The Alteration Of CD4~+CD25~+T Cells, CD8~+CD28~+T Cells And The Gene Foxp3 In Peripheral Blood Of BXSB Mice After Immunization With T-cell Vaccine

SLE(Systemic lupus erythematosus) is an autoimmune disease that involves multi-organ and multi-system.Its prevalence rate is as high as 70-100/100000 people. and if the population of our country is 1.6 billion,there are 112-116 ten thousand people suffering from SLE,and the desease is particularly prevalent in young women.SLE has many features,such as complex pathogenesis,high prevalence rate, the variety of clinical manifestion,difficulty of treatment and high mortality.Currently there are many ways of treatment for SLE.It is the preferred method to combine glucocorticoids with immunosuppressant drugs,generally larger doses are used at the beginning to control the illness in 1-2 weeks.And if necessary, we can use the pulse therapy of glucocorticoid or combined immunosuppressive agents.But in this course of treatment,because of large drug consumption and long time,it is prone to emerge side effects or complications,such as infections(viruses, bacteria,fungi,etc.),the risk of diabetes,gastrointestinal ulcers,perforation or gastrointestinal bleeding,and bone fractures or ischemic necrosis,mental disorders, many patients died last on account of these side effects.It has affected the survival and quality of patients' life seriously.At the same time,because the selectivity of the drug is not satisfactory,part of patients is still unable to effectively control condition. The pulse therapy of large dose immunoglobulin,plasma replacement therapy or the irradiation treatment to lymph node all over the body,all don't aim at etiological treatment directly,and its effect is temporal,so they also can not be widely applied. Although there are encouraging reports of hematopoietic stem cell transplantation, but it needs high technology and prices are also very expensive,so it is difficult to promote also.Therefore,the treatment of SLE is still recognized as a difficult medical problem at home and abroad,and there is no effective cure method.At 1981, TCV(T-cell Vaccine) concept was first proposed by BEU-NUN etc,when they researched Lewis rats experimental autoimmune encephalomyelitis(EAE) of active immunizating by myelin basic protein(MBP).At present some scholars at home and abroad have researched the TCV treatment on autoimmune disease related with T cell-mediated,such as rheumatoid arthritis,organs and tissues after transplantation, autoimmune thyroiditis,autoimmune myocarditis etc,and have some encouraging results.A few also had a pilot clinical trial,such as rheumatoid arthritis,has also made a preliminary efficacy.At present,it's regarded that T and B lymphocyte dysfunction are characteristics of SLE.Self-reactive T lymphocyte activation and the mediated autoimmune reaction is a central link in this disease.As long ago as 1990s,abroad scholars researched and shown that inhibitting self-reactive T-cell activity can reduce the severity of SLE disease,and extend the life span of mice.Therefore removing self-reactive T cells may be another new therapeutic approach for SLE.Autoreactive T lymphocyte which can recognize their own autoantigen is a normal part of the T-cell,existing in peripheral circulation,but does not cause autoimmune disease(AID).Idiotype-anti-idiotypic network theory confirms,the autoreactive T cells are constrainted by the anti-idiotypic regulatory T cells in the normal body,and are in a relatively small number and lower state.Once the steady state of immune was damaged,atuoreactive T cells can activate,proliferate and cause pathological damage.In response to this theory,the treatment theory of TCV is using inactivated autoreactive T cells to make the body produce large amounts of regulatory T cells to resist the pathogenic role of autoreactive T cells,the treatment is the new direction in the current treatment of autoimmune diseases.The CD4~+CD25~+T cells is an important regulatory T cells,and more and more studies have shown that recently,the abnormity of amount and function of CD4+ CD25~+ T cells related to many autoimmune diseases closely.Immunology has regard CD4~+CD25~+T cells as a pronoun for regulatory T cells.And CD4~+CD25~+ regulatory T cells play the role in the pathogenesis of SLE has gradually become the hot spot of research.Many studies