Effect of nonpeptide NK1 receptor antagonist L-703,606 on the edema formation in rats at early stage after deep partial-thickness skin scalding

OBJECTIVE@#To investigate the effect and the relevant potential mechanism of nonpeptide neurokinin 1 (NK1) receptor antagonist L-703,606 in the edema formation after burn injury.@*METHOD@#L-703,606 treatment was performed in Sprague-Dawley (SD) rats at early stage after deep partial-thickness skin scalding. One hundred and fifty two adult male SD rats were used in the study and randomly divided into sham scald (SS, n=8), scald control (SC, n=48), and L-703,606 treatment (LT, n=48) groups. The rats in SC and LT groups were subjected to 20% total body surface area (TBSA) deep partial-thickness skin scalding. Modified Evans blue extravasation, tracing electron microscopy by lanthanum nitrate and mean water content assay were employed to observe and detect the changes of vascular permeability, ultrastructure and edema formation in adjacent tissue to the wounds and in the jejuna of rats at early stage (72 h) after scald.@*RESULTS@#The pathological increase of vascular permeability in the periwound tissue and jejunum of rats in LT group were significantly lower than that in SC group (P<0.01), and recuperated earlier. Meanwhile, the changes of water contents of corresponding tissues in LT group were lighter than those in SC group (P<0.01). The ultrastructural changes of the microvessels in the peri-wound tissue of LT group showed that the junctions between microvascular endothelium cells were more narrow than those of SC group, moreover, and the number of opening and the engorgement and cavitation of the vascular endothelium cells decreased, the areosis and edema in perivascular tissue lightened, and the precipitation of the high eletron density lanthanum tracing agent in the interspace of the tissue decreased significantly in LT group.@*CONCLUSIONS@#It is concluded that nonpeptide NK1-receptor antagonist L-703,606 could lighten the vascular permeability and edema formation in the periwound tissue and jejunum, and accelerate the normalization process of pathological changes in the tissues of rats after scald.

Construction of the tissue engineering seed cell (HaCaT-EGF) and analysis of its biological characteristics

OBJECTIVE@#To construct the tissue engineering seed cell (HaCaT cell line) with stable expression of the human epidermal growth factor (EGF), and analyze the changes of its biological characteristics.@*METHODS@#PCDNA3.1-EGF eukaryotic expression vector was transferred into HaCaT cell, and G418 was utilized to select the HaCaT-EGF cell line. Using an inverted microscope, PCR, ELISA method to detect the changes of the cell morphology, the expression of the EGF gene and protein, and the mRNA expression levels of apoptosis related molecule Caspase-3, the cell cycle related protein cyclin D1.@*RESULTS@#The mRNA expression levels of the obtained HaCaT-EGF cell were more than 100 times higher than the level of ordinary HaCaT cell. The colony of the HaCaT-EGF cells was more focused and tight compared to the empty vector transfected HaCaT cells and normal HaCaT cells. The expression levels of apoptotic factor Caspase-3 and cyclin D1 in HaCaT-EGF cell were significantly higher than those in the empty vector HaCaT- pcDNA3.1 cell, and the differences were statistically significant (P0.05).@*CONCLUSIONS@#HaCaT-EGF cell can continuously secrete EGF, and the biological characteristic is stable. It can be used for tissue engineering experiment and is an ideal seed cell for constructing tissue engineered skin.

Effects of Angelica dahurica extract on biological behavior of dermal fibroblasts / 中华外科杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>At 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).</p><p><b>CONCLUSIONS</b>Angelica 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.</p>

