Role of mitogen-activated protein kinases in the formation of hypertrophic scar with model of lipopolysaccharide stimulated skin fibroblast cells

Objective: Hypertrophic scar is common in burn patients, but treating result could not meet the expectation of the patients and doctors. We have found that certain concentration level of lipopolysaccharide [LPS] stimulated normal fibroblast cells have statistically similar with fibroblast cells from hypertrophic scar on the phenotype level, and with this work we are trying to figure out which Mitogen-Activated Protein Kinase [MAPK] is affected and how it is affected

Methods: Experiments were conducted in May, 2017 at the first affiliated hospital of the Chinese PLA General Hospital, Beijing, China. We have cultured the cell line of human skin fibroblast cells and randomly divided cells into four groups: control group and three stimulation groups. We have rebuilt the LPS stimulated model of skin fibroblast cells in hypertrophic scar based on our previous work. Experimental groups were stimulated with 0.1ug/mL LPS concentration for 24 hours, 48 hours, and 72 hours, respectively. Then we performed western blot analysis of Erk, p-Erk, JNK, p-JNK, p38 and p-p38. We performed statistical analysis with SPSS 15.0

Results: LPS can up regulate the MAPK/p38 pathway [p<0.05] and down regulate the MAPK/Erk and MAPK/ JNK pathways [p<0.05]. The changes of phosphorylated protein are time-related, with longer stimulation duration, significant difference is increased [p<0.05]

Conclusion: MAPKs can play an important role in the formation of hypertrophic scar in the skin. Early intervention through the MAPKs could be a promising target in the prevention of the formation of hypertrophic scar

Changes of content and mRNA expression of gelsolin and proliferation activity of T-lymphocyte in spleen of mice with severe burn injury / 中华烧伤杂志

Objective@#To investigate the changes of content and mRNA expression of gelsolin and proliferation activity of T-lymphocyte in spleen of mice with severe burn injury, so as to determine the optimum intervention time of gelsolin.@*Methods@#Eighty male BALB/c mice were divided into sham injury group and burn group according to the random number table, with 40 mice in each group. Mice in burn group were inflicted with 15% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Immediately after injury, mice in burn group were hypodermic injected with 1 mL normal saline, with iodophor smeared on back once a day to prevent infection. Mice in sham injury group were sham injured without fluid infusion and smearing iodophor. At post injury hour (PIH) 0 (immediately), 8, 24, 48, and 72, spleen of 8 mice of each group were harvested aseptically, respectively. Proliferation activity of T-lymphocyte was determined with methyl-thiazolyl-tetrazolium colorimetry method; gelsolin content of spleen was determined with enzyme-linked immunosorbent assay; mRNA expression of gelsolin of spleen was determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with analysis of variance of factorial design, one-way analysis of variance, LSD test and Bonferroni correction.@*Results@#(1) There was no significant difference in proliferation activity of T-lymphocyte in spleen of mice in two groups at PIH 0 (P>0.05). Proliferation activity of T-lymphocyte in spleen of mice in sham injury group was significantly higher than that in burn group at PIH 8, 24, 48, and 72 (with P values below 0.05). There was no significant difference in proliferation activity of T-lymphocyte in spleen of mice in sham injury group at each time point post injury (F=0.756, P>0.05). Proliferation activity of T-lymphocyte in spleen of mice in burn group at PIH 8 was 0.12±0.04, significantly lower than that at PIH 0, 24, 48, and 72 in the same group (0.73±0.07, 0.56±0.07, 0.51±0.09, and 0.59±0.07, respectively, with P values below 0.05). (2) There was no significant difference in gelsolin content of spleen of mice in two groups at PIH 0 (P>0.05). Gelsolin content of spleen of mice in sham injury group was significantly higher than that in burn group at PIH 8, 24, 48, and 72 (with P values below 0.05). There was no significant difference in gelsolin content of spleen of mice in sham injury group at each time point post injury (F=1.083, P>0.05). Gelsolin content of spleen of mice in burn group at PIH 8 was (11.9±2.6) pg/mg, significantly lower than that at PIH 0, 24, 48, and 72 in the same group [(37.7±2.9), (19.9±4.0), (24.1±4.1), and (24.6±4.0) pg/mg, respectively, with P values below 0.05]. (3) There was no significant difference in mRNA expression of gelsolin of spleen of mice in two groups at PIH 0 (P>0.05). The mRNA expressions of gelsolin of spleen of mice in sham injury group were significantly higher than those in burn group at PIH 8, 24, 48, and 72 (with P values below 0.05). There was no significant difference in mRNA expression of gelsolin of spleen of mice in sham injury group at each time point post injury (F=0.413, P>0.05). The mRNA expression of gelsolin of spleen of mice in burn group at PIH 8 was 0.307±0.064, significantly lower than that at PIH 0, 24, 48, and 72 in the same group (0.944±0.023, 0.625±0.091, 0.744±0.104, and 0.821±0.072, respectively, with P values below 0.05).@*Conclusions@#Severe burn injury could induce decrease of proliferation activity of T-lymphocyte and content and mRNA expressions of gelsolin in spleen of mice, and all of them decreased into the lowest at PIH 8. Optimum intervention time of gelsolin for severe burn would be before PIH 8.

