Sub-cytotoxic concentrations of ionic silver promote the proliferation of human keratinocytes by inducing the production of reactive oxygen species / 医学前沿

Silver-containing preparations are widely used in the management of skin wounds, but the effects of silver ions on skin wound healing remain poorly understood. This study investigated the effects of silver ions (Ag) on the proliferation of human skin keratinocytes (HaCaT) and the production of intracellular reactive oxygen species (ROS). After treating HaCaT cells with Ag and/or the active oxygen scavenger N-acetyl cysteine (NAC), cell proliferation and intracellular ROS generation were assessed using CCK-8 reagent and DCFH-DA fluorescent probe, respectively. In addition, 5-bromo-2-deoxyUridine (BrdU) incorporation assays, cell cycle flow cytometry, and proliferating cell nuclear antigen (PCNA) immunocytochemistry were conducted to further evaluate the effects of sub-cytotoxic Ag concentrations on HaCaT cells. The proliferation of HaCaT cells was promoted in the presence of 10 and 10 mol/L Ag at 24, 48, and 72 h. Intracellular ROS generation also significantly increased for 5-60 min after exposure to Ag. The number of BrdU-positive cells and the presence of PCNA in HaCaT cells increased 48 h after the addition of 10 and 10 mol/L Ag, with 10 mol/L Ag markedly increasing the cell proliferation index. These effects of sub-cytotoxic Ag concentrations were repressed by 5 mmol/L NAC. Our results suggest that sub-cytotoxic Ag concentrations promote the proliferation of human keratinocytes and might be associated with a moderate increase in intracellular ROS levels. This study provides important experimental evidence for developing novel silver-based wound agents or dressings with few or no cytotoxicity.

Clinical application of artificial dermis combined with basic fibroblast growth factor in the treatment of cicatrix and deep skin wounds / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effects of artificial dermis combined with basic fibroblast growth factor (bFGF) on the treatment of cicatrix and deep skin wounds.</p><p><b>METHODS</b>The clinical data of 72 patients with wounds repaired with artificial dermis, hospitalized in our unit from October 2010 to April 2015, conforming to the study criteria, were retrospectively analyzed. The types of wounds were wounds after resection of cicatrices, deep burn wounds without exposure of tendon or bone, and wounds with exposure of small area of tendon or bone, in a total number of 102. Wounds were divided into artificial dermis group (A, n=60) and artificial dermis+ bFGF group (B, n=42) according to whether or not artificial dermis combined with bFGF. In group A, after release and resection of cicatrices or thorough debridement of deep skin wounds, artificial dermis was directly grafted to wounds in the first stage operation. After complete vascularization of artificial dermis, wounds were repaired with autologous split-thickness skin grafts in the second stage operation. In group B, all the procedures were exactly the same as those in group A except that artificial dermis had been soaked in bFGF for 30 min before grafting. Operation area, complete vascularization time of artificial dermis, survival of skin grafts, and the follow-up condition of wounds in the two groups were recorded. Data were processed with t test and Fisher's exact test.</p><p><b>RESULTS</b>(1) Operation areas of wounds after resection of cicatrices, deep burn wounds without exposure of tendon or bone, and wounds with exposure of small area of tendon or bone in the two groups were about the same (with t values from -1.853 to -0.200, P values above 0.05). Complete vascularization time of artificial dermis in wounds after resection of cicatrices, deep burn wounds without exposure of tendon or bone, and wounds with exposure of small area of tendon or bone in group B were respectively (15.6 ± 2.9), (14.7 ± 2.7), and (20.3 ± 4.4) d, and they were shorter by an average time of 2.7, 4.0, 7.4 d, respectively, as compared with those in corresponding types of wounds in group A [respectively (18.3 ± 4.7), (18.7 ± 4.2), and (27.7 ± 8.8) d, with t values from -2.779 to -2.383, P values below 0.05]. (2) The ratio of skin grafts with excellent survival in the three types of wounds in group B were higher than those in corresponding types of wounds in group A, but there were no statistically significant differences (with P values above 0.05). (3) Patients were followed up for 1 to 48 months, and there were no obvious cicatrices in skin graft sites and the donor sites during the following time.</p><p><b>CONCLUSIONS</b>Artificial dermis combined with bFGF can effectively shorten the vascularization time of artificial dermis in wounds after resection of cicatrices and deep skin wounds.</p>

