Subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 protein / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) protein in endothelial cells.</p><p><b>METHODS</b>Human umbilical vein endothelial cell strain ECV304 were cultured in vitro. The fusion protein of enhanced green fluorescent protein (EGFP)-EOLA1 expressing plasmid was constructed. Empty plasmid with EGFP at N side (pEGFP-N2) and fusion protein expressing plasmid EGFP-EOLA1 was respectively transfected into ECV304 cells with liposome. After being cultured for 48 hours, the expression levels of EGFP and fusion protein EGFP-EOLA1 in cells were detected with Western blot. The subcellular localization of EOLA1 protein was detected by laser scanning confocal microscope and immunoelectron microscopy.</p><p><b>RESULTS</b>The EGFP-EOLA1 coexpression plasmid was verified to be successfully constructed by enzyme cutting and gene sequencing. The fusion protein of EGFP-EOLA1 was observed to express in transfected cells through Western blot. Green fluorescence scattered all over the ECV304 cells transfected with empty plasmid and cells transfected with fusion protein expressing plasmid, and it gathered obviously in the nuclei in the latter cells. Immune deposits were observed in the matrix of cells transfected with fusion protein expressing plasmid but not in the cells transfected with empty plasmid.</p><p><b>CONCLUSIONS</b>EOLA1 protein is localized in the nucleus and the matrix of ECV304 cell, and it plays its role as a signal transduction factor.</p>

Relationship between learning and memory ability and expression of hippocampal N-methyl-D-aspartic acid (NMDA) in burn rats with depression / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe changes of learning and memory ability (LMA) in burn rats with depression, and study the relationship between LMA and expression of hippocampal NMDA.</p><p><b>METHODS</b>According to simple random method, 46 Wistar rats were divided into burn group (B, with 30% TBSA deep partial-thickness burn, n = 10), depression group (D, with moderate stress stimulation in chronic and unpredictable, n = 12), B + D group (with the same stress stimulation inflicted to B group after burn, n = 12), healthy control group ( HC, without treatment, n = 12). Changes in escape latency was examined in water maze test. Expression of hippocampal NMDA in CA1, CA2 regions and dentate gyrus were observed with immunohistochemistry.</p><p><b>RESULTS</b>Compared with that of HC group (22 +/- 20 s), water maze escape latency in B, D, B + D groups on 2 day after training prolonged (38 +/- 31, 41 +/- 36, 42 +/- 33 s, respectively, P < 0.05 or P < 0.01). Water maze escape latency in B + D group on 4th day after training was longer than that of other groups (P < 0.01). There was no obvious difference in positive expression of NMDA in CA1, CA2 regions among groups (P > 0.05). The positive count of NMDA in dentate gyrus in D group (198 +/- 14) and B + D group (191 +/- 6) were lower than that of HC group (224 +/- 23, P < 0.05 or P < 0.01), but there was no obvious difference between HC group and B group (219 +/- 25, P > 0.05).</p><p><b>CONCLUSIONS</b>Burn complicated with depression can reduce LMA, which may be due to a decrease in NMDA in dentate gyrus.</p>

