The Virus Titers And Expression Of PD-L1 And IFN-β In The Cardiac Myocytes And Cardiac Fibroblasts Of Mice After Infected The CVB3

Object: Viral myocarditis belongs to infectious diseases,that can be caused by the infection of a variety of virus especially Coxsackie virus B3(CVB3) of enterovirus which is the most common cause.Viral myocarditis can cause direct damage and body’s immune inflammatory damage after infected with CVB3.Viral myocarditis even can develop into dilated cardiomyopathy. At present there isn’t the effective and specific treatment of viral myocarditis in clinical specificity.Therefore,it is very imperative to explore the process and the treatment about viral myocarditis after infecting CVB3.However,there are 50% of the cells in the heart is fibroblasts,and the most functional cells is cardiac myocytes.So it is necessary to research the changes about CVB3 in cardiac myocytes and cardiac fibroblasts and the reaction about the two kinds cells after infecting CVB3 to providing the basis for the study of the development and treatment of viral myocarditis.Methods:(1)Cultured murine cardiomyocytes and cardiofibroblasts(C57BL/6):The heart of neonate mouse was cut into pieces,then typeⅡcollagenase plus trypsi-nase digestion method were used for isolating cell and then cardiacmyocytes and cardiacfibroblasts were collected to 6-well plates and cultured by different speed adherence into the CO2 incubator.(2)Cultured and observed the cardiacmyocytes and the cardiacfibroblasts of mice that infected with CVB3,then take the cell supernatant about 1day,2days,3days, 4days,5days,7days,9days later.(3)Take the cell supernatants to measure the viral titer by Hela cells.(4)Extraction of total cellular RNA:total cellular RNA was extracted in different timepoints after cardiacmyocytes infected with CVB3,(in 1days,3days,5days,7days,9days,72hours);total cellular RNA was extracted in different timepoints after cardiacfibroblasts infected with CVB3,(in 3hours,6hours,12 hours,24hours,48 hours,72hours) untreated cells controls for normal group,the RNA extracted was used PCR. Results:(1)Cultured cardiacmyocytes and cardiacfibroblasts of mice grows well,that is closed to the morphology cells in vivo growth.(2)After the infection of mice cardiomyocytes with coxsackievirus,take the supernatants at different time points to infect Hela cells,then the virus titer determination decline,even reaching a zero level of CCID50 on day 5.And the cardiacfibroblasts is same with cardiomyocytes after taking the supernatants at different time points to infect Hela cells,the virus titer determination decline, even reaching a zero level of CCID50 on day 3.(3)The results of the Real Time PCR showed that PD-L1 m RNA expression in cardiomyocytes infected with CVB3,begin to increases at 1 d,at 3 d reached a peak,at 5 d declined slightly,then declined below the normal level at 7 d and 9 d;of the m RNA expression of IFN-β declined at begin,at 3 d reached a peak then declined to normal level,and reached the highest level at 9 d.The results of the Real Time PCR showed that PD-L1 m RNA expression in cardiacfibroblasts infected with CVB3,declined at begin,then increased slightly,reached a peak at 24 h,then declined to the normal level at 48 h and 72 h;of the m RNA expression of IFN-β increased slightly at begin,at 12 h closed to normal level,then increased obviously and reached the highest level at 72 hours.Conclusion:(1)Infected with the CVB3 will mainly destroy the cardiomyocytes of mice,even induce apoptosis,but CVB3 can’t proliferate in cardiomyocytes and cardiacfibroblasts.(2)Normal cardiomyocytes and cardiacfibroblasts also forms expression of PD-L1 m RNA and IFN-β m RNA.(3)Infected with the CVB3 made m RNA expression of PD-L1 m RNA and IFN-β m RNA in cardiomyocytes and cardiacfibroblasts of mice changed at different time points.m RNA expression of IFN-β in the two kinds cells both increase obviously and the expression in cardiacfibroblasts is higher than cardiomyocytes.