Association Between MHC Gene Polymorphism And VMC

Objective The 4-6 weeks Balb/C mice were inoculated with COXB₃virus intraperitoneally for the model of acute viral myocarditis to explore the relationship between viral myocarditis disease progression and the major histocompatibility complex polymorphism,to clarify the immune genetic characteristics of viral myocarditis,providing a method for the futher study about viral myocarditis and human major histocompatibility complex polymorphism,as well as provide a possible way to explore the protective genetype of viral myocarditis for possible gene therapy and explore new therapeutic approaches.MethodsThe experiment was divided into animal experiment part and molecular experiment part.?I?animal experiment:Hela cells were maintained in Dulbecco's modified Eagle's medium?DMEM?supplemented with 10%fetal bovine serum,100 units/ml penicillin,and 100 ug/ml streptomycin,CVB₃ strain was propagated in Hela cells to increase the virulence,collected supernatant on the virus and calculate the half tissue infection dosage(50%tissue culture infective dosed,TCID₅₀)when the virus reached certain value,inoculated the mice with different concentration of virus,determined the appropriated dose of virus infection according to the level of mice serum CKMB and the myocardial tissue pathological changes;Select 120 mice,divided into virus group and control group randomly,while the virus intraperitoneal inoculation the determined dose of COXB₃ virus,observe the clinical changes,at the same time select two mice randomly from two groups to fetch blood and anatomy per day,test serum concentration of IFN-?,in accordance with the control group as a result,records the time about the serum IFN-?is higher than the normal range,reach the peak level and the fell to the normal range,at the same time observing the myocardial pathological change as the reference;?ii?molecular experiment part:80 mice were randomly selected and divided into virus group and the control group,each group then divided into 3 groups,record the change of weight and activity,collect the mice serum and heart tissue at the three points of IFN-?concentration changes,then myocardium was tested dynamically by immunohistochemical technique,and had comparative analysis with the method of hematoxylin and eosin staining,then designing five set of primers according to the reported the second exon nucleic acid sequence of literature of Balb/C mice H2-Eb area,IFN-?within genes as the reference,RT-PCR method to detect the gene expression in cardiac muscle,At the same time,identified the virus by real-time fluorescence quantitative PCR.Results?1?The selection of viral infection modulus:Hela cells,after infected 72 hours by Nancy COXB₃ strain showed shrinkage deformation,capsule rupture and disarrangement,and many cell fragments were seen floating in the culture medium.By observing cell CPE and reed-muench method,TCID₅₀0 was 10^-3,and the experiment showed the most appropriate infectious dose was 10³TCID₅₀.?2?Optimal virus concentration for modeling:1.General information:mice in group A began to show significant activity and weight loss at the 3rd day,followed by a death peak at the 7th day.Mice in group B and C began to show activity loss at the 4th day,but none died within 7 days.2.Serum CKMB level:at the 7^thh day after virus infection,serum CKMB level in group A was higher than that in group B and group C,and was significantly higher than that in group D.3.Myocardial tissue pathology:myocardial lysis was observed in the myocardial tissue pathology of mice in group A,and large necrotic lesions appeared.Macroscopically,no obvious necrosis was observed in the myocardium of group B and group C,and few inflammatory cell infiltration appeared invasion in mice cardiomyocytes,No inflammatory cell infiltration was observed in the myocardial stroma of group D.4.The appropriate amount of virus for modeling was 10³TCID₅₀0 according to the combination with changes in weight,serum CKMB level and myocardial pathological changes of mice.?3?Changes of serum IFN-?level in VMC mice:the serum IFN-?level in mice detected by ELISA method observed to start to increase at about the third day after inoculation,reach a peak level at about the 2ⁿdd week,and gradually decrease at about the3^rdd week.?4?Pathological integral in myocardial tissue:histopathological observation was implemented after myocardial tissue of mice with HE staining.After statistical analysis,the pathological integral of mice in the virus group at 3^rdd day,7^th,14^thh and 28^thh was significantly different from that of the control group?P<0.01?.?5?Designing the nucleic acid sequence of the second exon in the H2-Eb region of Balb/C mice in order to analyze the relationship between the expression of 4 groups of sequence-specific primers and the myocardial pathological changes:By computing the pathological integral and MudoEb5 positive results,the results showed that the correlation coefficients were:r=0.449,P<0.05,7 day,r=0.745,P<0.05,14 days r=0.712,P<0.05,14 days r=0.676,P<0.05,suggesting that the VMC mice pathological integral and MudoEb5 that there was a positive correlation between,simultaneously MudoEb7 expression level and the pathological integral of correlation analysis showed that there was a positive correlation as well.Conclusion IFN-level changes may occur during the progression of viral myocarditis infection,which is correlated with the degree of infection.H2 gene may be involved in immune regulation,leading to different susceptibility of myocardial tissue to virus.