Progress in treatment of refractory wound and its challenges we are facing / 中华烧伤杂志

Till now, though great progress has been made in the treatment of refractory wound, severe challenges are still awaiting to be solved. The strategy of treatment is generally either non-surgical treatment or surgical treatment. Non-surgical treatment includes physical therapy, negative pressure wound therapy, growth factor therapy, stem cell transplantation, gene therapy, application of new biological dressing, application of skin tissue engineering, three-dimensional bio-printing technology, biological therapy, and Chinese herbal medicine therapy. Surgical treatment mainly includes skin graft transplantation and a variety of skin flap transplantation. To my mind, comprehensive therapy with concept of precision treatment strategy should be advocated for treatment of refractory wound.

Effects of ulinastatin on immune function of spleen in severely burned rats and its mechanism / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effects of ulinastatin on immune function of splenic CD4(+) T lymphocytes and CD4(+) CD25(+) regulatory T lymphocytes (Tregs) and content of high mobility group box 1 (HMGB1) in peripheral blood of severely burned rats, and to analyze the possible mechanisms.</p><p><b>METHODS</b>Ninety-six male SD rats were divided into sham injury group, burn group, and ulinastatin group according to the random number table, with 32 rats in each group. Rats in sham injury group were sham injured on the back by immersing in 37 ℃ warm water for 12 s. Rats in burn group and ulinastatin group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back by immersing in 94 ℃ hot water for 12 s. Immediately after injury, rats in each group were intraperitoneally injected with saline (40 mL/kg), meanwhile rats in ulinastatin group were intraperitoneally injected with ulinastatin (4×10(4) U/kg), once per 12 h, till post injury hour 72. Eight rats of each group were respectively selected on post injury day (PID) 1, 3, 5, and 7 to collect abdominal aortic blood samples. Serum content of HMGB1 was detected by enzyme-linked immunosorbent assay (ELISA). And then, rats of the 3 groups were sacrificed immediately to collect spleens and separate CD4(+) CD25(+) Tregs and CD4(+) T lymphocytes. Flow cytometer was used to detect positive expression rates of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and forkhead-winged helix transcription factor p3 (Foxp3) in CD4(+) CD25(+) Tregs. Content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs, and content of interleukin 2 (IL-2), IL-4, and γ interferon (IFN-γ) in culture supernatant of CD4(+) T lymphocytes was detected by ELISA. The proliferative activity of CD4(+) T lymphocytes was determined by microplate reader. The sample number of above-mentioned experiments was 8 at each time point in each group. Data were processed with analysis of variance of factorial design and LSD test.</p><p><b>RESULTS</b>(1) Compared with that in sham injury group, serum content of HMGB1 of rats in burn group was significantly increased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, serum content of HMGB1 of rats in ulinastatin group was significantly decreased from PID 1 to 7 (with P values below 0.01). (2) Compared with those in sham injury group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in burn group were significantly increased from PID 1 to 7 (with P values below 0.01), peaking on PID 3 [(65±10)%, (76±10)%, and (28.2±4.4) pg/mL respectively]. These 3 indexes of rats in sham injury group on PID 3 were (45±7)%, (46±7)%, and (11.2±2.3) pg/mL respectively. Compared with those in burn group, the positive expression rates of CTLA-4 and Foxp3 in CD4(+) CD25(+) Tregs and content of IL-10 in culture supernatant of CD4(+) CD25(+) Tregs of rats in ulinastatin group were significantly decreased from PID 1 to 7 (P<0.05 or P<0.01), reaching the nadir on PID 7 [(43±6)%], PID 1 [(50±8)%], and PID 7 [(12.4±3.4) pg/mL] respectively. These 3 indexes of rats in burn group on PID 7, 1, and 7 were (58±8)%, (71±9)%, and (19.7±2.8) pg/mL respectively. (3) Compared with those in sham injury group, the content of IL-2 and IFN-γ in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased in burn group from PID 1 to 7, with P values below 0.01. Compared with that in burn group, the content of IL-2 and IFN-γ in culture supernatant of CD4(+) T lymphocytes of rats was significantly increased, while the content of IL-4 in culture supernatant of CD4(+) T lymphocytes of rats was significantly decreased in ulinastatin group from PID 1 to 7, P<0.05 or P<0.01. (4) Compared with that in sham injury group, the proliferative activity of CD4(+) T lymphocytes of rats in burn group was significantly decreased from PID 1 to 7 (with P values below 0.01). Compared with that in burn group, the proliferative activity of CD4(+) T lymphocytes of rats in ulinastatin group was significantly increased from PID 1 to 7 (with P values below 0.01).</p><p><b>CONCLUSIONS</b>Ulinastatin can weaken the immunosuppressive function mediated by splenic CD4(+) CD25(+) Tregs in severely burned rats, and improve proliferative function and secretory function of splenic CD4(+) T lymphocytes, which may be attributed to the inhibiting effect of ulinastatin on the release of HMGB1 in large amount.</p>