Influence of HSP70 on function and energy metabolism of mitochondria in intestinal epithelial cells after hypoxia/reoxygenation / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effect of recombinant adenovirus-mediated heat shock protein 70 (HSP70) on energy metabolism of mitochondria in intestinal epithelial cells (IEC-6) after hypoxia/reoxygenation injury .</p><p><b>METHODS</b>IEC-6 cells were transfected with HSP70 recombinant adenovirus vectors (Ad-HSP70) and empty adenovirus vectors. The expression of HSP70 protein was detected by Western blotting. Cultured IEC-6 cells were divided into: control group (without treatment), hypoxia/reoxygenation group (with challenge of hypoxia/reoxygenation) and Ad-HSP70 transfection group (with challenge of hypoxia/reoxygenation after Ad-HSP70 transfection). The activity of mitochondrial dehydrogenase was assessed by MTf method. The contents of cellular ATP, ADP , AMP and energy charge (EC)were determined by high-performance liquid chromatography (HPLC).</p><p><b>RESULTS</b>The expression of HSP70 protein in IEC-6 cells was significantly upregulated after Ad-HSP70 transfection compared with empty adenovirus vector transfection. Compared with that in control group, the activity of mitochondrial dehydrogenase was significantly lowered in IEC-6 cells in hypoxia/reoxygenation group (P < 0.01). The activity of mitochondrial dehydrogenase in Ad-HSP70 transfection group was significantly greater than that in hypoxia/reoxygenation group (P < 0.01). Compared with those in control group,the content of cellular ATP was significantly decreased in hypoxia/reoxygenation group, the contents of cellular ADP and AMP were significantly increased. The above cell energy indices in Ad-HSP70 transfection group was similar to those in control group (P > 0.05), which were ameliorated compared with those in hypoxia/reoxygenation group (P < 0.050 or P < 0.01). The cellular EC in hypoxia/reoxygenation group (0.615 +/- 0.060) was significantly lower than that in control group (0.748 +/- 0.012, P < 0.01) and Ad-HSP70 transfection group (0.736 +/- 0.028, P < 0.01).</p><p><b>CONCLUSION</b>Ad-HSP70 transfection in IEC-6 cells can upregulate the expression of HSP70, the content of cellular ATP and EC after hypoxia/reoxygenation, and protect mitochondrial function. Mitochondria may be one of main target organelles for HSP70 in protection of IEC against hypoxia/reoxygenation injury.</p>

Pay great attention to research on monitoring burn shock / 中华烧伤杂志

Improvement in early burn treatment has been realized, the mortality of burn shock has been decreased. However, the treatment of burn shock is still inadequate and occult hypoperfusion is usually occurred. This may be difficult to identify the appropriate resuscitation endpoint. The goal in management of burn shock is restoration of adequate tissue perfusion and normalization of cellular metabolism. Traditional endpoints, such as blood pressure, urine output are useful in managing mild and moderate burn shock. Additional endpoints that evaluate the adequacy of global and regional perfusion and oxygenation at the tissue level should be used in treatment of severe burn injury. Now the most useful parameters may be blood pressure, urine output, serum lactate, BE and CVP, SCVO2.

Achievements in burn surgery over the past 50 years in China / 中华烧伤杂志

This paper reflects briefly the main advancements of clinical and scientific research in the field of burn surgery over the past 50 years in China. It includes emergency care of massive burns, resuscitation, anti-infection, prevention and treatment of internal organ injury, metabolic and nutritional support, repair of wound and rehabilitation, and special types of burns. The article also covers the researches in pathology, microbiology, immunology, cell biology, molecular biology, and tissue engineering pertaining to burn injury.

Advancement in study of inhalation injury / 中华烧伤杂志

Inhalation injury is a major contributor to the morbidity and mortality associated with serious burns. The improvement in the understanding of smoke inhalation injury had been obtained in the last half century in China. The models of steam and smoke inhalation injury had been reproduced and a series of experimental studies had been performed. It was found that chemical bronchiotracheitis, pulmonary edema and alveolar collapse (atelectasis) were the primary pathologic findings after inhalation injury. The second inflammatory response would play an important role in the development of acute respiratory failure. The roles of some cytokines, inflammatory cells and pulmonary surfactants in the development of inhalation injury had been elucidated. The etiologic factors and the pathophysiologic changes in inhalation injury had been illustrated clearly. These basic science investigations had led to the advances in protective strategies for the complications of inhalation injury. Now the morbidity and mortality of inhalation injury have decreased markedly in China.