Changes and immune function mechanism of tumor necrosis factor-alpha in murine peritoneal macrophage after severe scald / 中国组织工程研究

BACKGROUND: Severe scald injury leads to a variety of disorders in the immune system. Activated macrophages are known to secrete many kinds of biologically active transmitter, but the relation between the functional disorder of the macrophages and signal transduction after burn injury has not been fully understood.OBJECTIVE: To observe the changes in nuclear factor(NF) -κκB activity and expressions of IκκB-α and tumor necrosis factor(TNF) -α in peritoneal macrophage of mice at different time points after severe scald injury and after the application of specific NF-κκB inhibitor pyrrolidine dithiocarbamate (PDTC), thereby to explore the mechanism of macrophage dysfunction in light of signal transduction.DESIGN: A randomized controlled experimental research.SETTING: State Key Laboratory of Trauma, Burn and Combined Injury.MATERIALS: The experiment was carried out in the State Key Laboratory of Institute of Burn Research, Third Military Medical University during the period from January to June, 1999, using 30 healthy clean-grade Kunming mice of inbred strain.INTERVENTIONS: Common scald injury models(with third degree burn of 15% total body surface area) were established in the mice, which were randomized into 6 groups according to different time points after the injury for observation, namely 0 hour(normal control group) and postburn 2, 6, 12,24 and 48 hours. Peritoneal macrophages were collected at these time points for examining TNF-α content using radio-immunoassay and NF-κκB activity by means of electrophoretic mobility shift assay(EMSA). The expressions of IκκB-α and TNF-α mRNA were determined by immunoblotting method and reverse transcription-PCR, respectively.MAIN OUTCOME MEASURES:Examinations of ① the content of TNF-α, ② NF-κκB activity,③ expression of IκB-α, and ④ expression of TNF-α mRNA.RESULTS: Macrophage secretion of TNF-α was enhanced postburn, reaching the peak level at 12 hours[(1085.65 ± 122.99) ng/L], which was significantly higher than that in the normal control group( t = 14.92, P ＜ 0.01) .Postburn NF-κκB activity significantly increased after the injury, peaking at 2 hours[ (56. 8 ± 7.3)RDU], which occurred much earlier than the peak of TNF-α secretion( t=13. 31, P ＜ 0.01 ). Compared with that in the normal control group, IκB-α expression decreased significantly 2 hours postburn ( t =4. 23, P ＜ 0. 01) to 0. 632 ±0. 086, followed by gradual increase to the peak level to 1. 161 ± 0. 097 24 hours after the burn injury( t = 7.06, P＜ 0. 01) and then by slight decrease to 1. 149 ±0. 167 till 48 hours(t = 4. 82, P ＜ 0.01) . Twelve hours after injury was the time point for intervention with PDTC application, when NF-κκB activity and TNF-α mRNA expression both decreased significantly( P ＜ 0.01 ).CONCLUSION: NF-κB activity and TNF-αmRNA expression decrease significantly after severe scald. At high levels, IκB-α and NF-κκB maintain an interaction for their restriction. After burn injury, NF-κκB signal transduction pathway is involved in the modulation of TNF-α expression in mouse peritoneal macrophage.

Alternation and modulation of tumor necrosis factor-alpha and protein kinase C in celiac macrophage of mouse after serious scalding / 中国组织工程研究