Effects of ulinastatin on immune function of patients with severe burn injury / 中华烧伤杂志

<p><b>OBJECTIVE</b>To primarily evaluate the effects of ulinastatin on immune function of patients with severe burn injury.</p><p><b>METHODS</b>Forty patients with severe burn admitted to our ward from March 2013 to October 2015, conforming to the study criteria, were divided into conventional treatment group (CT, n=20) and ulinastatin treatment group (UT, n=20) according to the random number table and patient's consent. After admission, patients in group CT received antishock treatment, antibiotic treatment, debridement, skin grafting, and nutrition support, etc. On the basis of the above-mentioned treatment, patients in group UT received intravenous drip of ulinastatin from first day after admission twice a day, with a dosage of 8×10(5) U every time, for 7 days in addition. Peripheral venous blood samples were collected from patients in groups CT and UT on post treatment day (PTD) 1, 3, 5 and 7, respectively. Twenty healthy volunteer were selected as health control group (HC), and peripheral venous blood samples were collected on the first day of the study. Percentage of CD4(+) CD25(+) regulatory T lymphocytes (Tregs) was determined by flow cytometer. The proliferative activity of T lymphocytes was detected by microplate reader (denoted as absorbance value). Content of interleukin 2 (IL-2) in culture supernatant of T lymphocytes, and content of IL-4 and γ interferon (IFN-γ) in serum were detected by enzyme-linked immunosorbent assay. Expression of human leukocyte antigen-DR (HLA-DR) on CD14(+) monocytes was determined by flow cytometer. Data were processed with analysis of variance for repeated measurement, chi-square test, and LSD-t test.</p><p><b>RESULTS</b>(1) Compared with that of volunteer in group HC, the percentage of CD4(+) CD25(+) Tregs of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 13.303 to 26.043, P values below 0.01). Compared with that in group CT, the percentage of CD4(+) CD25(+) Tregs of patients in group UT was significantly decreased on PTD 5 and 7 (with t values respectively 8.317 and 15.071, P values below 0.01). (2) The proliferative activity of T lymphocytes of patients in group CT on PTD 1, 3, 5, and 7 was respectively 0.71±0.11, 0.61±0.15, 0.54±0.12, and 0.67±0.17, which was significantly lower than that in group HC (1.21±0.22, with t values from 8.686 to 11.957, P values below 0.01). The proliferative activity of T lymphocytes of patients in group UT on PTD 3, 5, and 7 were respectively 0.81±0.11, 0.85±0.14, and 1.08±0.13, which was significantly higher than that in group CT (with t values from 4.808 to 8.568, P values below 0.01). (3) Compared with those of volunteer in group HC, content of IL-2 in culture supernatant of T lymphocytes of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 8.073 to 9.288, P values below 0.01), content of IL-4 in serum of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 18.926 to 41.451, P values below 0.01), and content of IFN-γ in serum of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 4.543 to 27.659, P values below 0.01). Compared with those in group CT, content of IL-2 in culture supernatant of T lymphocytes of patients in group UT was significantly increased from PTD 3 to 7 (with t values from 6.507 to 8.869, P values below 0.01), content of IL-4 in serum of patients in group UT was significantly decreased from PTD 3 to 7 (with t values from 6.922 to 8.843, P values below 0.01), and content of IFN-γ in serum of patients in group UT was significantly increased on PTD 5 and 7 (with t values respectively 5.369 and 13.521, P values below 0.01). (4) The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group CT on PTD 1, 3, 5, and 7 were respectively (28±6)%, (25±7)%, (25±7)%, and (39±10)%, which were significantly lower than the percentage of volunteer in group HC [(87±8)%, with t values from 16.323 to 25.645, P values below 0.01]. The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group UT on PTD 3, 5, and 7 were respectively (40±6)%, (42±9)%, and (49±10)%, which were significantly higher than those in group CT (with t values from 3.071 to 7.324, P values below 0.01).</p><p><b>CONCLUSIONS</b>On the basis of CT, additional ulinastatin intervention can decrease CD4(+) CD25(+) Tregs percentage, improve the immune function of T lymphocytes and T helper cells, and increase expression of HLA-DR on CD14(+) monocytes of patients with severe burn injury, thus improve the immune function of patients.</p>

Curative effect of autologous blister skin grafting on cicatricial depigmentation caused by deep burn / 局解手术学杂志