Effects of myrrh extract on proliferation and collagen mRNA expression of human fibroblasts in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effects of myrrh extract on biological characteristics of human dermal fibroblasts (Fb), and to explore its possible mechanisms in promoting wound healing.</p><p><b>METHODS</b>Normal Fb was isolated from human foreskin tissue and cultured in vitro. The third to fifth passages of Fb were used in the experiment. (1) Fb were planted onto 96-well plate and divided into control group, and 1 × 10(-4), 1 × 10(-3), 1 × 10(-2), 1 × 10(-1), 1, 10, 1 × 10(2) g/L myrrh water extract groups and myrrh ethanol extract groups according to the random number table. Fb in control group were cultured with DMEM medium containing 0.25% calf serum (briefly called low-concentration serum medium), and those in various concentrations of myrrh water extract and myrrh ethanol extract groups respectively with low-concentration serum medium containing corresponding concentration of 2 kinds of myrrh extract. After being cultured for 48 h, cell morphology was observed with inverted-phase contrast microscope, and Fb proliferation activity (denoted as absorbance value) was determined with MTT method. (2) Fb were respectively planted into flasks and dishes and divided into two groups according to the random number table. Fb in control group were cultured with low-concentration serum medium, and that in 1 g/L myrrh water extract group with low-concentration serum medium containing 1 g/L myrrh water extract. After being cultured for 72 h, Fb cell cycle and the type I and III collagen mRNA expression were respectively determined by flow cytometry and real-time fluorescent quantitative PCR. Data were processed with LSD-t test.</p><p><b>RESULTS</b>(1) Fb in all groups grew in long-spindle shape, but the cell fusion was much obvious in 1 g/L myrrh water extract group than in control group. Fb absorbance value in 1 × 10(-3), 1 × 10(-2), 1 × 10(-1), 1, 10 g/L myrrh water extract groups was respectively 0.378 ± 0.032, 0.402 ± 0.007, 0.390 ± 0.038, 0.453 ± 0.036, 0.390 ± 0.037, all higher than that in control group (0.332 ± 0.044, with t value respectively 2.24, 2.93, 2.69, 5.73, 2.71, P values all below 0.05). Compared with that in control group, Fb absorbance value in 1 × 10(-4) g/L myrrh water extract group was not statistically different (0.312 ± 0.048, t = 2.84, P > 0.05), while that in 1 × 10(2) g/L myrrh water extract group was significantly lower (0.154 ± 0.009, t = 7.17, P < 0.05). Fb absorbance values in 1 × 10(-3), 1 × 10(-1), 1, 10, 1 × 10(2) g/L myrrh ethanol extract groups were significantly lower than that in control group (with t values from 2.30 to 24.79, P values all below 0.05). (2) Compared with those in control group [(82.2 ± 7.9)% and (13.3 ± 2.3)%, (4.5 ± 0.8)%], the percentage of cells in G0/G1 phase in 1 g/L myrrh water extract group was obviously decreased [(74.3 ± 6.3)%, t = 6.77, P < 0.05], while those in S and G2/M phases increased [(16.6 ± 3.4)%, (9.1 ± 1.6)%, with t value respectively 7.53, 6.34, P values below 0.05]. Compared with those in control group (1.00 ± 0.05, 1.00 ± 0.06), the mRNA level of collagen III in 1 g/L myrrh water extract group was significantly up-regulated (1.38 ± 0.12, t = 3.81, P < 0.01), while that of collagen I was not statistically different (0.89 ± 0.08, t = 1.17, P > 0.05).</p><p><b>CONCLUSIONS</b>Myrrh water extract can notably promote the proliferation of Fb, accelerate the cell cycle of Fb, and up-regulate the mRNA expression of type III collagen in Fb, which may be related to its mechanisms in promoting wound healing.</p>

Different expression of protein in the supernatant of heat injured keratinocytes / 中华整形外科杂志

<p><b>OBJECTIVE</b>To compare the difference of protein expression in the supernatant of heat injured keratinocytes (KC) and normal KC.</p><p><b>METHODS</b>A model of heat injured KC was produced in vitro. The supernatant of normal KC and heat injured KC was collected after culture for 12 hours, and was ultrafiltered and lyophilized to get the protein. The protein sample was separated by immobilized pH gradient based two dimensional gel electrophoresis (2-DE). The gel was stained and the different expression of protein was analyzed using ImageMaster 2D analysis software.</p><p><b>RESULTS</b>(1) Average protein spots were 1,898 +/- 113, 1,877 +/- 97 in the supernatant of normal and heat injured KC and 1,118 protein spots could be used for statistical analysis. (2) Statistical result showed that 26 protein spots were significantly different between the two groups. 16 protein spots were higher in the supernatant of normal KC and then 10 protein spots were lower in the normal group. (3) 16 protein spots, which included 10 kinds of proteins, were identified successfully as different spots. Lower expression proteins were alpha-enolase, actin cytoplasmic 2, peroxiredoxin-4, phosphoglycerate mutase 1, G protein-regulated inducer of neurite outgrowth l in the supernatant of heat injured KC. Higher expression proteins in heat KC were purine nucleoside phosphorylase, tumor necrosis factor ligand superfamily member 10, proteasome subunit alpha type-7, UDP-glucose 6-dehydrogenase in the supernatant of heat injured KC.</p><p><b>CONCLUSIONS</b>The result indicated that there are some significant different expression proteins in the supernatant of normal KC and heat injured KC. These findings provide new data for screening major molecules of tissue repair and finding the mechanism of wound repair.</p>

