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BACKGROUND: Scar hypertrophy is always followed by the wound healing in burn and trauma. Endothelial cells play a key role in scar hypertrophy, so inhibitory growth of endothelial cells can relieve scar hypertrophy to a certain degree. OBJECTIVE: To construct a recombinant adenovirus vector expressing human endostatin (Ad/hEnd), and to investigate the cooperative effect of Ad/hEnd and keratinocyte on endothelial cell proliferation. DESIGN, TIME AND SETTING: Observational study, which was performed in the State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Burn Research, Southwest Hospital of the Third Military Medical University of Chinese PLA between September 2006 and May 2007. MATERIALS: pAdTrack-CMV and pAdEasy-1 were obtained from Stratagene Company, USA; 293 cell and Ecoli.DH5α were stored in our laboratory. METHODS: The endostatin gene sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR) based on mRNA of human fetal hepatic tissue and inserted into the adenovims shuttle plasmid pAdTrack-CMV to obtain recombinant plasmid pAdTrack-ES. After identification, positive recon was transformed into pAdeasy 1 recipient virus to screen positive clones. The adenovirus Ad/hEnd was generated from 293 cells and identified by PCR and fluorescence microscope. Then the keratinocytes were infected with Ad/hEnd, and co-cultured with endothelial cells by nest dish culture method. The content of endostatin was detected, and the non-transfection keratinocytes were used as the controls. MAIN OUTCOME MEASURES: Homologous recombination and identification of pAd/hEnd; generation and identification of Ad/hEnd; endostatin expression after 293 cell transfection; purification and titer measurement of Ad/hEnd; content of endostatin in culture solution; apoptotic percentage of endothelial cells; inhibitory ratio of endothelial cells. RESULTS: Ad/hEnd was constructed and the virus titer was generally up to 1.65×1012 PFU/L. Ad/hEnd-infected keratinocytes could effectively express and secrete endostatin of which the content reached 226 μg/L after 3 days of co-culture. The apoptotic percentage and inhibitory ratio of the endothelial cells co-cultured with Ad/hEnd-infected keratinocytes were significantly higher than those in control group (P<0.05). CONCLUSION: Ad/hEnd-infected keratinocytes co-cultured with endothelial cells can promote apoptosis and inhibit proliferation of endothelial cells through excretion of endostatin.
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Objective To investigate the protective effects of diazoxide on cardiomyocytes after severe burn injury Methods A total of 24 healthy Wistar rats were randomized into normal control group(Control), burn group(Burn) and diazoxide treated group(Diazo)( n =8) Rats in Burn and Diazo groups were inflicted with 30%TBSA Ⅲ degree burn and resuscitated with Ringer's solution intraperitoneally 30 min after burn Diazoxide was injected into rats in Diazo group at the dose of 10 mg/kg through the external jugular vein After rats were sacrificed at 6 h after burn, myocardial mitochondrial K + influx, respiratory function, Ca 2+ concentration ([Ca 2+ ]m), MDA content, serum CK and LDH levels were determined Results Mitochondrial K + influx of Diazo group was evidently higher than that in Control and Burn group Mitochondrial respiratory control rate(RCR) and ST 3 in Diazo group were higher than that in Burn group However, [Ca 2+ ]m, MDA, CK and LDH levels in Diazo group were significantly lower than those in Burn group Conclusion Diazoxide can attenuate the damage to cardiomyocytes after severe burn injury, which might be related to the opening of mitochondrial K + channel, inhibition of mitochondria from Ca 2+ overloading and decrease of free radical production
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Objective To investigate the changes of myocardial mitochondrial permeability transition pore(PTP) and its mechanism in the early stage after severe burns. Methods An experimental model of 30% TBSA full thickness skin scalding was established in rats. All rats were injected with deoxyglucose(DOG) before sacrifice. Myocardial mitochondrial DOG and cytochrome c content, Ca 2+ concentration([Ca 2+ ] m) and MDA content were determined. Results ① There were no obvious changes of mitochondrial DOG and cytochrome c content at 1 h after burns, but mitochondrial DOG increased evidently at 3, 6, 12 and 24 h after burns. Meanwhile, cytochrome c content was significantly lower than that of the control, being 68.8%, 50.0%, 77.1% and 72.9% of that in the control, respectively. ② [Ca 2+ ] m and MDA content were significantly higher than those of the control at 3, 6, 12 and 24 h after burns. ③ Mitochondrial DOG content was positively correlated with [Ca 2+ ] m and MDA content, respectively, after burns. Conclusion There is no obvious change in myocardial mitochondrial permeability transition pore, but PTP opening increases markedly at 3, 6, 12 and 24 h after burns. Mitochondrial Ca 2+ overloading and increase in free radicals may be the cause leading to PTP opening.