Objective To compare the treatment effect of autologous blister skin grafting with ReCell autologous chromocyte grafting on cicatricial depigmentation caused by deep burn.Methods Thirty-four patients with cicatricial depigmentation caused by deep burn who were admitted into hospital from May 2012 to February 2015 were included in this study.The total 61 depigmentation areas were randomly divided into two groups;32 areas from 18 patients were treated with autologous blister skin grafting,and the other 29 areas from 16 patients were trea-ted with ReCell autologous chromocyte grafting.In the autologous blister skin grafting treated group,epidermis from the depigmentation area was removed by grinding with a BY-II AM type epidermal graft vitiligo treatment equipment.Then the autologous blister skin was harvested with the suction blistering method and grafted onto the wound of depigmentation area.In the ReCell autologous chromocyte grafting treated group,split-thickness skin flap was harvested by electric dermatome.Then the donor skin was processed into chromocyte suspension with the ReCell assay kit and evenly sprayed onto the depigmentation areas.The wound healing time and the pigment recovery 3 months after surgery were observed.Results The wound healing time of autologous blister skin grafting treated group was significantly shorter than that of ReCell autologous chromocyte grafting treated group (P <0.05 ).The effective rate of pigment recovery 3 months after surgery in autologous blister skin grafting treated group was markedly higher than that of ReCell autologous chromocyte grafting treated group(P <0.05 ). Conclusion The autologous epidermal grafting treatment using grinding and suction blistering method is simple and easy to perform,marked-ly effective,with no suture scar and low surgical risk,thus serving as a promising and ideal therapeutic method for burn scar depigmentation.

Preparation of fusion protein between ZZ affinity peptide and alkaline phosphatase and its primary application / 中国免疫学杂志

Objective:To construct and express the fusion protein between ZZ affinity peptide and alkaline phosphatase and examine its biological activities.Methods:The alkaline phosphatase gene was cloned into pEZZ 18 vector containing ZZ peptide gene resulted in the pEZZ-AP recombinant vector.Then the vector was transformed and expressed in E.coli DH5α.And HisTrap affinity chromatography was employed to separate and purify the target protein.After analyzed by Western blot , ZZ-AP fusion protein was applied to immunocytochemistry as an alterative second antibody.Results:The result of SDS-PAGE showed that the fusion protein with a relative molecular weight was consistent with the theoretical values (62 kD).The fusion protein has rabbit IgG-binding ability and en-zymatic activity of alkaline phosphatase ,those were validated in Western blot;and it produced a good signal that was comparable in its staining pattern to that generated with goat anti-rabbit IgG-AP in immunocytochemistry.Conclusion: The ZZ-AP fusion protein was constructed successfully ,it has rabbit IgG-binding ability and enzymatic activity of alkaline phosphatase.We anticipate that the ZZ-AP fusion protein has a potential application in immunoassay field.

Site-specific biotinylation of EGFP with double biotin molecules based on Avi-tag technology / 中国免疫学杂志

Objective:To prepare the site-specific biotinylation of enhanced green fluorescence protein with double biotin molecules using Avi-tag technology.Methods:The EGFP gene was prepared by PCR and cloned into pdi-Avitag resulting the vector pEGFP-( Avitag) 2.The fusion protein EGFP-( Avitag ) 2 was expressed in E.coli DH5αand purified by employing IMAC.The site-specific biotinylation was implemented by BirA enzyme in vitro, and then was identified by competitive ELISA and Western blot.Results:The recombinant prokaryotic expression vector pEGFP-(Avitag)2 was correctly constructed,and EGFP-(Avitag)2 fusion was successfully expressed in E.coli DH5α.The results of competitive ELISA and Western blot showed that the EGFP-( Avitag) 2 could be site-specific biotinylation with double biotin molecules based on Avi-tag technology.Conclusion: The site-specific biotinylation of EGFP with double biotin molecules is successfully prepared,and we anticipate that can be used for BAS to improve the sensitivity and specificity of immunosensors.

