Effects of adipose-derived stem cells on renal injury in burn mice with sepsis / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effect of adipose-derived stem cells (ADSC) on renal injury in mice with burn injury and sepsis and its underlying mechanism.</p><p><b>METHODS</b>(1) Adipose tissue was collected from both inguinal regions of 5 C57BL/6J mice to isolate, culture and purify ADSC through enzyme digestion, density gradient centrifugation, and adherence method. Cells of the third passage were used in the experiment. The morphologic change in cells was observed and the growth curve of cells was determined. The expression of cell surface antigen phenotype was analyzed by flow cytometry, and the cells were identified by adipogenic and osteogenic differentiation. (2) Another 37 C57BL/6J mice were divided into normal control group (n = 5), saline group (n = 16), and group ADSC (n = 16) according to the random number table. The mice in saline group and group ADSC were injected with Pseudomonas aeruginosa after being subjected to 15% TBSA full-thickness burn on the back to reproduce septic burn model. Then the mice were injected with saline and ADSC through tail vein respectively. At post burn hour (PBH) 12, 24, 48, and 72, the pathological change in kidney tissue was observed, the levels of blood urea nitrogen and serum creatinine were determined, and the levels of TNF-α, IL-12, IL-10, and cyclooxygenase-2 (COX2) mRNA were determined with real-time fluorescence quantitative PCR in both groups. Above-mentioned indexes were also examined in the normal control group (without burn). Data were processed with multifactor analysis of variance and LSD- t test.</p><p><b>RESULTS</b>(1) Cells in the third passage were orderly arranged with the shape similar to fibroblasts. The percentages of CD90(+), CD105(+), CD34(-), and CD45(-) cells were all above 90%. The cells could differentiate into osteoblasts and adipocytes. The cells were identified to be ADSC. (2) From PBH 12 to PBH 72, the neutrophil infiltration gradually increased, and the structure of kidney tubules and glomeruli were deranged in saline group. The pathological change in kidney tissue in group ADSC was less serious than that of normal control group at each time point. From PBH 12 to PBH 72, the levels of blood urea nitrogen and serum creatinine in saline group were significantly higher than those of normal control group and group ADSC (P values all below 0.01). Compared with those of the normal control group, the levels of TNF-α and IL-12 mRNA were higher in group ADSC and saline group at PBH 24 (P values all below 0.05). At PBH 24, the level of TNF-α mRNA in group ADSC (1.58 ± 0.19) was lower than that of saline group (3.36 ± 0.30, P < 0.05). At PBH 24, the levels of IL-10 and COX2 mRNA in group ADSC (2.89 ± 0.47, 4.90 ± 0.59) were higher than those in normal control group (1.00 ± 0.15, 1.00 ± 0.27) and saline group (1.32 ± 0.38, 1.57 ± 0.38, P values all below 0.05).</p><p><b>CONCLUSIONS</b>ADSC can decrease the levels of blood urea nitrogen and serum creatinine, promote the production of anti-inflammatory cytokines IL-10 and COX2, and reduce the release of the pro-inflammatory cytokines TNF-α and IL-12 to offer protective effects against renal injury in burn mice with sepsis.</p>

Reproduction of a mouse model of deep partial-thickness scald and determination of hypoxia in the wound / 中华烧伤杂志

<p><b>OBJECTIVE</b>To reproduce a stable mouse model of deep partial-thickness scald and to determine the hypoxia status in the wound.</p><p><b>METHODS</b>(1) A homemade scald-producing apparatus with constant steam (92 °C) emission was used to reproduce scald injury on the back (2 cm in diameter) in 80 male BALB/c mice for different duration (2, 4, 6, and 8 s), with 20 mice for each scald duration. The nozzle was aligned perpendicularly to the back of mice, 2 cm above the skin surface. The gross condition of wound was observed with naked eyes immediately after injury. Skin samples of 5 mice with different burn duration were harvested 0, 12, 24, and 48 h after scald for histopathological observation with hematoxylin and eosin staining, to screen the scalding time and time for biopsy of scalded skin to determine proper scalding time for the experiment. (2) Model of deep partial-thickness scald was reproduced with the desired scalding time as shown in the preliminary experiment in another 5 BALB/c mice. The hypoxia status in subcutaneous tissue was observed with immunohistochemical staining 72 h after scald. Another 20 BALB/c mice were divided into normal control group (n = 5, without scald) and deep partial-thickness scald group (n = 15, scalded for a suitable duration as determined in the preliminary experiment) according to the random number table. The subcutaneous oxygen content in wound center, the margin of the wound, and the normal skin adjacent to the wound was detected with laser Doppler transcutaneous oxygen tension 72 h after scald, with 5 mice in each region. Data were processed with one-way analysis of variance.