This study was aimed to further investigate the function of platelet collagen receptor-glycoprotein VI and to screen its specific inhibitor. The extracellular domain of platelet glycoprotein VI (GPVI) in E. coli was expressed by recombinant technology, the extracellular domain cDNA of GPVI was amplified from pBluescript KS(-)-GPVI plasmid by PCR. Proved by sequencing, the expression vector pET-20b(+)-GPVI was constructed, which was then transformed into E. coli (BL21(DE3)pLysS) and induced by IPTG. The recombinant GPVI was purified on Ni-NTA resin column and renatured in PBS containing GSH and GSSG. The anti-penta His McAb and anti-GPVI polyclonal antibody were used to identify the recombinant GPVI in Western blotting. Collagen binding test was conducted to investigate the biological activity of recombinant GPVI. The results showed that the recombinant GPVI was expressed in E. coli and successfully purified, which was confirmed to be similar to the native GPVI in Western blotting. The recombinant GPVI can bind the type I collagen in dose-dependent manner. In conclusion, the recombinant GPVI can be achieved in E. coli and restore its native characteristics after renaturation.
Blood Platelets ; metabolism ; Blotting, Western ; Escherichia coli ; genetics ; Humans ; Integrin alpha2beta1 ; Platelet Membrane Glycoproteins ; biosynthesis ; genetics ; Protein Binding ; Receptors, Collagen ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo study the expression of the estrogen receptor (ER) and type II collagen in the mandibular condylar cartilage of the human embryo.

METHODSThe expression and localization of ERalpha, ERbeta, and type II collagen in the mandibular condylar cartilage of the human embryo were examined through hematoxylin-eosin (HE) and immunohistochemistry staining.

RESULTSType II collagen was primarily localized in the transitional and hypertrophic layers of the condylar cartilage. ERa was mostly expressed in the transitional and hypertrophic cartilaginous layers of the condylar cartilage. ERa was evenly distributed in the cell, whereas ERbeta was localized in the nuclei. No expression of type II collagen and ER was found in the fibrage and the proliferative layer although minimal expression was found in the calcified cartilage.

CONCLUSIONThe distribution of ER and type II collagen in the mandibular condylar cartilage was consistent. Estrogen can selectively combine with different subtypes of ER that regulate the ability of the condylar cartilage cells to secrete type II collagens.

Cartilage ; Collagen ; Collagen Type II ; Embryo, Mammalian ; Estrogen Receptor alpha ; Humans ; Immunohistochemistry ; Mandibular Condyle ; Receptors, Estrogen

PURPOSE: Proliferative chondrocytes and prehypertrophic chondrocytes secrete significant amounts of type II collagen in an extracellular matrix. In contrast, hypertrophic chondrocytes secrete type X collagen. In addition, fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) also appear to play an important role during differentiation. Accordingly, the current study identified and characterized the chondrocytes and FGFR mRNA expressed at different stages of differentiation. METHODS: Chondrocytes were isolated from the caudal one-third portion (LS) of the sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and the lower portion of the proximal tibial growth plate (Ti). Chondrocytes from the LS, USP, USC, and Ti of 17-day-old chick embryo sterna and tibia were cultured and type II and type X collagen mRNA and FGFR1, FGFR2, and FGFR3 mRNA were isolated and analyzed by Northern blotting. RESULTS: Generally, the cells were larger in size after two days of culture than after seven days of culture and the cells from the USC and Ti were larger and more mature than those from the LS and USP. Type II collagen genes were found to be expressed in all the chondrocyte types, while type X collagen was strongly expressed in the USC and Ti. Therefore, the LS was identified as a resting or proliferative zone, the USP as a postproliferative or prehypertrophic zone, and the USC or Ti as a hypertrophic zone. FGFR1 was expressed only in hypertrophic chondrocytes in proportion to the culture time, FGFR2 was not expressed in any of the chondrocyte types, and FGFR3 was expressed in all the chondrocyte types. CONCLUSION: As such, it is possible that the different receptors play distinct roles during chondrocyte differentiation.
Animals ; Blotting, Northern ; Chick Embryo* ; Chondrocytes* ; Collagen ; Collagen Type II ; Collagen Type X ; Extracellular Matrix ; Fibroblast Growth Factors* ; Fibroblasts* ; Growth Plate ; Receptors, Fibroblast Growth Factor* ; RNA, Messenger ; Tibia