Effects of sivelestat on acute lung injury in dogs with severe burn-blast combined injury / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe and study the effects of sivelestat on acute lung injury in dogs with severe burn-blast combined injury.</p><p><b>METHODS</b>Thirty-two male beagle dogs of clean grade were divided into 4 groups: uninjured group (U), combined injury control group (CIC), combined injury+low dose of sivelestat group (CI+LS), combined injury+high dose of sivelestat group (CI+HS), with 8 dogs in each group. Except for the dogs in group U which were not injured, the dogs in the other 3 groups were inflicted with severe burn-blast combined injury. According to the Parkland formula, the dogs in groups U and CIC were infused with physiological saline, and the dogs in groups CI+LS and CI+HS received sivelestat with the dosage of 0.5 and 2.0 mg·kg(-1)·h(-1) respectively in addition. The 24 h continuous intravenous infusion was carried out for 2 days. At post injury hour (PIH) 6, CT scanning was conducted to observe the lung damage. At PIH 2, 6, 12, 24, and 48, mean arterial pressure (MAP), respiratory rate (RR), extra vascular lung water (EVLW), pulmonary vascular permeability index (PVPI), PaO2, and PaCO2 were measured; the contents of neutrophil elastase (NE), IL-8, and TNF-α were determined by ELISA. At PIH 48, all the dogs were sacrificed, and the lung tissues were harvested to measure the wet to dry lung weight ratio. The same examination was carried out in the dogs of the group U at the same time points. Data were processed with analysis of variance of repeated measurement and LSD test.</p><p><b>RESULTS</b>(1) CT images showed some exudative lesions in the dogs of groups CIC and CI+LS but not in the dogs of groups U and CI+HS. (2) No statistically significant differences were observed in MAP at each time point between every two groups (with P values above 0.05). The RR values in group U were significantly different from those of the other 3 groups at all time points (with P values below 0.05). The values of EVLW and PVPI in 3 combined injury groups were significantly different from those in group U at PIH 6, 12, 24, and 48 (with P values below 0.05). The values of RR and EVLW in group CI+LS were significantly different from those in group CI+HS at PIH 12, 24, and 48 (with P values below 0.05). The values of PVPI in group CI+LS were significantly different from those in group CI+HS at PIH 24 and 48 (with P values below 0.05). (3) The levels of PaO2 and PaCO2 showed significant differences between group U and the other 3 groups at each time point (with P values below 0.05). The levels of PaO2 in group CI+LS were significantly different from those in CI+HS group at PIH 12, 24, and 48 (with P values below 0.05). The level of PaCO2 showed significant differences between group CI+LS and group CI+HS at PIH 24 and 48 (with P values below 0.05). (4) The contents of NE (except for PIH 2), TNF-α, and IL-8 showed significant differences between group U and the other 3 groups at each time point (P < 0.05 or P < 0.01). At PIH 2, 6, 12, 24, and 48, the contents of NE in groups U, CIC, CI+LS, and CI+HS were respectively (69 ± 21), (83 ± 24), (80 ± 20), (75 ± 17), (72 ± 27) pg/mL; (66 ± 24), (196 ± 20), (231 ± 26), (252 ± 25), (266 ± 22) pg/mL ; (71 ± 22), (180 ± 27), (214 ± 21), (194 ± 24), (218 ± 20) pg/mL; (68 ± 22), (136 ± 24), (153 ± 22), (146 ± 26), (150 ± 28) pg/mL. NE values in group CI+HS were statistically different from those in groups CIC and CI+LS at PIH 6, 12, 24, and 48 (with P values below 0.05). The contents of TNF-α in group CI+LS were statistically different from those in groups CIC and CI+HS at PIH 24 and 48 (with P values below 0.05). The contents of IL-8 in group CI+LS were statistically different from those in group CI+HS at PIH 24 and 48 (with P values below 0.05). (5) At PIH 48, the wet to dry lung weight ratio of group CIC was statistically different from that in group CI+LS or group CI+HS (with P values below 0.05); there was also difference between group CI+LS and group CI+HS (P < 0.05).</p><p><b>CONCLUSIONS</b>Sivelestat, especially in a high dose, exerts a protective effect in acute lung injury after burn-blast combined injury through improving the index of blood gas analysis, ameliorating pulmonary edema, and lowering the production of pro-inflammatory mediators.</p>

Influence of lipopolysaccharide on collagen metabolism of normal skin fibroblasts of human / 中国医师杂志

ObjectiveTo observe the influence of lipopolysaccharide (LPS) on collagen metabolism of normal human skin fibroblasts and its biological role in the formation of hypertrophic scar.Methods Fibroblasts were isolated and cultured in vitro,and then exposed to different doses of LPS (0.005,0.01,0.05,0.1,0.5,1.0 μg/ml) from E.coli.055:B5 respectively.The expression of proccllagen type Ⅰ,Ⅲand collagenase mRNAs was tested by RT -PCR.Fibroblasts from hypertrophic scar tissue obtained from the same patients in the same culture passage were used as control.ResultsCompared with control group,the expression of procollagen typeⅠ,Ⅲ mRNAs in normal skin fibroblasts increased (0.323 ± 0.041,0.303 ± 0.063,0.391 ± 0.071,0.344 ± 0.086,0.488 ± 0.059,0.401 ± 0.087,0.616 ± 0.107,0.434 ±0.084,0.823 ±0.092,0.542 ± 0.082),while the expression of collagenase mRNAs of normal skin fibroblasts depressed(0.598 ± 0.068,0.556 ± 0.049,0.441 ± 0.043,0.372 ± 0.083,0.260 ± 0.027 ).When LPS was set to the concentration of 0.005 μg/ml,it showed a concentration dependent manner.However,when the concentration of LPS was set to 0.5 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts began to decrease (0.451 ± 0.063,0.374 ± 0.072,0.360 ± 0.062).When the concentration of LPS was set to 1.0 μg/ml,the expression of procollagen type Ⅰ,Ⅲ mRNAs (0.162 ± 0.025,0.171 ± 0.061 )were inhibited and the expression of collagenase mRNAs began to increase (0.444 ±0.114).When the concentration of LPS was set to 0.1 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts(0.823 ±0.092,0.542 ±0.082,0.260 ±0.027)was similar to that of hypertrophic scar tissue fibroblasts(0.829 ±0.049,0.569 ±0.038,0.277 ±0.059).ConclusionsThis result supported that LPS may be an important factor in collagen metabolism of normal skin fibroblasts and it plays an important role in hypertrophic scar formation.