Effect of heat injured keratinocytes supernatant on biological behavior of fibroblasts / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effect of the supernatant of heat injured keratinocytes (KC) on biological behavior of the dermal fibroblasts (Fb).</p><p><b>METHODS</b>Human dermal Fb were isolated and cultured. A model of heat injured KC (HaCaT) was reproduced in vitro. Supernatant of normal KC and the supernatant of KC culture 12 hours after heat injury were collected and diluted with non-serum DMEM in 1:1 volume ratio to make normal KC conditioned medium (NKCM) and heat injury KC conditioned medium (HKCM) respectively. Fb was respectively treated with non-serum DMEM and 2 kinds of conditioned medium. (1) The proliferation of Fb was detected with MTT method at post culture hour (PCH) 12, 24, 36, 48. (2) The apoptosis of Fb was determined by flow cytometry at PCH 12 (Fb were heat injured in advance; Fb without heat treatment was used as control). (3) At PCH 24, expression of a-SMA in Fb cytoplasm was determined with immunofluorescence method; expression of a-SMA mRNA in Fb was determined with real-time quantified PCR. Data were processed with one-way analysis of variance, and pairwise comparison among groups with LSD-t test.</p><p><b>RESULTS</b>(1) The proliferation of Fb: the absorbance value of Fb cultured with HKCM at PCH 12, 24, 36, 48 was respectively higher than that of Fb cultured with non-serum DMEM (with t value respectively 1.89, 2.35, 2.02, 1.94, and P values all below 0.01). There were significant statistical differences between the absorbance values of Fb cultured with HKCM and those of Fb cultured with NKCM at PCH 12, 24, and 48 (at PCH 12, t = 1.83, P < 0.01; at PCH 24, t = 2.91, P < 0.05; at PCH 48, t = 1.83, P < 0.05). (2) Apoptosis of Fb cultured with HKCM was diminished as compared with that of Fb cultured with NKCM and of Fb without treatment (t = 3.31, P < 0.05; t = 1.47, P < 0.01). (3) The expression of alpha-SMA (red fluorescence) in Fb cultured with non-serum DMEM or NKCM was less as seen under fluorescence scope, and it was obviously increased in Fb cultured with HKCM. (4) The relative expression amount of alpha-SMA mRNA in Fb cultured with HKCM was 1.32 +/- 0.06, which was higher than that both in Fb cultured with NKCM (1.14 +/- 0.07, t = 2.51, P < 0.05) and in Fb cultured with non-serum DMEM (1.00 +/- 0.09, t = 1.77, P < 0.05).</p><p><b>CONCLUSIONS</b>The supernatant of KC 12 hours after heat injury can obviously promote the proliferation of Fb, inhibit its apoptosis and accelerate transdifferentiation of Fb to myofibroblasts.</p>