OBJECTIVEThis study further explores the stromal cell-derived factor-1 (SDF-1)/chemokine receptor 4 (CXCR4) signaling axis mechanism in temporomandibular joint osteoarthritis (OA) by detecting the changes in CXCR4, interleukin (IL)-6, and collagen X expression in the ATDC5 cell line stimulated by the cyclic tensile strain and SDF-1.

METHODSInsulin-transferrin-selenium (ITS) was used to induce ATDC5 cells to differentiate into chondrocyte-like cells. After three weeks, the cells were divided into two groups: those with and without cyclic tensile strain. These groups were further divided into the negative control and SDF-1 groups. Strain force of 20% was applied. After 12 h, the total proteins were extracted from cells of the four groups, and Western blot analysis was used to detect the changes in CXCR4, IL-6, and collagen X expression.

RESULTSSDF-1 could enhance CXCR4, IL-6, and collagen X expressions in the chondrocytes, and 20% tensile strain force could further upregulate the three factors.

CONCLUSIONUnder abnormal tensile force, SDF-1 can upregulate its specific receptor CXCR4, thus increasing its-binding efficiency and resulting in the activation of the SDF-1/CXCR4 axis. This condition enhances the expressions of IL-6 and other inflammatory factors and directly damages to cartilage tissue. Such damage directly promotes chondrocyte hypertrophy, which enhances collagen X expression.

Cell Differentiation ; Cell Line ; Chemokine CXCL12 ; Collagen ; Humans ; Interleukin-6 ; Receptors, CXCR4 ; Signal Transduction ; Stromal Cells

OBJECTIVETo observe the expressions of discoidin domain receptor 2 (DDR2) in different phases of alcoholic liver fibrosis (ALF) in a rat model and to study the possible association between DDR2 and collagen deposition in ALF.

METHODSAfter an ALF rat model was established by alcohol gastrogavage and an olive oil diet, the liver histopathology was observed in different phases of the development of fibrosis. The expressions of DDR2 mRNA and protein were also detected by RT-PCR and Western blot respectively to make a dependability analysis with the index of ALF.

RESULTS(1) The expressions of DDR2 mRNA and protein increased gradually along with ALF aggravation. In the normal control group, they were respectively 1.023+/-0.132 and 0.321+/-0.027; in the model 1 group (week 12) they were 3.644+/-1.686, 0.476+/-0.046; in the model 2 group (week 16) they were 8.337+/-2.387, 0.738+/-0.057; and in the model 3 group (week 20) they were 15.730+/-4.569, 0.997+/-0.049. The differences of DDR2 mRNA (F = 21.74, P less than 0.01) and protein (F = 10.38, P less than 0.01) among these four groups were significant. (2) The expressions of DDR2 had a positive correlation with collagen type I, III, IV contents and the serum index of ALF, especially with type III and IV collagen and serum hexadecenoic acid.

CONCLUSIONThe expression of DDR2 in this ALF model correlates closely with collagen deposition in the liver, suggesting that it may play an important role in ALF pathogenesis.

Animals ; Collagen ; metabolism ; Discoidin Domain Receptors ; Disease Models, Animal ; Liver Cirrhosis, Alcoholic ; metabolism ; pathology ; Male ; Rats ; Rats, Wistar ; Receptor Protein-Tyrosine Kinases ; metabolism ; Receptors, Mitogen ; metabolism