</p><p><b>RESULTS</b>(1) The wound of mice with different scald durations was pale, clean, and no exudate was observed right after injury. (2) The burn depth developed gradually along with the scalding time and sample harvesting time, and it became stable 24 h after scalding. A deep partial-thickness injury was observed in the dermis of mice scalded for 4 s and harvested 24 h after scald, and it was shown that the external hair sheath was still present, and it was determined to be a deep partial-thickness scald. (3) Dense staining of pimonidazole (hypoxia) was found in deep partial-thickness scald wound 72 h after scald, especially in the marginal zones of the wounds. The partial oxygen pressure in the wound center, wound margin, and normal skin around the wound was respectively (36.2 ± 3.2), (37.0 ± 1.4), (37.4 ± 2.7) mm Hg (1 mm Hg = 0.133 kPa), showing no statistically significant difference among them (F = 74.705, P > 0.05), but they were significantly lower than that of the control group [(53.1 ± 2.4) mm Hg, with F values respectively 82.377, 91.375, 100.531, P values all below 0.05].</p><p><b>CONCLUSIONS</b>Deep partial-thickness scald model can be reproduced in (20.0 ± 1.0) g male BALB/c mice by scalding with 92 °C hot steam for 4 s, and the depth of wound becomes stable 24 h after scalding. Hypoxia can be found in the scalded wounds, especially in the marginal zones of the wounds.</p>

Influence of negative pressure wound therapy on the angiogenesis of wounds in diabetic rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the influence of negative pressure wound therapy on the angiogenesis of wounds in diabetic rats.</p><p><b>METHODS</b>Diabetes model was reproduced by intraperitoneal injection of 20 g/L streptozotocin in the dosage of 65 mg/kg in 40 SD rats. Two weeks later, rats were divided into control group (C) and negative pressure group (NP) according to the random number table, with 20 rats in each group. A piece of full-thickness skin in the center of the back of each rat in the size of 2 cm×2 cm was excised to produce a wound. Immediately after injury, wounds in group C were given conventional dressing change; wounds in group NP were treated with continuous negative pressure (-16.0 kPa) therapy for four hours a day, which lasted for seven days. (1) Blood glucose and body weight of rats in two groups were respectively measured by glucose meter and electronic scale before treatment, and 1 and 2 week (s) after. (2) Wound blood flow was detected by laser Doppler perfusion imager before treatment and on post treatment day (PTD) 1, 3, 7, with 5 rats at each time point. (3) On PTD 3 and 7, respectively, five rats from each group were sacrificed. The wound tissue was excised and divided into two parts. The angiogenesis in the left part tissue was observed with immunohistochemical staining. The microvessel density was calculated. (4) The full-thickness skin excised before treatment and the right part tissue freeze on PTD 3 and 7 were collected. On PTD 1 and 14, wound tissue was excised in the above-mentioned method. The mRNA levels of the vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (Fit-1), angiopoietin 1 (Ang-1), Ang-2, and tyrosine kinase receptor 2 (Tie-2) were determined with real-time fluorescence quantification PCR. Data were processed with two-way analysis of variance or LSD-t test.