Involvement of JNK signal pathway in hypoxia related upregulation of calcium/calmodulin-dependent serine protein kinase in endothelial cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the expression of calcium/calmodulin-dependent serine protein kinase (CASK) induced by short-term hypoxia, and to explore the role of JNK pathway in this signal event.</p><p><b>METHODS</b>EA. hy926 cells were cultured in normoxic condition for 0, 12, 24, 48, 72 h after being exposed to hypoxic condition for 3 h, then the cellular lysates were extracted. CASK promoter luciferase reporter recombinant was constructed and transfected into EA. hy926 cells for 48h. Cellular lysates were extracted 1, 3, 6, 12 h after hypoxia treatment and were used to detect firefly luciferase activity and rinella luciferase activity with luminometer. EA. hy926 cells were cultured under hypoxic condition for 1, 3, 6, 12 h or under normoxic condition, then the cell lysates were extracted and used to detect phospho-JNK with Western blot. EA. hy926 cells were pretreated with different concentrations of JNK specific inhibitor SP 600125 (0, 10, 100 nmol/L and 1,10 micromol/L) 1h before hypoxic treatment of various duration, and the cell lysates were extracted to detect CASK expression with Western blot.</p><p><b>RESULTS</b>CASK expression was obviously elevated by hypoxia, and the high expression sustained for 72 h when the hypoxic cells were cultured in normal conditions, and it was significantly higher than that of normal controls. Dual luciferase reporter assay showed that CASK promoter activity was significantly increased after hypoxia (0.010 +/- 0.003, P < 0.01), and it reached the peak 12 hrs after hypoxia (0.192 +/- 0.023, P < 0.01). The phosphorylation of JNK was enhanced with the prolongation of hypoxic time. CASK protein expression was suppressed by JNK specific inhibitor SP600125 in a dose dependent manner, and it decreased to the lowest level with 10 micromol/L SP600125 pretreatment.</p><p><b>CONCLUSION</b>JNK signal pathway is involved in short-term hypoxia related CASK upregulation.</p>

Some aspects worth concern in the management of burn injury / 中华烧伤杂志

Although the outcome of burn patients has been improved, many aspects of management of severe burn patients remain controversial. Here we focus on the management of hypermetabolism and the resuscitation of respiratory function. Currently, the fluid resuscitation method shifts from insufficient fluid regimen to excessive fluid loading. The benefit of colloid infusion and restrictive blood transfusion need to be authenticated by further clinical trial, and the best form of fluid resuscitation has yet to be identified. The respiratory management of burn patients had been improved. Early tracheostomy, ventilation with low tidal volume and bronchoalveolar toilet are recommended. Many potential beneficial treatment strategies have been identified by recent research in the metabolic response to burn injury. Although immunomodulation therapy is promising, most of them are not clinical viable,and further clinical research is warranted.

The influence of terbutaline on VEGF gene expression in rat astrocytes after norepinephrine and burn serum induction / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of adrenoreceptor beta-agonists terbutaline on gene expression of vascular endothelial growth factor (VEGF) in rat astrocyte after induction by norepinephrine (NE) and burn serum.</p><p><b>METHODS</b>The sera of normal and burn rats were separated for use. Primary astrocytes of brain tissue were isolated from neonatal 1-3 d rats and cultured and divided into following groups: (1) CONTROL GROUP: with 10% normal rat serum in the culture medium. (2) NEl, NE2, NE3 groups: with 10% burn rat serum and 10, 20, 50 micromol/L NE in the culture medium, respectively. (3) TBN1, TBN2, TBN3 groups: with 10% burn rat serum and 10, 20, 50 micromol/L NE and 10, 20, 50 micromol/L terbutaline in the culture medium, respectively. The mRNA and protein expression of VEGF in each group were determined with real-time PCR and Western blotting, respectively.</p><p><b>RESULTS</b>There was a low protein expression of VEGF in control group, but it increased slightly in NE1 group, and then increase gradually in NE2, NE3 groups, and it was obviously increased in TBN1, TBN2, TBN3 groups. The mRNA expression of VEGF in NE1, NE2, NE3 groups were [(13.26 +/- 0.03), (10.37 +/- 0.04), (14.87 +/- 0.55) copies/g], respectively, which were significantly higher than that of control [(5.72 +/- 0.12) copies/g, P < 0.01]. In addition, the expression of VEGF mRNA in TBN1, TBN2, TBN3 groups was higher than that in control group, and expression of VEGF mRNA [(13.39 +/- 0.19), (15.77 +/- 0.11), (16.00 +/- 0.07) copies/g] was gradually increased, which showed obvious difference between TBN2 and NE2, and also between TBN3 and NE3 groups (P < 0.01).</p><p><b>CONCLUSION</b>Terbutaline can increase gene expression of VEGF in rat astrocytes after induction by NE and burn serum.</p>