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AIM：To investigate the change in myocardial mitochondrial Ca2＋ concentration (［Ca2+］m) and its mechanism in the early stage of severe burn. METHODS：An experimental model of 30%TBSA full-thickness skin scalding was reproduced in rats. ［Ca2+］m, cytosolic Ca2＋ concentration (［Ca2＋］c) and mitochondrial Ca2＋ transport velocity were determined. RESULTS: ① ［Ca2+］m increased evidently at 1st hour postburn, and continuously at 3rd hour, reached the peak at 6th hour postburn, then, it decreased at 12th and 24th hour, but remained in higher level than that of the control. ② There was no significant difference in ［Ca2＋］c between 1st hour postburn and the control, but ［Ca2＋］c increased evidently at 3rd, 6th, 12th, 24th hour postburn. ③ mitochondrial Ca2＋ uptake velocity at 1st hour postburn was higher than that of control, and Ca2＋ release velocity didn't change obviously, but both of them were decreased at 3rd, 6th, 12th, 24th hour postburn. ④ ［Ca2+］m was positive correlated with ［Ca2＋］c after burn, and negative correlated with mitochondrial Ca2＋ release velocity at 3rd, 6th, 12th, 24th hour postburn, respectively. CONCLUSION: There was obvious Ca2＋ overload in myocardial mitochondria after severe burn, the mechanism of which might include ascent of ［Ca2＋］c and disorder of Ca2＋ transport in mitochondria.
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AIM: To study the role of mitochondrial nitric oxide synthase (mtNOS) in the damages of myocardial mitochondria during the early stage after severe burns.METHODS: An experimental model of 30% TBSA full-thickness skin scalding was reproduced in rats. Myocardial mitochondria were isolated from control and burned rats at 1, 3, 6, 12 and 24 h postburn. The mitochondrial respiratory function, content of mitochondrial calcium([Ca 2+ ] m) and activities of mtNOS and cytochrome c oxidase were determined. RESULTS: (1) Myocardial mitochondrial respiratory control rate(RCR) at 1 h was evidently higher than that of control, but at 3, 6, 12 and 24 h postburn, it was significantly lower than that of the control. The changes in ST 3 is parallel to those of RCR, and ST 4 was evidently increased only at 3 h postburn. (2) [Ca 2+ ] m was higher at all time points postburn and the activity of mtNOS was higher significantly only at 3, 6, 12 and 24 h than that of the control. The activity of cytochrome c oxidase at the 3, 6, 12 and 24 h was low comparing to the control. (3) After severe burns, RCR was negatively correlated with mtNOS activity( r=0.9347, P
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AIM: To study the effects of nitric oxide (NO) on mitochondrial damage caused by exogenous calcium. METHODS: Normal myocardial mitochondria were divided into three groups; L-arginine control group (CG), Ca 2+-damaged group (DG) and L-NAME-preserved group (PG). Mitochondria of all groups were incubated at 30℃ with reaction medium containing 20 ?mol/L EDTA, 100 ?mol/L CaCl 2 and 1 ?mol/L L-NAME with 100 ?mol/L CaCl 2 respectively. Then the NO- 2/NO- 3 contents, mitochondrial viability and membrane potential were investigated.RESULTS: The NO- 2/NO- 3 contents of DG was obviously higher than that of CG and PG, meanwhile, there was no obvious difference between CG and PG. Mitochondrial viability and membrane potential of DG were significantly lower than that of CG and PG, and negatively related to NO- 2/NO- 3 contents (r=-0.5297, P
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AIM: To study the effects of nitric oxide (NO) on mitochondrial damage caused by exogenous calcium. METHODS: Normal myocardial mitochondria were divided into three groups; L- arginine control group (CG), Ca2 + - damaged group (DG) and L - NAME - preserved group (PG). Mitochondria of all groups were incubated at 30℃ with reaction medium containing 20μmol/L EDTA, 100μmol/L CaC12 and 1 μmol/L L- NAME with 100μmol/L CaCl2 respectively. Then the NO2-/NO3- contents, mitochondrial viability and membrane potential were investigated. RESULTS: The NO2-/NO3 contents of DG was obviously higher than that of CG and PG, meanwhile, there was no obvious difference between CG and PG. Mitochondrial viability and membrane potential of DG were significantly lower than that of CG and PG, and negatively related to NO2-/NO3- contents ( r = - 0.5297, P < 0.01; r = -0.6041, P < 0.01 ). But, the mitochondrial viability and membrane potential of PG were still lower than that of CG. CONCLUSION: Exogenous calcium could activate mitochondrial nitric oxide synthase resulting in NO production and the latter play an important role in decreasing mitochondrial viability and membrane potential.
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AIM:To investigate the change in myocardial mitochondrial Ca 2+ concentration ([Ca 2+ ] m) and its mechanism in the early stage of severe burn. METHODS:An experimental model of 30%TBSA full-thickness skin scalding was reproduced in rats. [Ca 2+ ] m, cytosolic Ca 2+ concentration ([Ca 2+ ] c) and mitochondrial Ca 2+ transport velocity were determined. RESULTS: ① [Ca 2+ ] m increased evidently at 1st hour postburn, and continuously at 3rd hour, reached the peak at 6th hour postburn, then, it decreased at 12th and 24th hour, but remained in higher level than that of the control. ② There was no significant difference in [Ca 2+ ] c between 1st hour postburn and the control, but [Ca 2+ ] c increased evidently at 3rd, 6th, 12th, 24th hour postburn. ③ mitochondrial Ca 2+ uptake velocity at 1st hour postburn was higher than that of control, and Ca 2+ release velocity didn't change obviously, but both of them were decreased at 3rd, 6th, 12th, 24th hour postburn. ④ [Ca 2+ ] m was positive correlated with [Ca 2+ ] c after burn, and negative correlated with mitochondrial Ca 2+ release velocity at 3rd, 6th, 12th, 24th hour postburn, respectively. CONCLUSION: There was obvious Ca 2+ overload in myocardial mitochondria after severe burn, the mechanism of which might include ascent of [Ca 2+ ] c and disorder of Ca 2+ transport in mitochondria. [