Protective effect of melatonin on oxidative stress inducing hair follicle injury in scald rat / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of melatonin on residual hair follicle cells of scald rats at early stage.</p><p><b>METHODS</b>Eighteen male Sprague-Dawley rats were randomly divided into scald group, treatment group, sham group , with 6 rats in each group. The rats in scald group and treatment group were subjected to 30% TBSA partial thickness scald on the back, and were resuscitated with balanced solution after 1 hour, while those in sham group were immersed in water at 37 degrees C for 25 s to simulate scald, and did not receive fluid replacement. Rats in treatment group were intraperitoneally injected with 10 mg/kg melatonin solution at 1 minute, 8 hours and 12 hours after scald, while those in sham group and scald group were given equal volume of 1% alcohol sodium-isotonic saline instead. Tissue samples were harvested at 6, 12 and 24 post scald hours (PSH) for determination of MDA and GSH levels. Apoptosis of residul hair follicle was detected by TUNEL method and immunohistochemistry of caspase-3.</p><p><b>RESULTS</b>The level of MDA in scald group at each time point was much higher than that in sham group (P < 0.01) and treatment group (P < 0.05), and it peaked at 12 PSH. The changes in GSH were just opposite to that of MDA. Under fluorescence microscope, the residual hair follicle cells were blue, and the apoptotic cells appeared green. The apoptosis rate in scald group at 6, 12, 24 PSH was obviously higher than that in sham (P < 0.01) and treatment groups (P < 0.05), which was (20.2 +/- 3.4)% vs (4.3 +/- 2.3)% vs (10.9 +/- 3.2)%, (31.2 +/- 3.6)% vs (5.1 +/- 2.5)% vs (19.1 +/- 3.7)%, (22.4 +/- 2.7)% vs (4.1 +/- 2.4)% vs (13.1 +/- 3.4)%, respectively. The score of caspase-3 positive cell in scald group was higher than those in sham group (P < 0.01) and treatment group (P < 0.05).</p><p><b>CONCLUSIONS</b>There is obvious correlation between oxidative stress and apoptosis rate of hair follicle cells in rats with partial thickness scald. Early administration of melatonin may have anti-apoptosis ability for residual hair follicle cells by attenuation of oxidative stress.</p>

Reproduction of a model of heat injured keratinocyte in vitro and observation on its apoptosis rate / 中华烧伤杂志

<p><b>OBJECTIVE</b>To reproduce a model of heat injured KC in vitro and explore its apoptosis rate of KC due to heat injury at different temperature.</p><p><b>METHODS</b>Human KCs were cultured in vitro, and they were incubated at 37, 41, 43, 45, 48, and 51 degrees C respectively for 10 mins in water bath. Trypan blue staining and Hoechst 33258 fluorescence staining were used respectively to determine necrosis and apoptosis of KC. Rates of apoptosis and necrosis of KC were analyzed quantitatively by flow cytometer. The proliferation activity of KC after heat injury was detected by MTT test.</p><p><b>RESULTS</b>The results of trypan blue staining, Hoechst 33258 fluorescence staining, and flow cytometer demonstrated that number of apoptotic and necrotic KC increased gradually along with a rise of water bath temperature. The rates of apoptosis and necrosis of KC were respectively (12.3 +/- 3.2)% and (14.1 +/- 1.6)% at 45 degrees C, (27.7 +/- 5.1)% and (58.0 +/- 4.2)% at 48 degrees C. Rate of KC necrosis reached up to (83.0 +/- 5.3)% at 51 degrees C. Inhibition of KC growth reached a stationary phase when the injurious temperature reached 45 degrees C as observed with MTT test.</p><p><b>CONCLUSIONS</b>Heat injury can induce apoptosis and growth inhibition of KC in vitro. Incubating KC at 45 degrees C for 10 mins is a good condition to reproduce a model of heat injured KC in vitro. This model may be used to study the biological character and apoptosis of KC after burn injury.</p>

Effects of insulin on the growth factor secreting function of adipose-derived stem cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs).</p><p><b>METHODS</b>ADSCs 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>Insulin 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.</p>

