OBJECTIVETo explore the damage effects and expression of vascular endothelial growth factor (VEGF) exposed with different low-temperatures on rat dermal microvascular endothelial cells (DMVECs).

METHODSPrimary DMVECs were obtained by discontinuous Percoll gradient centrifugation. The DMVECs were identified by phase contrast microscope and immunofluorescence studies for CD31 antigen. Applied 28 degrees C, 12 degrees C and 0 degrees C to interfere with rat DMVECs as cold-exposure model. The changes of cells morphology were observed under invert microscope. The membrane integrity was determined by lactate dehydrogenase (LDH) activity. RT-PCR was used to examine the expression of vascular endothelial growth factor mRNA in cells.

RESULTSThe monolayer of cultured PMVECs displayed the shape of pavingstone. CD31 antigen and binding BSI results by fluorescence microscope identified the cultured cells were DMVECs. After 24 h cold exposure, the cell morphology of 0 degrees C group was shrunken, the other groups were "Fibroblast-like". The LDH activity (U/L) in the medium of 28 degrees C, 12 degrees C and 0 degrees C groups was 54.17 +/- 3.02, 64.66 +/- 3.03, 82.13 +/- 10.91 respectively, which was significantly higher than that of 37 degrees C group (12.23 +/- 3.0, P < 0.01). The VEGF mRNA expression level was up-regulated in 28 degrees C group and 12 degrees C group versus control group (P < 0.05), but unchanged in 0 degrees C group.

CONCLUSIONThe rat DMVECs injury severity are deteriorated with temperature decreasing, and VEGF might be involved in the regulation of membrane permeability in this period.

Animals ; Animals, Newborn ; Cells, Cultured ; Cold Temperature ; Dermis ; blood supply ; Endothelial Cells ; cytology ; metabolism ; Endothelium, Vascular ; cytology ; Rats ; Rats, Wistar ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo observe the effects of Angelica dahurica extracts on the biological characteristics of human dermal fibroblasts in vitro and to preliminary explore its possible therapeutic mechanism for wound healing.

METHODSThe optimal concentration of Angelica dahurica extracts was identified by analysing of proliferation activity of human normal fibroblasts (Fb) that treated with different concentration of Angelica dahurica extracts through thiazole blue (MTT) colorimetric assay. Cell cycle, collagen I and collagen III mRNA levels of the optimal Angelica dahurica extracts treated Fb were detected by flow cytometry (FCM) and real-time PCR techniques.

RESULTSAt concentrations of 5 × 10(-4) to 5 × 10(-2) g/L, the Angelica dahurica extracts significantly enhanced the proliferation of Fb. The most significant concentration was 5 × 10(-3) g/L (t = 5.79, P < 0.01), at which an increased percentage of G1 to S and S to G2 phase cells (t = 11.2, 5.69, 2.44, P < 0.05) as well as an increased level of collagen I (1.61 ± 0.26 vs. 1.00 ± 0.16) and collagen III mRNA (3.36 ± 0.40 vs. 1.00 ± 0.14) were obtained compared to the control group (t = 6.69, 7.64, P < 0.01).

CONCLUSIONSAngelica dahurica extracts can notably promote the proliferation of Fb and accelerating the cell cycle of Fb as well as up-regulating the expression of collagen I and collagen III, which may enhance the process of wound healing.

Angelica ; chemistry ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; metabolism ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Plant Extracts ; pharmacology

OBJECTIVETo explore the optimal method for extracting human serum that retains rich growth factors.

METHODSHuman serum was isolated by centrifugation of coagulated whole blood or by Anitua's method, and the concentrations of PDGF-AB and transforming growth factor-β1 (TGF-β1) in the serum samples were measured. Human dermal fibroblasts were cultured in the presence of 10% feral bovine serum or 10% human serum obtained, and the cell morphology, viability and proliferative activity of the cells were evaluated.

RESULTSThe fibroblasts grew well in all the media with good viability. The cells cultured in the presence of human serum isolated by centrifugation of coagulated whole blood, which had the richest content of growth factors, showed the greatest cell number and cell viability among the groups (P<0.05), a result consistent with the growth curve and MTT curve.

CONCLUSIONCentrifugation of coagulated whole blood retains high contents of growth factors in human serum to better promote cell growth, and is simple, cost-effective and most efficient for serum isolation.

