Coxsackievirus Infection Induced Cytopathy And Cytokine Secretion In Astrocyte

Background and aimsCoxsackievirus is one of the most common pathogens arouse human infection , the infection can cause many diseases and symptoms such as respiratory tract infection, hand, foot and mouth disease, it's manifested by fever, herpes angina, more serious are myocarditis, pericarditis and neurological disorders. In recent years, several cases with the main features of aseptic meningitis encephalitis were outbreak in China. The patient's cerebrospinal fluid levels of cytokines in abnormal increase. Glial cells, particularly microglia and astrocytes in the central nervous system (CNS) are the main source of cytokine production, but what function of the glial cells undertake when the Coxsackievirus infected CNS still unclear, so far. Therefore, the main purpose of this study is to explore the astrocytes in the Coxsackievirus infection and the pathological changes that occur related to immune reactions such as cytokines in order to define infection of glial cells in the role, understand the CNS after infection of Coxsackievirus pathogenesis of the necessary experimental basis.We construct chemokine IP-10 (CXCL10) eukaryotic expression vector using gene cloning method for further study of chemokine IP-10 biological functions.Materials and MethodsOne day postnatal BALB / C mice pup, the brains were removed, the meninges were stripped off, and tissues were cut into small cubes in sterile conditions, rapidly stripping the dura, made blunt separation of mixed brain cell suspension. After culture 20-25 days, combined with a mild trypsin digestion method and orbit shaking method to purify astrocytes. Thereafter, infected astrocyte with Coxsackievirus A16 and B3, respectively. The infection of astrocytes was detected by virus replication, virus growth curve, cytopathic effect, cell survival, the change of cytokine gene transcription and cytokine secretion. We construct chemokine IP-10 (CXCL10) eukaryotic expression vector using gene cloning method and then transfected into astrocyte cells for overexpression of IP-10 study on the state of cell survival.Results and conclusions1. Successfully isolated and purified astrocytes and microglia. Detected by immunofluorescence and flow cytometry cells more than 90% purity, and this method was mature and high repeatability to meet experimental requirements.2. Coxsackievirus B3 can infect astrocytes and produce offspringvirus. Coxsackievirus A16 is not sensitive to astrocytes and can not produce offspringvirus.3. Coxsackievirus B3 infection in astrocytes can cause significant cytopathic effect (CPE), virus titer of the supernatant reached a peak at 24h post infection. Coxsackievirus A16 does not cause CPE in astrocytes.4. Coxsackievirus B3 infection in astrocytes increased the pro-inflammatory cytokines IL-1, IL-6 and TNF-αand the chemokine CCL5, IP-10 of the mRNA expression; Coxsackievirus A16 also increased pro-inflammatory cytokines such as IL-1, IL-6 and TNF-αand the chemokine CCL5, IP-10 mRNA levels, but lower than Coxsackievirus A16 did.5. ELISA quantification analysis of supernatant IP-10 content revealed that Coxsackievirus B3 infection of astrocytes can significantly increase IP-10 expression compared to Coxsackievirus A16; Inactivated Coxsackievirus B3 can also be induced astrocyte to secrete an amount of IP-10, but lower than activated Coxsackievirus B3.6. Constructed IP-10 (CXCL10) eukaryotic expression vector and transfected astrocyte cells arouse overexpression IP-10.