BACKGROUND: The mechanism by which mutant cartilage oligomeric matrix protein (COMP) induces a pseudoachondroplasia phenotype remains unknown, and the reason why a mutation of a minor protein of the growth plate cartilage causes total disruption of endochondral bone formation has not yet been determined. The current study was performed to investigate the effects of mutated COMP on the synthesis of the cartilage-specific major matrix proteins of Swarm rat chondrosarcoma chondrocytes. METHODS: The Swarm rat chondrosarcoma chondrocytes transfected with a chimeric construct, which consisted of a mutant gene of human COMP and an amino acid FLAG tag sequence, were cultured in agarose gel. Formation of extracellular proteoglycan and type-II collagen by the cells was evaluated by immunohistochemical staining and measuring the (35)S-sulfate incorporation. RESULTS: No difference was observed for the detection of type-II collagen among the cell lines expressing mutant COMP and the control cell lines. Histochemical staining of sulfated proteoglycans with safranin-O showed that lesser amounts of proteoglycans were incorporated into the extracellular matrix of the chondrocytes transfected with the mutant gene. (35)S-sulfate incorporation into the cell/matrix fractions demonstrated markedly lower radiolabel incorporation, as compared to that of the control cells. CONCLUSIONS: Mutation of COMP has an important impact on the processing of proteoglycans, rather than type-II collagen, in the three-dimensional culture of Swarm rat chondrosarcoma chondrocytes.
Aggrecans/analysis/*biosynthesis ; Animals ; Cells, Cultured ; Chondrocytes/*metabolism ; Chondrosarcoma/metabolism ; Collagen Type II/*biosynthesis ; Extracellular Matrix/*metabolism ; Extracellular Matrix Proteins/*genetics ; Glycoproteins/*genetics ; Humans ; Mutation ; Rats ; Transfection

Cells termed myofibroblasts are prominent in the injury response of all epithelial tissues. They exhibit proliferation, migration, production of collagen and other extracellular matrix (ECM) molecules, and contraction, all for containing the injury and closing the wound. When the injury is limited in time, the final stage of the repair involves a dismantling of the cellular apparatus and restoration of normal tissue structure. With multiple cycles of repair, however, there is net accumulation of ECM, to the detriment of tissue structure and function. Repair-related ECM coalesces into fibrous bundles and, over time, undergoes changes that render it resistant to degradation. The result is a scar. In the skin, a scar may have cosmetic importance only. In the liver, however, extensive scarring is the setting for unregulated growth and neoplasia; also, fibrous bands disrupt normal blood flow, leading to portal hypertension and its complications. With regard to therapy for fibrosis, the first consideration is elimination of the injury factor. However, given that many liver diseases do not have effective therapies at present, strategies targeting fibrogenesis per se are under development. The main source of myofibroblast-like cells and ECM production in the liver is the perisinusoidal stellate cell, which responds to injury with a pleiotypic change termed activation. Activation is orchestrated by cytokines and the ECM itself. Among the cytokines involved in this process, transforming growth factor-beta (TGF-beta) is particularly prominent. The early changes in ECM include de novo production of a specific "fetal" isoform of fibronectin, which arises from sinusoidal endothelial cells. It is stimulated by TGF-beta and acts directly on stellate cells to promote their activation. Based on these and other advances in understanding the fundamentals of the injury response, several strategies now exist for altering fibrogenesis, ranging from agents that block TGF-beta to traditional Chinese herbal extracts. Arrest of fibrogenesis, even with underlying cirrhosis, is likely to extend life or prolong the time to transplant. Whether it reduces the risk of hepatocellular carcinoma remains to be proven. Although TGF-beta antagonists are effective anti-fibrogenic agents, they will require detailed safety testing because of the finding that several forms of epithelial neoplasia are associated with altered regulation of TGF-beta.
Animal ; Carcinoma, Hepatocellular/*etiology/pathology ; Chronic Disease ; Extracellular Matrix/metabolism ; Extracellular Matrix Proteins/biosynthesis ; Fibronectins/biosynthesis ; Fibrosis/complications/drug therapy ; Human ; Liver/cytology ; Liver Cirrhosis/*complications/pathology ; Liver Neoplasms ; Transforming Growth Factor beta/*physiology

OBJECTIVETo improve the biological property of artificial skin.

METHODSWe have ameliorated Hansburgh and Middelkoop's method of manufacturing artificial dermis. The type I collagenase and Dispase were used to isolated neonate prepuce' dermis fibroblast. The gel dermis was constructed by compounding the fibroblast and collagen swelling solution. The property of the collagen gel dermis was measured.

RESULTSThe neonate prepuce's dermis fibroblast had property of high proliferation, high activation of the dermis, and it could secrete abundant extracellular matrix (ECM).

CONCLUSIONThe collagen gel dermis is an useful dermis substitute.

