To investigate the optimum transfection condition in transfecting human keratinocytes with plasmid-liposome complexes in vitro,the cultured human keratinocytes at 60% ～ 100% confluences were exposed to the eukaryotic expression plasmid,pCMV*SPORT-β-gal,coated with LipofectAMINE in different DNA/liposome mixing concentration ratios.After cultured for another 48 hours following the ends of 6 ～ 24 hours exposures to the DNA-liposome complexes,the transfected human keratinocytes were visualized by β-galactosidase staining.Then,the transfection efficiency was determined by calculating the rate of β-galactosidase staining positive cells.β-galactosidase expression was showed clearly in human keratinocytes transfected with the DNA-liposome complexes.The highest efficiency was achieved with cultured cells at 80% and 90% confluences,demonstrating by the transfection rates of (31.35±1.35)% and (32.32±2.47)% respectively.Meanwhile,the essential transfection conditions for these efficiencies were in coating pCMV*SPORT-β-gal DNA of 1.5μg/100μL with LipofectAMINE of 12.5μL/100μL,and exposing the cells to the DNA-liposome complexes for 8 hours.These results indicate that LipofectAMINE could effectively transfer eukaryotic expression plasmid into human keratinocytes in vitro,for which the optimization of transfection conditions involve in cells growth state,DNA/liposome mixing concentration ratio,and exposure time of cells to DNA-liposome complexes.

ObjectiveTo investigate the effects of bFGF on human hypertrophic scar in a nude mouse model. MethodsHuman hypertrophic scars excised in operation for burned patiens were grafted onto the back of nude mice subcutaneously and the mice were randomly divided into: control group, collagenase group, bFGF group, and bFGF plus collagenase group. Two weeks after grafting, preparations were injected into the scars for three times. The size, hardness, and morphological changes of the scars were examined. Biopsies were done 3 weeks after first injection and the histological changes were examined. ResultsIt was found that in bFGF group the size of the grafted scars reduced to 1/2～1/3 of their original size, the majority of the coarse and dense scar collagen bundles resolved and became soft loosed lump. In bFGF plus collagenase group, two thirds of the grafted scar disappeared. ConclusionTopical injection of bFGF into hypertrophic scar degrades scar collagen.

rdial interstitial fibrosis.

OBJECTIVETo lower down the antigenicity of heterogenous swine acellular dermal tissue, and to explore the feasibility of clinical using it as a composite graft for human patients.

METHODSSplit-thickness skin was harvested from healthy swines and then processed by two methods. The swine acellular dermal matrix (sADM) was prepared by removing cells from the skin with trypsin and Triton X-100. Then the cross-linked sADM (sADM(1)) and non-cross-linked sADM (sADM(0)) were embedded subcutaneously in rabbits and also transplanted onto the burn wounds of patients. The histological changes and also transplantation results were observed.

RESULTS(1) In animals with sADM(0) embedded subcutaneously, the grafted tissue was invaded immediately by host cells with obvious inflammatory reaction and tissue degradation. But there was less inflammatory reaction, and with no obvious skin degradation and contraction with sADM(1). (2) In ten burn patients with III degree burn wounds and one patient with wound in chest after scar removal, sADM and ultra-thin skin (UTS) composite graft were grafted on the wounds with autologous thin skin (ATS) and autologous razor-thin or UTS as the control. Nineteen pieces of composite skin of sADM with UTS were grafted on the wounds with survival rate of 78.9%, exhibiting no evident difference with that of ATS. When sADM(0) and UTS were grafed, there exhibited remarkable early inflammatory reaction and wound contraction with similar external appearance with that of UTS. Whereas when sADM(1) and UTS were grafted, there appeared less early inflammatory reaction and wound contraction, resulting in an even appearance and soft to touch similar to that with ATS. But ulceration occurred, with exposure of sADM(1), exposure and severe macrophage reaction to foreign body in 6 wounds of 3 cases 12.8 +/- 6.9 weeks after sADM(1) and UTS grafting.

CONCLUSIONGrafting of sADM as a dermal substitute of composite skin could alleviate early post-grafting immune reaction and improve UTS grafting results. But the delayed graft rejection couldn't be avoided.

Animals ; Burns ; surgery ; Dermatologic Surgical Procedures ; Dermis ; immunology ; transplantation ; Humans ; Rabbits ; Skin ; immunology ; injuries ; Skin Transplantation ; methods ; Skin, Artificial ; Swine ; Time Factors ; Transplantation, Heterologous ; Wound Healing

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