Analysis of the early adaptive response of endothelial cells to hypoxia via a long serial analysis of gene expression.

Activation of endothelial cells in humans is an early event in the response to hypoxia that may contribute to the endothelium's endogenous capacity to reduce tissue injury. To better understand the mechanism underlying this process, we utilized Long Serial Analysis of Gene Expression to study the transcriptome of human vein umbilical endothelial cells (EA.hy926) shortly after the induction of hypoxia. Of over 13,000 genes detected in each pool, 112 showed obvious differences in expression. Metabolic processes such as protein biosynthesis and proteolysis, aminoglycan metabolism, ribonucleotide biosynthesis, adenosine salvage, and lipid metabolism were reinforced. Pro-proliferation and pro-apoptotic states suggest the co-existence of pro- and anti-injury forces in endothelium shortly after the induction of hypoxia. Other adaptive responses include reinforced angiogenesis and vasodilation. Additionally, gene transcription in the endothelium shortly after the induction of hypoxia was regulated independently of HIF-1alpha. Our efforts to elucidate the adaptive response at an early post-hypoxia stage should contribute to further investigation of the protective processes that occur in the endothelium and has potential clinical implications.

[Long non-coding RNA PCAT-1 expression in oral squamous cell carcinoma and its clinical significance].

PURPOSE: To investigate the correlation between the expression of lncRNA PCAT-1 and clinicopathological features of oral squamous cell carcinoma. METHODS: In this study, 112 oral tissue specimens, including 60 oral squamous cell carcinoma, and 52 non-cancer oral tissues were collected from Department of Stomatology in Anhui Provincial Hospital during 2013 to 2016. Reverse transcription quantitative PCR (RT-PCR) was performed to detect the expression of PCAT-1 and c-Myc. The correlation between the levels of PCAT-1 and clinical features (age, gender, high risk habit, histological stage, cervical lymphatic metastasis, differentiation) were analyzed. Graphpad Prism 7.0 software package was used for statistical analysis. RESULTS: Significant different levels of PCAT-1 were found between the high risk habit (alcohol and tobacco) group and low risk habit group, between patients with cervical lymphnode metastasis and patients without cervical lymphnode metastasis. The expression of PCAT-1 and c-Myc were up-regulated in OSCC, and there were positive correlation between the expression of PCAT-1 and c-Myc; up-regulated expression of PCAT-1 may be associated with higher morbidity of OSCC. CONCLUSIONS: PCAT-1 is overexpressed in oral squamous cell carcinoma and is associated with higher morbidity of OSCC.

Apoptosis in cardiac myocytes during the early stage after severe burn.

BACKGROUND: Cardiac dysfunction after severe burn is associated with postburn myocardial injury. We hypothesize that myocyte apoptosis is triggered and presented as the pathologic basis of postburn myocardial injury during the early stage after severe burn, and that apoptosis may be related to inflammatory responses in the postburn myocardium. METHODS: Rats with 40% total body surface area full-thickness burn were used. The following functions were measured at several time points after the burn injury: myocyte apoptosis (TUNEL staining, DNA ladder, and caspase-3 activity assay); mRNA levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (reverse transcriptase-polymerase chain reaction [RT-PCR]); activities of myeloperoxidase and p38 mitogen activated protein (MAP) kinase (Western blots); and left cardiac function. RESULTS: TUNEL positive myocytes appeared as early as 6-hour and their numbers showed further increases at 12-hour and 24-hour postburn; DNA fragmentation was clearly observed, and caspase-3 activity was significantly increased in the myocardium after burn. Infiltration of neutrophils, evidenced by the levels of myeloperoxidase activity, expression of TNF-alpha, and p38 MAP kinase activity in the heart, were all significantly increased within 24-hour after burn. Cardiac function was decreased after burn, which approximately paralleled the increased amount of cardiac apoptosis. CONCLUSION: These results demonstrate that cardiomyocyte apoptosis progressively develops during the early stage after severe burn, which may in part contribute to burn-induced cardiac dysfunction. Myocardial inflammatory responses, evidenced by the increased infiltration of neutrophils, as well as production of TNF-alpha probably because of the activation of p38 MAP kinase, may be involved in burn-induced cardiomyocyte apoptosis.

