Effects Of Interaction Between HUVECs Infected With DENV-2 And Human Macrophages On Expression Of Major Adhesion Molecules

Objective:To study the effects of adhesion molecules on dengue virus?DENV-2?-infected human umbilical vein endothelial cells?HUVECs?and macrophages.Methods:DENV-2-infected C6/36 cells and RNA was extracted,the partial base sequence of DENV-2 NS1 was amplified by RT-PCR and identified by agarose gel.The virulence of DENV-2 was detected by TCID50.Monocytes were extracted by horizontal density gradient centrifugation and stimulated with M-CSF factor for 5-7 days.CD14 and CD11b molecules on cells surface were identified by flow cytometry.HUVECs and human macrophages were infected with 10³TCID50DENV-2,then infected-cells were co-cultured.The relative expression of DENV NS-1,VCAM-1,ICAM-1 and E-selectin mRNA detected by QPCR.The secretion of VCAM-1,ICAM-1 and E-selectin in culture supernatant were analyzed at different time by ELISA.Results:The partial gene fragment of DENV-2 NS1 was amplified by RT-PCR,and a bright band appeared between 400-500 bp after 1.5%agarose gel electrophoresis,which proved the virus was DENV-2.According to the TCID50,the DENV-2 virulence is 10^-5.5/0.1ml.The purity of macrophages was?92.15±1.24?%.The expression of DENV NS-1 mRNA in HUVECs increased and then reached the peaking at 24h?1.25±0.16?,in macrophages decreased first then increased at 24h?5.34±0.23?.The expression of DENV NS1 mRNA in HUVECs of co-culture group was higher than that infected-HUVECs group.The expression of ICAM-1 mRNA in DENV-2-infected HUVECs was up-regulated at 4h and 72h,down-regulated at 8h,12h,24h and 48h,the relative expression of VCAM-1 mRNA increased first then decreased,and peaked at 8h?1.6±0.04,P<0.0001?,E-selectin mRNA was decreased,the expression was up-regulated at 4h and 8h,down-regulated at 12h,24h,48h and72h.The relative expression of ICAM-1 mRNA in DENV-2-infected macrophages was higher than the uninfected group at 4h?2.09±0.01,P<0.0001?,8h?2.04±0.01,P<0.0001?,but no VCAM-1 mRNA was detected.The relative expression of ICAM-1and VCAM-1 mRNA in HUVECs of co-culture group were up-regulated after 8h and higher than infected-HUVECs alone,and peaked at 24h.The E-selectin mRNA in HUVECs was the highest at 4h?1.20±0.08?,but the relative expression of each group was lower than the co-culture group.The secretion of ICAM-1 in the co-culture supernatant of DENV-2-infected HUVECs was higher than the independent infection group of HUVECs.E-selectin in the co-culture group increased first then decreased and reached a peak at 24h?6.12±1.03?,but the expression of VCAM-1 in the culture supernatant was not detected.Conclusion:DENV-2 could infect HUVECs and human macrophages,NS1 mRNA are up-regulated in HUVECs after co-culture with infected-macrophages.Activated macrophages could enhance DENV-2-infected HUVECs and increase the expression of adhesion molecules.