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Article in Chinese | WPRIM | ID: wpr-325633


<p><b>OBJECTIVE</b>Previous studies have suggested that under hypoxic conditions hypoxia inducible factor-1 alpha (HIF-1 alpha) contributes to the progression of neonatal pulmonary hemorrhage (NPH) by increasing the expression of endothelin-1 (ET-1) gene. RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene-silencing mediated by double-stranded RNA. This new gene-silencing technique has recently been shown to be a powerful approach for studying gene function. The aim of this study was to construct the small interfering RNA (siRNA) eukaryotic expression vectors specific to human HIF-1 alpha gene (pSIREN-Shuttle HIF-1 alpha siRNAin order to observe its silencing effect on HIF-1 alpha under hypoxic conditions.</p><p><b>METHODS</b>Six potential siRNA target sites (a-fspecific to human HIF-1 alpha gene were designed and synthesized to two complementary oligonucleotides (A-F) for each siRNA target site. Using a gene recombination technique, the recombinant expression vectors (A-F') were constructed by cloning the double strands oligonucleotide into RNAi-Ready pSIREN vector. The recombinant vectors were then transfected into the cultured human umbilical vein endothelial cells (HUVECs). After 48 hrs of culture, the cells were treated with CoCl2 (100 mu M) for 3 hrs. Expression of HIF-1 alpha mRNA and protein was detected using RT-PCR and Western blot. ET-1 level in cell culture supernates was detected using ELISA.</p><p><b>RESULTS</b>The recombinant HIF-1 alpha siRNA eukaryotic expression vectors A'-F'respectively aiming at sites (a-f) were constructed successfully. Compared to the non-transfection group, liposome-mediated gene transfection of pSIREN-Shuttle HIF-1 alpha siRNA expression vectors into HUVECs obviously down-regulated the mRNA and protein levels of HIF-1 alpha, and partly decreased the ET-1 level in the B' and D' transfection groups.</p><p><b>CONCLUSIONS</b>The specific pSIREN-Shuttle HIF-1 alpha siRNA expression vectors B' and D' aiming at b and d sites can inhibit the expression of HIF-1 alpha, thus decreasing the level of its target gene ET-1. This may be helpful to study the relationship between HIF-1 alpha and neonatal pulmonary hemorrhage in vivo in future.</p>

Base Sequence , Endothelin-1 , Genetic Vectors , Genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , Molecular Sequence Data , RNA, Small Interfering , Genetics
Article in Chinese | WPRIM | ID: wpr-262705


<p><b>OBJECTIVE</b>To study the effect of NADPH oxidase on hypoxia-inducible factor (HIF)-1alpha and endothelin (ET)-1 expression in human umbilical endothelia cells (HUVECs) and its possible mechanism.</p><p><b>METHODS</b>Twenty-five bottles of HUVECs culture fluid were randomly assigned into five groups: group A (normoxic control), group B (hypoxic), group C (NADPH oxidase inhibitor apocynin + normoxic), group D (H2O2 which can degrade HIF-1alpha rapidly+hypoxic) and group E (H2O2+apocynin+normoxic), with five bottles in each group. The culture supernates were collected and the total protein was extracted 3 hrs after treatment. Western Blot and ELISA were used to detect the HIF-1alpha protein expression in HUVECs and the ET-1 level in the culture supernates respectively.</p><p><b>RESULTS</b>There was a lower expression of HIF-1alpha protein (0.336 +/- 0.012) and lower ET-1 levels (5.87 +/- 2.22 pg/mL) in group A. The HIF-1alpha protein expression in groups B and C (0.773 +/- 0.018 and 0.888 +/- 0.022) and ET-1 levels (95.38 +/- 8.06 and 33.67 +/- 4.21 pg/mL) were noticeably higher than in group A (P < 0.05). The groups D and E had the HIF-1alpha protein expression levels similar to group A, but the ET-1 levels in group D (108.43 +/- 8.38 pg/mL) and group E (109.66 +/- 5.80 pg/mL) were significantly higher than in group A (P < 0.05).</p><p><b>CONCLUSIONS</b>Hypoxia or apocynin can increase the HIF-1alpha and ET-1 expression in HUVECs. H2O2 can inhibit the HIF-1alpha expression but increase the ET-1levels. It is speculated that NADPH oxidase as an oxygen sensor regulates the HIF-1alpha expression by changing the intracellular redox reaction and that except HIF-1, H2O2 might contribute to ET-1 synthesis and release.</p>

Blotting, Western , Cell Hypoxia , Cells, Cultured , Endothelial Cells , Metabolism , Endothelin-1 , Genetics , Metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Metabolism , NADPH Oxidases , Physiology
Article in Chinese | WPRIM | ID: wpr-640175