Inhibition of p38 MAP kinase improves survival of cardiac myocytes with hypoxia and burn serum challenge.

This study was aimed to investigate the effects of SB203580, the specific p38 mitogen-activated protein (MAP) kinase inhibitor, on cardiac myocyte survival and secretion of cytokines in an in vitro model of hypoxia and burn serum challenge. Results demonstrated that hypoxia and burn serum induced a persistent activation of p38 MAP kinase in primary cultured neonatal rat cardiomyocytes during the 12h period of stimulation, concomitant with a time-dependent increased expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide (iNOS), a progressively developed oxidative stress reflected by malondialdehyde (MDA) production, and myocytes injury evidenced by the increased levels of released lactate dehydrogenase (LDH) and the decreased myocyte viability. Furthermore, hypoxia and burn serum resulted in a significant increase in myocyte apoptosis, which may account for the impairment of myocyte viability as observed. SB203580 abolished p38 MAP kinase activation, blunted the upregulation of TNF-alpha, iNOS and the subsequent nitric oxide (NO) production, reduced oxidative stress, and alleviated hypoxia and burn serum-induced myocytes injury or apoptosis. These results demonstrated for the first time that inhibition of p38 MAP kinase improves survival of cardiac myocytes with hypoxia and burn serum challenge possibly via reducing the production of cytokines, such as TNF-alpha and NO, and the subsequent oxidative stress, providing strong evidence that the excessive inflammatory cytokines produced by cardiomyocytes themselves may be sufficient to cause myocardial injury after burn.

Induction of heat shock protein 70 by sodium arsenite attenuates burn-induced intestinal injury in severe burned rats.

The present study was designed to assess the effects of induced heat shock protein 70 (HSP70) on intestinal injury after severe burn. Wistar rats were randomly divided into four groups: control group, burn group (B group), sodium arsenite pretreatment group (SA group), and sodium arsenite+quercetin pretreatment group (SA+Qu group). Plasma endotoxin and d-lactic acid content were determined at 3, 6, 12, 24, and 48h after severe burn. Samples of small intestine were obtained for histologic assessment of intestinal mucosal injury and the expression of HSP70 was assayed by Western blot. Apoptosis of the intestinal epithelial cells was examined by the TUNEL method. Results showed that SA pretreatment significantly increased expression of HSP70 in the small intestine. SA pretreatment attenuated the burn-induced increase in plasma endotoxin and d-lactic acid content, intestinal injury scores and the percentage of apoptotic intestinal epithelial cells. Co-administration of quercetin with SA abolished the SA-induced HSP70 over-expression and the beneficial effects of SA. Our findings suggest increasing expression of HSP70 induced by SA pretreatment attenuates burn-induced intestinal injury apparently by preventing apoptosis.

Involvement of p38 MAP kinase in burn-induced degradation of membrane phospholipids and upregulation of cPLA2 in cardiac myocytes.

This study was aimed to evaluate the role of p38 mitogen-activated protein (MAP) kinase in the degradation of membrane phospholipids and the regulation of cytosolic phospholipase A2 (cPLA2) in cardiac myocytes after burn trauma. In an in vivo study, rats were randomized into four groups: (1) sham-burn group, (2) burn group (40% total body surface area full-thickness burn), (3) burn + SB203580 group, and (4) burn + vehicle group. The rats from each group were killed at varying times after burn to examine the p38 MAP kinase activation (by means of Western blot analysis and immunohistochemical assay), the expression of cPLA2 (by means of reverse transcriptase polymerase chain reaction), the level of cardiac membrane phospholipids, and the level of the remaining creatine kinase-MB (CK-MB) isoenzyme in the heart. These studies showed that burn resulted in a significant decrease in the level of cardiac membrane phospholipids from 3 to 24 h after burn, which was paralleled with a persistent activation of p38 MAP kinase and an increased expression of cPLA2 in the heart. SB203580, a selective inhibitor of p38 MAP kinase, inhibited the activation of cardiac p38 MAP kinase, suppressed the burn-induced upregulation of cPLA2 and the increased PLA2 activity, and prevented burn-induced decrease in the levels of the cardiac membrane phospholipids and the remaining creatine kinase-MB isoenzyme. In addition, the in vitro treatment of cardiac myocytes with SB203580 also abolished the upregulation of cPLA2 and the disturbance of phospholipid homeostasis elicited by hypoxia and burn serum challenge. Taken together, these results have demonstrated for the first time that p38 MAP kinase is involved in burn-induced membrane phospholipids degradation in cardiac myocytes, at least in part through the regulation of cPLA2.