Influence of lipopolysaccharide on the cell cycle and the mRNAs expression of procollagen type Ⅰ,Ⅲ of normal human skin fibroblasts / 中国医师杂志

Objective To observe the influence of lipopolysaccharide (LPS) on the cell cycle and the mRNAs expression of procollagen type Ⅰ , Ⅲ of normal human skin fibroblasts. Methods Purified dermal fibroblasts were exposed to different doses of LPS(0. 005 ～ 1.0 μg/ml) from E. coli. Then the cell cycle of fibroblasts at logarithmic stage at day 7 after LPS administration was assayed with flow cytometry.The expression of procollagen type Ⅰ , Ⅲ and collagenase mRNAs was tested by RT-PCR. Results The percentage of S phase cells in cell cycle of normal human skin fibroblasts increased when LPS concentrations were changed from 0. 005 to 0. 1 μg/ml, and the increase showed a concentration dependent manner. However, when the concentration of LPS was 0. 5 μg/ml, the percentage of S phase cells began to decrease, but still higher than normal control. When LPS concentration reached 1.0 μg/ml, the percentage of S phase cells were lower than normal control. The expression of procollagen type Ⅰ , Ⅲ mRNAs of normal skin fibroblasts increased when LPS was challenged to the concentration of 0. 005 μg/ml, and the influence showed a concentration dependent manner. However, when the concentration of LPS was 0. 5 μg/ml, the influence of LPS on the expression of procollagen type Ⅰ , Ⅲ of normal skin fibroblasts began to decrease.When the concentration of LPS reached 1.0 μg/ml, the expression of procollagen type Ⅰ , Ⅲ mRNAs were inhibited. Conclusions LPS promoted the proliferation and collagen synthesis of normal human skin fibroblasts within a certain range of low doses, while high doses of LPS might inhibit the proliferation and collagen synthesis of normal human skin fibroblasts.

Effect of carbachol on the expression of la on monocyte and lymphocyte apoptosis rate of the burned rats with delayed resuscitation / 中国医师杂志

Objective To explore the changing trend of Ia on monocyte, lymphocyte apoptosis rate, TNF-α and IL-6 in abdominal aorta of burned rats with delayed resuscitation and the influence of application of carbachol on them. Methods Adult male Wistar rats were randomly divided into normal control group(n =8), scald group(n =48) and scald with carbachol treatment group(n =48). In latter two groups, rats were inflicted with 30% total body surface area (TBSA) full-thickness scald and delayed fluid resuscitation. All scald rats were sacrificed at the 6th hours or 1st, 2nd, 3rd, 7th, 14th day after scald, with 8 rats at each time point. Expression of Ia antigen on monocyte and lymphocyte apoptosis rate were determined by direct immunofluorescence on a flow cytometer, and TNF-α and IL-6 was measured by ELISA. Results Expression of la on monocyte was obviously lower than that of controls. The lowest levels were recorded on the 6th hours and 1st day after scald. Subsequently, Ia was elevated gradually, but still lower than that of normal rats(P <0. 01). After administration of carbachol, Ia expression was obviously promoted, compared with the simple scald group (P <0. 01). Lymphocyte apoptosis rate, TNF-α and IL-6 was higher than that of controls(P <0. 01). After administration of cavachol, , lymphocyte apoptosis rate and TNF-α and IL-6 was obviously down-regulated on the 6th hours, 1st day, 2nd day and 3rd day after scald injury, compared with the simple scald group (P < 0. 01 or 0. 05). Conclusion After severe burn with delayed fluid resuscitation, there is a low la expression, high lymphocyte apoptosis rate and increased releasing of proinflammatory cytokine. Immune function was suppressed. Carbacho] could improve the immune function of scald rats with delayed fluid resuscitation.

Treatment strategies for mass burn casualties transferred from a distance-clinical experience / 解放军医学杂志