have found that,the number of the CD4~+CD25~+ regulatory T cells in SLE patients is decrease,and the function is abnormal.Following the improvement of SLE disease,CD4~+CD25~+ T cells will also be resumed.So the CD4~+CD25~+ T cells can be used as an indication to monitor the development of SLE disease.Therefore,we prepare the TCV and use it to immune the BXSB rat,then detect the leves of the CD4~+CD25~+T cells in the peripheral blood at arranged time, thereby speculate the potential therapeutic effect of the TCV in SLE desease.In addition,the relationship between transcription factor Foxp3 and CD4~+CD25~+ T cells are also researchful hotspot.Both home and abroad a number of researches indicate that Foxp3 is the function-related gene of CD4~+CD25~+ regulatory T cell.As a relatively specific signs of CD4~+CD25~+ regulatory T cells,the Foxp3 expression is the premise of cell development and function.Furthermore research shows that expression of Foxp3 decline in peripheral blood of SLE patients,and its expression level has dependent relationship with CD4~+CD25~+ regulatory T cells.As a result,Foxp3 gene detection and correlation analys is a new research area in SLE immunology.In the situation of stimulated by dual signal,T cells is in response to activation and proliferation.The MHC combines and creates first signal on TCR/CD3 complex and antigen presenting cell,then while CD28 and the corresponding ligands CD80 or CD86 are combined costimulatory signal is create.This situation results in the activation of T lymphocytes to trigger immune cells.When Salomon B injected lupus mice with the CD28 antagonists,he found that the generation of dsDNA antibody is related to CD28-CD86 costimulatory molecules.Furthermore the injection of CD28 antagonist can reduce the incidence of disease and extend the life span of lupus mice. It can be seen that CD28-CD86 costimulatory pathway is very important at SLE pathogenesis.Another purpose of this paper is by detecting the CD8~+CD28~+T cells change of T cells vaccine BXSB mouse,to analyze the relationship between SLE illness and changes of cell subsets in SLE pathogenesis.Some domestic scholars have suggested that,the level of CD8~+CD28~+T cell is decreased in peripheral blood of SLE patients.This indicates that CD8~+CD28~+T cell is possibly an index to reflect or monitor the state of SLE.But the mechanism about CD8~+CD28~+ T cell in SLE desease has reported very few.At home few scholars do experimental study of T cell vaccination in systemic lupus erythematosus.With the progress in the fields of immunology of SLE pathogenesis,this paper research the changement of CD4~+CD25~+ T cells and related gene Foxp3,CD8~+CD28~+ T cells T-cell about vaccine BXSB rats,and dicuss the application prospects of T-cell vaccine therapy.Objective1.To research the diversify of the CD4~+CD25~+T cells and the CD8~+CD28~+T cells in peripheral blood of BXSB mice after vaccined by TCV,and then explore the mechanism of this treatment. 2.To research the expression of Foxp3 gene in BXSB mice after immunization with TCV,and analyse the relationship between Foxp3 gene and CD4~+CD25~+ regulative T cells.Accordingly we analyse the possible mechanism of T-cell vaccine in the level of gene.Methods1.AnimalsTwelve 2.5-month-old BXSB male mice,weight 22-27g,provided by medical college of Beijing university,and breeded in SPF laboratory of NanFang hospital.2.Methods(1) The extraction of T cellsApply the generally method in many studies recently to prepare the TCV,that is to extract T cells from spleen of BXSB mice.Every procedure abide by the aseptic technique,and follow the instruction of agentia.(2) The preparation of TCVWe use Con A(2.5ug/ml) to promote the caryocinesis of T cells extracted from spleen,then use mitomycin C to deal with cells,and adjust the concentration of cells to finish the reserve of TCV.(3) Immune BXSB mice under skin with prepared TCV.Get 1×10~7 cells from prepared TCV to immune BXSB mice first,and then respectively repeat immune after 1 and 2 weeks.(4) Detection:Flow cytometry was used to measured the level of CD4~+CD25~+T cells,CD8~+CD28~+T cells and gene Foxp3 before immunization and at 1,2,4 weeks after first immunization.