The effect of inhibiting EOLA1 expression in ECV304 cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the effect of inhibiting the expression of endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) on proliferation of human umbilical vein endothelial cell line ECV304.</p><p><b>METHODS</b>After constructing and transfecting EGFP-EOLA1 fusion protein expressive vector into ECV304 cells, the transfected cells was cultured in M199 containing G418 for 5 weeks to screen the cell line stable expression EGFP-EOLA1 fusion protein. Oligonucleotides targeting EOLA1 at different sites were synthesized and inserted into pSinencer3.1/H1 vector. Then, the recombinant vector was transfected into the cultured ECV304 cells and the inhibiting effect to target gene EOLA1 was investigated by observing the green fluorescence in transfected cells under inverted fluorescent microscope and by Western blot assay. The proliferation of ECV304 cells was numbered when the expression of EOLA1 in ECV304 cells was inhibited by RNA interference.</p><p><b>RESULTS</b>The ECV304 cell line stably expressing EGFP-EOLA1 fusion protein was constructed and the siEOLA1 interfere vectors can knock down EOLA1 gene expression specially. When blocking the expression of EOLA1 in ECV304 cells,the proliferation of cells slowed down.</p><p><b>CONCLUSION</b>EOLA1 maybe has a role on the proliferation of cells.</p>

Effect of nuclear factor-kappaB activation on expression of proinflammatory cytokines in rat lung tissues in early stage of burn injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of nuclear factor-kappaB (NF-kappaB) activation on the expression of proinflammatory cytokines in the lung tissues of rats with early-stage burn injury.</p><p><b>METHODS</b>Wistar rats were randomly divided into 3 groups, namely the normal control, burn, burn and PDTC treatment groups, and in the latter two groups, the rats were subjected to 35% TBSA full-thickness burns. Activation of pulmonary NF-kappaB at 1, 3, 6, 12, and 24 postburn hour (PBH) was tested by electrophoretic mobility shift assay , and the expressions of pulmonary tumor necrosis factor alpha (TNF alpha) and interleukin-8 (IL-8) mRNAs at 3, 6, 12, and 24 h were detected by RT-PCR.</p><p><b>RESULTS</b>Compared to that of the control group, activity of pulmonary NF-kappaB in burned rats was markedly increased within 1 PBH and kept increasing till 24 h. Expressions of pulmonary TNF alpha and IL-8 mRNAs increased gradually, reaching the peak level at 6 PBH, and PDTC could effectively inhibit pulmonary NF-kappaB activation and expression of the pulmonary cytokines induced by the burn injury.</p><p><b>CONCLUSION</b>Severe burn injury may activate pulmonary NF-kappaB, which ultimately leads to secretion of cytokines in the lung tissues.</p>

