OBJECTIVETo explore the role of EGF in regulating HaCaT apoptosis through peroxisome proliferator-activated receptor beta (PPARbeta).

METHODSCultured HaCaT cells were divided into different groups with different additives in culture medium as control (normal culture), TNF-alpha (with addition of 10 ng/mL TNF-alpha), EGF (with addition of 20 ng/mL EGF), EGF + TNF-alpha (cells were treated with 10 ng/mL TNF-alpha for 60 mins after the exposure to 20 ng/mL EGF for 4 hs) groups. Conjugation activity and transcription activity of PPARbeta of HaCaT cells in each group were detected by electrophoretic mobility shift assay (EMSA) and luciferase gene analysis (LGA). Protein expression of PPARbeta of HaCaT cells after transfected by missense oligonucleotide (scrODN) and antisense oligonucleotide (asODN) was determined by Western blot. Caspase-3 activity and apoptosis rate were detected by flow cytometry.

RESULTSConjugation and transcription activity of PPARbeta DNA were enhanced as shown in EMSA and LGA. Compared with that of cells in groups transfected by scrODN, protein expression of PPARbeta in cells of groups transfected by asODN was obviously inhibited as shown in Western blot. Caspase-3 activity of cells in TNF-alpha and EGF + TNF-alpha groups transfected by asODN was stronger than that of cells in TNF-alpha and EGF + TNF-alpha groups transfected by scrODN (P < 0.01). Apoptosis rate of cells in control, EGF, TNF-alpha, and EGF + TNF-alpha groups which were transfected by scrODN was (7.31 +/- 0.45)%, (7.43 +/- 0.21)%, (39.78 +/- 0.65)%, (28.34 +/- 0.54)% respectively, and that in those groups transfected by asODN was (8.22 +/- 0.51)%, (7.83 +/- 0.67)%, (46.78 +/- 0.48)%, (44.69 +/- 0.83)%. Apoptosis rate of cells in TNF-alpha and EGF + TNF-alpha groups transfected by asODN was respectively higher than that in TNF-alpha and EGF + TNF-alpha groups transfected by scrODN (P < 0.01).

CONCLUSIONSEGF inhibits HaCaT KC apoptosis caused by TNF-alpha in a PPARbeta-dependent manner.

Apoptosis ; drug effects ; Cell Culture Techniques ; Cell Line ; Epidermal Growth Factor ; pharmacology ; Humans ; PPAR-beta ; genetics ; metabolism ; Transcription, Genetic ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors

OBJECTIVETo explore influence of oxidative stress reaction on apoptosis rate and expression of apoptosis-related genes bax and bcl-2 of enterocytes in severely burned rats with delayed resuscitation on the plateau.

METHODSOne hundred and twenty rats subjected to 30% TBSA full-thickness scald on the back were devided into plateau experimental group (PE, altitude 3840 m) and Lanzhou experimental group (LE, altitude 1517 m). Then LE and PE groups were subdivided into Lanzhou immediate fluid resuscitation group (LIFR, with immediate intraperitoneal injection of isotonic saline after scald, 40 mL/kg), Lanzhou delayed fluid resuscitation group [LDFR, with intraperitoneal injection of isotonic saline at 6 post scald hour (PSH), 40 mL/kg], and plateau immediate fluid resuscitation group (PIFR, with immediate intraperitoneal injection of isotonic saline after scald, 40 mL/kg), plateau delayed fluid resuscitation group (PDFR, with intraperitoneal injection of isotonic saline at 6 PSH, 40 mL/kg). Another 12 rats were divided into Lanzhou sham scald group (LS) and plateau sham scald group (PS), with 6 rats in each group. Rats in LS and PS groups were sham scalded in a water bath for 15 s without fluid infusion. Rats were sacrificed at 6, 12, 24, 48, 72 PSH for collection of small intestine samples to determine the contents of malonaldehyde (MDA) and total hydrosulfide (TSH). The apoptosis of enterocytes was determined by TUNEL, and the expression of bax and bcl-2 in epithelial cells were observed by immunohistochemical method. Intestinal sample of LS and PS groups were collected to determine the contents of MDA and TSH.