[The effect of inhibiting EOLA1 expression in ECV304 cells].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: EOLA1 maybe has a role on the proliferation of cells.

The mechanism of lipopolysaccharide infiltration through HUVEC membrane surface in direct endotoxin injury.

In this study, the HUVEC's cellular biomechanical properties of HUVEC (elastic modulus K1, K2 and viscoefficient mu) were determined with micropipette aspiration system and analyzed after being directly damaged with lipopolysaccharide (LPS). The phospholipid compositions of HUVEC membrane were analyzed with high-performance capillary electrophoresis and PLA2 activity was determined to research the modification and metabolism of HUVEC membrane phospholipid. Infiltration of LPS on HUVEC membrane was studied by observation with confocal microscopy and fluorescent microscopy. Results showed that LPS direct injuring HUVEC can cause the changes of HUVEC biomechanical properties and membrane lipid contents; HUVEC directly damage by LPS could also activate HUVEC phospholipase A2 (PLA2), influencing membrane lipid metabolism; LPS could directly infiltrate and intercalate HUVEC membrane, causing and membrane contents variation. Based on these experimental results, the mechanism of lipopolysaccharide infiltration VEC membrane surface in direct LPS injury was studied and analyzed in view of the cellular biomechanical mechanism.

[Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation].

OBJECTIVE: To amplify the full-length cDNA and characterize the structure and biological function of a novel expression sequence tag ST55 (GenBank Accession No. BM121646). METHODS: Rapid amplification of cDNA ends was used to clone the full-length of cDNA of ST55 in this study. Then, its tissue distribution was checked by Northern blots, and the associated protein was screened by GAL 4-based yeast two-hybrid. The effect of stable transfection of the cDNA on cell proliferation was evaluated in ECV304 cells. RESULTS: A full-length 1404 bp cDNA was cloned, and it was accepted as a novel human mRNA by GenBank (No. AY074889), named endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1). Bioinformatic analysis found that the EOLA1 encoded 158 amino acids, 17.89 kDa protein, and mapped to chromosome Xq27.4 with 5 exons. EOLA1 expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. The interaction between EOLA1 and MT2A was confirmed by co-immunoprecipitation experiments. Stable transfection of EOLA1 was noted to stimulate ECV304 cell proliferation (P < 0.05). CONCLUSION: The findings suggest that EOLA1 is a novel gene and the interaction of EOLA1 and MT2A may play an important role in cell protection in inflammation reaction.

[Effects of burn patients' serum on IkappaBalpha degradation and NF-kappaB activation in endothelial cells in vitro].

OBJECTIVE: To investigate the effects of the serum from burned patients (burn serum) on inhibitor of kappaBalpha (IkappaBalpha) degradation and nuclear factor-kappaB (NF-kappaB) activation in the endothelial cells in order to explore the role of burn serum on endothelium activation. METHODS: Cultured endothelial cells (ECV-304) were stimulated by the serum from healthy volunteers, burn serum and burn serum with pyrrolidine dithiocarbamate (PDTC), respectively. Activation of endothelial NF-kappaB at 30, 60, 120, 240 min after stimulation was tested by electrophoretic mobility shift assay (EMSA) and the degradation of endothelial IkappaBalpha at these time points determined by Western blotting. RESULTS: Compared with that in the control group, cytosolic IkappaBalpha degradation in the endothelial cells occurred within 30 min after burn serum stimulation, and peaked at 60 min, followed by gradual recovery in the cytoplasm till reaching the pre-stimulation level at 2 h. Meanwhile, the activity of endothelial NF-kappaB was markedly increased, reaching the peak at 30 to 60 min with subsequent gradual decrease 2 h after stimulation. PDTC could effectively inhibit endothelial IkappaBalpha degradation and activation of NF-kappaB induced by the burn serum. CONCLUSION: Burn serum might induce the degradation of IkappaBalpha to activate NF-kappaB, which ultimately leads to cytokine release from the endothelium.