</p><p><b>RESULTS</b>(1) No significant difference was observed between two groups in blood glucose level and body weight as a whole or at each time point (with F values respectively 0.667, 0.176, t values from 0.311 to 0.707, P values all above 0.05). (2) The difference in the overall wound blood flow of rats between two groups was significant (F = 24.66, P < 0.05). On PTD 1, 3, 7, values of wound blood flow of rats in group NP were (179 ± 24), (219 ± 12), (192 ± 30) perfusion unit, significantly higher than those of rats in group C[(127 ± 16), (179 ± 8), (144 ± 17) perfusion unit, with t values respectively 3.71, 5.57, 2.77, P < 0.05 or P < 0.01]. (3) The difference in the overall microvessel density in the wound of rats between two groups was significant (F = 33.25, P < 0.05). On PTD 3, the microvessel density in the wound of rats in group NP was (80 ± 12) per 100-time visual field, which was significantly higher than that of group C[(38 ± 4) per 100-time visual field, t = 9.257, P < 0.05]. On PTD 7, the microvessel density in the wound of rats in two groups were close (t = 1.159, P > 0.05), but the vessels in group NP were regularly arranged with spacious lumen, while the vessels in group C were disorderly arranged with narrow lumen. (4) On PTD 1, 3, mRNA expression levels of VEGF, Fit-1, and Ang-1 in group NP were obviously higher than those in group C (with t values from 1.28 to 11.60, P values all below 0.01). On PTD 7, the mRNA expression level of Ang-1 (27.59 ± 3.55) in group NP was obviously higher than that in group C (19.87 ± 1.86, t = 7.23, P < 0.001), while the mRNA level of its antagonist Ang-2 (5.79 ± 0.61) in group NP was obviously lower than that in group C (17.62 ± 0.85, t = 19.88, P < 0.001). On PTD 3, 7, 14, mRNA levels of Tie-2 in group NP were obviously lower than those in group C (with t values from 8.92 to 15.60, P values all below 0.01).</p><p><b>CONCLUSIONS</b>Negative pressure wound therapy may promote wound angiogenesis by enhancing the expression of Ang-1 and lowering the expression of Ang-2 in diabetic rats.</p>

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>

Effects of Wnt/beta-catenin signaling on the phenotype change of human dermal fibroblasts and its mechanism / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the role of Wnt/beta-catenin signaling in the phenotype change of normal skin fibroblasts (NFb) into myofibroblasts and the underlying mechanism.</p><p><b>METHODS</b>NFb were isolated by collagenase digestion and cultured. (1) Experiment one. NFb were divided into four groups according to the random number table. Cells in control group were cultured with serum-free DMEM nutrient solution (briefly called nutrient solution). Cells in TGF-beta1 group were cultured with nutrient solution containing 10 ng/mL recombinant human TGF-beta1 (the same concentration for following experiments). Cells in Wnt3a group were cultured with nutrient solution containing 150 ng/mL Wnt3a (the same concentration for following experiments). Cells in TGF-beta1 + Wnt3a group were cultured with nutrient solution containing TGF-beta1 and Wnt3a. The mRNA and protein expression levels of beta-catenin and alpha-smooth muscle actin (alpha-SMA) were determined by real-time fluorescent quantitative PCR and Western blotting at post culture hour (PCH) 48. (2) Experiment two. NFb were divided into four groups according to the random number table. Cells in control group and TGF-beta1 group were treated as those in the corresponding groups in experiment one. Cells in SB415286 (glycogen synthase kinase-3beta inhibitor) group were cultured with nutrient solution containing 10 micromol/L SB415286 (the same concentration for following experiments). Cells in TGF-beta1 + SB415286 group were cultured with nutrient solution containing TGF-beta1 and SB415286. The mRNA and protein expression levels of alpha-SMA were determined by real-time fluorescent quantitative PCR and Western blotting, and the alpha-SMA-positive myofibroblasts were detected by immunofluorescence cytochemical staining at PCH 48. The experiments were all repeated for three times. Data were processed with analysis of variance and LSD- t test.</p><p><b>RESULTS</b>(1) Experiment one. There was no statistically significant difference among four groups in beta-catenin mRNA level (F = 0.302, P = 0.823). There were statistically significant differences among four groups in beta-catenin protein level (F = 16.713, P = 0.001). The protein level of beta-catenin was higher in TGF-beta1 group (0.73 +/- 0.12) and Wnt3a group (0.82 +/- 0.17) than in control group (0.34 +/- 0.11, with t values respectively 3.028, 3.727, P < 0.05 or P < 0.01). The protein level of beta-catenin in TGF-beta1 + Wnt3a group (1.23 +/- 0.21) was higher than that of the other three groups (with t values respectively 6.911, 3.883, 3.184, P values all below 0.01). There were statistically significant differences among four groups in alpha-SMA mRNA level (F = 31.830, P = 0.001). Compared with that of control group, the expression level of alpha-SMA mRNA was up-regulated in TGF-beta1 group and down-regulated in Wnt3a group (with t values respectively 6.759, 2.535, P < 0.05 or P < 0.01). The expression