The Study Of Autophagy Induced By DENV-2 And The Effect On Viability Of HUVECs

Objective:To investigate the autophagy induced by dengue virus type 2?DENV-2?and the effect of DENV-2 on viability of primary human umbilical vein endothelial cells?HUVECs?.Methods:The NS1 partial sequence?413bp?of DENV-2 was identified by RT-PCR,TCID₅₀ was used to determine the titration of DENV-2.The expression of CD31 on HUVEC surface and the factor?related antigen in cytoplasmic were identified by flow cytometry and immunohistochemistry.The identificationof HUVECs were detected via Immunohistochemistry and flow cytometry.HUVECs were infected with DENV-2NGC,and the relative expression of NS1 in HUVECs was detected via Real time PCR.HUVECs were treated with rapamycin?RAPA?as autophagy inducers,or pretreated with chloroquine?CQ?as autophagy inhibitor to detect the changes in autophagy flux,The formation of autophagosomes induced by DENV-2 in HUVECs was observed by Laser confocal microscopy.The viability of HUVECs were detected via CCK-8.The expressions of LC3 and P62 were detected by Western Blot.Results:RT-PCR amplification of NS1 partial sequence by agarose electrophoresis,the product length of about 400bp,with the expected 413bp,confirmed that the virus was DENV-2.Flow cytometry CD31 surface molecule expression rate was 99.86%.HUVEC was detected by Immunohistochemistry of factor VIII related antigen.Brownish yellow granules can be seen incytoplasm,which consistent with the characteristics of HUVEC.The NS1 gene was expressed in DENV-2 infected HUVECson all time points.HUVECs were cultured on the second day at the logarithmic growth stage.The vitality of HUVECs were declined which were infected with DENV-2;Compared with the untreated group,the cell viability cells was not decreased significantly on 24h,compared with the untreated group,the vitality of HUVECs were decreased significantly on 36h and 48h?p<0.05?,and the vitality of HUVECs were decreased82.46%and 78.47%,related to the untreated group,respectively.Infected with DENV-2,the expression of LC3-?was increased significantly.There is a significant difference in the expression of P62 as autophagy substrate was decreased significantly compared with untreated group,infected with DENV-2 on 36 h and 48 h?p<0.05?,similar to the result of the group treated with RAPA?p>0.05?.There was a significant difference in the expressions of LC3-?and P62 were obviously higher in the group treated with CQ,compared with untreated group on 36 h and 48 h?p<0.05?;Infected with DENV-2,Laser confocal microscopy show that the expression of LC3-?was significantly higher than that in the untreated group on 36h,which was more obvious in the group treated with RAPA.The group treated with DENV-2 and CQ had a decreaseed vitality of HUVECs as 13.61%compared with the group treated with DENV-2 in 36h,and the results showed significant difference?p<0.05?.Conclusion:HUVECs were infected effectively by DENV-2.Autophagy was induced by the infection of DENV-2 in HUVEC.The growth of the HUVECs were inhibited by infection of DENV-2,and the autophagy induced by DENV-2 make a protective effect on HUVECs.