The Effect Of Proliferation And Osteopontin Expression In Rat Myocardial Fibroblasts Infected By CVB3

Objective: To observe cell proliferation and OPN expression in ratmyocardial fibroblasts infected with Coxsackievirus group B3(CVB3).To evaluate possible mechanism that OPN may be associatedwith the development of viral myocarditis.Methods: Neonatal rat cardiac fibroblasts were isolated andcultured with an improved method and the cells were identified withtheir morphology and immunocytochemical stain. Fibroblasts wereinfected by CVB3 with different multiplicity of infection (MOI), cellproliferation was estimated by MTT assay and OPN expression weredetermined by RT-PCR and Western Blotting.Results:1. Digestion with pancreatin and collagenaseⅡwhich canincrease the survival rate. The staining of monoclonal antibodies againstVimentin was positive, while the staining was negative for theantibodies anainst a-Smooth Actin.2. The primary cultured rat myocardial fibroblasts were infectedwith different MOI(0,0.2PFU/cell,0.5PFU/cell,1.0PFU/cellrespectively). The results of OD values were 0.35±0.05,0.37±0.04,0.66±0.07,0.32±0.07 respectively in defferent viruses tite.The results ofOD values of fibroblasts which was infected by CVB3 with 0.5PFU/cellwas higher than those of other groups, the differences were significant(P＜0.05).3. The primary cultured rat myocardial fibroblasts were infectedwith CVB3 wich multiplicity of infection (MOI) was 0.5 PFU/cell. Theresults of OD values were 0.36±0.06,0.47±0.06,0.56±0.04,0.72±0.04,0.64±0.02 respectively in control groups(12h)and viral infectgroups(12h,1d,2d,3d).These results suggest that the OD values offibroblasts started to increase at 12h when infected by CVB3, peaked at2d, and then decreased, the difference were significant each other(P＜0.05)4. The gray scale values of the OPN/β-actin in control groups(12h,1d,2d,3d)were 0.38±0.06,0.35±0.02,0.41±0.07,0.30 ±0.06 respectively with RT-PCR, there were no significant differenceeach other(P＞0.05). The gray scale values of the OPN/β-actin in viralinfect groups(12h,1d,2d,3d) were 0.56±0.06,0.72±0.05,0.98±0.06,0.86±0.02 respectively with RT-PCR.These results suggestthat the OPN expression started to increase at 12h when infected byCVB3, peaked at 2d, and then decreased, the difference were significanteach other(P＜0.05)5.The gray scale values of the OPN/β-actin in controlgroups(12h)and viral infect groups (12h,1d,2d,3d) were 0.26±0.04,0.36±0.03,0.52±0.06,0.76±0.06,0.62±0.03 respectivelywith Western-Blotting. These results suggest that the OPN expressionstarted to increase at 12h when infected by CVB3, peaked at 2d, andthen decreased, the difference were significant each other (P＜0.05).6.To carry out correlation analysis with the OPN expression andthe OD values,correlation coefficient of Pearson r=0.951, P=0.000 andr=0.954, P=0.000 respectively.They have positive correlation.Conclusions:1. CVB3 can cause proliferation of myocardial fibroblasts in adefinite virus tite.2. CVB3 can cause cell proliferation and up-regulate the OPNexpression in rat myocardial fibroblasts in time-dependent way indefinite time.3 There is a positive correlation between cell proliferation andOPN expression .It suggested that OPN expression combining with cellproliferation of myocardial fibroblasts may play an important role in themechanism of viral myocarditis.