[Role of mitogen - activated protein kinases signal pathway in cardiomyocyte injury induced by serum after hypoxia and burn injury].

OBJECTIVE: To observe the activation and explore the role of three major mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38 kinase and c-Jun NH(2)-terminal protein kinase (JNK), in cardiomyocytes injury induced by serum after hypoxia and burn injury. METHODS: Phosphorylation of the three major MAPKs in primary cultured neonatal rat cardiomyocytes were determined by Western blotting. Contents of released lactate dehydrogenases (LDH) and death-rate of myocytes treated with serum after hypoxia and burn injury, SB203580+hypoxia and burn serum, PD98059+hypoxia and burn serum were observed respectively. RESULTS: Exposing rat neonatal cardiomyocytes to hypoxia and burn serum resulted in a rapid and prolonged activation of p38 kinase and ERK. Phosphorylation degree of p38 kinase, ERK1/2 was increased. Myocytes treated with SB203580 (10 micromol/L), a selective inhibitor of p38 kinase, resulted in a significant decline in LDH leakage leaking and cell death. However, with pretreatment of cell with PD98059(25 micromol/L), an inhibitor of ERK, LDH leakage and cell death were increased. CONCLUSION: Serum obtained after hypoxia and burn injury activate p38 kinase and ERK, but not JNK, in cardiomyocytes. p38 kinase pathway might play a role in mediating cardiomyocytes injury, whereas ERK plays a protective role.

[Expression of heat shock protein 70 in intestinal mucosa during the early stage after severe burns and its significance].

OBJECTIVE: To study the expression of heat shock protein 70 (HSP70) in intestinal mucosa during the early stage after severe burn injury and its significance. METHODS: With a model of 30% total body surface area (TBSA) full-thickness burned rats, the expression and distribution of HSP70 and heat shock factor-1 (HSF1) in intestinal mucosal were determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry at 3, 6, 12, 24 and 48 hours postburn. RESULTS: The expression of HSP70 mRNA and protein in intestinal mucosa increased markedly at 3 hours after severe burns, peaked at 6 and 12 hours, and lasted for 48 hours postburn (all P<0.01). Following a slight decline at 3 hours postburn, the expression of HSF1 increased gradually, and reached a highest level at 48 hours postburn (all P<0.01). CONCLUSION: The expression of HSP70 and HSF1 markedly increase in intestinal mucosa following severe burn injury. It is suggested that the self-protective mechanism of cells might explain the increase of HSP70 expression.

Improvements of postburn renal function by early enteral feeding and their possible mechanisms in rats.

AIM: To investigate the protective effects of early enteral feeding (EEF) on postburn impairments of renal function and their possible mechanisms. METHODS: Wistar rats with 30 % of total body surface area (TBSA) full-thickness burn were adopted as the experimental model. The effects of EEF on the postburn changes of gastric intramucosal pH (pHi), endotoxin levels in portal vein, water contents of renal tissue, and blood concentrations of tumor necrosis factor (TNF-alpha), urea nitrogen (BUN), creatinine (Cr), as well as the changes of clearance of creatinine (CCr) were dynamically observed within 48 h postburn. RESULTS: EEF could significantly improve gastric mucosal acidosis, reduce portal vein endotoxin levels and water contents of renal tissue, as well as blood concentrations of TNF-alpha after severe burns (P<0.01). The postburn elevations of BUN and BCr were not found to be recovered by EEF. However, the CCr in EEF group was greatly increased by 4.67-fold compared with that of the non-feeding burned control (16.43+/-2.90 vs. 3.52+/-0.79, P<0.01). CONCLUSION: EEF has beneficial effects on the improvement of renal function in severely burned rats, which may be related to its increase of splanchnic blood flow, decrease of the translocation of gut-origin endotoxin and the release of inflammatory mediators.

Effects of Panax notoginseng saponins on myocardial Gsalpha mRNA expression and ATPase activity after severe scald in rats.