Influence of hypoxia on the proliferation and activity of human umbilical vein endothelial cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of hypoxia on the proliferation and activity of human umbilical vein vascular endothelial cells (EA. hy926).</p><p><b>METHODS</b>EA. hy926 cells were cultured in vitro and divided into normal control and hypoxia groups. The cells in hypoxia group were placed into hypoxic jar and treated with mixed gases(94% N2 +5% CO2 + 1% O2) for 1,3,6 and 12 hours. Then the total proteins were extracted for the determination of the expression of vascular endothelial growth factor (VEGF) and proliferation cell nuclear antigen (PCNA). The cell cycle and growth curve were determined with flow cytometry and MTT method, respectively.</p><p><b>RESULTS</b>The expression of PCNA protein began to increase at 3 post-hypoxia hour (PHH), peaked at 6 PHH, but without obvious difference compared with that at 12 PHH. The expression of VEGF began to increase at 1 PHH, peaked at 6 PHH, and decreased at 12 PHH, though it was still markedly higher than that of normoxia at 12 PHH. MTT results showed that the cell activity began to increase at 1 PHH, and it was still to increased at 3 PHH, then decreased at 6 PHH, and it was lower than that in control group at 12 PHH. The number of cells in G0/G1 phase was decreased, but the cells in S and G2/M phase was increased at 1, 3, 6 PHH when compared with those in normal controls. The proliferation index (PI) of cells in hypoxia group at 1PHH (43 +/- 9)%, 3PHH (39 +/- 11)%, 6 PHH (40 +/- 11))% were higher than that before hypoxia (32 +/- 9)% and 3 (39 +/- 11) % and 6 hours (40 +/- 11)% after hypoxia (P < 0.05). The PI was obviously lower at 12 PHH (27 +/- 4))% compared with that of cells under normoxic condition (P < 0.05).</p><p><b>CONCLUSION</b>Short-term hypoxia is beneficial to promote the proliferation of the cells, but this effect will be inhibited with the prolongation of hypoxia.</p>

Effect of escharectomy on rats'pulmonary NF-?B activation in early stage of burn injury / 中华急诊医学杂志

Objective To investigate the effect of escharectomy on rats' pulmonary NF-?B activation and the expression of pulmonary proinflammatory cytokines in early stage of burn injury.Method Wistar rats were randomly divided into three groups:group A(control group),group B(postburn without escharectomy),group C(escharectomy at early stage of burn injury).Thermal-injuried rats underwent 35% TBSA full-thickness burns. Activation of pulmonary NF-?B at 12 hours and 24 hours postburn was tested by electrophoretic mobility shift assay (EMSA),and at the same time expressions of pulmonary TNF-?mRNA were measured by reverse transcription polymerase chain reaction(RT-PCR)and release of pulmonary TNF-?were assayed by enzyme-linked immunosorbent assay(ELISA).Results Compared with control group,activity of pulmonary NF-?B in group B was markedly increased,reached(19.56?1.36)?10~4 A at 12 hours and(15.23?1.94)?10~4 A at 24 hours,which was higher than that in group A[(4.36?0.38)?10~4 A,P

Investigation and analysis of the self-esteem level and social adaptation ability of hospitalized burn patients / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the self-esteem level and social adaptation ability of hospitalized burn patients in our burn ward.</p><p><b>METHODS</b>One hundred and twenty hospitalized burn patients in our burn ward were enrolled in the study and evaluated according to their sex, severity of burn injury and education level. Their self-esteem level and social adaptation ability were scored with the Felling of Inadequacy Scale and Abbreviated Burn Specific Health Scale.</p><p><b>RESULTS</b>The general score of self-esteem of the patients with mild burns( 183+/-23) was obviously lower than that with moderate and severe burns (167+/-21 and 154 +/-24) , ( P <0.01). The self-esteem level of burn patients was different in different sex and education level. Among the self-esteem scores, male burn patients presented evidently higher scores of self evaluation, social ability, appearance, as well as the general score than those in the female ( P < 0.05). Moreover, the self evaluation score and study ability was higher in those with higher education level than those with lower education. Furthermore, the score of social adaptation ability was higher in the patients with mild burns than that in patients with moderate and severe burns ( P < 0. 01). The social adaptation ability and psychological function were much higher in male patients than those in female patients, but the former were weaker than the latter in regard to the body function. The psychological function, social relationship and general condition of the patients with lower education were better than those with higher education ( P <0. 05 ).</p><p><b>CONCLUSION</b>There existed difference in the self-esteem and social adaptation ability in different burn patients during different periods.</p>