RESULTSAfter being scalded, content of MDA in intestinal tissue of rats in LDFR group and PDFR group was respectively greater than that in LIFR group and PIFR group (P < 0.05 or P < 0.01). Intestinal tissue content of MDA of rats in LDFR group (9.8 +/- 4.0 nmol/mg) and PDFR group (10.2 +/- 1.3 nmol/mg) was respectively greater than that in LIFR group (9.5 +/- 2.7 nmol/mg) and PIFR group (9.6 +/- 1.1 nmol/mg) (P < 0.05) at 24 PSH. After being scalded, intestinal content of TSH of rats in PE group and PDFR group was respectively smaller than that in LE group and LIFR group (P < 0.05). A multitude of brown positive apoptotic cells were observed in PDFR at 6 and 12 PSH. Absorbance values in LDFR group, PIFR group, and PDFR group were higher than that in LIFR group at each time point (P < 0.05 or P < 0.01). A multitude of bax positive cells were observed in intestinal mucous membrane in PDFR at 6 and 12 PSH. Expression of bal-2 was negative in PE group, and LIFR and LDFR groups at 6, 12 PSH, and it was weakly positive in LIFR and LDFR groups at 24, 48, 72 PSH.

CONCLUSIONSEnhancement of bax expression and weakening of bcl-2 expression in enterocytes are induced by the severe oxidative stress reaction in intestinal mucous membrane after burn with delayed resuscitation on the plateau, which may be one of the important reasons in inducing an increase in apoptosis of enterocytes.

Animals ; Apoptosis ; Burns ; metabolism ; Endothelial Cells ; cytology ; metabolism ; Enterocytes ; cytology ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Oxidative Stress ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; Resuscitation ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo evaluate the economic significance of Meek skin grafting and automicrografting combined with large piece of allogenous skin (micrografting in brief) in the treatment of patients with extensive deep burn.

METHODSTwenty-four patients with extensive deep burn admitted to the First Affiliated Hospital of Wenzhou Medical College were divided into Meek skin grafting group and micrografting group, with 12 patients in each group. Statistical comparison between Meek skin grafting group and micrografting group in respect of wound healing time, consumption of each special dressing, total cost of hospitalization, rehabilitation cost during convalescence was made. Then the cost and effect value was compared between two groups.

RESULTSThe wound healing time, consumption of each special dressing, total cost of hospitalization and rehabilitation cost in Meek skin grafting group was (14.4 +/- 1.9) d, yen(16 590 +/- 521), yen(421 628 +/- 145), yen(39 571 +/- 225), respectively, and that in micrografting group was (25.6 +/- 4.2) d, yen (136 441 +/- 356), yen(539 526 +/- 686), yen(55 853 +/- 794), respectively. The difference between two groups were statistically significant (P < 0.01).

CONCLUSIONSIn a definite range of burn size, Meek skin grafting has a lower therapeutic cost and better therapeutic effects as compared with micrografting.

Adolescent ; Adult ; Burns ; surgery ; Female ; Humans ; Male ; Middle Aged ; Skin Transplantation ; economics ; methods ; Surgical Flaps ; Young Adult

OBJECTIVETo investigate the effect of Thymosin and growth hormone(GH) on inflammatory response in burn rats or burn rats with sepsis.

METHODSSixty-four SD rats were randomly divided into normal control group (NC, without treatment), sepsis group (S, with injection of LPS), sepsis + Thymosin group (ST, with successive injection of Thymosin and LPS), sepsis + GH group [SGH, with successive injection of recombinant human GH (rhGH) and LPS], burn group, burn + sepsis group (BS, with injection of LPS after burn), burn + sepsis + Thymosin group (BST, with successive injection of Thymosin and LPS after burn), burn + sepsis + GH (BSGH, with successive injection of rhGH and LPS after burn), with 8 rats in each group. Specimens of spleen tissues were harvested to determine HLA-DR in lymphocyte and evaluate inflammatory cell infiltration (score). Specimens of peripheral blood were collected to determine Toll-like receptor 4 (TLR4) level in monocyte and serum level of TNF-alpha, IL-4, IL-6, IL-10.