The aim of this study was to investigate the changes of myocardial Gsalpha mRNA expression and ATPase and the effects of Panax notoginseng saponins (PNS) on that after burns in rats. Wistar rats were exposed to a 95 degrees C water bath for 10s to produce 30% TBSA skin-full-thickness scalds. Myocardial Gsalpha mRNA level, cAMP content and adenyl cyclase (AC) activities were determined with dot blotting hybridization, radioimmunoassay and indirect method, respectively. The ATPase activities in plasma membrane of cardiomyocytes and erythrocytes were measured colorimetrically. At 3, 6, 9 hours after scalds, the myocardial Gsalpha mRNA expression decreased significantly (P<0.01). PNS (100, 200 mg kg(-1), i.p.) markedly increased these levels (P<0.01). The elevation was correlated significantly with PNS dose (r=0.95, P<0.05). At 3, 6, 9, 12, 24, 48 hour after the scalds, the myocardial cAMP content and AC basal activity was reduced significantly. PNS (100, 200 mg kg(-1), i.p.) increased cAMP content and enhanced AC activity markedly in comparison with the 3rd hour postburn group. The activities of (Ca(2+)-Mg(2+))-ATPase and (Na(+)-K(+))-ATPase in plasma membrane of myocardial cells and red blood cells in scald group were significantly lower than those in the normal control group (P<0.01). PNS (100 mg kg(-1), i.p.) improved these indexes significantly after scalds (P<0.01 or 0.05). These data suggested that the effects of PNS on myocardium in burned rats involved its action to increase myocardial Gsalpha mRNA expression and AC activity, cAMP content as well as ATPase activities.

Calcium induced the damage of myocardial mitochondrial respiratory function in the early stage after severe burns.

OBJECTIVE: To explore the role of Ca(2+) in the damage to myocardial mitochondrial respiratory function in 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 in the 1st, 3rd, 6th, 12th and 24th hour post-burn. The mitochondrial respiratory function, contents of mitochondrial calcium ([Ca(2+)](m)), activities of mtPLA(2), mtNOS, F(0)F(1)-ATPase and cytochrome c oxidase were determined. RESULTS: (1) At the 1st hour post-burn, [Ca(2+)](m) was increased significantly and the myocardial mitochondrial respiratory function was significantly reinforced. At the same time, mitochondrial respiratory control rate (RCR) was elevated and positively correlated with [Ca(2+)](m) (r=0.8415, P<0.01). At the 3rd, 6th, 12th and 24th hour post-burn, [Ca(2+)](m) increased further to a higher level, however, the mitochondrial respiratory function was decreased from the peak value at 6h, and RCR was negatively correlated with [Ca(2+)](m). (2) The activities of mtNOS and mtPLA(2) were higher significantly at the 3rd, 6th, 12th and 24th hour post-burn than that of the control. After severe burns, mtNOS and mtPLA(2) activities were both positively correlated with [Ca(2+)](m) (r=0.8945, P<0.05; r=0.9271, P<0.01, respectively). (3) The F(0)F(1)-ATPase synthetic activity increased at the 1st hour post-burn, but it decreased to 51.4, 44.9, 77.6 and 87.4% of that of the control at the 3rd, 6th, 12th and 24th hour post-burn respectively. The F(0)F(1)-ATPase hydrolytic activity decreased at the 1st hour post-burn and increased at the 3rd, however, it decreased again at the 6th, 12th and 24th hour post-burn. The activity of cytochrome c oxidase at the 3rd, 6th, 12th and 24th hour was low compared to the control. CONCLUSIONS: The changes of [Ca(2+)](m) were involved in damage to or regulation of mitochondrial respiratory function after severe burns. Appropriate increase of [Ca(2+)](m) reinforced the mitochondrial respiration at 1st hour after of burn injury, but Ca(2+) severe overload impairing F(0)F(1)-ATPase and cytochrome c oxidase directly, or, indirectly by activation of mtPLA(2) and mtNOS, might play an important role in damage to myocardial mitochondrial respiratory function at later stages after severe burns.

Cloning of differentially expressed genes following lipopolysaccharide stimulation in human umbilical vein endothelial cells.

OBJECTIVE: To clone the differentially expressed genes in human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide (LPS). METHODS: Two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on HUVEC cultured in either standard media or treated for 6 hours with LPS (100 ng/ml). To restrict the number of false-positive clones, colony dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced. RESULTS: These analyses have identified both novel and known genes whose expression is influenced by LPS. The known genes include a group related to proinflammatory events, a group related to cellular apoptosis and proliferation, a group related to protein synthesis and cytoskeletal rearrangment, and a group related to energy metabolism and signal transduction. CONCLUSIONS: SSH is a powerful technique of high sensitivity for the detection of differential gene expression in HUVEC stimulated by LPS.