The role of nuclear factor-kappaB in the burn serum-induced expression of intercellular adhesion molecules-1 in endothelial cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the role of nuclear factor-kappaB (NF-kappaB) in the burn serum induced expression of intercellular adhesion molecules-1 (ICAM-1) in endothelial cells.</p><p><b>METHODS</b>Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and divided into control (with normal serum stimulation), burn serum (B, with burn serum stimulation) and PDTC (with burn serum and PDTC stimulation) groups. The NF-kappaB activity in endothelial cells was determined with electrophoretic mobility shift assay (EMSA) at 0.5, 1.0, 2.0, 4.0 post-stimulation hour (PSH). The expression of ICAM-1 at 3.0, 6.0, 12. 0, 24.0 PSH was detected by flow cytometry.</p><p><b>RESULTS</b>The NF-kappaB activity in endothelial cells in burn serum group and PDTC group were markedly higher than that in control group (P <0.01), and it reached the peak at 1.0 PSH [(21.03 +/- 4.87), (7.44 +/- 0.60) x 10(4) A], respectively, then gradually decreased. But it was obviously lower in PDTC group than that in burn serum group ( P <0. 01 ). The expression of ICAM-1 was gradually increased in both burn serum group and PDTC group, reaching the peak level at 12.0 PSH [(327 +/- 37), (142 +/- 31) mean fluorescence intensity], respectively, which were significantly higher than that in control group (P <0.01). But it was evidently lower in PDTC group than that in burn serum group at 12.0 and 24.0 PSH (P <0.01).</p><p><b>CONCLUSION</b>Burn serum can initiate the synthesis and release of adhesion molecules in endothelial cells through activation of NF-kappaB, indicating that NF-kappaB plays an important role in the process of burn serum induced endothelial secretion of adhesion molecules.</p>

Effect of NF-kappaB activation on the early expression of tumor necrosis factor alpha and myocardial dysfunction in burned rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effect of NF-kappaB activation on the early expression of proinflammatory cytokines in myocardium and early myocardial dysfunction in burn rats.</p><p><b>METHODS</b>One hundred and seventy Wistar rats were enrolled in the study and randomly divided into three groups, i. e. control( C, n = 20, with isotonic saline solution) , burn ( B, n = 90, with isotonic solution after burns) and pyrrolidine dithiocarbamate (PDTC, n =60, with isotonic saline and 250 mg/kg PDTC after burns) groups. The rats in B and PDTC groups were inflicted with 35% TBSA full-thickness burns on the back. The activity of myocardial NF-kappaB was determined by electrophoretic mobility shift assay at 1 , 3, 6, 12,24 postburn hours (PBH), with expression of integral absorbance ( A ) value . The expression of myocardial tumor necrosis factor alpha ( TNF-alpha) mRNA was assessed by reverse transcription polymerase chain reaction( RT-PCR) and in situ hybridization at 6, 12 PBH, with expression ofA value. The left ventricular systolic pressure( LVSP) , the left ventricular end diastolic pressure (LVEDP) ,the maximum rate of rise of left ventricular pressure ( +/- dp/dt max) were also observed at 3, 6, 12,24 PBH. RESULTS The activity of myocardial NF-KB in B group was markedly increased at 1 PBH [ (20. 3+/-3. 4) x 104A ] ,which was obviously higher than that in C group (2. 2 +/- 0. 4) x 104A , P <0.01]. It peaked at 3 PBH, and was still evidently higher than that in C group at 24 PBH ( P <0. 01). The expression of TNF-alpha mRNA was obviously higher than that in C group at 3 PBH ( P < 0. 01) , peaking at 6 PBH, and it was mainly expressed in myocardium. The expression of LVSP and +/- dp/dt max were lower, but LVEDP was higher than that in C group during 3 -24 PBH ( P <0.01).</p>

