Study On The Role Of CD8～+T Lymphocyte And Granzyme B And The Effect Of Perforin Antibody On The Expression Of Granzyme B In Viral Myocarditis

ObjectivesTo explore the role of CD8⁺T lymphocyte and Granzyme B in viral myocarditis, and to study the effect of perforin antibody on the expression of Granzyme B in viral myocarditis. Methods85 four-week-old Bald/c male mice were randomly divided into 3 groups, including normal control group (Gr1), viral control group (Gr2) and perforin antibody therapy group (Gr3). The mice in Gr1 were inoculated with 0.15ml Eagle reagent, and the mice in Gr2 and Gr3 with 0.15ml TCID₅₀10⁹/ml CVB₃. The mice in Gr3 were inoculated with perforin antibody (0.1mg/Kg) on hour 6 and day 3 post inoculation (p.i.). Three normal mice and eight infected mice in Gr2 were sacrificed on day 7, 10,14,21,28 after inoculation. The surplus mice in Gr3 were sacrificed on day 10 post inoculation.The heart were resected for the following examination:1) Histopathologic examination of the heart were observed by optical microscope ,and the myocardial histopathologic scores were counted;2) The expression of CD8⁺ T lymphocyte and Granzyme B proteins in myocardium were determined by immunohistochemistry; 3)Terminal transferase-mediated Dutp-biotin nick end labeling(TUNEL) assay was used to quantify cardiomyocyte apoptosis.The percents of CD8⁺ T lymphocyte were counted by optical microscope,and the areas of Granzyme B were measured by theLeica Qwin V₃ system.Results1. There were on pathological changes in myocardium in normal control group (Gr1), and the incidence of myocarditis was 100% in each virus-infected group. Obvious focal degeneration and necrotic lesions appeared in the myocardium with inflammatory cell infiltrations on day 7, and reached the peak on day 10, the recovered gradually on day 28.2. The cardiomyocyte apoptosis in myocardial tissues increased apparently from day 7 after infection (13.91 ± 1.77) %, peaked on day 10-14 p.i. (18.99± 2.77) %,and a few apoptotic cardiomyocytes were detected on day 28 in virus control group(9.04±1.34) %.3.The cardiac myocyte apoptosis rates had a significantly positive correlation with myocardial histopathologic scores (r=0.83, P<0.01).4.The protein expression of CD8⁺T lymphocyte in myocardial tissues increased apparently from day 7 after infection (28.86±1.46) %, peaked on day 10-14 p.i (66.66±1.95) %., then gradually decreased on day 21 in virus control group (32.86±1.56) %.5.The protein expression of CD8⁺ T lymphocyt had a significantly positive correlation with myocardial histopathologic scores and cardiac myocyte apoptosis rates (r=0.88, P<0.01; r=0.86, P<0.01).6.The protein expression of Granzyme B in myocardial tissues increased apparently from day 7 after infection (46.41±3.72) %, peaked on day 10-14 p.i. (67.13±1.82) %, then gradually decreased on day 21 in virus control group (28.01±1.66) %.7.The protein expression of Granzyme B had a significantly positive correlation with myocardial histopathologic scores and cardiac myocyte apoptosis rates ((r=0.85, P<0.01; r=0.83, P<0.01).8. The protein expression of Granzyme B in perforin antibody therapy group (6.98±1.34)% was significantly decreased compared with that in virus control group (67.13±1.82) % (P<0.01) . Conclusions1. Cardiomyocyte apoptosis was detected in murine viral myocarditis, and was highly correlated with the changes of myocardial histopathologic scores. Apoptosis is the important manifestation of myocyte injury in VMC.2. CD8⁺ T lymphocyte had a significantly positive correlation with the myocardial histopathologic scores. It suggested that CD8⁺ T lymphocyte was the main cell that injured myocardium.3. CD8⁺T lymphocyte had a significantly positive correlation with the cardiac myocyte apoptosis rates. It might lead to myocyte injury by apoptosis in VMC.4. Granzyme B had a significantly positive correlation with the myocardial histopathologic scores. It suggested that Granzyme B played a major role in myocyte injury in VMC.5. Granzyme B had a significantly positive correlation with the cardiac myocyte apoptosis rates. Apoptosis induced by in Granzyme B played a important role in VMC.6. Granzyme B in myocardial tissue in PFP antibody therapy group was significantly decreased, this could provide some new therapy methods about VMC.7.The main passway that Granzyme B entered the cardiomyocytes is the pores produced by PFP, but Granzyme B could enter the cardiomyocytes by other ways.