(5) Statistical analyseWe use SPSS 11.5 to analyse the results of this study.Results1.The alteration of CD4~+CD25~+T cell after immunization with TCV 1.1 The level of CD4~+CD25~+T cell in different periods before and after immunization with TCV.Mauchly globular test statistics W=0.429,P=0.261.This study show that globular hypothesis isn't rejected,so applicating univariate analysis needn't correction ofε(epsilon).So the diversity of CD4~+CD25~+T cell between before and after immunization with T cell vaccination has statistical significance.(F=16.441, p=0.000).1.2 The comparison of the level of CD4~+CD25~+T cells between preimmune and after immunization with TCV.CD4~+CD25~+T cells appear different levels in respective period after immunizaition with TCV,and the levels are higher than preimmune.And the comparison between preimmune and first week after immunization has no statistical significance(P=0.075).The diversity between preimmune and second/forth week after immunization have statistical significance(both P is 0.000).1.3 The comparison of the level of CD4~+CD25~+T cells among different periods after immunization with TCV.In both second and forth week the level of CD4~+CD25~+T cells are higher than first week after immunization(P is respectively 0.002,0.003).And the diversity between the forth week and the second week has no statistical significance (P=0.559).2 The alteration of the gene Foxp3 after immunization with TCV2.1 The level of the gene Foxp3 in different periods before and after immunization with TCV.Mauchly globular test statistics W=0.409,P=0.231.This study show that globular hypothesis isn't rejected,so applicating univariate analysis needn't correction ofε(epsilon).So the diversity of the level of the gene Foxp between before and after immunization with TCV have statistical significance.(F=15.388, P=0.000).2.2 The comparison of the level of the gene Foxp3 between preimmune and after immunization with TCV.The mean of the gene Foxp3 in respective period after immunizaition with TCV is higher than preimmune,and the comparison between preimmune and first week after immunization has no statistical significance(P=0.251).The diversity between preimmune and second/forth week after immunization have statistical significance(P is respectively 0.003,0.002).2.3 The comparison of the level of the gene Foxp3 among different periods after immunization with TCV.In both second and forth week the level of the gene Foxp3 are higher than first week after immunization(P is respectively 0.004,0.002).And the diversity between the forth week and the second week has no statistical significance(P=0.143).3 The correlation analysis between levels of the gene Foxp3 and CD4~+CD25~+T cells.The correlation analysis show that,the Spearmon coefficient correlation is 0.631(r_s=0.631),P=0.000(two-tailed).So positive correlation exists between gene Foxp3 and CD4~+CD25~+T cells.4 The altration of CD8~+CD28~+T cell after immunization with TCV4.1 The level of CD8~+CD28~+T cell in different periods before and after immunization with TCV.Mauchly globular test statistics W=0.498,P=0.0.375.This study show that globular hypothesis isn't rejected,so applicating univariate analysis needn't correction ofε(epsilon).So the diversity of CD8~+CD28~+T cell between before and after immunization with T cell vaccination has statistical significance.(F=16.172, P=0.000).4.2 The comparison of the level of CD8~+CD28~+T cells between preimmune and after immunization with TCV.CD8~+CD28~+T cells appear different levels in respective period after immunizaition with TCV,and the means of the level of post-immune all are higher than preimmune.And the diversity between preimmune and post-immune has statistical significance(P is respectively 0.010,0.001,and 0.001).4.3 The comparison of the level of CD8~+CD28~+T cells among different periods after immunization with TCV.In both second and forth week the level of CD8~+CD28+T cells are higher than first week after immunization(P is respectively 0.025,0.007).And the diversity between the forth week and the second week has no statistical significance (P=0.074)Conclusion1.Immunization of TCV can promote the level of CD4~+CD25~+T cells and CD8~+CD28~+T cells in peripheral blood of BXSB mice,so we can presume that,the TCV can intervene with the status of immunity in body of BXSB mice,and this may be the main mechanism of TCV.2.Immunization of TCV can enhance the expression of Foxp3 gene,this indicated that TCV may possibly produce a marked effect in level of gene.