level of alpha-SMA mRNA in TGF-beta1 + Wnt3a group was lower than that of TGF-beta1 group (t = 4.532, P < 0.01). The protein levels of alpha-SMA in control, TGF-beta1, Wnt3a, and TGF-beta1 + Wnt3a groups were respectively 0.83 +/- 0.17, 1.43 +/- 0.20, 0.53 +/- 0.12, and 0.89 +/- 0.14 (F = 16.597, P = 0.001). Compared with that of control group, the protein level of alpha-SMA was up-regulated in TGF-beta1 group and down-regulated in Wnt3a group (with t values respectively 4.582, 2.291, P < 0.05 or P < 0.01). The protein level of alpha-SMA in TGF-beta1 + Wnt3a group was lower than that of TGF-beta1 group (t = 4.123, P < 0.01). (2) Experiment two. There were statistically significant differences among four groups in alpha-SMA mRNA level (F = 34.101, P = 0.001). The alpha-SMA mRNA level in SB415286 group was lower than that of control group (t = 2.511, P < 0.05). The alpha-SMA mRNA level in TGF-beta1 + SB415286 group was lower than that of TGF-beta1 group (t = 3.587, P < 0.01). There were statistically significant differences among four groups in alpha-SMA protein level (F = 11.381, P = 0.003). The alpha-SMA protein level was lower in SB415286 group than in control group (t = 2.364, P < 0.05). The alpha-SMA protein level was down-regulated in SB415286 +TGF-beta1 group as compared with that of TGF-beta1 group (t = 2.556, P < 0.05). There were few alpha-SMA-positive fibroblasts in control group. Compared with that of control group, the expression of alpha-SMA was significantly increased in TGF-beta1 group (t =11.198, P < 0.01), and the expression of alpha-SMA was down-regulated in SB415286 group. Meanwhile, the expression of alpha-SMA in TGF-beta1 + SB415286 group were significantly lower than that of TGF-beta1 group (t = 5.902, P < 0.01).</p><p><b>CONCLUSIONS</b>The Wnt/beta-catenin signaling might be involved in the fibroblasts-myofibroblasts transition, and it negatively regulate the TGF-beta1 -mediated profibrotic effects.</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>

Efficacy of vacuum sealing drainage in mice infected with Pseudomonas aeruginosa and its mechanism / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effect of vacuum sealing drainage (VSD) on the proliferation of Pseudomonas aeruginosa (PA) in infected wound, and to explore its possible mechanism.</p><p><b>METHODS</b>Full-thickness skin wounds each with area of 1 cm x 1 cm were produced on the back of 40 C57 BL/6 mice, and then they were contaminated with wild type PA strains PAO1 marked with target gene of bacterial luciferase luxCDABE (PAO1-lux), they were dressed for 24 hours to reproduce PA infection model. Then mice were divided into experiment [E, with treatment of VSD (pressure value at -16.625 kPa)] and control (C, with treatment of conventional dressing change) groups according to the random number table, with 20 mice in each group. The fluorescence intensity of PAO1-lux and blood flow in wound was respectively measured by in vivo optical imaging system and laser Doppler perfusion imager before treatment and at post treatment hour (PTH) 24. The expression levels of IL-1beta and vascular endothelial growth factor (VEGF) mRNA in wound edge were determined by real-time fluorescence quantitative RT-PCR before treatment and at PTH 24. The specimens of wound edge tissue were collected for observation of pathological change at PTH 24. Data were processed with t test.</p><p><b>RESULTS</b>There were no obvious difference in fluorescence intensity of PAO1-lux and blood flow in wound between E and C groups before treatment (with t value respectively 0.03, 0.50, P values all above 0.05). The fluorescence intensity of PAOl-lux and blood flow in wound in E group at PTH 24 [(2.69 +/- 0.75) photons x s(-1) x cm(-2) x sr(-1) and (96 +/- 9) PU] was respectively lower and higher than that inC group [(5.18 +/- 0.96) photons x s(-1) cm x (-2) x sr(-1) and (70 +/- 11) PU, with t value respectively 3.54, 3.13, P values all below 0.05]. The expression levels of IL-1beta and VEGF mRNA in both groups before treatment were similar (with t value respectively 0.19, 0.07, P values all above 0.05). The expression levels of IL-1beta and VEGF mRNA in E group at PTH 24 was respectively 4.72 +/- 0.37, 2.68 +/- 0.39, all markedly higher than those in C group (2.24 +/- 0.50, 1.22 +/- 0.13, with t value respectively 6.90, 6.12, P values all equal to 0.00). The number of inflammatory cell infiltrating the wound edge in E group at PTH 24 was increased by nearly 77% as compared with that in C group.</p><p><b>CONCLUSIONS</b>Compared with conventional dressing change, VSD can reduce the amount of Pseudomonas aeruginosa in full-thickness skin defect wound at the early stage, it may be related with an increase in blood flow and number of inflammatory cells in wound tissue, promoting expression of IL-1beta and VEGF mRNA.</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>