OBJECTIVE: To evaluate the expression of Toll-like receptor (TLR) 2, 4 and 9 and investigate the effects of IL-17 on the expression of TLRs in experimental rheumatoid arthritis (RA) model. METHODS: After induction of collagen-induced arthritis (CIA) by type II collagen in DBA1 mice, phosphate-buffered saline (PBS, PBS group) or IL-17 (IL-17 group) was injected to both knee joint at day 28 and 32. At day 35, mice were sacrificed and knee joints were isolated. Synovial mRNA expressions of TLR-2, 4 and 9 determined by real-time RT-PCR were compared among normal DBA1 mice (normal group), PBS and IL-17 group. RESULTS: Synovial TLR-2, 4, and 9 mRNA expressions of IL-17 and PBS group were significantly higher than normal group, and those of IL-17 group were higher than PBS group. CONCLUSION: Synovial TLR-2, 4 and 9 expression was enhanced in CIA and up-regulated by local overexpression of IL-17. These results suggest that TLRs play a roles on CIA and IL-17 induced aggravation of arthritis in CIA.
Animals ; Arthritis ; Arthritis, Experimental* ; Arthritis, Rheumatoid ; Collagen Type II ; Interleukin-17 ; Knee Joint ; Mice ; RNA, Messenger ; Toll-Like Receptors*

Fibromatosis of the breast is a rare tumor. We describe here a case of mammary fibromatosis in a 37-year-old woman. The mass from the right breast was 3 cm at the greatest dimension. The lesion was poorly circumscribed, firm and white-gray on the cut surface. Histologically, the lesion infiltrated into the lobules of the breast, and the tumor was composed of relatively uniform fibroblasts and collagen. Neither mitotic activity nor cellular atypia was seen. On the immunohistochemistry, the cells were positive for vimentin and they were focally positive for smooth muscle actin. Staining results for estrogen receptor and progesterone receptor were negative.
Actins ; Adult ; Breast Neoplasms ; Breast* ; Collagen ; Estrogens ; Female ; Fibroblasts ; Fibroma* ; Humans ; Immunohistochemistry ; Muscle, Smooth ; Receptors, Progesterone ; Vimentin

Retinoids are compounds with pleiotropic functions, selectively targeting certain skin structures. They are vitamins which must be derived from diet because retinol(vitamin A) is not synthesized in the body, and also they are hormones with intracrine activity, because retinol is transformed into molecules that bind to nuclear receptors, exhibit their activity, and are subsequently inactivated. Enhanced healing of full-thickness skin wounds has been demonstrated in early wound healing studies. OBJECTIVE: Our purpose is to observe the chemical and histologic effects of topical retinoid derivatives(0.05%) applied directly to the dermal wound of guinea pig. METHODS: We prepared five kinds of retinoid derivative, applied each one on the wound on back of guinea pig. After 4 and 8 weeks, each wound was observed under microscope and analyzed for the amount of the collagen. RESULTS: Among the newly developed retinoid derivatives, PVP-RA(ester form) was the most effective in wound healing of the dermal damaged guinea pig. CONCLUSION: Retionid derivatives were effective in dermal wound healing if well prepared.
Animals ; Collagen ; Diet ; Guinea Pigs ; Polyethylene* ; Polyvinyls* ; Receptors, Cytoplasmic and Nuclear ; Retinoids ; Skin ; Tretinoin* ; Vitamin A ; Vitamins ; Wound Healing* ; Wounds and Injuries*