Cell Separation ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Dermis ; cytology ; metabolism ; Extracellular Matrix Proteins ; biosynthesis ; Fibroblasts ; cytology ; Humans ; Infant, Newborn ; Male ; Skin, Artificial ; Tissue Engineering

AIMTo investigate the changes in the extracellular matrix protein expression and the morphology of seminiferous tubules in the testis of 88 azoospermic men.

METHODSThe patients were of the following categories: (1) 22 cases of Sertoli-cell-only syndrome, (2) 20 cases of spermatogenic arrest, and (3) 46 cases with hypospermatogenesis. Testicular sections were immunohistochemically stained for fibronectin, vimentin, laminin and collagen type IV. The seminiferous tubular diameter and the connective matrix zone (CMZ, the acellular zone between the basement membrane [BM] and the peritubular cells) thickness were measured. Seminiferous tubules were typed according to the thickness of the connective matrix in the lamina propria. The predominant tubule type and the Johnsen and Silber scores were determined.

RESULTSThe mean tubular diameter were 119 +/- 27, 117 +/- 20, and 140 +/- 38 microm for Groups 1, 2, and 3, respectively. Both the laminin and the type IV collagen were localized to the epithelial BM and peritubular cells. In most of the tubules, BM and peritubular cells were separated by a homogenous acellular layer, the CMZ, in which laminin, type IV collagen, fibronectin and vimentin were not present. It is perceived that the worse the testicular histology, the higher the thickness of the CMZ.

CONCLUSIONIn testis with no or low sperm production, the diameter of the seminiferous tubules is decreased, the thickness of the seminiferous tubular wall is increased and a CMZ is formed between the peritubular cells and the BM. The thickness of CMZ is increasing with the advancement of testiclar deterioration. The most important morphologic predictive factor for spermiogenesis is the predominant

Adult ; Collagen Type IV ; analysis ; biosynthesis ; Extracellular Matrix Proteins ; analysis ; biosynthesis ; Fibronectins ; analysis ; biosynthesis ; Humans ; Immunohistochemistry ; Laminin ; analysis ; biosynthesis ; Logistic Models ; Male ; Middle Aged ; Oligospermia ; metabolism ; pathology ; Seminiferous Epithelium ; metabolism ; pathology ; Spermatogenesis ; Testis ; chemistry ; metabolism ; pathology ; Vimentin ; analysis ; biosynthesis

Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood except the suppression of osteoblast function. To investigate the effect of simulated weightlessness on mRNA level of extracellular matrix proteins, osteoblasts were rotated in horizontal plane as a model of simulated microgravity. Primordial osteoblasts of rats were grown for 2 d and then rotated for 24, 48 and 72 h, respectively. After isolating total RNA in cells, reverse transcription polymerase chain reaction (PT-PCR) was made to examine the mRNA level of osteopontin (OPN) and osteonectin (ON). Meanwhile, the levels of alkaline phosphatase (ALP) and osteocalcin (BGP) in the cultured medium were measured to evaluate the calcific function of cell. The expression of OPN and ON mRNA fell significantly after rotating for 24, 48 and 72 h, respectively. The contents of BGP descended significantly, meanwhile, the activity of ALP also showed a degressive tendency. Horizontal rotation decreased the expression of ON and OPN as well as diminished the secretion of BGP and ALP, which affected the calcific function of osteoblast. The results obtained suggest that depression of extracellular matrix proteins expression plays a key role in bone loss during weightlessness.
Animals ; Animals, Newborn ; Cells, Cultured ; Extracellular Matrix Proteins ; biosynthesis ; genetics ; Osteoblasts ; cytology ; metabolism ; Osteonectin ; biosynthesis ; genetics ; Osteopontin ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Rotation ; Sialoglycoproteins ; biosynthesis ; genetics ; Skull ; cytology ; Weightlessness Simulation

OBJECTIVETo investigate the effects of specific RNA interference (RNAi) to Notch ligand Delta1 on proliferation and differentiation of human dental pulp stem cells (DPSC).

METHODSDPSC were infected by lentivirus vectors carrying Delta1-RNAi. DPSC were divided into three groups, DPSC/Delta1-RNAi group, DPSC/wt group and DPSC/vector group as control. Cell counting kit-8 (CCK-8) assay and flow cytometry were used to evaluate the proliferation of DPSC. Expression of proliferating cell nuclear antigen (PCNA) was examined with immunohistochemical staining. All groups were cultured in an odonto-inductive medium and were observed under microscope. The number of mineralization nodules was counted after Alizarin red staining. Alkaline phosphatase (ALP) activity and the expression of dentin sialophosphoprotein (DSPP) were detected by ALP activity assay and Western blotting.

RESULTSCompared with DPSC/wt or DPSC/vector separately, proliferating rate and S-cycle of DPSC/Delta1-RNAi was significantly lower. The S phase and proliferation index (PI) decreased markedly from 22.32 ± 2.35 and 33.68 ± 4.19 (DPSC/Delta1-RNAi) to 5.44 ± 0.91 and 16.00 ± 6.07 (DPSC/wt). The PCNA staining of DPSC/Delta1-RNAi was evidently weaker. DPSC/Delta1-RNAi group had more calcified cell nodules than the other two control groups, and ALP activity and DSPP expression of DPSC/Delta1-RNAi group increased markedly.