BACKGROUND: Serious scalding leads to dysfunction of each aspect in immune system, and activated macrophage can secret many bioactive transmitters. The relationship between macrophage dysfunction and signal conduction after scalding is unclear at present.OBJECTIVE: To observe the alternation of tumor necrosis factor- alpha(TNF-α) at different time points after scalding and the activity of nuclear factor κB(NF-κB) and alternation of protein kinase C (PKC) after the application of specific modulator H-7 to explore whether PKC participates in the modulation of TNF-α in macrophage on signal conduction level for the clarification of some mechanisms of macrophage dysfunction.DESIGN: A randomized controlled experimental study by employing experimental animals as subjectsSETTING: An institute of burn research of a military medical university MATERIALS: The study was conducted in the Laboratory (state) of the Institute of Burn Research, Third Military Medical University of Chinese PLA between January and December 1999. Experimental animals were 32 healthy clean inbreeding Kunming white mice.METHODS: 15% Ⅲ scalding was created in mice for the establishment of routine scalding model. Mice were randomly divided into 6 groups according to different time points before or after scalding, I.e. 0(normal control group), 2, 6, 12, 24, or 48 hours group. Celiac macrophages were collected for the detection of TNF-α content by radioimmunoassay, NF-κB activity by electrophoretic mobility shift analysis (EMSA), and membrane or plasma PKC activity by isotope analysis.MAIN OUTCOME MEASURES: ① TNF-α content; ② NF-κB activity; ③Membrane or plasma PKC activity RESULTS: After scalding, macrophage excessively secreted TNF-α and reached its peak of (1 085.65 ± 122.99) ng/L at 12 hours, which was significantly higher than that of control group( t = 14.92, P ＜ 0.01 ).Compared with control group, membrane PKC activity increased after scalding, which significantly heightened to(231.80 ± 31.66) nmol/min · g at 2hours( t = 7. 930, P ＜ 0.01 ), slightly decreased to close to normal level of (174.29±16.80) nmol/min· gat 6hours(t=2.531, P ＜0.05), and rapidly elevated at 12 hours [512. 10 ±33.42) nmol/min · g] and 24 hours [ (454.70 ± 21.06) nmol/min · g] to reach its peak of(530.49 ± 28.54)nmol/min. G at 48 hours( t = 29.42, 28.03, 30. 19, P ＜ 0. 01 ). Correlation analysis of the alternation between TNF-α and membrane PKC indicated a significant positive correlation( r = 0. 796 4, P ＜ 0. 05) . As indicated by EMSA image, NF-κB activity significantly elevated after scalding. Twelve hours after scalding was set as modulation point, NF-κB activity was significantly inhibited by the application of H-7.CONCLUSION: The secretion of TNF-α and the activities of PKC and NF-κB are significantly activated in celiac macrophage after scalding, and PKC-NF-κB signal pathway participates in the modulation of TNF-α expression, which provide experimental data for the modulation of immune function and rehabilitative intervention during scalding.serious scalding.

Construction and sequencing of recombinant lentiviral plasmid affecting human CCL20 gene by RNA interfering / 第三军医大学学报

Objective To design the small interference RNA (siRNA) specific to human CCL20 gene by RNA interfering technique, construct its recombinant lentiviral expression vectors, and identify these vectors by DNA sequencing. Methods According to Tuschl’s principle, the siRNA was designed and converted into cDNA of shRNA (small hairpin RNA) of siRNA for CCL20 gene. The cDNA was synthesized and inserted into plasmid pHSER-dsRNA-GFP-SIN which was linearized by restriction endonucleases SpeⅠ and SalⅠ. The recombinant plasmid was transformed into competent E. coli. DH5? cells. The positive recombinant colony was selected by ampicillin medium agar and identified by DNA sequencing. Results Two recombinant lentiviral plasmids of siRNA specific to CCL20 gene were constructed successfully. Their DNA sequence analysis completely coincided with their designed sequences. Conclusion Lentiviral vector-based siRNA expression plasmids against CCL20 gene have been successfully constructed and identified. They will be further used for interfering CCL20 mRNA transcription and lay the foundation for CCL20 gene modified human keratinocyte stem cells.

The Effects of Total Saponins of Panax Notogineseny (PNS) on NF-κB Activity and TNF-α mRNA Expression of Peritoneal Macrophages after Severe Scald / 医药导报

Objective:To explore the mechanism of total saponins of panax notogineseny (PNS) on secretion of TNF-α by macrophages and optimal dosage of PNS in vitro, through observation of the effects of different PNS dosages on nuclear factor-κB (NF-κB) activity and TNF-α mRNA expression of murine peritoneal macrophages (PMΦs) after severe scald.　Methods: The experimental model of 15% TBSA full-thickness scalded mice with vapor was used and PMΦ collected. NF-κB activity was measured by EMSA and TNF-α mRNA by RT-PCR. 　Results: There was a significant increase of the NF-κB activity and TNF-α mRNA expression in the PMΦs following scald, which was inhibited by application of PNS. It was also found that the effects of PNS was dosage-dependent within a certain range of concentrations, with the inhibition effect most obvious at 0.8 mg*mL-1.　Conclusion:PNS probably decreases TNF-α mRNA expression by inhibiting the NF-κB activity of PMΦs.

