| Myocarditis is mainly induced by a cardiotropic variant of coxsackievirus group B. Myocardial pathology injury is charactered by myocardium necrosis, fibrosis and Infiltrated by a mumber of mononuclear cells. It is clear that the direct lesion to myocardial cell by virus is transitory. Myosin released by the damaging myocardial cell cause advanced selfimmunity as selfantigen. Myocarditis can be induced in susceptible animal by immunization with purified myocardial myosin or synthetic myosin heavy chain fragment (eg aa735-747, aa947-960) emulsfied in FCA. We purified myosin from the porcine myocardium. Myosin αheavy chain is identified as the major component by Western blot. Balb/c developed obvious myocarditis after immunized with purifed porcine myosin emulsfied in FCA. Histopathological examination is characterized by infilation of mononuclear inflammatory cells and focal cardiomyocyte necrosis. Increased plasma cardiac Troponin I was detected in mice immunized with cardiac myosin. High titer of myosin antibodies was detected in mice plasma using indirect ELISA. Flow cytometric classification of th , Ts cells sand TCRVβ8.1&8.2 positive, TCRVβ10 positive T lymphocytes in mice peripheral blood and spleen was done. th, Ts, Th/Ts and TCRVβ10 positive T lymphocytes did not change significantly between mice immunized with cardiac myosin and the control group, while TCRVβ8.1&8.2 positive T lymphocytes increased significantly. Plasma concentration of TGF-β1 in mice immunized with cardiac myosin decreased significantly compared with the control.A 681bp fragment of cardiac myosin heavy chain was cloned by molecular cloning methods. To construct the retrovirus vector that secrect the myosin heavy chain fragment. We further constructed the IL-2-myosin fusion gene. Expression of myosin fragment was verified by RT-PCR, dot blot, immune electron microscope and immunohistochemistry.Normal Balb/c mice were used as bone marrow donor. Mice were injected with 150mg/kg 5-FU. 4 days later, the bone marrow cells were harvested. After erythrocyte lysis in 0.17M ammonium chloride, cells were cultured in RPMI 1630 media supplemented with 10ng/ml IL-3, Il-6, SCF and 10% heat -inactiveated FCS. After 48h, bone marrow cells were cocultured with a subconfluent monolayer of PA317 cells in the same media, which contains 4ug/ml polybrene. After a further 48h, non-adherent bone marrow cells were harvested. Cells were infused into irradiated (6Gy) Balb/c recipients. Two groups were transplanted. The gene transferred group mice were transplanted with bone marrow cells infected with pLNC-hIL-2-myosin retrovirus. The control group mice were transplanted with pLNCX retrovirus infected bone marrow cells. Another group of mice were bred as normal control. Mice were bred and maintained in ultra-clean lab. Eight weeks later, mice were immunized with purified porcine myosin emulsfied in FCA. Mice were killed on day 18 after the first immunization. Heart was fixed in 10% formalin. The degree of myocarditis in gene transfer group decreased significantly compared with the control group and the normal control. The positive rate of plasma cTnI also decreased significantly, while the titer of myosin antibodies did not change significantly. Flow cytometric test showed that Th1/Th2 in peripheral blood and spleen of the gene transfer group decreased significantly compared with the control and normal control group. Supernatant IL-4 concentration of spleen mononuclear cells stimulated with myosin for 72 hours was much higher in the gene transfer group compared with the control and the normal control group, while the concentration of IFN-γ was much lower than the control and the normal control group. Th1-Th2 immune deviation was obvious in the gene transfer group. 3H-TDR incorporation test was done to determine the proliferative response of mice spleen mononuclear cells after stimulated with myosin for 72 hours. The proliferative response of gene transfer group decreased significantly compared with the control group and the normal control group, w...
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