This paper is to introduce our experiences in treating 2 batches of 13 burn victims transferred from remote areas on postburn days 3 and 4. Methods Thirteen burn victims of 2 mass casualties were transferred to our burns institute from remote areas on postburn days 3 and 4 on June 27, 2001 and June 2, 2002, respectively. There were 4 males and 9 females, age ranged from 20 to 43 years, with a mean age of 31.1±6.2 years. The mean total burn area was 74.3%±24.7% TBSA (range, 25% to 97%). Among them, 10 patients suffered from serious burn with mean total burn area involving 86.0%±11.5% TBSA (range, 60% to 97%), and mean full-thickness burn of 63.9%±26.3% TBSA. Four patients also manifested signs of severe inhalation injury, and 6 patients with moderate inhalation injury. In three patients with mean total burn area covering 35.5%±10.0% TBSA (range, 25% to 45%), with mean full-thickness burn of 15.3%±5.0%, were al having moderate inhalation injuries. Among these 13 patients, 3 were having high body temperature (39℃), while 3 manifested hypothermia. The heart rate was 140-160/min, and respiratory rate 26 to 32/min in 6 patients. Abdominal distension or loss of bowel sound were found in 4 patients. Low white cell and platelet count were found in some patients. In 13 cases, liver function, renal function, myocardiac enzyme, and coagulation function were abnormal. Results Among 13 burn victims, one patient died of myocarditis on postburn day 29, and another one died of hepatic failure (history of chronic hepatitis B) on postburn day 45 with only 2% TBSA of burn wound remained open. Conclusion Burns victims occurred in mass casualties who were transferred from remote areas to our Burns Institute were all in critical condition, usually with multiple complications, demanding most meticulous care. Our strategies in this regard consisted of dispatch of experienced surgeons and nurses to the referring hospitals and the airport to receive the patients to offer appropriate care to them during the journey,organization of the medical staff so that each of them was ordained specific function, thus conditions of the patients were evaluated immediately and appropriate treatment started expeditiously for those lethal complications on arrival. Timely and exact comprehensive treatments were prerequisite to save the patients’ life. Adequate metabolic support should be emphasized, and coagulant of anticoagulant treatment should be carried out when indicated.

Respiratory support with high frequency jet ventilation in severely burned patients with inhalation injury during early postburn stage / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effects of respiratory support with high frequency jet ventilation (HFJV) in severely burned patients with inhalation injury during early postburn stage.</p><p><b>METHODS</b>Twenty severely burned patients with TBSA of 79.6 +/- 29.3% and inhalation injury were enrolled in the study. Nineteen cases received tracheostomy after admission and only one received nasal intubation. All the patients underwent HFJV to correct hypoxia. The changes in blood gas analysis, respiratory rate and pulse were recorded before and 11 days after the ventilation.</p><p><b>RESULTS</b>Tracheostomy was performed on 2.7 +/- 2.4 postburn days (PBDs), and HFJV was given during 4.4 +/- 2.9 PBDs. PaO(2) was evidently higher during 1 - 3 days after HFJV than that before the ventilation (P < 0.01) and remained at high level for 1 week after HFJV. There was no change in PaCO(2), respiratory rate and pulse during the ventilation.</p><p><b>CONCLUSION</b>HFJV was beneficial in improving oxygenation and without any obvious side effects during the early management of severely burned patients with inhalation injury. This might be an optimal respiratory support pattern.</p>

The Influence of Composite Transplantation of Two Kinds of Acellular Xeno-dermis and Micro-autograft on Wound Healing / 中国医师杂志

Objective To investigate the influence of composite transplantation of two kinds of acellular xeno-dermis (pigskin) and micro-autograft on wound healing. Methods Seventy-two Sprague-Dawley rats with full-thickness skin defect on the back were randomly divided into three groups, and then compositt skin I(acellular dermal xeno-matrix+autologous particulate skin +selective acellular xeno-dermis) was adopted to cover the skin defect in the experimental group I, composite skin II(acellular dermal xeno-matrix+autologous particulate skin + glutaraldehyde xeno-skin) was adopted to cover the skin defect in the experimental group II, and auto-particulate skin was adopted in the control group. The area of wound healing and the rates of wound contraction were calculated.Tissue samples were harvested and examined by means of histology, immunohistochemistry and electron microscopy. Results There were continuous epidermis and basement membrane, and the dermal matrix was a kind of matrix with normal structure and organization of collagen and without any cellular components in selective acellular xeno-dermis. There was full differentiation of epithelium, orderly collagen arrange and basal membrane of the skin could be identified by immunohistochemical stain in compound skin grafting group I. The healing effect of compound skin grafting group I was better than compound skin grafting group II and auto-particulate skin grafting group. Conclusion The composite grafts constructed by selective acellular xeno-dermis (pigskin) could be a potential new type of composite skin substitute for the repair of full-thickness skin defect.

Effect of low molecular weight heparin on hypercoagulability in extensive severe burn casualties / 解放军医学杂志

Objective To observe the effect of low molecular weight heparin(LMWH)on hypercoagulability in extensive severe burn patients.Methods 9 patients were assigned as LMWH treatment group(TG),and another 12 burn patients with the similar extent of burn injury admitted in the same period were assigned as routine treatment group(RTG).All the treatments were the same except 5000U LMWH was given via subcutaneous injection per 12 or 6 hours to the TG group for 3-7days.The patients in normal control group(NCG)consisted of patients receiving plastic surgery.The following coagulation parameters were determined before and after the heparin treatment:prothrombin time(PT),thrombin time(TT),activated partial thromboplastin time(APTT),international normalized ratio(INR),fibrinogen(Fib)and platelet(PLT).Results INR,APTT and PLT in both TG and RTG groups were significantly lower compared with that in NCG before treatment,and Fib in two former groups were higher than that in NCG.Compared with those values before treatment,Fib decreased and PLT increased significantly after LMWH treatment in TG.However,there were no significant changes in other parameters in TG after LMWH treatment.Severe side effects such as haemorrhage were not found in patients in TG.Conclusion LMWH could ameliorate hypercoagulability following severe burns.

