Experimental Study Of Function And Expression Of Osteopontin And Anti-sense Osteopontin In Viral Myocarditis

Background: Myocarditis is a common cardiovascular disease in clinical practice, which is one of the main reasons of sudden death. Usually, chronic myocarditis leads to dilated cardiomyopathy (DCM). Many researchers considered that virus infection is one of the primary causes contributed to myocarditis at present. About 50 percent of viral myocarditis (VMC) cases are associated with Coxsackievirus B (CVB) infection. The pathogenesis of VMC is not clarified thoroughly and there are no effective therapies against VMC so far. Osteopontin (OPN) is a secreted arginine-glycine-(RGD)-containing phosphoprotein with cell adhesive and aspartate chemotactic properties in vitro and in vivo. It is closely associated with infiltrating macrophage in tumors and it can directly stimulate macrophage migration, which has made it a key target as a molecule likely to be important in mediation tumor metastasis. The aim of study is to research the effect of ANOPN gene on viral myocarditis.Methods:1. Effect of osteopontin on viral myocarditisIn this study we first established VMC models through BALB/c mice infected with coxsackie virus B3, 80 pure bred male Balb/c mice aged 4 to 6 weeks and weighted 18 to 20 grams, were divided into control group (n=20) and model group (n=60) randomly. VMC model was founded by injecting intraperitoneally 0.1ml CVB3 Nancy solution, while the control group was injected with 0.1ml MEM culture fluid excluding virus. All mice were feeded normally. The model mice were sacrificed by snipping carotids with aether anaesthesia on day 4, day 7, day 9, day 14 and day 21 respectively, after injecting CVB3 0.8-1.0ml blood were extracted by burettes. The serums were separated and stored at 4℃, preparing for the test of virus counteracting antibody. Then all mice were killed by cutting necks, obtaining hearts and putting them into 4% formaldehyde solution for biopsy after blotting up blood with filter paper and being weighed. The twenty mice of control group were dealed with by the same methods on day 21. After virus inoculation until day 0, 4, 7, 9, 14, we examined the contents of OPN in the cadiocyte nucleus, virus titers of hearts, score for myocardial necrosis and the infiltration of mononuclear cells (IMNC). Based on the results, we further studied the effects of CVB3 infection time in OPN of the cadiocyte nucleus and score for myocardial necrosis and OPN of the cadiocyte nucleus and score for myocardial necrosis and cellular infiltration. Our study would provide new theoretic base and experimental evidence for prevention and therapy of VMC. We found that the model mice displayed tarnishable furs, reduced movements, been weary spirits and poor appetites, vaulted backs, showed indifference to stimulation or tended to irritation and been lower temperature since the third day. Death occured on day 4, and reached the top from day 7 to day 10 (18 mice died, the ratio was 58%). The symptoms mentioned above were alleviative gradually and nodeath happened from day 13. Among 60 mice in model group, 31 died. The death rate was 51.7%. None of the control mice had any symptom and death. The livability was 100%. The serum anti-CVB3 antibody concentrations of the model group were much higher than that of the control mice at each time spot (P<0.01), and the serum antibody concentrations reached high point on day 7 and day 14. Pathological characteristics included myocardial necrosis, lymphocytes and monocytes soakage, which were the most obvious on day 7 and day 14. Above data indicated that myocarditis model of Balb/c mice was established successfully by injecting CVB3 intraperitoneally. The expression of OPN was detected in myocardial tissue at the 0, 4, 7, 9 and 14 days post CVB3 infection quantitatively by semiquantitative analysis RT-PCR. The results showed that the expression of OPN increased slightly at the fourth day post infection, then the expression was increased continuously, and the expression of OPN was climbed a peak at the 9th day post infection. The expression of OPN at 4, 7, 9 and 14 days were higher than that of uninfected control (P<0.01). It indicated that the expression of OPN was dependent of the infection time within 14 days post infection in myocardial tissue in vivo.Linear correlation analysis showed the contents of OPN in the cadiocyte nucleus with score for myocardial necrosis and cellular infiltration myocardial necrosis, the infiltration of mononuclear cells (IMNC) is positive correlation remarkably, but virus titers of hearts no correlation with the contents of OPN in the cadiocyte nucleus. It suggested that OPN may be involved in the infiltration of mononuclear cells in myocardium of VMC.2. Construction of the mammalian expression recombinant pcDNA 3.1-ANOPNA OPN gene recombinant expression vector plasmid was constructed by RT-PCR from rat osteosarcoma cell line ROS 17/2.8 and cloned into a mammalian expression vector pcDNA3.1(+) at the sites of Xho I and HindⅢ, and named pcDNA3.1-ANOPN. The recombinant was cleaved with appropriate endoneuclear and sequenced. The results showed that the inverse direction of the insert was found to be correct, while no rearrangement was found.3. Experimental study of anti-sense osteopontin in viral myocarditis In order to research effects of ANOPN on the onset of VMC, myocardium was injected with myocardial tissue pcDNA3.1-ANOPN. 200 pure bred male Balb/c mice were divided into control group (n=20) and model group (n=180) randomly. The establishment of model and heart tissue sampling were the same as that in part one. The morphological characteristics of heart were observed under optical microscope after staining with hematoxylin-eosin and myocardial histopathplogic scores were counted through semi-quantity. The expressions of mRNA of OPN were detecded by reverse transcription polymerase chain reaction (RT-PCR) and we examined the virus titers of hearts, score for myocardial necrosis and cellular infiltration. The result of the experiment was displayed compared with the control group, there was a significant remission in the myocatdial pathological change integration pcDNA3.1-ANOPN treatment group and linear correlation analysis showed the contents of OPN in the cadiocyte nucleus with score for myocardial necrosis and cellular infiltration myocardial necrosis, the infiltration of mononuclear cells (IMNC) is positive correlation remarkably, but virus titers of hearts no correlation with the contents of OPN in the cadiocyte nucleus.Conclusion:In summary, our study suggested that: We constructed the mammalian expression recombinant pcDNA3.1-ANOPN possess some curative effects on viral myocarditis model mice.Intervention with OPN could relieve VMC. The positive correlation in the dependablity analysis between the expression of the OPN and the pathological integral in the musculature of the VMC mice demonstrates that OPN educes an important effect on the developing of the VMC. The result that the expression of the OPN obviously increases in the musculature of the VMC mice compared with the control group and has a close correlation with the damage proceed and severity degree of musculature shows that OPN educes an important effect on the immunologic injury and it can be as a indicatrix to infect the level of myocardial cellular necrosis and VMC. The OPN molecule maybe become an effective measure for the protect and the treatment of the VMC.The study has elucidated the role of OPN in pathogenesis of VMC, providing a potential pathway in prevention and therapy of VMC and giving a new idea to research on other inflammatory related diseases.