The protective effect of intensive insulin treatment on the myocardium in severely scalded rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the protective effect of intensive insulin treatment on the myocardium of severely scalded rats, and to primarily explore its mechanism.</p><p><b>METHODS</b>Eighteen SD rats were divided into three groups, with 6 rats in each group. The rats in burn and intensive insulin group were inflicted with 30% TBSA full-thickness injury on the back. Isotonic saline containing 0.12 U/ml insulin solution, and 100 g/L glucose solution were infused into the rats in the intensive insulin group to keep plasma glucose at the level of 4.0 - 6.6 mmol/L (the total fluid amount was 2 ml x kg(-1) x 8h(-1)). In sham burn group,fluid was given according to physiological demand. The same amount of isotonic saline was infused into the rats in burn group. The venous blood was obtained for the detection of plasma glucose contents, and the left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) were recorded via aortic ventricle cannula before scald and at 1, 2, 3, 4, 5, 6 post-scald hours (PSH). The tissue of the left ventricle was harvested at 6 PSH for the detection of troponin T expression in myocardiocytes.</p><p><b>RESULTS</b>Plasma glucose level was increased to (7.6 +/- 1.7) mmol/L - (8.4 +/- 4.7) mmol/L in burn group during 1-6 PSH, which was significantly higher than that in intensive insulin group (4.5 +/- 0.9) mmol/L - (5.2 +/- 1.3) mmol/L, P < 0.01). Compared with the intensive insulin group, LVSP was markedly decreased in the burn group (60 +/- 11 mm Hg vs 72 +/- 8 mm Hg, P < 0.05) at 1 PSH,whereas LVEDP was increased significantly (21.3 +/- 11.3 mmHg vs 11.7 +/- 5.2 mmHg, P < 0.05). Intensive insulin treatment could significantly inhibit the loss of troponin T protein in myofilaments of myocardium.</p><p><b>CONCLUSION</b>Intensive insulin treatment possesses a protective effect on myocardia function after severe burns, and it may be related to its preventive effect on the loss of contractile protein in cardiocytes.</p>

Primary observation on construction of skin with human hair follicle bulge cells with tissue engineering technique / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of fabricating tissue engineering skin with human hair follicle bulge cells (HFBCs) to repair full-thickness skin wound.</p><p><b>METHODS</b>HFBCs and dermal papilla cells (DPCs) isolated from human fetal hair follicles by collagenase digestion were cultured, purified and passaged. PGA-collagen scaffolds as bioengineered dermis were randomly divided into A and B groups. The HFBCs and DPCs (1 : 2) were seeded in scaffolds of group A and the equal amount of DPCs was seeded in scaffolds of group B as control. Then the keratinocyte sheets were seeded onto the surfaces of the scaffolds as bioengineered epidermis. The tissue engineering skins were then transplanted to repair the full-thickness wound on the back of nude mice. The wound healing process was observed and the plant histological changes of the transplanted engineered skin was observed with light microscope on 2, 4, 6 post-operation weeks (POW).</p><p><b>RESULTS</b>The full-thickness defect of nude mice in A and B groups could be effectively repaired by bioengineered skins. On 2 POW, integral epidermal and dermal structures were observed in the wounds in A and B groups, with thin epithelial layer and basement membrane. On 4 POW, epithelial layer became thickening and rete pegs formation was observed in basement membrane in A group, but only thickening of epithelial layer was observed in B group. On 6 POW, rete pegs structure was seen to descend and hair-follicle-like structure was formed, while only thickened epithelial layer with flat basement membrane were formed in B group.</p><p><b>CONCLUSION</b>From the composite skin engineered with PGA-collagen hybrid scaffolds and keratinocytes, HFBCs and DPCs could effectively repair the full-thickness skin defect of nude mice. The hair follicle stem cells participate in the process of anatomic repair of wound, and might be able to induce the repair of skin structure and function.</p>

