Phosphorylation of beta-catenin tyrosine residue 654 plays an important role in the epithelial to myofibroblast transition (EMT). Introducing mimic peptide of tyrosine residue 654 domain of beta-catenin into cells may influence phosphorylation of beta-catenin tyrosine residue 654. To deliver this mimic peptide into renal epithelial cells, we used penetratin as a vector, which is a novel cell permeable peptide, to deliver hydrophilic molecules into cells. A tyrosine 654 residue domain mimic peptide of beta-catenin (PM) with fused penetratin was constructed, purified and then detected for the penetration of the mimic peptide into human renal tubular epithelial cells (HK-2). The results showed that purified fusion mimic peptide could efficiently and rapidly translocate into human renal tubular epithelial cells. It is concluded that a cell-permeable peptides mimic of tyrosine residue 654 domain of beta-catenin was successfully obtained, which may provide a useful reagent for interfering the human renal tubular epithelial-mesenchymal transition.
Carrier Proteins/*metabolism ; Epithelial Cells/cytology ; Epithelial Cells/*metabolism ; Fibroblasts/cytology ; Fibroblasts/metabolism ; Kidney Tubules/*cytology ; Peptides/metabolism ; Permeability ; Phosphorylation ; Tyrosine/*metabolism ; beta Catenin/*metabolism

Bone Marrow Cells ; cytology ; Fibroblasts ; cytology ; metabolism ; Fibrosis ; Humans ; Myocardium ; pathology

OBJECTIVETo explore the influence of different thawing temperatures on the morphology and type I collagen metabolism of the human fibroblasts processed at - 10 degrees C in vitro.

METHODSIn vitro cultured human fibroblasts were randomly divided into control, 20 degrees C thawing, and 37 degrees C thawing groups. After being frozen at -10 degrees C, the cells in the latter two groups were thawed at 20 degrees C and 37 degrees C, respectively. The cell proliferation was assessed with MTT method and was expressed by absorption under 570nm (A ), The morphological change of the cells was observed with inverted phase contrast microscope, the change in the intracellular content of collagen was determined with immunohistochemistry, and the extracellular content of collagen was assayed with ELISA.

RESULTSIn 20 degrees C thawing group, the absorbance decreased at first and increased thereafter, and they were obviously lower than that before freezing (0.95 +/- 0.16, P < 0.05 or 0.01). Cell dehydration and shrinking, cytoplasm loss and increased ratio of cytoplasm to nucleus were found in the survived fibroblasts. The cells proliferated actively at 72 and 96 hours after injury, with increased mitotic index and disordered arrangement. Compared with that before freezing (96.4 +/- 2.9) , the extracellular collagen content increased at first, decreased thereafter, and increased again slowly later (P < 0.05), while the intracellular collagen content decreased at first and increased thereafter (P < 0.05). The collagen metabolism in 37 degrees C thawing group was no difference compared with that in control group. Some cells undergone a floating period before adhering to the culture dish walls.

CONCLUSIONCell dehydration after low temperature treatment could protect the cells from damage. Proper thawing temperature could be beneficial to the cell resuscitation. Comparing with slow thawing, rapid thawing could minimize the cell damage.

Cells, Cultured ; Collagen Type I ; metabolism ; Cryopreservation ; Fibroblasts ; cytology ; metabolism ; Freezing ; Humans

Epithelial-mesenchymal transition (EMT) is a process by which fully differentiated epithelial cells undergo a phenotypic conversion and assume a mesenchymal cell phenotype, including elongated morphology, enhanced migratory and invasiveness capacity, and greatly increased production of extracellular matrix (ECM) components. The EMTs associated with wound healing, tissue regeneration, and organ fibrosis are termed as type 2 EMT. Over the past two decades, emerging evidence suggested that injured epithelial cells, via type 2 EMT, may serve as important sources of fibroblasts and contribute to organ fibrosis, such as kidney, liver, lung and eyes. There is perhaps no doubt that adult epithelial cells can undergo EMT in vitro in response to transforming growth factor (TGF)-β1 and other inflammatory or pro-fibrotic stimuli. However, whether type 2 EMT really occurs in vivo, whethers it is actually a source of functional and activated interstitial fibroblasts and whether it contributes to tissue fibrosis have already been the subjects of heated debate. In this review, we will describe the main features of EMT, the major findings of type 2 EMT in vitro, the evidences for and against type 2 EMT in vivo and discuss the heterogeneity and pitfalls of the techniques used to detect EMT during fibrotic diseases. We suggest that in order to ascertain the existence of type 2 EMT in vivo, different proper phenotype markers of epithelial and mesenchymal cells should be jointly used and cell lineage tracking techniques should be standardized and avoid false positives. Finally, we believe that if EMT really occurs and contributes to tissue fibrosis, efforts should be made to block or reverse EMT to attenuate fibrotic process.
Animals ; Epithelial-Mesenchymal Transition ; physiology ; Fibroblasts ; cytology ; metabolism ; Fibrosis ; metabolism ; pathology ; Humans

OBJECTIVETo investigate the influence of high mobility group box 1 (HMGB1) on the expression of interleukin 6 (IL-6), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) on periodontal ligament fibroblasts.