The influence of 18% TBSA fullthickness scalding on murine splenic T lymphocyte phosphoinositide-specific PLC signal pathway and its function / 中国免疫学杂志

Objective:To investigate the changes of postburn activity on murine splenic T lymphocyte phosphoinositide-specific PLC signal pathway, and look for the relationship between the postburn and T cell function suppression, IL-2 and IL-10 secretion.Methods:The experimental model was 18% TSBA (total body surface area) fullthickness scalded mice by vapor. The activities of G-protein, PTK (membrane, cytoplasmic) PKC (membrane, plasmic),PI-PLC and cytoplasmic free calcium concentration were detected at different postburn periods, moreover T lymphocyte proliferating function, IL-2 and IL-10 secretion were examined.Results:Compared with control group, membrane GTPase and plasmic PI-PLC enzyme were suppressed after scalded, calcium concentration lowered down significantly, the activities of PTK and PKC were complex, membrane PKC activity elevated after decreasing, those of plasmic PKC were just on the contrary, and the total activity of membrane and plasmic PKC was not stable; Membrane PTK activity decreased in the postburn early stage, then increasing.T cells proliferating function and IL-2 production marginally reduced, and the depressed levels of IL-2 production and T cells proliferating activity were positive parallel with the activities of G-protein and Ca ++. Cytoplasmic PKC activity lowered down after elevating, which was just negative linearly correlated with IL-10 secretion.Conclusion:Inhibition of G-protein ?PTK and Ca ++ activities in phosphoinositide-specific PLC signal pathway was the main reason which resulted in the decrease of IL-2 secretion, suppressed T cell proliferation and the dual-directional changes in IL-10 secretion.

Preliminary application of the grey system forecast in buru medicine / 第三军医大学学报

The GM(1,1)model of the grey system forecast was used to process and analyze the total annual admission of patients,the total number of annual patient death,and the annual mortality rate of the burn patients of the Institute in the 5-year-period from 1986 to 1990.3 corresponding forecast models were established respectively.The forecast values by these models were quite well accordant with the actual values in the period from 1986 to 1990.The forecasts of 1991 by these models are that the total annal admission of patients will approach that of 1989,which was the highest one in the last 5-year-period,and the total number of annual patient deatb and annual mortality rate will be slightly higher than that of 1990,which was the lowest one in the last 5-year-period.The grey system forecast will be beneficial for the work-planning and the personnel arrangement of our Institute.

Postburn Kinetic Changes of Stress Hormones and Splenocyte Immune Functions in Rats / 第三军医大学学报

After the major burn a multitude of immunologic alterations develop. Although the roles of suppressor cells and serum inhibitory factors in the mechanism of postburn immunosuppression are well known, the role of the neuroendo-crine response is still not understood. In this paper determining stress hormones and splenocyte immune functions are simultaneously focused on in deeply burned rats with 25% BSA. The changes of plasma corticosterone (B) and catecholamine are dominant within 24 h postburn(PB), and soon return to normal. The endogenous activity of splenocytes (SBT) decreases at 12 h PB. while the ability of spleno-cytes responsing to ConA in vitro (MSBT) is almost unchanged. The pathological lesions of spleen may be affected by increasing levels of plasma B. When most of the stress hormones return to normal at 72 h PB the SBT and MSBT become different. This may be influenced by many other factors rather than the neuroendocrine system. The differences between SBT and MSBT suggest thai SBT may reflect the more real condition of lymphocytes in vivo than MSBT in vitro. Following burns the roles of streess hormones actually participate in the immune regulation such as plasma B and catecholamine act in the early postburn stage.