Résumé
OBJECTIVE@#To establish a micellar electrokinetic capillary chromatography-based method for identification and quantitative detection of interleukin-12 (IL-12) and analysis of its unfolding process.@*METHODS@#An uncoated fused-silica capillary (inner diameter 50 μm) with a total length of 48.5 cm (40 cm to the detector) was used for the experiment. The factors influencing the separation efficiency of IL-12 were analyzed, and a standard curve of IL-12 concentration was established. The mixture of IL-12 and anti-IL-12 antibody was incubated in a water bath at 38 ℃ for 40 min, and capillary electrophoresis was then performed under the same conditions. The results were compared with those of IL-12 and anti-IL-12 antibody to identify IL-12. IL-12 and dithiothreitol (DTT) were incubated at 60 ℃ in water bath for different lengths of times, and the unfolding process of IL-12 was analyzed based on electrophoresis results of IL-12 in different states.@*RESULTS@#A micellar capillary electrophoresis on-line sweep method was established with 80 mmol/L borate (pH=9.3) containing 30 mmol/L sodium dodecyl sulfate (SDS) as the buffer solution. This system showed a good linear relationship between the peak area and the mass concentration of IL-12 with a linear correlation coefficient of 0.9991 within the linear range of 2 to 120 ng/L. As the incubation time of IL-12 and DTT prolonged, the disulfide bond of IL-12 gradually opened and resulted in distinct changes in the protein peak.@*CONCLUSIONS@#This capillary electrophoresis-based method is simple and sensitive for IL-2 analysis and allows rapid detection of changes in IL-12 content in the setting of tumors and analysis of the possible causes.
Résumé
AIM: To investigate the effects of protein C activator (PCA) from Agkistrondon acutus venom ( AAV) on the tension of thoracic aorta rings isolated from the rats with sepsis.METHODS:The model of sepsis was es-tablished by intraperitoneal injection of lipopolysaccharide ( LPS) .SD rats were randomly divided to 6 groups ( n=6 ):sham group, LPS group, PCA intervention group (LPS+PCA, PCA at doses of 0.1 mg/kg, 0.3 mg/kg and 0.6 mg/kg) and LPS+polymyxin B (at dose of 0.2 mg/kg) group.Using perfusion experiment in vitro, the tension of the aortic rings was measured by biological signal analytical system.RESULTS:The values of MABP, HR, LVDP and ±dp/dtmax were significantly lower in LPS group than those in sham group and LPS+PCA groups.Compared with sham group, the relaxa-tion response to acetylcholine ( ACh) and the contractile response of aorta rings induced by phenylephrine ( Phe) were sig-nificantly decreased in LPS group, which were increased significantly in PCA intervention group ( especially at dose of 0.6 mg/kg) compared with LPS group.The dose-response curve of aorta contraction with denuded endothelium induced by Phe shifted down significantly in LPS group compared with sham group, and no significant difference between LPS group and PCA intervention group was observed.Also no statistical difference was found in non-endothelium dependent relaxation of aortic rings induced by sodium nitroprusside among the groups.Pretreatment of N-nitro-L-arginine methl ester and methyl-ene blue increased the contraction amplitude of aortic rings induced by Phe.CONCLUSION:PCA from AAV effectively reverses the hypoergia of the vessels in rats with sepsis through protecting vascular endothelium, the mechanism of which may be mediated by inhibiting NO-GC-cGMP signal transduction pathway.