Effects of interferon-gamma on the transforming growth factor beta/Smad pathway in keloid-derived fibroblasts / 中华烧伤杂志

<p><b>OBJECTIVE</b>To elucidate the effects of interferon-gamma (IFN-gamma) on the transforming growth factor beta (TGF-beta)/Smad pathway in keloid-derived fibroblasts (KFb), and to investigate the underlying mechanism in the treatment of pathologic scar with IFN-gamma.</p><p><b>METHODS</b>Keloid tissue of 3 patients were obtained, and then KFb were separated and cultured in vitro. KFb from passages 3 to 5 were used for the study. (1) KFb were divided into control group (incubated with serum-free DMEM), TGF-beta(1) group (treated with 10 ng/mL TGF-beta(1)), IFN-gamma group (treated with 100 ng/mL IFN-gamma), and TGF-beta(1)+IFN-gamma group (incubated with 10 ng/mL TGF-beta(1) combined with 100 ng/mL IFN-gamma). The expression level of mRNA and protein of connective tissue growth factor (CTGF), alpha smooth muscle actin (alpha-SMA) protein and expression of alpha-SMA positive KFb were detected by real-time fluorescent quantitation RT-PCR (FQ-RT-PCR), Western blot and immunofluorescence cytochemical staining. (2) Another sample of KFb was obtained and treated with 10 ng/mL IFN-gamma. The expression level of Smad 3 and Smad 7 protein was detected by Western blot before and 1, 2, 4, 6, 8 h post stimulation (PSH). The expression level of Smad 3 and Smad 7 mRNA was assessed by FQ-RT-PCR before stimulation and 30 mins post stimulation and at PSH, 1, 2, 4, 6, 8. (3) Another sample of KFb was obtained and divided into 1, 10 and 100 ng/mL IFN-gamma groups based on the concentration of IFN-gamma, treated for 4 hours; KFb without IFN-gamma treatment was set up as control group. The expression levels of the protein and mRNA of Smad 3 and Smad 7 were measured by FQ-RT-PCR and Western blot.</p><p><b>RESULTS</b>(1) The level of mRNA and protein of CTGF in IFN-gamma group (0.017 +/- 0.009 and 1.198 +/- 0.004) was respectively lower than that in control group (0.024 +/- 0.013 and 1.229 +/- 0.011, P < 0.05). The level of mRNA and protein of CTGF in TGF-beta(1)+IFN-gamma group (0.634 +/- 0.138 and 1.204 +/- 0.010) was respectively lower than that in TGF-beta(1) group (1.331 +/- 0.298 and 1.727 +/- 0.004, P < 0.01). The fluorescence intensity of alpha-SMA positive KFb (0.922 +/- 0.059) and the expression level of alpha-SMA protein (0.3051 +/- 0.0031) in IFN-gamma group decreased significantly than those in control group (1.055 +/- 0.005 and 0.4513 +/- 0.0094, P < 0.01). The fluorescence intensity of alpha-SMA positive KFb (1.129 +/- 0.004) and the expression level of alpha-SMA protein (0.6734 +/- 0.0098) in TGF-beta(1)+IFN-gamma group decreased significantly than those in TGF-beta(1) group (1.270 +/- 0.005 and 1.3842 +/- 0.0024, P < 0.01). (2) The expression level of Smad 3 mRNA and protein at the first time point after IFN-gamma treatment increased temporarily then decreased gradually, and mRNA expression level reached the nadir at PSH 4, it rose gradually later, though it was still lower at PSH 8 than that before treatment (P < 0.01); protein expression level at PSH 8 was significantly lower than that before treatment (P < 0.01). The expression level of Smad 7 mRNA and protein increased gradually to the maximum at PSH 2 and 4 respectively, then decreased but was still higher at PSH 8 than that before treatment (P < 0.05). (3) Compared with those in control group, the expression levels of Smad 3 mRNA and protein in 1, 10 and 100 ng/mL IFN-gamma group were significantly lower, the expression levels of Smad 7 mRNA and protein were significantly higher (P < 0.05 or P < 0.01). The higher concentration of IFN-gamma, the more significant differences were observed.</p><p><b>CONCLUSIONS</b>IFN-gamma can down-regulate the expression of Smad 3 while up-regulate the expression of Smad 7 in a time- and dose-dependent manner, and reduce the expression level of CTGF and alpha-SMA in the basic state or induced by TGF-beta(1), which shows a significant inhibitory effect on the TGF-beta/Smad signal pathway. This may be an important mechanism in the treatment of pathologic scar by IFN-gamma.</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>