950 mL/L for 2 hours.Gross anatomical changes and histological changes(HE staining)of lungs were observed,VEGF expression was detected by immunohistochemical method.Results Two rats in hypothermia-hypoxia group and 4 rats in rewarming-reoxygenating group died while none in control group.Lungs of hypothermia-hypoxia group and rewarming-reoxygenating group represented edema and punctiform,local and diffuse pulmonary hemorrahge.Histopathological changes included pulmonary edema,alveolar septum broken,pulmonary alveoli fusion and pulmonary hemorrahge.More severe pathological change could be found in rewarming-reoxygenating group.Optical density value of VEGF expression in 3 groups were 0.29?0.06,0.36?0.05,0.22?0.05,respectively,there were significant diffe-rences of VEGF expression between 3 groups(F=15.64 P

Article in Chinese | WPRIM | ID: wpr-639676


0.05).Conclusions Neither the new nor old SIRS diagnostic criteria had a high conforning rate with neonatal critical illnesses;There was no significant difference between them in each clinical item.It shows that the new SIRS diagnostic criteria is not superior to the old one,therefore we should improve the neonatal SIRS diagnostic criteria in the future.

Chinese Journal of Pediatrics ; (12): 446-449, 2004.
Article in Chinese | WPRIM | ID: wpr-340306


<p><b>OBJECTIVE</b>To observe the phenomenon of pulmonary hemorrhage (PH) induced by exogenous endothelin-1 (exET-1) and the antagonizing effect of exogenous calcitonin gene-related peptide (exCGRP) in newborn rats.</p><p><b>METHODS</b>(1) To study the exET-1 induced PH: 100 newborn Wistar rats were randomly assigned into control group (group A, n = 10) and experiment groups (20 rats in each of groups B, C, D and E and 10 in group F). Thirty microl of normal saline and different concentrations of exET-1 in saline (ranged from 2 x 10(-6) mol/L to 10 x 10(-6) mol/L) were dripped into the rats' trachea through intubation for control group and the experiment groups, respectively. (2) To study the antagonizing effect of calcitonin gene-related peptide against endothelin: 50 rats were randomly assigned into control group (group D(1), n = 10) and experiment groups D(2), D(3), D(4) and D(5) (10 rats in each group), and were treated with 30 microl of normal saline as control and 4 x 10(-6) mol/L exET-1 via tracheal dripping. Twenty microl of exCGRP (concentrations ranged from 6.7 x 10(-8) mol/L to 6.7 x 10(-6) mol/L) were given by dripping to rats in groups D(3) to D(5) 30 minutes after the administration of exET-1. (3) The rats were sacrificed 3 hours after the first tracheal dripping and the gross anatomical and histological (HE staining) changes in lungs were observed.</p><p><b>RESULTS</b>(1) Following the treatment with exET-1, the rats showed cyanosis and dyspnea rapidly. The severity of respiratory symptoms varied in a dose dependent fashion with the concentrations of exET-1. The symptoms were relieved in the survived rats in about 30 minutes. The rats of all exET-1 treated groups presented with different degree of PH and group D (treated with 4 x 10(-6) mol/L of exET-1) had the highest incidence (diffuse PH 30%, focal PH 25%, spotty PH 25% and 80% in total), with a mortality of 20%. Rats in group E and F had lower incidence of PH (50% and 20%) but higher mortality (35% and 60%). (2) After the administration of different concentrations of exCGRP, the skin of the exET-1 treated rats turned ruddy rapidly with a significantly decreased incidence of PH and all the rats survived. The best protective effect was observed with the concentration of 6.7 x 10(-6) mol/L, and the incidence of PH was reduced to 20% (focal PH 10%, spotty PH 10%).</p><p><b>CONCLUSION</b>A significant increase of the endogenous ET-1 in hemorrhagic lung tissue caused by rewarming and reoxygenation following hypothermia and hypoxia had been confirmed. Administration of intratracheal exET-1 could induce pulmonary hemorrhage. This suggests that a significant increase of endogenous ET-1 in lung tissue may be one of the mechanisms in pathogenesis of PH caused by rewarming and reoxygenation following hypothermia and hypoxia. Endotracheal administration of exCGRP showed protective antagonizing effect against PH induced by exET-1. The authors speculate that the exCGRP has the potential to treat or even prevent PH caused by a significant increase of the endogenous ET-1.</p>

Animals , Animals, Newborn , Calcitonin Gene-Related Peptide , Pharmacology , Dose-Response Relationship, Drug , Endothelin-1 , Toxicity , Female , Hemorrhage , Lung , Pathology , Lung Diseases , Male , Random Allocation , Rats , Rats, Wistar
Article in Chinese | WPRIM | ID: wpr-639144


95 mL/L O2) for 2 hours.The gross anatomical and histological changes(HE staining)in lungs were observed,VEGF mRNA expressions were studied by reverse transcription polymerase chain reaction(RT-PCR).Results Lungs of experimental groups represented edema,inaddition,punctiform,local and diffuse pulmonary hemorrhage were observed in groups of HH,HHR and HHRO2.Histopathological changes included pulmonary alveoli and interstitial edema,spacer breaking,pulmonary alveolidilating,fusion and hemorrhage,in which the most severe cases involved in group HHRO2.VEGF 188 mRNA expression increased significantly in group H and HH(P