METHODSHuman periodontal ligament fibroblasts were stimulated with HMGB1 at concentrations of 10, 30, and 100 ng x mL(-1) for 24 h. RT-PCR and Western blot analysis were performed to check mRNA and protein expression of IL-6, RANKL and OPG on the cells.

RESULTSThe ratio of RANKL/OPG was increased at both mRNA and protein level after HMGB1 stimulation at 10, 30, 100 ng x mL(-1). Inflammatory cytokine IL-6 was upregulated by HMGB1 at the concentration of 100 ng x mL(-1).

CONCLUSIONIncreased ratio of RANKL/OPG and IL-6 on periodontal ligament fibroblasts suggests that HMGB1 might play a role in the pathogenesis and progression of periodontal disease.

Cells, Cultured ; Fibroblasts ; metabolism ; HMGB1 Protein ; metabolism ; Humans ; Interleukin-6 ; metabolism ; Osteoprotegerin ; metabolism ; Periodontal Ligament ; cytology ; RANK Ligand ; metabolism

Cell adhesion is a basic and very important tissue in the field of tissue engineering. Fibronectin and integrins are the most important elements to cell adhesion. Some surface receptors of fibroblast can also conjugate with type I collagen in extracellular matrix (ECM) directly. Laminin receptors on the surface of fibroblast bound to laminin also play a role in cell adhesion. In this paper are reviewed a number of related articles. The structures and function of fibronectin and integrins are discussed in detail; the tendon cell's adhesion structures are also discussed. Yet, there was scarcely any paper on the effects which the preservation of tissue engineered products may have on cells' adhesion fo ECM. Therefore, researching on cell adhesion and finding a way of preservation that has no or very little adverse effect on cell adhesion is an important topic. Results from expected advanced researches on cell adhesion may probably find promising applications in the field of tissue engineering.
Cell Adhesion ; Extracellular Matrix ; metabolism ; Fibroblasts ; cytology ; Fibronectins ; metabolism ; Humans ; Integrins ; metabolism ; Laminin ; metabolism ; Tendons ; metabolism ; Tissue Engineering

Cells, Cultured ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Lung ; cytology ; metabolism ; Macrophages, Alveolar ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Silicon Dioxide ; toxicity ; Silicosis ; metabolism

OBJECTIVETo investigate the effect of different dynamic tensional and compressive stress on the mRNA expression of collagen type I and fibronectin in cultured human periodontal ligament fibroblasts (hPDLF), and explore the regularity of functional change in hPDLF.

METHODSA new cyclic strain loading apparatus was used for mechanically loading. Cells cultured in vitro were loaded with three levels (1000 microstrain, 2000 microstrain, 4000 microstrain) of tensional and compressive forces and collected at different time (0 h, 0.5 h, 1 h, 4 h, 8 h,12 h) course after strain loading. The quantity of collagen type I and fibronectin mRNA was analyzed by means of quantitative real-time PCR with special primers of up- and down-regulated genes. Data were analyzed using SPSS version 10.0 software.

RESULTSDifferent magnitude and different kinds of mechanical forces as well as the force application time significantly changed the expression of collagen type I and fibronectin mRNA in hPDLF.

CONCLUSIONSDynamic mechanical forces could regulate the expression of collagen type I and fibronectin mRNA in hPDLF. Collagen type I and fibronectin participated in the mechanical signal transduction in human periodontal ligament fibroblasts.

Cells, Cultured ; Collagen Type I ; metabolism ; Fibroblasts ; metabolism ; Fibronectins ; metabolism ; Humans ; Periodontal Ligament ; cytology ; metabolism ; RNA, Messenger ; genetics ; Stress, Mechanical

OBJECTIVETo investigate the origin of myofibroblasts in oral submucous fibrosis.

METHODSThe oral keratinocytes and fibroblasts were isolated and cultured. The expression of the alpha-smooth muscle actin in the fibroblasts was examined by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR).

RESULTSNo difference was found in expression of alpha-smooth muscle actin between the fibroblasts that were directly stimulated by arecoline and the control. The expression of alpha-smooth muscle actin in the keratinocyte and fibroblast-cocultured group was higher than in the control group, and higher in fibroblasts cocultured with keratinocytes preprocessed by arecoline than in fibroblasts cocultured with keratinocytes without preprocessed by arecoline.

CONCLUSIONSThe differentiation of myofibroblasts from fibroblasts in oral submucous fibrosis might be induced by the interaction of arecoline and keratinocyte.

Actins ; metabolism ; Arecoline ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Fibroblasts ; cytology ; metabolism ; Humans ; Keratinocytes ; cytology ; Mouth Mucosa ; cytology ; Oral Submucous Fibrosis ; metabolism ; pathology

Animals ; Arthritis, Experimental ; metabolism ; pathology ; Breast Neoplasms ; pathology ; Cadherins ; metabolism ; physiology ; Cell Movement ; Female ; Fibroblasts ; cytology ; pathology ; Humans ; Macrophages ; cytology ; pathology ; Neoplasm Invasiveness ; Synovial Membrane ; cytology ; metabolism ; pathology