RESULTSCompared with those in NC group, serum level of IL-10 in S group decreased obviously, while other indices increased obviously (P < 0.01). The levels of HLA-DR and TLR4 and serum level of TNF-alpha were similar between SGH and ST groups (P > 0.05). Compared with those in SGH group [(2.87 +/- 0.04) score, and IL-6 (0.0083 +/- 0.0018) microg/mg, IL-4 (0.0102 +/- 0.0021) microg/mg, IL-10 (0.0310 +/- 0.0027) microg/mg, respectively], degree of inflammatory cell infiltration (1.50 +/- 0.76) score and serum levels of IL-6, IL-4, IL-10 of rats in ST group decreased obviously (0.0064 +/- 0.0012, 0.0058 +/- 0.0024, 0.0230 +/- 0.0021 microg/mg, respectively, P < 0.01). The levels of HLA-DR, TLR4 and inflammatory cell infiltration degree of spleen in B group were respectively higher than those in NC group and lower than those in BS group. Compared with those in NC group, serum levels of TNF-alpha, IL-6 in B group increased significantly, while IL-4, IL-10 showed an opposite tendency. There was no obvious difference between BST and BSGH groups in serum levels of HLA-DR and IL-6 (P > 0.05). Compared with those in BST group, inflammatory cell infiltration degree in spleen and the levels of TLR, TNF-alpha obviously decreased (P < 0.01), while IL-4 and IL-10 levels increased in BSGH group (P < 0.01).

CONCLUSIONSInhibitive effects between Thymosin and GH on extensive inflammatory reaction were similar with or without trauma, and GH has better effect as compared with Thymosin when with trauma.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Burns ; immunology ; Human Growth Hormone ; pharmacology ; Inflammation ; immunology ; Male ; Rats ; Rats, Sprague-Dawley ; Sepsis ; immunology ; Thymosin ; pharmacology

OBJECTIVETo study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs).

METHODSADSCs were isolated from human abdominal adipose tissue and cultured. The immunophenotype and adipose induced-differentiation were identified, and the third generation cells were collected. The collected cells were assigned to 1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry.

RESULTSThe secretion amounts of VEGF and HGF of ADSCs in 1 x 10(-8) and 1 x 10(-7) mol/L insulin groups [(471 +/- 41, 762 +/- 66 ng/L), (643 +/- 64, 930 +/- 67 ng/L), respectively] were significantly higher as compared with those in control group (286 +/- 47, 577 +/- 84 ng/L) (P < 0.05 or P < 0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1 x l0(-6) mol/L insulin group (P > 0.05). The supernatant fluid of ADSCs' nutrient medium of 1 x 10(-8), 1 x 10(-7) mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 x 10(-7) mol/L group (P < 0.05 or P < 0.01).

CONCLUSIONSInsulin in the concentrations of 1 x 10(-8) and 1 x 10(-7) mol/L can notably promote ADSCs' function of secreting VEGF and HGF.

Adipocytes ; cytology ; drug effects ; secretion ; Cells, Cultured ; Fibroblasts ; cytology ; Hepatocyte Growth Factor ; metabolism ; Humans ; Insulin ; pharmacology ; Stem Cells ; cytology ; drug effects ; secretion ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo study the effect of hirudin on the function of human hyperplastic scar fibroblasts (HSFBs).

METHODSHSFBs were cultured in vitro. Hirudin solution in the concentration of 1, 10, and 50 kU/L was respectively added into DMEM culture medium to form 1, 10, and 50 kU/L hirudin groups, with 9 wells in each group. HSFBs cultured without hirudin were set up as control group. Cell inhibition rate, secretion level of TGF-beta1 from cells, and expression levels of mRNA of type I and III precollagen were determined at 24, 48, and 72 h after culture.

