Study Of Synergy Between LPS And LTA And The Mechanism Of It On Vascular Endothelial Cells

Objective:To study the influence of activating vascular endothelial cells(VEC) with LPS(lipopolysaccharide) and LTA(lipoteichoic acid) by incubating HUVEC(human umbilical vein endothelial cells).To investigate the synergy between LPS and LTA and the mechanism of transmembrane signal transmission for studying the synergy between intestinal bacterial translocation and gram-positive bacteria in intensive care unit.Method:This paper consists of two parts.1.THE FIRST PART.Incubated the ECV304cell with the low-dose LPS.The incubation time was6h,8h,10h,12h,24h. Toll-like receptor2(TLR2) which was set in membrane was observed by immunocytochemistry (ICC) method. Choosed the best activating time of the most expression of TLR2on the membrane.Based on the previous trials, ECV304were derived into three groups on random:the control group,the low-dose LPS group and the antibody group. After cultured with LPS or LPS+anti-TLR4mAb,The cell proliferation was measured by MTT test. And then the expressions of TLR2mRNA and TLR4mRNA was evaluated by real-time quantitative PCR.2.THE SECOND PART. ECV304were pretreated by LPS or LPS+anti-TLR4mAb at first and then cultured by LTA. The culture supernatants were collected. Observed the production of IL-8and vWF of the supernatant by enzyme-linked immuno sorbent assay(ELISA). And then tried to observed the influence of LPS and the effect of TLR4on VEC ingury and activation.Results:At the first part, ECV304without LPS stimulation remained monolayer, attachment-inhibited growth.Boundaries were explicit.ECV304cell were altered to become long cord-like in shape and arrayed in disorder by low-dose LPS cultured. Cells was dying individually12hours after LPS cultured. Especially24hours after LPS cultured,some nuclear fragmented and cells died and then rose up.Observed by ICC,ECV304could express little TLR2,the expression levels were up-regulated by low-dose LPS.The best activating time is8hour. The cell proliferation was not changed by LPS and LPS+anti-TLR4mAb.The expression levels of TLR2mRNA was not changed in the low-dose LPS group for4hour and was up-regulated for8hour. The expression levels of TLR2mRNA was up-regulated in the antibody group for8hour,but did not differ than in the low-dose LPS group. The expression levels of TLR4mRNA was up-regulated in the low-dose LPS group and antibody group for4and8hours. But the expression levels of TLR4mRNA in antibody group was lower than the low-dose LPS group for8hour.At the second part,the levels of IL-8and vWF from the culture supernatants rose up by incubated with LPS or LTA. Pretreated by LPS for8hour at first and then cultured by LTA for24hour,The levels of IL-8and vWF was significantly higher than incubated with LPS or LTA. The levels of IL-8and vWF was no influence when ECV304was incubated with LPS+anti-TLR4mAb.Conclusion:There was the synergy between LPS and LTA on VEC ingury and activation. The mechanism of the synergy was tied to TLR2up-ragulation.TLR4is not excluded from mediating the up-regulation of TLR2.Anti-TLR4m.Ab has no antagonism to the synergy between LPS and LTA. The mechanism of the synergy are still to be further investigated.