COMPARISON AND ANALYSIS OF THE INFLUENCE OF DIFFERENT STIMULATING FACTORS ON PROTEIN METABOLISM OF RAT SKELETAL MUSCLE / 解放军医学杂志

Objective To investigate the influence of different stimulating factors on protein metabolism of rat skeletal muscle. Methods Three kinds of animal models were used in this study as follows: burn injury group (group B, n =40), endotoxin challenge group (group E, n =40), and burn injury followed by endotoxin challenge group (group B+E, n =40). The proteolytic rate of extensor digitorium longus (EDL) muscle was determined by amino acid auto analyzer after incubation in a muscle incubation system. Results The proteolytic rate of EDL muscle of rats was increased markedly in those three groups, especially the myofibrillar proteolytic rate. The protein breakdown of muscle in group E was higher than that of group B, and it was even much higher in group B+E. Conclusions As compared among burn injury, endotoxin challenge and burn injury followed by endotoxin challenge, the change in muscle proteolytic rate is correlated to seriousness of the injuries

EXPERIMENTAL STUDY ON CHANGE IN UBIQUITIN TRANSCRIPTION EXPRESSION AFTER EARLY ESCHARECTOMY IN SEVERELY BURNED RATS / 解放军医学杂志

Objective To investigate the mechanism of changes in skeletal muscle proteolytic rate after escharectomy during early stage of severe burn in rats. Methods One hundred and fifty six Wistar rats were subjected to 30% total body surface area (TBSA) full thickness burn on the back, and they were randomly divided into normal control group (group C), burn injury group (group B), escharectomy during early stage (1 PBD) group (group S)and escharectomy after early stage (4 PBD) group (group N). The extensor digitorium longus (EDL) muscle was collected from each rats using a technique that injury to the muscle was avoided, and the proteolytic rate of EDL was determined after in vitro muscle incubation with sufficient oxygen supply with amino acid automatic analyzer. Transcriptional expression of ubiquitin was assessed by Northern Blot. Results The myofibrillar proteolytic rate (MPR) was markedly increased at each time points after scald, and the MPR in group S was significantly decreased compared with group N. The expression of ubiquitin mRNA markedly increased in the three groups compared with group C, and was decreased in Group S. There was a significant positive correlation between MPR and expression of ubiquitin mRNA. Conclusion Escharectomy during early stage can alleviate protein catabolism in skeletal muscle, and may be related to decreased activity of ubiquitin proteasome pathway

Studies on the preparation and end result of porcine acellular dermal matrix without cytotoxicity / 解放军医学杂志

Objective To prepare porcine acellular dermal matrix(PADM) without cytotoxicity,to evaluate its biological safety,and investigate its end result in vivo.Methods A piece of porcine split-thickness skin of 0.3-0.4mm was obtained,the epidermis was removed by hypertonic saline immersion,and then the skin was immersed in sodium hydroxide solution,and dermal cells were removed by shaking water bath at normal temperature to harvest PADM.After cross-linking and sterilization,bacteria and porcine virus were examined in the PADM.The PADM were embedded beneath the Sprague-Dawley(SD) rat's skin,and then the general and histological changes were observed after transplantation.Result The obtained PADM was soft and elastic,easy to be moulded,and convenient for operation.No cellular component was found in PADM,the collagen was regularly arranged and elastic fibers were abundant.Bacteria and virus examination of all specimens was negative.No immunologic rejection of PADM was found after being embedded beneath the skin of SD rat.The PADM adhered to the wound firmly,and it was difficult to detach 3 weeks after implantation,and the general structures of PADM beneath rat skin were discernible 24 weeks after implantation.The PADM was infiltrated by inflammatory cells in the early stage,fibroblasts and capillary vessels increased in number along with the time,and collagen fibers gradually gained a regular and compact arrangement in PADM.Conclusions The PADM prepared by hypertonic saline/sodium hydroxide method is a simple preparation process,and there is no cytotoxicity.It has a high biological safety and can be used as dermal scaffold with long-term existence in vivo.

Construction of the fusion protein between EGFP and IgG-binding peptide / 中国海洋药物

Objective Making the fusion protein of IgG-binding peptide with enhanced green fluorescent protein(EGFP) and determining its bioactivity.Methods The enhanced green fluorescent protein(EGFP) gene was cloned into pEZZ 18 vector containing ZZ peptide gene to construct expression vector pSpA-EGFP-His.The fusion protein was expressed in E.coliDH5? and its bioactivity was detected by competitive ELISA and fluorescence properties.Results The fusion protein migrated at approximately 42kD in SDS-PAGE,which correspond to the theoretical molecular weight.The spectra of SpA-EGFP fusion protein was similar to what was reported.SpA-EGFP competed with SpA-Peroxidase to bind IgG.Conclusion The plasmid pSpA-EGFP-His correctly expressed in E.coli.The fusion protein retains the bifunctional effects of EGFP and IgG-binding activity.