulated mechanical stress to joints, little is known about the underlying molecular mechanism. To apply approaches from mouse genomics, this study created experimental mouse OA models by producing instability in the knee joints. METHODS: The models were of four types: severe, moderate, mild, and medial, depending on the severity and direction of instability imposed by combinations of ligament transection and menisectomy. OA development was evaluated by X-ray and histology by Safranin-O staining, and quantified using our original gradings. Expressions of type II, IX and X collagens and matrix metalloproteinase (MMP)-2, -3, -9 and -13 were further examined by immunohistochemistry and in situ hybridization (ISH). RESULTS: The severe, moderate and mild models exhibited OA development in the posterior tibial cartilage. The severe model showed cartilage destruction at 2 weeks and osteophyte formation at 4-8 weeks after surgery; however, the mild model showed only a partial cartilage destruction at 8 weeks. The grading confirmed that the OA disorders progressed depending on the severity of joint instability. In the medial model, the OA development in the medial tibial cartilage was similar to that in the posterior cartilage of the mild model. Among the collagens and MMPs, type X collagen and MMP-13 were markedly induced and colocalized in the early stage OA cartilage. CONCLUSION: We established four types of mouse models exhibiting various speeds of OA progression. By applying a mouse genomics approach to the models, molecular backgrounds in various stages of OA development can be clarified.