Study Of The Mechanism On Recombination Interleukin-12 Therapeuty Mice Viral Myocarditis

Viral myocarditis is a common disease of pediatric cardiovascular medicine. The clinical manifestations varying it can be asymptomatic onset. gradually develop into heart failure. Fulminant onset shall also be quickly developed into acute heart failure. It is also a major cause of dilated cardiomyopathy. Abroad, it has become a major cause of heart transplantation in young people. VMC is therefore an important disease threat to the health of children.Treatment of viral myocarditis plagued clinicians. In addition to the traditional method -improve myocardial energy metabolism and immune status, such as gamma globulin conditioning means to reduce myocardial injury, no clear-cut and effective treatment methods. Many experts had tried different approaches attempting to make a breakthrough in treatment. In recent years, with the deeply study in cytokines, any experts will focus more on the treatment of myocarditis using cytokine. recombinant cytokines IL-2 and interferon-2a have been applied in the clinical.The molecular weight of IL-12 is 70kD (P70) and it is dimer heterogeneous molecular. The two sugar chains of the molecular weight of 40 kD (P40) and the 35kD (P35) are connected by disulfide form. IL-12 can enhance cell-mediated cytotoxicity. It makes sensitized T cells and NK cells to the differentiation and proliferation. IL-12-induced T cells and NK cells produce many cytokines:such as IFN-γ, IFN-α, GM-CSF, M-CSF, IL-13, IL-8 and IL-2 and so on, and IL-12 plays roles by them. It is one of the most important physiological functions of IL-12 induced NK cells to produce IFN-γ. In the early, IL-12 inhibits viral replication and activation of cytotoxic T lymphocytes by induction IFN-γ. IL-10 is the main inhibitor of IL-12, IL-12 stimulation of T cells induced by its own generation, to block IL-12 gene expression by inhibiting IL-12 production. In our study, we investigate the effects of recombinant IL-12 in the treatment of viral myocarditis in mice. By investigating the changes in the course of treatment of some cytokines, NK cell function, perforin expression and apoptosis, we explore the mechanisms for the treatment of viral myocarditis using recombinant IL-12.Materials and methodsGroup: inbreeding BALB/C male mouse, divided to 6 groups trabantly.Group A: blank group, which were intraperitoneal injected by NS for 5 days. Group B:virus control group, which were intraperitoneal injected by CVB3 virus for 5 days.Group C:medicine control group, which were intraperitoneal injected by CVB3 virus and IFN-γ(400IU per mouse) for 5 days. Group D:low dose therapy group, which were intraperitoneal injected by CVB3 virus and rmIL-12 (1ng per mouse) for 5 days. Group E: middle dose therapy group, which were intraperitoneal injected by CVB3 virus and rmIL-12(10ng per mouse) for 5 days. Group F:big dose therapy group, which were intraperitoneal injected by CVB3 virus and rmIL-12(100ng per mouse) for 5 days.Experiment one: compare the mortality between groups after rmIL-12 treating, and record everyday.Experiment two: grouping ditto, 15 mice each group. We inject virus on the first day. On the sixth day, we take 4 mice from each group and weigh them. Blood taken from the eyes were detect the IL-10 and IFN-γ. Mouse was luxatioed and sacrificed after taken blood. Half of hearts were marked to do pathology and detect apoptosis. Other hearts were detected the virus titre. On the tenth day, fifteen day, we do the same work.1. On the fifth day, tenth day (after inoculate virus), IL-10 and IFN-γwere detected by the ELISA.2. On the condition of asepsis, taking out of mouse's heart, grinding, making organize suspension, adding the stock solution and the 10-1~10-6 to cultivation with amnion cell. We watch cenotaphic effect everyday, and determine the virus toxicity.3. On the condition of asepsis, taking out of mouse's spleen, grinding, centrifuging, schizolysising the red cell, making Yak-1 cell target cell, detecting the cytoactive of the NK cell by the MTT.4. Expression of the perforin in the mouse's cardiac muscle. Fresh hearts were taken out, grinded, added Trizol fluid, extracted RNA, and detect the expression of the perforin by the RT-PCR. 5. Mouse's heart were fixed by the 10% formalin, paraffin imbedded, paraffin section, stained by the TUNEL,mounted,detected the apoptosis by the microscopy.Result: Positive cell, also can be called apoptosis cell, has the yellow granules in the cellular nucleus.6. Preparation of the paraffin section with HE stain. The cellular nucleus can be seen in blue color and the cytoplasm was pink on the microscope. Comparing the inflammation between the myocarditis.Statistics analysis:We do the data analysis by the SAS statistics software, ANOVA, P<0.05 has the statistical significance.RESULTExperiment 1: Except the blank group, middle dose group has the lowest mortality, while the big dose group has the highest one.Experiment 2: We can see that IFN-γand IL-10 in the serum of the big dose group are much higher than the one of the middle dose group (P<0.05), by comparison of the cytokine IFN-γ,IL-10 in each group rmIL-12 after treating. The peak of the IFN-γis earlier than the IL-10.2. Comparison the virus toxicity in each group before and after IL-12 treatingOn the third day of inoculation of the heart grinding fluid, we can see: cell of the blank group grows well, while the one of control group have cytopathic effect. Cell of the virus control group have CE in the 10-4 dilution, which shows that cell aggregation and quassation. The same CE shows in the10-2 dilution, middle dose group and big dose group.3. Comparison the activity of NK cell in each group before and after IL-12 treatingDifferent dose and time of treatment infect the activity of NK cell. There is difference between the virus control group and middle dose group and big dose group (P<0.05), but no significant difference between IFN control group and little dose group (P>0.05). On the 5th day and 10th day, NK cell activity increased with the rmIL-12's increasing, and got top on the 10th day.4. Detection the expression of the porforin in each group before and after IL-12 treating.Experimental result: no expression of perforin in the blank control group. The expression amount is biggest in the middle dose group and big dose group, less in the little dose group and IFN control group.5. Comparison the myocardial damage before and after treatingComparison the weight and weight rate in mouse's hearts: there is difference in the virus control group and little dose group, alse in INF and other groups.Comparison the inflammation change of the myocardial in each group (HE stain): the one in middle dose group is lest, while big dose group has the most serious one.6. Comparison of the apoptosisThe myocardial cell's apoptosis of the big dose group is most seriously, more seriously of middle dose group, and the ones in the little dose group and the INF treating group and virus comparing group are the same, none in the blank group.CONCLUSION1. rmIL-12 increase the release of IFN-γ, which has a function of inhibit the virus replication, by the way of increasing the NK cell activity. All above lead to virus titer decreasing of rmIL-12 group with decreasing of rmIL-12 treating dose.IL-10 can inhibit IFN-γ's overdose excretion. From each group's pathology result we can see: NK cell'activity increasing and IFN-γ's excretion can inhibit virus replication, decrease myocardial inflammation. For middle dose group has the less inflammation, NK cell can lead to myocardial cell damage mouse's mortality rate increasing by its killing function.2. By increasing the NK cell's activity, rmIL-12 increases the release of the porforin, which infect the apoptosis by Fas and FasL. Appropriately increasing the apoptosis of the infect myocardial cell can relieve the cellular damage, but higher one can increase the damage and mouse's mortality.