The protective effect of early insulin treatment on vascular endothelial cells in severely scalded rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of insulin on vascular endothelial cells of rats at early post-burn stage,and its mechanism.</p><p><b>METHODS</b>Adult male Sprague-Dawley rats were randomly divided into 3 groups: i. e, sham scald group (n = 7), scald group (n = 7) and treatment group (n = 7). The rats in the latter 2 groups were subjected to 30% TBSA full-thickness burns with 94 degrees C water, and the sham scald rats were treated with 37 degrees C water. Intra-peritoneal injection of 40 ml/kg isotonic saline solution and subcutaneous injection of 3 units/kg insulin were given to the rats in treatment group after being subjected to 30% TBSA full-thickness burns. Subcutaneous injection of equal amount of isotonic saline was given to the sham and burn groups. The changes in vascular endothelial cell structure were observed with electron microscopy at 24 post-scald hours(PSH). Meanwhile, the blood glucose contents, the serum levels of nitric oxide (NO) and nitric oxide synthetase (NOS) were determined with oxidase method and colorimetric method, respectively.</p><p><b>RESULTS</b>The injury of arterial endothelial cells in the treatment group was obviously alleviated compared with that in burn group. The blood glucose content in the treatment group (7.1 +/- 0.7 mmol/L) was significantly lower than that in scald group (8.2 +/- 1.0 mmol/L, P < 0.05), though it was much higher in both groups than that in sham scald group (4.9 +/- 0.8 mmol/L, P < 0.01) at 24 PBH. The serum content of NO, total NOS and cNOS in treatment group were obviously higher than those in scald group (P < 0.01), but there was no obvious difference in iNOS content between the two groups(P > 0.05).</p><p><b>CONCLUSION</b>Insulin exhibits protective effect on vascular endothelial cells in severely scalded rats at the early post-burn stage, and it is attributed to its promotion of cNOS level leading to NO production.</p>

Protective effect of insulin on oxygen-radical induced hepatic injury in severely scalded rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the protective effect of insulin on oxygen-radical induced hepatic injury in severely scalded rats in early stage of severe scald.</p><p><b>METHODS</b>Eighty-four male Sprague-Dawley rats were randomly divided into three groups: i. e, normal group, saline group, and insulin group, with 28 rat in each group. The rats in the latter two groups were subjected to 30% TBSA full-thickness scald on the back, and received intra-peritoneal injection of 40ml/kg isotonic saline, and subcutaneous injection of 3 IU/kg insulin, respectively. The total anti-oxygen capability (T-AOC), the expression of superoxide dismutase (SOD), reactive oxygen species (ROS) and intercellular adhesion molecule (ICAM-1) in hepatic tissue, and serum alanine transaminase (ALT) were determined in each group at 6, 12, 24, 48 post-scald hours (PSH) with corresponding methods.</p><p><b>RESULTS</b>The hepatic T-AOC and SOD content were obviously decreased, while the ROS content were markedly increased at 6 PSH in saline group compared with that in normal group (P < 0.05 or P < 0.01). The expression of ICAM-1 and serum content of ALT were significantly higher than that in normal group at 12 PSH and 48 PSH (P < 0.01). At 24 PSH, the hepatic T-AOC (386 +/- 75) U/g and SOD content (210 +/- 39 ) U/g were obviously higher in insulin group than those in saline group [(124 +/- 18), (111 +/- 9) U/g, respectively, P < 0.01), but the ROS content (154 +/- 29 ) U/g was much lower than that in saline group [(351 +/- 41) U/g, respectively, P < 0.01]. At 48 PSH, the serum content of ALT and hepatic expression of ICAM-1 in insulin group exhibited obvious difference when compared with those in saline group (P < 0.01). Meanwhile, Pathological examination showed that hepatic injury was alleviated by insulin administration after scald.</p><p><b>CONCLUSION</b>Insulin administration early after severe scald exhibits protective effect on liver function by improving anti-oxygen radical ability of rat liver.</p>

Primary study on the culture of human fetal follicle bulge cells and their differentiation into sebaceous gland / 中华烧伤杂志

<p><b>OBJECTIVE</b>To develop a rapid and reproducible method for the culture of human fetal hair follicle bulge cells, and observe the plasticity of its differentiation into sebaceous gland in vitro.</p><p><b>METHODS</b>The bulge cells isolated from fetal human hair follicles by enzymatic digestion (digestion method) and manual microdissection (conventional method) were cultured and passaged respectively, the efficiency and biological features of cells were investigated , the clone forming efficiency was assayed by MTT, and the expression of K19 was further compared by immunocytochemistry (ABC). The morphological change and the expression of EMA of bulge cells were also observed after induction.</p><p><b>RESULTS</b>By conventional method, 8-10 bulges were harvested in one hour, 40%-50% of their cells were found to adhere to the culture plate after culturing for 48h, and they became confluent after 14 days. In comparison, about 100 bulges were harvested in one hour by digestion method, the adherence efficiency of their cells was 30% after cultivation for 12h and became confluent after 7 days. The cells grew larger with time, with irregular shape and droplets of lipid around the nucleus. The clone forming efficiency of bulge cells cultured by digestion method was (18.2 +/- 2.1) %, which was much higher than that of cells obtained by conventional method[ (12.7 +/- 3.4) %, P < 0.05]. Immunocytochemistry staining showed that positive staining of K19 was observed in most of the bulge cells, with a large amount of brown granules in the cytoplasm.</p><p><b>CONCLUSION</b>Human hair follicle bulge cells can be efficiently cultured and multiplied in vitro, and they retained the characteristics of stem cells. And they have the potential to differentiate into sebaceous glands by induction in vitro.</p>