Influence of delayed rapid fluid resuscitation on oxygen metabolism in dogs with burn shock / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of delayed rapid fluid resuscitation on oxygen metabolism in dogs with burn shock.</p><p><b>METHODS</b>Twenty-four mongrel dogs inflicted with 40% TBSA full thickness scald were enrolled in the study and randomly divided into burn control (C), delayed even fluid replacement (E), and delayed rapid fluid replacement (R) groups, with 8 dogs in each group. The changes in oxygen delivery (DO(2)), oxygen consumption (VO(2)), oxygen extraction (O(2)ext) and blood base deficit (BD), and lactate (LA) were determined before scalding and at 2, 6, 8, 12, 24, 36 and 48 post scalding hours (PSHs).</p><p><b>RESULTS</b>The DO(2) in each group was decreased obviously after scalding and was evidently lower than that before injury (P < 0.01), while the O(2)ext value markedly increased compared with that before scalding (P < 0.01). After fluid resuscitation, DO(2) and VO(2) in E and R groups increased, but O(2)ext decreased. The values of DO(2), VO(2) and O(2)ext showed significant differences between R and E groups at 8 PSH (R group vs E group, DO(2): 7.35 +/- 0.21 L.min(-1).m(2) vs 5.32 +/- 0.96 L.min(-1).m(2), P < 0.01; VO(2): 2.02 +/- 0.58 L.min(-1).m(2) vs 1.71 +/- 0.38 L.min(-1).m(2), P < 0.01); The blood BD levels in each group were remarkably lower after scald than that before scald (P < 0.01), and they gradually increased after fluid replacement. The blood BD level in R group at 8 PSH (-6.5 +/- 0.7 mmol/L) was obviously higher than that in E group (-9.3 +/- 1.4 mmol/L, P < 0.01). The blood LA level in each group were evidently higher than that before scald (P < 0.01), and they decreased after fluid replacement. The blood LA level in R group at 8 PSH (2.30 +/- 0.20 mmol/L) was obviously lower than that in E group (2.67 +/- 0.30 mmol/L, P < 0.01)</p><p><b>CONCLUSION</b>Rapid fluid replacement could improve tissue oxygen metabolism, which was beneficial to the correction of tissue oxygen supply when fluid resuscitation was delayed.</p>

Purification of human endothelial overexpressed lipopolysaccharide-associated factor 1 protein / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of obtaining of a highly pure protein of human endothelial overexpressed lipopolysaccharide-associated factor 1 (EOLA1) with metal chelation chromatography.</p><p><b>METHODS</b>Inclusion bodies of the E. coli transformed with EOLA1 gene were extracted and washed with BugBuster Protein Extraction Reagent. The primary purified products were purified by His. Bind Resin Chromatography under denaturing condition and dialyzed for renaturation, and then were analyzed with SDS-PAGE, Western blotting and peptide mass fingerprinting (PMF).</p><p><b>RESULTS</b>EOLA1 was mainly expressed in E. coli as insoluble inclusion bodies. The protein content in the primary extracted inclusion bodies accounted for over 75%, and it accounted for more than 90% after chromatography and renaturation. It was indicated by PMF that the targeted protein peptide overlaid many of designed protein peptide.</p><p><b>CONCLUSION</b>The method of EOLA1 protein purification and renaturation was convenient and efficient, and by this method sufficient amount of highly pure EOLA1 protein could be obtained for the preparation of EOLA1 monoclonal antibody and for the study of its gene function.</p>

