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

The growth of fibroblasts on the acellular dermal matrix (ADM) was studied. The fibroblasts isolated from the skin of an adult New Zealand Rabbit were cultured in vitro and identified subsequently. After the cells were inoculated on the ADM as seeds, the adhesion rate and the growth ability were examined, and cellular morphology was assayed with DAPI fluorescent staining and Scanning electron microscope (SEM). The possibilities of applying ADM as cells carrier or deliverer in the field of transplantation were evaluated. The result revealed that pure fibroblasts were isolated through the specific method. Skin fibroblasts could adhere to ADM easily, and the adhesion rate was 96.78%, displaying no significant difference (P > 0.05) when compared with that rate of the control holes. The cells on the scaffolds and those on the control holes showed similar growth tendencies, but the activity of the former was lower (P < 0.01). The integral nucleus with blue fluorescence could be observed on the ADM under fluorescence microscope. The number of fibroblasts scaled up with the cultured time, The results of SEM showed that the state of cell was good and the fibroblasts were fused into a layer after being cultured for 5-10d. So rabbit fibroblasts can attach, survive, grow and proliferate on the ADM in a healthy way. It is entirely possible to use ADM as an appropriate scaffold material for the culture of fibroblasts and as a material for transplantations.
Animals ; Biocompatible Materials ; Cell Adhesion ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dermis ; cytology ; Fibroblasts ; cytology ; Rabbits ; Skin ; cytology ; Skin, Artificial ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVETo investigate the relationships between skin photoaging and point mutations in mitochondrial DNA (mtDNA) control region for replication of dermal fibroblast.

METHODSCultured dermal fibroblasts were treated by 8-methoxypsora len /ultraviolet A (8-MOP/UVA). mtDNA was extracted by one-step-method and th e PCR products of D-loop and adjacent transcription promoter (DLP(6)) fragment of mtDNA control region for replication were detected by polymerase chain reaction-single strain conformation polymorphism and direct sequencing.

RESULTSAfter treated by 8-MOP/UVA, point mutations of 414 T-->G of DLP(6) fragment of mtDNA control region for replication largely accumulated.

CONCLUSIONAccumulation of point mutations of DLP(6) fragment of mtDNA control region for replication may be closely associated with skin photoaging.

Adult ; Binding Sites ; Cells, Cultured ; DNA Replication ; DNA, Mitochondrial ; drug effects ; radiation effects ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; radiation effects ; Humans ; Male ; Methoxsalen ; pharmacology ; Photosensitizing Agents ; pharmacology ; Point Mutation ; Ultraviolet Rays

Autologous multipotent stem cells are most relevant cells for regenerative medicine and show prosperous future in the treatment of human diseases. Previous reports have indicated that multipotent stem cells (MSCs) can be obtained from bone marrow and adipose tissues. In this study, we proved that dermis may be another source of these cells. MSCs were isolated from the dermis of newborn rats one day old by adhesion competition and successive culture. These cells conserved the ability to differentiate to osteoblasts, chondrocytes and adipocytes by induction media containing dexamethasone. After long term of more than 6 months, till 25th generation, the cells still maintained the characteristics of stem cells: high activity of self-renewal and multipotency. Mixed collagen matrix from dermis could promote the growth of dermis-derived multipotent stem cells and collagen sponge stent could promote their three-dimensional growth in vitro.
Animals ; Biomechanical Phenomena ; Cell Culture Techniques ; methods ; Cells, Cultured ; Collagen ; pharmacology ; Dermis ; cytology ; Multipotent Stem Cells ; cytology ; drug effects ; physiology ; Rats ; Rats, Wistar

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

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

OBJECTIVETo investigate the effects of preemptive freezing with different temperature and cross-linking methods on the ultrastructure of collagen membrane and its influence on human fibroblast proliferation.

METHODSBovine collagen type I solution in concentration of 10 g/L was preliminarily frozen at -20 degrees C or - 80 degrees C for 12 hours, and lyophilized at -70 degrees C for 48 hours. The diameter of apertures in collagen membranes prepared with two different preliminary temperatures were observed by scanning electron microscope (SEM) and compared. The preliminary freezing temperature of - 80 degrees C was used for the following study. The apertures of collagen membrane performed with cross-linking glutaraldehyde and ultraviolet (UV) radiation cross-linking with glutaraldehyde (double cross - linking) after preliminary freezing were also compared. The proliferation of human fibroblasts inoculated in above cross-linking collagens were assessed by MTT assay, in terms of absorption value.

RESULTSThe mean diameter of apertures of collagen membrane pre-frozen at -20 degrees C was (172 +/- 374 microm, while that at -80 degrees C was (99 +/- 24) microm. The apertures of collagen membrane were reduced in size after glutaraldehyde cross-linking, while those of double cross-linking showed no change in size. There was obvious difference in absorption value of fibroblasts 8 days after seeding between above two cross-linking methods (1.534 +/- 0.013 for glutaraldehyde cross-linking, 3.778 +/- 0.010 for double cross-linking, P < 0.05).

CONCLUSIONThe collagen membrane after preliminary freezing at - 80 degrees C and double cross-linking with UV radiation and glutaraldehyde may be used as a dermal skeleton substitute.

Animals ; Cattle ; Cells, Cultured ; Coculture Techniques ; Collagen ; ultrastructure ; Cross-Linking Reagents ; chemistry ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; radiation effects ; Freezing ; Glutaral ; chemistry ; Humans ; Skin, Artificial ; Tissue Scaffolds ; Ultraviolet Rays

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

Cordycepin (3'-deoxyadenosine) has been shown to exhibit many pharmacological activities, including anti-cancer, anti-inflammatory, and anti-infection activities. However, the anti-skin photoaging effects of cordycepin have not yet been reported. In the present study, we investigated the inhibitory effects of cordycepin on matrix metalloproteinase-1 (MMP-1) and -3 expressions of the human dermal fibroblast cells. Western blot analysis and real-time PCR revealed cordycepin inhibited UVB-induced MMP-1 and -3 expressions in a dose-dependent manner. UVB strongly activated NF-kappa B activity, which was determined by I kappa B alpha degradation, nuclear localization of p50 and p65 subunit, and NF-kappa B binding activity. However, UVB-induced NF-kappa B activation and MMP expression were completely blocked by cordycepin pretreatment. These findings suggest that cordycepin could prevent UVB-induced MMPs expressions through inhibition of NF-kappa B activation. In conclusion, cordycepin might be used as a potential agent for the prevention and treatment of skin photoaging.
Aging/physiology ; Cells, Cultured ; Deoxyadenosines/*pharmacology ; *Dermis/cytology/drug effects/physiology/radiation effects ; Dose-Response Relationship, Drug ; Enzyme Induction/drug effects ; Fibroblasts/drug effects/metabolism/radiation effects ; Gene Expression Regulation, Enzymologic ; Humans ; Infant, Newborn ; Male ; *Matrix Metalloproteinase 1/antagonists & inhibitors/biosynthesis/genetics/radiation effects ; Matrix Metalloproteinase 3/antagonists & inhibitors/*biosynthesis/genetics/radiation effects ; NF-kappa B/*antagonists & inhibitors/genetics/metabolism ; Skin/physiopathology/radiation effects ; *Ultraviolet Rays