The therapeutic effect of nanometer silver impregnated dressing on gunshot wounds after being immersed in brine and tapwater in rabbits / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the therapeutic effect of nanometer silver impregnated dressing on gunshot wounds after being immersed in brine and tapwater in rabbits.</p><p><b>METHODS</b>Rabbits were randomly divided into two groups after receiving gunshot wounds in both lower limbs. In group 1, the wounded limbs on the experimental side were immersed in brine for 5 h; in group 2, the wounded limbs on experimental side were immersed in tapwater for 5 h. All the wounds were treated with nanometer silver impregnated dressing on the experimental sides, while those of the control sides were treated with vaseline dressing. Biopsy was done after 30 min and 1, 2, 3, 4, 5 h, respectively.</p><p><b>RESULTS</b>In group 1, the onset of inflammation around the wounds of the experimental sides was delayed, the inflammatory response was less serious, and the wounds were dry with less exudation compared to the controls. The mean healing time of the entry wounds on experimental and control sides was (29.4 +/- 6.6) d and (36.3 +/- 6.0) d (P < 0.01), respectively, and that of the exit wounds on experimental and control sides was (20.1 +/- 6.0) d and (27.3 +/- 5.7) d (P < 0.01), respectively. In group 2, only one of the experimental wounds showed mild inflammation, while all of the control wounds showed serious inflammation with much exudation. The mean healing time of the entry wounds on experimentsides was (13.0 +/- 1.52) d, while that on control sides was (16.0 +/- 3.10) d (P < 0.01). The mean healing time of exit wounds on experimental sides was (11.0 +/- 2.75) d, and those of the control sides was (15.6 +/- 2.85) d (P < 0.01).</p><p><b>CONCLUSION</b>The nanometer silver impregnated dressing can control infection and accelerate healing in gunshot wounds in rabbits.</p>

Experimental study and clinical application of composite skin grafting / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore an ideal skin substitute with its appearance and texture similar to normal skin, to repair wounds with full-thickness skin defect.</p><p><b>METHODS</b>Composite skin (CS) in question was composed of allo/xenogeneic acellular dermal matrix (ADM) and razor thin autoskin. One step skin grafting was employed in the experimental study and clinical trial. Razor thin autoskin alone was used as the control in the study. Changes in the antigenicity of ADM and the reformation of basement membrane (BM) structure at epidermis-dermis junction (EDJ) of ADMs were studied at designated time points after the grafting with biochemical and immunohistochemical methods. Fifty-three patients with full thickness skin defects due to various causes, including scar excision were grafted with CS, and survival rate and long-term result were observed.</p><p><b>RESULTS</b>The grafted CS survived satisfactory. The reformation of the basement membrane structure was clearly observed at the 28th post-graft week. The basement membrane cells grew with polarization in an undulating arrangement. There was reformation of dermal papillae and ridges. The antigenicity of allo-ADM was obviously lower than that of xeno-ADM. Sixty-five out of 70 pieces of CS grafting (92.9%) survived totally, two of them survived partially, and three failed due to infection. The longest follow-up period was 8 and a half years. The grafted CS appeared similar to the normal skin in regard to the texture and color, especially allo-ADM, and no evident rejection reaction was seen.</p><p><b>CONCLUSION</b>ADM possessed very low antigenicity, thus serving a lasting framework after grafting. In addition, it could serve as a "dermal template" for the induction of tissue regeneration.</p>