CONCLUSIONSDelta1-RNAi induced by the lentivirus vectors may inhibit DPSC proliferation and differentiation. Notch-Delta signal pathway plays an important role in self-renewal and differentiation.

Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Dental Pulp ; cytology ; metabolism ; Epithelial Cells ; Extracellular Matrix Proteins ; biosynthesis ; Genetic Vectors ; Homeodomain Proteins ; Humans ; Lentivirus ; Phosphoproteins ; biosynthesis ; RNA Interference ; Sialoglycoproteins ; biosynthesis ; Signal Transduction ; Stem Cells ; metabolism

Diagnosis, Differential ; Drugs, Chinese Herbal ; therapeutic use ; Extracellular Matrix Proteins ; biosynthesis ; Humans ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; drug therapy ; metabolism ; pathology ; Medicine, Chinese Traditional ; Phytotherapy

Circular RNAs (circRNAs) are currently classed as non-coding RNAs that, unlike the better known canonical linear RNAs, form a covalently closed continuous loop without 5′ or 3′ polarities. With the development of high throughput sequencing technology, a large number of circRNAs have been discovered in many species. More importantly, growing evidence suggests that circRNAs are abundant, evolutionally conserved, and relatively stable in cells and tissues. Strikingly, recent studies have discovered that circRNAs can serve as microRNA sponges, interact with RNA-binding protein, and regulate gene transcription, as well as protein translation. Osteoarthritis (OA) is the most common chronic degenerative joint disease. CircRNAs are differentially expressed in OA cartilage. Moreover, some circRNAs are involved in multiple pathological processes during OA, mainly extracellular matrix degradation, inflammation, and apoptosis. In this review, we briefly delineate the biogenesis, characteristics, and biofunctions of circRNAs, and then, focus on the role of circRNAs in the occurrence and progression OA.
Apoptosis ; Cartilage ; Cartilage, Articular ; Extracellular Matrix ; Inflammation ; Joint Diseases ; MicroRNAs ; Osteoarthritis* ; Pathologic Processes ; Porifera ; Protein Biosynthesis ; RNA* ; RNA, Untranslated ; RNA-Binding Proteins

OBJECTIVETo study the gene expression changes in extracellular matrix of condylar cartilage following disc anterior displacement of rabbit TMJ.

METHODSThe right sides of 28 joints in 40 rabbits were subjected to surgical operation of disc displacement. The condylar Collagen II and Aggrecan mRNA expression were detected by in situ hybridization.

RESULTSCollagen II and Aggrecan mRNA mainly expressed in the lower zone of condylar chondrocyte. Aggrecan mRNA decreased faster than collagen II following disc displacement, and adjusted to normal later.

CONCLUSIONAnterior disc displacement leads to alteration of extracellurar matrix gene expression in the condylar chondrocyte, which means the start of remodeling.

Aggrecans ; Animals ; Bone Remodeling ; Cartilage, Articular ; cytology ; Chondrocytes ; metabolism ; Collagen Type II ; biosynthesis ; genetics ; Extracellular Matrix ; metabolism ; Extracellular Matrix Proteins ; biosynthesis ; genetics ; Female ; In Situ Hybridization ; Lectins, C-Type ; Male ; Mandibular Condyle ; cytology ; metabolism ; Proteoglycans ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rabbits ; Temporomandibular Joint ; cytology ; metabolism ; Temporomandibular Joint Disc ; surgery ; Temporomandibular Joint Disorders ; etiology ; genetics ; metabolism

OBJECTIVETo study the expression of tenascin in normal and fibrotic rat liver.

METHODSLiver fibrosis induced in rat with CCl(4) were divided into three stages: the stage of hepatic injury (4 weeks), early stage of hepatic fibrosis (8 weeks) and later stage of hepatic fibrosis (12 weeks). Tenascin expression in liver tissue was observed by immunohistochemical method and in situ hybridization using digoxigenin-labelled DNA probe.

RESULTSIn normal rat liver there was a weak staining for tenascin. In both liver injury stage and early stage of hepatic fibrosis, both mRNA signal and immunostaining for tenascin were significantly increased as compared to that in normal liver. In later stage of hepatic fibrosis, mRNA signal and immunostaining for tenascin were decreased compared with that in early stage of hepatic fibrosis. The cellular source of tenascin in liver mainly restricted in mesenchymal cells.

CONCLUSIONSTenascin is a component of the extracellular matrix of liver tissue. Plays a role in early matrix organization during liver fibrogenesis.

Animals ; Carbon Tetrachloride ; Disease Models, Animal ; Extracellular Matrix Proteins ; biosynthesis ; genetics ; Image Processing, Computer-Assisted ; Immunohistochemistry ; In Situ Hybridization ; Liver Cirrhosis ; chemically induced ; metabolism ; Male ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Tenascin ; biosynthesis ; genetics