Animals ; Cattle ; Cell Proliferation ; Cells, Cultured ; Culture Media ; Dermis ; cytology ; Fibroblasts ; cytology ; Humans ; Platelet-Derived Growth Factor ; metabolism ; Serum ; Transforming Growth Factor beta1 ; metabolism

AIMTo study the intervention of cetirizine on monocyte chemoattractant protein-1 (MCP-1) in different cutaneous inflammation models.

METHODSHistamine and IFN-gamma stimulated dermal fibroblast cells and HaCaT cells to mimic cutaneous inflammation. Expression of MCP-1 was assessed by means of RT-PCR and ELISA.

RESULTSCompared with the control group of dermal fibroblast (DF) cells and HaCaT cells, MCP-1 mRNA was significantly upregulated by histamine (10 micromol x L(-1)) and IFN-gamma (20 ng x mL(-1)). The protein secretions of MCP-1 were increased 3.5 fold and 8.4 fold in DF cells, respectively. The similar tendency was observed in HaCaT cells. The enhancing effects of histamine and IFN-gamma on MCP-1 protein production were significantly inhibited by cetirizine (1 and 10 micromol x L(-1)) in DF and HaCaT cells.

CONCLUSIONCetirizine may exert the anti-inflammatory effect of skin via inhibiting MCP-1 expression.

Cell Line ; Cells, Cultured ; Cetirizine ; pharmacology ; Chemokine CCL2 ; biosynthesis ; genetics ; Dermatitis ; metabolism ; Dermis ; cytology ; Fibroblasts ; cytology ; metabolism ; Histamine ; pharmacology ; Histamine H1 Antagonists, Non-Sedating ; pharmacology ; Humans ; Interferon-gamma ; pharmacology ; Keratinocytes ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the influence of epidermis from different sources on the proliferation and metabolism of fibroblasts (Fb), and to explore its cause.

METHODSIn a co-culture system, normal Fb (A group) and cicatricial Fb(B group) from 10 patients with scar during proliferative stage were co-cultivated with own normal skin epidermis (NSE), respectively, without direct contact. In control groups (C group), cicatricial Fb was cultured alone. Normal Fb and cicatricial Fb from 10 patients with scar during maturation period were co-cultured with own normal skin epidermis as mentioned above, and divided into D, E and F groups. The cell number of FB, the amount of type I and III procollagen (PC I, PC III) in the supernatants and the PC I to PC III ratio were determined.

RESULTSTo compare the C with A group and the F with D group, Fb in C and F groups exhibited increased cell number and PC I , PC III amounts (P < 0.05), and decreased ratio of PC I to PC III (P < 0.05). To compare the B with C group, PC III contents in the cell supernatant was increased (P < 0.05), and the ratio of PCI to PC III decreased in B group (P < 0.05), there were no obvious difference in Fb cell number and the amount of PC I contents between B and C group. To compare the E with F group, the cell number of Fb, as well as PC I and PC III contents in cell supernatant were obviously decreased in E group (P < 0.05), but no obvious decrease was observed in the ratio of PC I and PC III. To compare the B with A group and the E with D group, the cell number and the PC I and PC III contents in B and E groups were evidently increased, while the ratio of PC I to PC III decreased markedly (2.20 +/- 0.27 vs 1.16 +/- 0.21 in A, B group, P < 0.05; 2.18 +/- 0.14 vs 1.93 +/- 0.26 in D, E group, P < 0.05).

CONCLUSIONNormal epidermis may play an important role in preventing hypertrophic scar by producing some bioactive substances.

Cell Line ; Cell Proliferation ; Cicatrix, Hypertrophic ; pathology ; Coculture Techniques ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Dermis ; cytology ; Epidermis ; cytology ; Extracellular Matrix ; Fibroblasts ; cytology ; metabolism ; Humans ; Wound Healing

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

OBJECTIVETo investigate the effect of metformin in protecting against advanced glycation end products (AGEs)-induced apoptosis in human primary dermal fibroblasts.

METHODSFibroblasts were exposed to 100, 200, or 300 µg/mL AGEs, 300 µg/mL bovine serum albumin (BSA), or 300 µg/mL AGEs and 1 mmol/L metformin for 24, 48, or 72 h. The exposed cells were examined for cell apoptosis using a cell counting kit. The expressions of caspase-3, Bax and Bcl-2 protein in the fibroblasts treated for 72 h were detected with Western blotting.