Establishment of rat model of scalding with high pressure steam / 中华烧伤杂志

<p><b>OBJECTIVE</b>To establish a rat model of scalding with controllable depth and area by high pressure steam.</p><p><b>METHODS</b>High pressure steam apparatus consisting of an autoclave and a self-made scalding frame was employed in the study. The rats were inflicted with scalding with 0.12 Mpa (1 Mpa = 7500 mmHg) high pressure steam on the back through a hole of 2.6 cm in diameter for 3, 4, 5, 6, 7, 8, 9 and 10 seconds, with five wounds at each time point. The tissue samples were harvested at 24 post injury hour (PIH) for pathomorphological examination. The depth of scald was measured, and injury to the sweat gland and hair follicles injury, the hair growth in scalded area, and the wound healing condition were observed with Photoshop software.</p><p><b>RESULTS</b>There was positive correlation between the scalding depth and scalding time. The injury time for superficial and deep partial thickness burn and full thickness burn were 3, 5 and 7 seconds respectively. The wound healing time was similar even the scalding became more and more serious when injury time increased from 7 to 10 seconds.</p><p><b>CONCLUSION</b>The scalding depth and area in this model could be controlled, and the degree of scald injury could be graded accurately with easy manipulation. The result showed that it was an ideal model of skin burn wound.</p>

The in vitro isolation, culture and transfection of human fetal epidermal stem cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection.</p><p><b>METHODS</b>The HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal fibroblasts. The cultured cells were identified by immunohistochemistry staining of keratin-19 and integrin-beta1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial growth factor 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope.</p><p><b>RESULTS</b>HFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-19 and integrin-beta1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence.</p><p><b>CONCLUSION</b>HFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen. It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector.</p>

Long-term in vitro culture of the fibroblasts from the deep partial thickness burn wound in burn patients / 中华烧伤杂志

<p><b>OBJECTIVE</b>To establish a long-term in vitro culture of the fibroblasts obtained from burn wounds.</p><p><b>METHODS</b>Skin samples were harvested from normal volunteers and the deep partial thickness burn wound in burn patients on the 5th, 10th, 21st, 28th and 35th postburn days (PBDs). The non-dermal tissue was removed from the samples and primed by chlorhexidine solution in concentration of 2.5 g/L. The skin sample was then digested by trypsin-EDTA in concentration of 1.25 g/L and was centrifuged before the cells were harvested and cultured. When the cells grew nearly to form sheet, multiple passage culture, freezing storage and revivification were carried out with routine methods. The cell morphology was continuously observed during the culture. And the cell doubling time was calculated.</p><p><b>RESULTS</b>The wound-origin fibroblasts exhibited higher purity and better activity. The cellular growth features and gross morphology kept stable during primary and secondary culture, and during freezing storage and after revivification. The cells kept their activity above 80% of their original after many times of revivification.</p><p><b>CONCLUSION</b>The establishment of the in vitro culture of fibroblasts from burn wounds might be useful in the exploration of the pathogenesis and therapeutic measures of scars.</p>

An experimental study on the effects of wild type p16 gene on the proliferation and metabolism of human keloid fibroblasts / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effects of wild type p16 gene on the proliferation and metabolism of human keloid fibroblasts.</p><p><b>METHODS</b>Eukaryotic expression vector pcDNA3-p16 was constructed and imported into KFb by gene transfection mediated by liposome. And the positive clones were screened by G418. The transfected and untransfected KFbs were stained by Immunocytochemical method. The expression of p16 protein was observed. The changes of the proliferation and DNA synthesis of KFb before and after transfection were observed and compared by drafting cell growth curve and by (3)H-TdR incorporation method.</p><p><b>RESULTS</b>The recombinant vector pcDNA3-p16 was successfully constructed and identified by enzyme digestion. The positive clones were identified by G418 selection for 10 days from transfected KFb and with p16 protein expression. The growth rate of transfected KFb slowed down obviously and its DNA synthesis decreased significantly (P < 0.05) when compared with those of normal KFb.</p><p><b>CONCLUSION</b>p16 gene might inhibit the growth and DNA synthesis of KFb.</p>