The Relationship Between MRNA Expression Level Of Perforin Of Peripheral Blood Lymphocytes And Children Viral Myocarditis

At present thought the viral myocarditis is one kind of common disease due to a viral infection, which main pathological change include the myocyte denature and necrosis and the inflammatory infiltrates and the fiber effusion. Since 20 century 70 s, there is an increasing tendency of incidence all over the world. The incidence of viral myocarditis after acute viral infection is about 5%. In most cases, Clinical presentation of patients is light-middle, but part of patients may present with sudden death due to acute burned-out myocarditis. Some present with chronic consisting infection, or have serious sequelae: refractoriness arrhythmia and chronic cardiac functional insufficiency. Recently lots of study has proved that this disease may progress over time to dilated cardiomyopathy. Although a lot of work has been done, the mechanism involved are incompletely understood, it is believed the disease is related to direct attack to heart by virus and the damage by own immune system. Most scientists consider that cell immunologic deficiency is main pathogenesis of viral myocarditis, especially in end stage. Autoimmunity myocarditis induced by viral infection result in the damage of myocytes through the CMC. The effector cells which infiltrate and damage the myocytes mainly are the active CTL and NK cells, they infiltrate cardiac muscle cells and express the perforin in different phase. This paper is concentrate in the expression level of perforin of peripheral blood lymphocyte, disclose the relationship between them, and provide the basis for the diagnosis as well as the treatment.Perforin is a kind of protein that exists in CTL and NK cells, molecular weight 62-66KD, the structure and function similar to complement C9. The express level of perforin in un-active cytotoxic precursor cells is low, but in active cytotoxic cells, the express level appears high apparently. Inside the body, perforin expression has been found in patients'CTL cells who suffered with lymphocyte choroid plexus cephalitis meningica virus infection, autoimmunity disease such as rheumatoid arthritis or cancer. Beside this, in the heart transplantation, when the rejection happened, the expression of perforin increased obviously, this can be regard as a single of the acute rejection.A lot of research has showed that, perforin can make many target cells dissolve nonspecifically, the ways of dissolution for target cells may include: (1) the cell membrane's pore forms that increase the permeability of the cell membrane and result in the necrosis of target cells. When effector cells combine with target cells, they release the perforin, numbers of perforin insert the cell membrane and concentrate to a pore like a channel pass through the cell membrane with the 15-20nm diameter, that increase the permeability of the cells and induce the penetrable dissolution of target cells. (2) The pore in the cell membrane is non-selective ionic channel that allow large numbers of Ca2+internal flow and cause Ca2+over loading. The latter not only cause DNA degradation, impairment of mitochondria and so on, but effect on the cytoskeleton directly and kill the cells. (3) Granzyme kill the target cells(program death). As the cell membrane has the function of self-restoration, the effect of target cells death induced by perforin is limit, meanwhile, granzyme can assist to kill target cells. Granzyme and perforin are all exist in cytoplasm granule of effector cells. Granzyme can not enter into cells alone until perforin forms the hole in the membrane of the target cells, it can pass through these holes into the cells, activate endonucleases system and kill the cells through making the DNA degrade. Thus perforin has been considered as the main effecter molecular in the process of killing the target cells in CTL or NK cells in CMC.According to Seko and Young'papers, perforin really can mediate damaging cardiac muscle cells in VMC. The mediating course likes this. NK or CTL cells will release perforin out of the cell by producing cell granules, after the cells are infected and activated by virus and soaked into cardiac muscle. Perforin arrive at the surface of cardiac muscle cells through the slit between the effector cells and the cardiac muscle cells, insert into the membrane of the cardiac muscle cells, and aggregate to form the tunnel trans-membrane of cardiac muscle cells, as will result in the death of cardiac muscle cells. The result of immunity-organization chemistry checking showed perforin clang to the surface of cardiac muscle cells ,and electric-microscope also testify the trans-membrane pore (about 15-20nm diameter), and can exclude the result of complement compound's damage.Felzen acted on mice ventricle muscle cells with the perforin and granzyme A that are purred from CTL cells. The results showed the cardiac muscle cells shrinking, deformation and dissolution with perforin operation, but no effect appeared only using granzyme A. While using both of them can cause more serious damage of cardiac muscle cells. So, the author believe that the pore in the cardiac muscle cell membrane caused by PFP is an un-optional ionic channel that allow large numbers of Ca2+internal flow and cause Ca2+overloading, at last the cardiac muscle cells necrosis. In the same time, granzyme A also be essential in CMC, the mechanism can be expressed as promoting DNA degrade, and accelerating polymerization of perforin. Because of that, the pore of cell membrane has bigger diameter and higher conductance after granzyme A acts on.But as we see, the action of perforin in children viral myocarditis has not been reported, in the same time, the mechanism which damage cardiac muscle cells should be illuminated. The detection of perforin include biological activity determination and quantitative determination, the former include haemolyticus experiment and destruction of grave cells'experiment, the content of perforin is low in normal peripheral blood, so this method could not apply in the clinical judge as normal detection method. At present, there are no such works in the whole country, and the latter define quality of PFP using molecular biological method. This research detects relative amount of PFP mRNA in peripheral blood lymphocyte of 52 children who suffered viral myocarditis and 40 healthy children with RT-PCR method, it is found that the relative amount of PFP mRNA in peripheral blood lymphocyte of viral myocarditis is higher than that in 40 normal children. The two group difference has significant statistical meaning, that is 1.083±0.145, 0.599±0.161, P<0.01. According to the above results, the increasing of expression level of PFP mRNA is related to viral myocarditis. And it suggests that perforin maybe the main effecter molecular which cause cardiac muscle damage.Compared with the congener study both here and abroad, the following results have been achieved in our research. Firstly we detect the expression of perforin mRNA of lymphocyte in viral myocarditis children'peripheral blood with semi-quantitated method. Based on the contrast analysis of PFP express level between cases and normal people, the positive relativity between perforin and viral myocarditis is found out. We will lay the foundation for illustration of the VMC'mechanism through this research. At the same time, the result has also provided the basis for the diagnosis, interdiction of illness stage and treatment of viral myocarditis in the future. The semi-quantitative RT-PCR method about perforin is built up, and PFP express level could reflect CMC state perfectly. PFP has become the symbol of lymphocyte which has killing function. Many research center has regarded PFP as the index of monitoring heart transplant rejection, kidney, small intestine etc and it could be used to instruct the application of immuno- suppressant.Up to the present, there still are no quite effective measures to cure viral myocarditis. With the deep comprehension of perforin function in viral myocarditis, the measure, which is to intervene perforin effect, maybe has certain effect to cure viral myocarditis. (1) Gene therapy. It has been indicated that antisense oligodeoxynu- cleotides of perforin could decrease 65% proteinaceous express of perforin, and cytotoxic effect of T cell mediation decrease 69% at the same time. (2) Immunotherapy. Seko et al. considered that applying antibody to cure the myocardial damage induced by perforin is possible because perforin molecule could get to cardiac muscle cell by the cell outside space.