Influence of nuclear factor-kappaB activation on the expression of cytokines in monocytes stimulated by burn serum / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effects of NF-kappaB activation on the expression of cytokines in monocytes stimulated by burn serum, so as to explore the mechanism in monocyte activation by burn serum.</p><p><b>METHODS</b>Peripheral blood monocytes (PBMCs) isolated from healthy volunteers were employed as the target cells. The cells were stimulated by serum from healthy volunteers (control), by serum from burn patients (burn serum), and by burn serum with addition of PDTC (pyrrolidine dithiocarbamate). Activation of monocytic NF-kappaB before stimulation and at 0.5, 1.0, 2.0 and 4.0 poststimulation hours (PSH) was assessed with electrophoretic mobility shift assay (EMSA). Expression of TNF-alpha and IL-8 mRNA at 1.0, 2.0, 4.0, 6.0 PSHs was assayed with in situ hybridization (ISH). Meanwhile, the contents of TNF-alpha and IL-8 in the supernatants were assayed by enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The monocytic NF-kappaB activity in burn serum group increased significantly and reached the peak level at 1 PSH [(30.2 +/- 3.5) x 10(4) integration gray scale value] after the PBMCs were stimulated by burn serum, and it was obviously higher than that in control group [(4.4 +/- 0.8) x 10(4) integration gray scale value], (P < 0.01). It gradually decreased and returned to the pre-stimulation state at 2 PSH. The monocytic NF-kappaB activity in PDTC group decreased to [(6.8 +/- 0.9) x 10(4) integration gray scale value at 1 PSH] after the stimulation. The expression of TNF-alpha mRNA of the monocytes and the TNF-alpha level in the supernatant of the cultured PBMCs reached peak level at 1 PSHs after being stimulated by burn serum, and they were obviously higher than those in control group (P < 0.01). While the expression of IL-8 mRNA and the IL-8 level in the supernatant of the cultured PBMCs reached peak level at the 4 PSHs after being stimulated by burn serum, which were obviously higher than those in control group (P < 0.01) too. In addition, the synthesis and release of TNF-alpha (peaked at 1 PSH: 0.52 +/- 0.06 microg/L) and IL-8 (peaked at 4 PSH: 239 +/- 20 ng/L) in the supernatant of PBMCs in PDTC group were evidently higher than those in control group [(0.13 +/- 0.07) microg/L, < 156 ng/L] (P < 0.01).</p><p><b>CONCLUSION</b>Burn serum can induce the activation of NF-kappaB which lead to the synthesis and release of cytokines from PBMC. This result indicates that the activation of NF-kappaB plays important role in the secretion of cytokines from PBMCs induced by the burn serum.</p>

Influence of human telomerase reverse transcriptase gene transfection on the proliferation of human embryonic fibroblasts / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of human telomerase reverse transcriptase (hTERT) gene transfection on the proliferation of human embryonic fibroblasts (hEF).</p><p><b>METHODS</b>hEFs were cultured in vitro. Sense recombinant eukaryotic plasmid (pIRES2-EGFP-hTERT) and pIRES2-EGFP vacant vector were transfected into hEF respectively with Lipofectin reagent, and were named as hEF-hTERT and hEF-EGFP. The hTERT, Id1, PCNA and I, III type collagen expression in these cells were detected by Western blot. Then the cell cycle and growth curve were measured and plotted with flow cytometry and MTT method, respectively.</p><p><b>RESULTS</b>1. The expression of hTERT, Id1, PCNA, type I and III collagen in hEF-hTERT were much higher than that in hEF and hEF-EGFP. 2. As shown in the growth curve, the OD value of hEF-hTERT at 4 to 6 days after culture was obviously higher than that of hEF and hEF-EGFP (P < 0.05), while no difference existed between hEF and hEF-EGFP from 1 to 6 days after culture (P > 0.05). 3. The cell number in G0/G1 phase in hEF-hTERT was less than that in hEF and hEF-EGFP. The cell number of hEF-hTERT in S and G2/M phase and its proliferation index (57.47%) increased when compared with that in hEF-EGFP (13.13%) and hEF (17.38%), but there was no difference between hEF and hEF-EGFP.</p><p><b>CONCLUSION</b>Exogenous hTERT gene transfection could promote the proliferative capacity of hEF.</p>