Isolation And Culture Of Primary BMECs And AS From Tree Shrew And Preliminary Study On The Characteristics Of ZIKV Infection

ZIKV(Zika virus,ZIKV)belongs to the flavivirus genus of the Flaviviridae family.ZIKV infection is related to central nervous system damage,including microcephaly and other nervous system abnormalities caused by congenital infection and adult meningoencephalitis.However,the pathogenic mechanism of ZIKV is not completely clear,and the mechanism of ZIKV invading the nervous system is not clear.Some studies believe that ZIKV crosses the blood-brain barrier and invades the nervous system,infects neural progenitor cells and neural precursor cells,and weakens or hinders neurons.Differentiation,which leads to nerve damage.The mechanism by which ZIKV crosses the blood-brain barrier is not clear.Studies have found that the permeability of ZIKV across the blood-brain barrier is not reduced.It may invade the nervous system through an exocytosis-dependent replication pathway and transcytosis,but it has not been confirmed.evidence.The blood-brain barrier is a selective diffusion barrier composed of brain microvascular endothelial cells(BMECs),pericytes(pericyte)and astrocytes(astrocytes,AS).This study is based on previous studies and passed Isolate tree shrew brain microvascular endothelial cells(BMECs)and astrocytes(AS)in vitro,explore the characteristics of ZIKV infection on primary cells of tree shrew brain tissue,and establish an in vitro barrier culture model to verify that ZIKV is permeable across the blood-brain barrier Whether sex has changed.First,isolate and culture tree shrew microvascular endothelial cells(BMECs)and astrocytes(AS)and perform immunofluorescence staining for identification;secondly,perform in vitro experiments of ZIKV infection of BMECs and AS,real-time PCR detection of cells and culture supernatant Draw the virus proliferation curve and select multiple time points to detect the expression levels of cytokines IL-6,IL8,IFN-α,IFN-βmRNA and the blood-brain barrier after ZIKV infection of BMECs Related protein claudin3,MMP-9,MMP-12,Cav-1,Mfsd2a mRNA expression;finally,BMECs and AS were used to establish an in vitro barrier culture model,and the integrity of the in vitro barrier culture model was evaluated after ZIKV infection and mock infection.The results showed that:(1)The tree shrew BMECs and AS isolated and cultured by the method described in this article have high purity and good activity,which can be used in the next experimental study;(2)The proliferation dynamic curve shows that ZIKV can rapidly proliferate in BMECs and AS.ZIKV infection with BMECs reached a peak of 106 copies/mL at 48 hours,and ZIKV infection with AS reached a peak of 106 copies/mL at 60 hours;(3)After ZIKV infection with BMECs,pPCR detected increased levels of cytokine IL-6 and IL8 mRNA and blood-brain barrier related protein claudin3 mRNA levels The expression was up-regulated at 0-6h,down-regulated at 6-12h,up-regulated at 12-48h,MMP-9 and MMP-12 remained flat,Cav-1 was down-regulated at 0-12h after infection,and up-regulated at 12-48h,Mfsd2a at 0-after infection 6h down-regulation,6h-48h up-regulation;(4)ZIKV infection AS cytokine IL-6,IL8,IFN-α,IFN-β mRNA expression levels were up-regulated;(5)In vitro barrier culture mode permeability evaluation results showed that,ZIKV was infected with the monolayer cell culture model established by BMECs.ZIKV RNA was detected in the lower chamber at different time points,and the permeability of the monolayer cell culture model did not change at different time points.ZIKV infected BMECs and AS co-culture model,24h When the permeability is significantly reduced.Conclusion:This study successfully established a method for the isolation and culture of tree shrew BMECs and AS;ZIKV is susceptible to tree shrew BMECs and AS.ZIKV can replicate and release progeny viruses in tree shrew BMECs and AS;ZIKV infection can activate BMECs Local immune response to AS and AS without destroying the tight junctions and endothelial permeability of BMECs.It is speculated that it may enter the blood-brain barrier through transcytosis;ZIKV crosses BMECs and infects astrocytes,which may lead to reduced permeability.