Expression Of CD80 And CD86 In The Cardiomyocytes And Cardiac Fibroblasts Of Mice After CVB3 Infection

Object: Viral myocarditis is a myocardial disease caused by viral infection,the most common reason is Coxsackie virus B3(CVB3)infection.The pathogenesis of myocarditis is currently supporsed to the virus directly injury and the body’s immune response.Viral myocarditis can be developed to dilated cardiomyopathy,which shows a high mortality and high disability rate in children.Until now there is still no effective treatment methods available.So it is necessary to study the pathogenesis and treatment of viral myocarditis caused by CVB3 infection.In addition to the most functional cardiac muscle cells in the heart,there are 50% of the fibroblasts cells in the heart.So the reaction of cardiac myocytes and fibroblasts in the stimulation of CVB3 need to be studied,which might provide a basis for the study of the pathogenesis of viral myocarditis and to find effective treatment methods.Methods:1 Cultivation of cardiomyocytes and cardiac fibroblasts cells in vitro: the heart of newborned C57BL/6 mice were taken out aseptically,and then placed them in high-glucose DMEM growth medium which does not contain the serum and antibiotics.Firstly,the tissues were pipetted repeatedly to remove tissues outside the hearts,then re-adding a small amount of high-glucose DMEM growth medium which does not contain the serum and antibiotics.After the tissues were to cut into pieces with sterile scissors,and digested by trypsin and collagenase in turn,the suspension were filtered with copper mesh.The filtrate was collected into a centrifugal tube and centrifugate.Centrifugate again after discard the supernatant and then discard the supernatant again.Then add high-glucose DMEM growth medium which contain the serum and antibiotics into the centrifugal tube.Put these cells into six-well plates after they dispersed.After 90 minutes,the unattached cells were removed to a new six-well plates and then add fresh high-glucose DMEM growth medium which contain the serum and antibiotics into the remained wells(this is a method which rely on the different adherence speed to isolate and culture cardiomyocytes and cardiac fibroblasts of mice).Finally,put them into the CO2 incubator and culture at 37℃.2 Coxsackie virus were inoculated into the cardiomyocytes and cardiac fibroblasts culture of mice and the CPE were observed under microscope.3 Observation of the expression of CD80 and CD86 on cellx: total cellular RNA was extracted at different time points from cardiomyocytes and cardiac fibroblasts which were infected with Coxsackie virus according to the mannul of Trizol.In this study,the time points were set as 1 day,3 days,5 days,7 days,9 days after the cardiomyocytes were infected with Coxsackie virus;similarly,the time points set for the cardiac fibroblasts were 3 hours,6 hours,12 hours,24 hours,48 hours and 72 hours after Coxsackie virus infection,.Those cells which were not infected with the virus were used as control group,the RNA extracted was used for Real Time PCR amplification to observe the expression of CD80 and CD86.Results:1 The cardiomyocytes and cardiac fibroblasts of mice could be cultured in vitro and in good condition.2 Observed the cells with Coxsackie virus infection and found that the CPE was appeared in some of the cardiomyocytes of mice,but only appeared in a few of cardiac fibroblasts.3 After the Real Time PCR,the results showed that: CD80 mRNA expression in cardiomyocytes infected with CVB3 suddenly dropped at 1d,then began to rise,reached the peak at 3d,then began to decline,5d,7d,9d continued to decline,the whole process were below the normal level;the expression of CD86 mRNA was decreased significantly at 1d,and then began to rise,reached the peak in 3d,then began to fall,5d,7d,9d continued to decline,the whole process was below normal level.The results of the Real Time PCR also showed that: after infected by Coxsackie virus,CD80 mRNA expression in cardiac fibroblasts was continued to decline in 3h,6h,12 h,24h,24 h fell to the lowest point,then began to rise,48 h,72h and continued to rise,the whole process was below normal level;the expression of CD86 mRNA was continued to decline in 3h,6h,6h to the lowest level,but suddenly increased in 12 h,reached the highest peak,and then decreased in 24 h,but increased in 48 h,reached the second peak,it was in the normal level,but decreased in 72 h.The result showed that the lower expression of CD80 and CD86 might be the reason that immune injury was lighter and virus directly damage was heavier for adult in the early of infection with CVB3.Conclusion:1 After infected with CVB3,virus mainly damaged to some of the cardiomyocytes of mice in the early stage,and the damage of cardiac fibroblasts was not obvious,the cardiomyocytes and cardiac fibroblasts of mice in vitro cultured with proliferation.2 The expression of CD80 and CD86 mRNA was expressed in both cardiaomyocytes and cardiac fibroblasts in normal mice.3 After infected with CVB3,the expression of CD80 and CD86 mRNA in the cardiaomyocytes and cardiac fibroblasts of mice had the fluctuation,and the expression decreased obviously.Thus we could say that in the early stage of infection,the low expression of CD80 and CD86 could combine with cytotoxic T lymphocyte-associated antigen 4(CTLA-4),and regulated negative and this regulation could inhibit the proliferation of T cells.T cells could not play the immune response,which resulted in the development of the disease.