Periodontal ligament(PDL) cells have been known as playing an important roles in periodontal regeneration and gingival fibroblasts are also important to periodontal regeneration by forming connective tissue attachment. There were rare studies about the gene expression patterns of PDL cells and gingival fibroblasts, therefore in this study, we tried cDNA microarray-based gene expression monitoring to explain the functional differences of PDL cells and gingival fibroblasts in vivo and to confirm the characteristics of PDL cells. Total RNA were extracted from PDL cells and gingival fibroblasts of same person and same passages, and mRNA were isolated from the total RNA using Oligotex mRNA midi kit(Qiagen) and then fluorescent cDNA probe were prepared. And microarray hybridization were performed. The gene expression patterns of PDL cells and gingival fibroblasts were quite different. About 400 genes were expressed more highly in the PDL cells than gingival fibroblasts and about 300 genes were more highly expressed in the gingival fibroblasts than PDL cells. Compared growth factor- and growth factor receptor-related gene expression patterns of PDL cells with gingival fibroblasts, IGF-2, IGF-2 associated protein, nerve growth factor, placental bone morphogenic protein, neuron-specific growth- associated protein, FGF receptor, EGF receptor-related gene and PDGF receptor were more highly expressed in the PDL cells than gingival fibroblasts. The results of collagen gene expression patterns showed that collagen type I, type III, type VI and type VII were more highly expressed in the PDL cells than gingival fibroblasts, and in the gingival fibroblasts collagen type V, XII were more highly expressed than PDL cells. The results of osteoblast-related gene expression patterns showed that osteoblast specific cysteine-rich protein were more highly expressed in the PDL cells than gingival fibroblasts. The results of cytoskeletal proteins gene expression patterns showed that alpha-smooth muscle actin, actin binding protein, smooth muscle myosin heavy chain homolog and myosin light chain were more highly expressed in the PDL cells than gingival fibrobalsts, and beta-actin, actin-capping protein(beta subunit), actin- related protein Arp3(ARP) and myosin class I(myh-1c) were more highly expressed in the gingival fibroblasts than PDL cells. Osteoprotegerin/osteoclastogenesis inhibitory factor(OPG/OCIF) was more highly expressed in the PDL cells than gingival fibroblasts. According to the results of this study, PDL cells and gingival fibroblasts were quite different gene expression patterns though they are the fibroblast which have similar shape. Therefore PDL cells & gingival fibroblasts are heterogeneous populations which represent distinct characteristics. If more studies about genes that were differently expressed in each PDL cells & gingival fibroblasts would be performed in the future, it would be expected that the characteristics of PDL cells would be more clear.
Actins ; Carrier Proteins ; Collagen ; Collagen Type I ; Collagen Type V ; Connective Tissue ; Cytoskeletal Proteins ; DNA, Complementary* ; Epidermal Growth Factor ; Fibroblasts* ; Gene Expression Profiling ; Gene Expression* ; Humans ; Insulin-Like Growth Factor II ; Muscle, Smooth ; Myosin Heavy Chains ; Myosin Light Chains ; Myosins ; Nerve Growth Factor ; Oligonucleotide Array Sequence Analysis* ; Osteoblasts ; Periodontal Ligament* ; Receptors, Fibroblast Growth Factor ; Receptors, Platelet-Derived Growth Factor ; Regeneration ; RNA ; RNA, Messenger

Ullrich's disease is a congenital muscular dystrophy clinically characterized by generalized muscle weakness, multiple contractures of the proximal joints, and hyperextensibility of the distal joints. All the patients develop rigidity of spine, often assoicated with scoliosis, failure to thrive, and early and severe respiratory involvement, irrespective of their levels of motor function. Intellectual development is normal. The biopsied muscles show dystrophies including remarkable variation in the fiber size, notably proliferated endomysial connective tissues, and a lot of degenerated and regenerated fibers. The expression of merosin and dytrophin is normal. Recent studies have demonstrated that collagen VI is deficient in the muscles of the patients with Ullrich's disease, and some result from recessive mutations of the collagen VIalpha 2 gene(COL6A2). And a marked reduction of fibronectin receptors in the extracellular matrix of skin and cultured fibroblasts of these patients is also reported. These results suggest that collagen VI deficiency may lead to the reduction of fibronectin receptors and that any abnormalities of cell adhesion may be involved in the pathogenesis of the disease. A case of Ullrich's disease has not been reported yet in Korea. So, we describe a male patient with Ullrich's disease with a brief review of the literature.
Cell Adhesion ; Collagen ; Connective Tissue ; Contracture ; Extracellular Matrix ; Failure to Thrive ; Fibroblasts ; Humans ; Integrin alpha5beta1 ; Joints ; Korea ; Laminin ; Male ; Muscle Weakness ; Muscles ; Muscular Dystrophies ; Receptors, Fibronectin ; Scoliosis ; Skin ; Spine