RESULTSAGEs exposures caused significant dose- and time-dependent apoptosis in the fibroblasts. A 72-h exposure to 300 µg/mL AGEs resulted in obviously increased apoptosis of the fibroblasts compared to the control group (0.72 ± 0.02 vs 1 ± 0.04, P<0.05), and metformin significantly decreased AGEs-induced apoptosis (0.98 ± 0.02 vs 0.72 ± 0.02, P<0.05). The expressions of caspase-3 and Bax protein were significantly increased (P<0.05) and Bcl-2 protein expression was decreased (P<0.05) with a lowered Bcl-2/Bax ratio in AGEs-treated fibroblasts (P<0.05), and such changes were significantly reversed by metformin treatment (P<0.05).

CONCLUSIONMetformin can antagonize AGEs-induced apoptosis in human dermal fibroblasts by regulating the expressions of caspase-3, Bax and Bcl-2.

Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cells, Cultured ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; Glycation End Products, Advanced ; adverse effects ; Humans ; Metformin ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

This study was conducted to identify the effectiveness of platelet-rich plasma (PRP) and efficacy of intralesional injection as a method of application to acute cutaneous wounds in dogs. Healthy adult beagles (n = 3) were used in this study. Autologous PRP was separated from anticoagulant treated whole blood in three dogs. Cutaneous wounds were created and then treated by intralesional injection of PRP in the experimental group, while they were treated with saline in the control group on days 0, 2 and 4. The healing process was evaluated by gross examination throughout the experimental period and histologic examination on day 7, 14 and 21. In PRP treated wounds, the mean diameter was smaller and the wound closure rate was higher than in the control. Histological study revealed that PRP treated wounds showed more granulation formation and angiogenesis on day 7, and faster epithelialization, more granulation formation and collagen deposition were observed on day 14 than in control wounds. On day 21, collagen deposition and epithelialization were enhanced in PRP treated groups. Overall, PRP application showed beneficial effects in wound healing, and intralesional injection was useful for application of PRP and could be a good therapeutic option for wound management in dogs.
Animals ; Collagen/metabolism ; Dermis/cytology/injuries/physiology ; Dogs ; Epidermis/cytology/injuries/*physiology ; Female ; Granulation Tissue/cytology ; Injections, Intralesional/veterinary ; Male ; Neovascularization, Physiologic ; *Platelet-Rich Plasma ; Regeneration ; Treatment Outcome ; *Wound Healing ; Wounds and Injuries/therapy/*veterinary

OBJECTIVETo investigate the role of substance P in the formation of hypertrophic scar.

METHODSDermal fibroblasts derived from human normal skin were cultured with substance P alone or together with selective non-peptide NK-1 tachykinin antagonist, L-703, 606 oxalate salt. The effect of substance P on proliferation of fibroblasts was measured by MTT assay. Furthermore, the TGF-beta 1 mRNA expression in the fibroblasts was determined by in situ hybridization and image analysis.

RESULTSSubstance P enhanced fibroblast proliferation dose-dependently, which showed the maximum rate when the concentration of substance P was 25 ng/ml or higher in the culture media. By 48 hours cultured with 25 ng/ml of substance P, the fibroblasts expressed TGF-beta 1 mRNA more significantly than the fibroblasts without substance P. The effects of substance P on both fibroblast proliferation and TGF-beta 1 mRNA expression could be antagonized by L-703, 606 oxalate salt.

CONCLUSIONThe results suggest that substance P may play an important role in phenotype changes of fibroblasts in skin scarring.

Cell Division ; Cells, Cultured ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; metabolism ; Gene Expression ; drug effects ; Neurokinin-1 Receptor Antagonists ; Quinuclidines ; pharmacology ; RNA, Messenger ; Substance P ; metabolism ; pharmacology ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1 ; Up-Regulation

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin-producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis-derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal-endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic beta-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin-induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.
Animals ; Biological Markers/metabolism ; *Cell Culture Techniques ; *Cell Differentiation ; Cell Proliferation/drug effects ; Cell Separation ; Cells, Cultured ; Dermis/*cytology/drug effects ; Diabetes Mellitus, Experimental/*surgery ; Female ; Fibroblasts/*cytology/drug effects ; Genitalia, Female/*cytology ; Glucose/metabolism ; Hepatocyte Nuclear Factor 3-beta/metabolism ; Homeodomain Proteins/metabolism ; Humans ; Insulin/pharmacology/secretion ; Insulin-Secreting Cells/*cytology/metabolism ; *Islets of Langerhans Transplantation ; Mesenchymal Stem Cells/*cytology/drug effects/metabolism ; Mice ; Mice, Nude ; Niacinamide/pharmacology ; Recovery of Function ; SOXF Transcription Factors/metabolism ; Sodium Selenite/pharmacology ; Trans-Activators/metabolism ; Transferrin/pharmacology