[The relationship between angiotensin-converting enzyme gene polymorphism and heart rate variability in cerebral stroke].

OBJECTIVE: To identify the relationship between angiotensin-converting enzyme (ACE) gene polymorphism and heart rate variability in cerebral stroke patients. METHODS: An insertion/deletion(I/D) polymorphism of the ACE gene was analyzed by polymerase chain reaction in 43 normal subjects, 46 patients with ischemic cerebral stroke, and 40 patients with brain hemorrhage; their heart rate variability(HRV) parameters such as time domain and power spectral component were analyzed. RESULTS: The frequency of DD genotype and the frequency of deletion alleles in cerebral stroke groups were significantly higher than those in control groups (P<0.01), and the measured components of HRV, including total power (TP), low frequency power (LF), high frequency power (HF), LF/HF, and choas, were higher in the patients with the ACE DD genotype when compared with those in the patients with the ACE II, ID genotypes; there was significant difference in effective rate (P<0.05). CONCLUSION: The above related parameters of HRV were correlated with heritability, suggesting that the cerebral stroke patients with the ACE DD genotype are at high risk of cerebrogenic cardiac autonomic nerve function disturbances.

[Expression of genes related to inflammatory response in myocardium of rats after burn injury].

OBJECTIVE: To observe expression of genes related to inflammatory response in myocardium of rats after burn injury. METHODS: After 40 percent total body surface area (TBSA) of rat was burned gene expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), induced nitric oxide synthase (iNOS) and cytoplastic phospholipids A(2) (cPLA(2)) in myocardium was detected with reverse transcription-polymerase chain reaction (RT-PCR) at different time after burn. Meanwhile, changes of contractile and diastolic ability of left ventricle were determined with physiological recorder. RESULTS: TNF-alpha and cPLA(2) mRNAs were increased dramatically at 1 hour after burn and maintained at high level up to 24 hours later (P<0.01), IL-1beta gene expression was up-regulated at 3 hours after burn (P<0.01) and declined to normal level 12 hours later, herein any significant increase of iNOS mRNA in myocardium in rats was not found except decrease in iNOS mRNA could be detected at 1 hour after burn. As expected, severe impairment of myocardium was occurred at 3 hours after burn, represented by declining in both contractile and diasotolic ability of lefe ventrical. CONCLUSION: Over expression of TNF-alpha, cPLA(2) and IL-1beta mRNA in myocardium in rats may be partial cause of local myocardial uncontrolled inflammatory response and then partially contributed to cardiac impairment after burn.

[Protection on proliferation of intestinal epithelial cells against hypoxia-reoxygenation through recombinant heat shock protein 70 adenovirus transfection].

OBJECTIVE: To investigate the protecting effect on proliferation of intestinal epithelial cells against hypoxia-reoxygenation through injury by transfection of recombinant adenovirus with whole long human heat shock protein 70 (HSP70) gene. METHODS: The recombinant adenoviruses were constructed with whole long human HSP70 gene. Cultured intestinal epithelial cells (IEC-6 cells) were divided into four groups: control group without transfection and 24 hours, 48 hours, 72 hours after transfection groups. The expression of human HSP70 gene in transfected cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) after 48 hours transfection. Cells in different groups after 1 hour were treated with hypoxia followed by 1 hour of reoxygenation, then cell viability, growth curve and proliferative index were evaluated in vitro. RESULTS: The expression of human HSP70 gene in transfected IEC-6 cells was positive, while it was negative in control group. After treatment with hypoxia-reoxygenation, the cell viability in transfected groups was significantly higher than that of control (P<0.01), the proliferative index was also improved (P<0.05). CONCLUSION: The proliferation ability of intestinal epithelial cells (IEC-6 cells) could be promoted by HSP70 gene transfection, which therefore could protect the IEC-6 cells against hypoxia-reoxygenation injury.

[Subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 protein].

OBJECTIVE: To study the subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) protein in endothelial cells. METHODS: 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. RESULTS: 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. CONCLUSIONS: EOLA1 protein is localized in the nucleus and the matrix of ECV304 cell, and it plays its role as a signal transduction factor.