RESULTSInhibition rates of HSFBs growth was respectively (29.3 +/- 0.9)%, (30.1 +/- 0.3)%, and (45.2 +/- 1.9)% when cultured with 10 kU/L hirudin for 24, 48, and 72 hs, which were higher than those in control group [(0.0 +/- 0.0)%, P < 0.05]. There was statistically significant difference between control group and 1 and 50 kU/L hirudin groups in the inhibition rates of HSFBs at some time points (P < 0.05). Secretion level of TGF-beta1 of HSFBs in 1, 10, 50 kU/L hirudin groups was respectively (228.5 +/- 1.8), (210.5 +/- 11.1), and (168.5 +/- 14.1) pg/mL when cultured for 48 hs, of which the last 2 figures were significantly lower than that of control group [(265.0 +/- 1.5) pg/mL, P < 0.05]. Hirudin in the concentration of 10 and 50 kU/L could inhibit the expression of mRNA of type I and III precollagen in HSFBs.

CONCLUSIONSHirudin solution in the concentration of 10 and 50 kU/L can inhibit the proliferation of HSFBs and secretion of TGF-beta1 and collagen in certain degree.

Cells, Cultured ; Cicatrix, Hypertrophic ; pathology ; Fibroblasts ; cytology ; drug effects ; secretion ; Hirudins ; pharmacology ; Humans ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo establish an effective method of transfecting human marrow mesenchymal stem cells (MSC) with human vascular endothelial growth factor 165 (VEGF 165) gene.

METHODSMSCs isolated and cultured in vitro were divided into transfection group (pShuttle-CMV/VEGF 165 plasmid was transfected into MSCs through liposome-mediating method), empty plasmid group (pShuttle-CMV vehicle was transfected into MSCs as control), liposome group (liposome was transfected into MSCs as control) and control group (normal culture). Expressions of mRNA and protein of MSCs were determined by RT-PCR, enzyme-linked immunosorbent assay and Western Blot. Sensitivity to MSCs on VEGF plasmid transfection was detected by MTT test.

RESULTSExpression level of VEGF 165 gene mRNA in transfection group, empty plasmid group, liposome group, and control group was respectively 0.89 +/- 0.03, 0.34 +/- 0.04, 0.40 +/- 0.03, and 0.30 +/- 0.03, and the difference between transfection group and the other three groups was statistically significant (P < 0.01). Content of VEGF protein in transfection group, empty plasmid group, liposome group, and control group was respectively (778 +/- 35), (543 +/- 24), (561 +/- 28), (571 +/- 23) pg/mL, and the difference between transfection group and the other three groups was statistically significant (P < 0.01). In the transfection group, expression level of VEGF protein peaked on 7(th) day after transfection, which was decreased gradually later. In transfection group, expression level of VEGF 165 protein was obviously higher than that of the other three groups (P < 0.01), and no inhibitory effect of VEGF plasmid transfection on MSCs proliferation was found.

CONCLUSIONSThe method for transfecting human VEGF 165 gene into MSCs is established in this research, through which target gene and protein can express effectively.

Bone Marrow Cells ; cytology ; Cell Culture Techniques ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; Transfection ; Vascular Endothelial Growth Factor A ; genetics

OBJECTIVETo establish the tridimensional culture method for tissue-engineered skin to observe the histomorphological change in human immortal KC strain (HacaT)cocultured with xenogenic acellular dermal matrix (ADM).

METHODSThe ADM was prepared from SD rats by a modified method. HaCaTs were cultured in defined KC-serum free medium. HaCaTs in log growth phase were inoculated on ADM at the cell density of 2 x 10(5)/cm(2). They were submergedly cultured for 5 days and then changed to air-liquid phase culture for another 5 days. ADM and growth of HaCaTs on day 1 and 5 after cocultured with ADM were observed with scanning electron microscope. The histological change in ADM and HaCaTs on day 1, 5, and 10 after cocultured with ADM were examined by HE staining.

