The Study Of Lipopolysaccharide-induced Apoptosis In Cultured HUVEC

PerfaceEdotoxin(Lipopolysaccharide in Gram's-Negative bacillus cell wall)plays a vital role in sepsis and septic shock.Endotoxin injures endothelia cells through the chain reaction of cytokines,result in multiple organ failure(MOF).Nowadays,the pathogenesis of sepsis and MOF are still unclear,most of scholars think that are related with the apoptosis of endothelia cells and excess response of host or uncontrolled of immunological network et al.The apoptosis of endothelia cells is the main causative agent of sepsis.As some researches indicate,endothelium is far more than a barrier,playing critical roles in regulating the levels of cellular metabolites,vascular tone and hemostasis,as well as the ingress and egress of leukocytes.Bacterial infections are associated with activation of an innate immune response that includes the expression of proinflammatory cytokines and chemokines.Some of these inflammatory cytokines (e.g.,TNF-α,IL-1)have profound effects on endothelial function,including their regulation of vascular tone,permeability,and leukocyte diapedesis.During times of overwhelming sepsis,these inflammatory mediators trigger septic shock,a syndrome associated with endothelia cells failure and death.So it is necessary to study the apoptosis of endothelia cells in sepsis.Protected ECs is benefit to prevent MOF in sepsis or sepsis shock.In this study, we tested the rate of apopsis in HUVEC cultured with different LPS concentrations at different time points to investigate the possible mechanisms of sepsis and research the new idea of therapy. Materials and Methods1.Materials(1)CellsHUVEC ECV-304 strain(2)ReagentDMEM/F-12 medium,Lipopolysaccharide,Fetal Bovine Serum,Annexin V-FITC kit,MTT,DMSO.(3)ApparatusCell culture super-clean bench,CO₂ incubated trunk,Low temperature super centrifuge,profound hypothermia box,Flowcytometer,inverted phase contrast microscope.2.Methods(1)Culture of cellsThe human umbilical vein endothelial cells(ECV304)were cultured at 37℃,5% CO₂ humidified environment and changed the culture medium every 24 hours.We harvested the cells when the cells had grown 24 hours.(2)Groups of experimentHUVEC were cultured in culture cuvette.When cells were at subconfluence,the were preincubated in fresh medium and then were devided into following groups at random on lps concentrations according the results of MTT experiment.Such as lps at 6.25,12.5,25 and 50ug/ml.The cells were incubated to different time points(4h,12h,24h and 48h).Each group have 3 samples.(3)apoptosis of HUVEC were assessed by flowcytometer(4)statistical analysisAll data were analyzed by software package of SPSS11.0.Results were summarized with mean±standard deviation.Statistical comparisons between groups were performed by the SNK and two-way ANOVA.Values of P＜0.05 were considered significant.Results1.Effect of Morphology in ECs by LipopolysaccharideAfter HUVEC were exposed to lipopolysaccharide,the morphology of HUVEC is like apoptosis.2.Test cell's inhibited growth ratio through MTT methodAll different concentrations of LPS inhibited HUVEC's growth.The inhibited growth ratio showed significant concentration-dependence.Four concentrations (6.25,12.5,25 and 50ug/ml)were considered significant(P＜0.05).3.Test the apoptosis ratio of HUVEC by flowcytometerAfter HUVEC were exposed to lps at different concentrations for different time points(4h,12h,24h,48h),the apoptosis ratio was higher compared with control group.At the time 4h,the ratio was unsignificant(P＞0.05)among the four lps group.Likewise, after exposed to lps at same concentration for different time points(4h,12h,24h),the ratio was significant(P＜0.05),except in 48h.Conclusion1.HUVEC's growth were inhibited by lipopolysaccharide in vitro.2.HUVEC's growth were inhibited by lipopolysaccharide showed significant concentration-dependence.3.The apoptosis ratio of HUVEC were induced by lipopolysaccharide showed significant concentration-dependence and time-dependence.