RESULTSThe gross appearance of ADM was white with smooth and soft texture, and intact collagen bundles without cellular residue. HaCaTs adhered and stretched out pseudopodia on the surface of the ADM on day 1 after combined culture, and a monolayer of cells was formed on day 5, growing into 3-6 layers of cells on day 10 with a tendency to grow into ADM.

CONCLUSIONSSD rats ADM is benefit for the adhesion of HaCaTs and the permeation of nutrient solution, from which an engineered multiple-layered human skin can be obtained within 10 days.

Animals ; Cells, Cultured ; Coculture Techniques ; Dermis ; cytology ; Humans ; Keratinocytes ; cytology ; Rats ; Rats, Sprague-Dawley ; Skin, Artificial ; Tissue Engineering ; methods

OBJECTIVETo compare the differences of the clinical effects, side effects and treatment-related cost between two kinds of negative-pressure wound therapy (NPWT).

METHODSForty-four inpatients with acute, subacute, and chronic wounds were divided into simplified NPWT group (A group) and conventional NPWT group (B group) according to the random number table. Wounds of patients in A group were treated with gauze + continuous suction with hospital central negative pressure (-10.64 kPa) for 24 hs; wounds of patients in B group were treated with sponge + interrupted suction with a purpose-designed suction appliance (-16.63 kPa) for 24 hs. Gross wound condition, treatment time, survival rates of skin graft and flap, changes of bacterial species on wound, treatment cost, and ratio of side effects between two groups were compared.

RESULTSThere was no significant difference between A and B groups in respect of gross wound condition, treatment time [A group (29 +/- 12) d, B group (26 +/- 13) d, P > 0.05], changes of bacterial species, survival rates of skin graft [A group (98 +/- 4)%, B group (98 +/- 4)%, P > 0.05] and flap (A group 98%, B group 100%, P > 0.05). Treatment cost of A group yen(374 +/- 134) was obviously lower than that of B group yen(9825 +/- 4956) (P < 0. 01), while more side effects were observed in A group (33.3%) than that in B group (5.0%) (P < 0.05).

CONCLUSIONSBoth simplified NPWT and NPWT with purpose-designed appliance can effectively improve wound healing. The simplified method may cause many side effects and has a potential risk of inciting nosocomial infection, but it can be conveniently employed with a low cost. In contrast, the cost of using purpose-designed appliance should be cut down to meet the aim of generalization.

Adolescent ; Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Negative-Pressure Wound Therapy ; methods ; Wound Healing ; Young Adult

OBJECTIVETo study the effects of negative-pressure wound therapy (NPWT) on the treatment of complicated and refractory wounds.

METHODSSixty-seven patients with complicated or refractory wounds admitted to our hospital from September 2005 to November 2008 were randomly divided into NPWT group (n = 35) and conventional treatment (CT) group (n = 32). Wounds of patients in NPWT group were treated with interrupted suction under a pressure of -16.63 kPa for 24 hs, or continuous suction under a pressure of -10.64 kPa for 24 hs. Wounds of patients in CT group were covered with petrolatum gauze overlaid with isotonic saline gauze and dry gauze. Duration of treatment, times of operation, treatment cost, and the process of healing were compared between two groups.

RESULTSThe duration of treatment, treatment cost and times of operation of patients in NPWT group were obviously less or fewer than those of CT group (P < 0.05). Wounds of patients in NPWT group were mainly healed by themselves (40.0%) or healed after free skin grafting (40.0%). While wounds in patients in CT group healed mainly after tissue flap transplantation (66.7%) or free skin grafting (23.3%).

CONCLUSIONSCompared with CT, NPWT can shorten the length of hospital stay, reduce operation frequency and treatment cost, and it is easier to carry out in the surgery of treating complicated and refractory wounds, which is worth generalization.

Adult ; Aged ; Diabetic Foot ; surgery ; Female ; Humans ; Male ; Middle Aged ; Negative-Pressure Wound Therapy ; Pressure Ulcer ; surgery ; Wound Healing