| BackgroundSystemic lupus erythematosus (SLE) is a common autoimmune disease involving multiple body systems and organs, clinical variety, and the high incidence of secondary infection, diagnosis and treatment difficulties, is a serious hazard to human health. SLE is widely distributed around the world. The prevalence rate of the world is about 17.0/10 million -100.0/10 million, with the rate of women is particularly high. According to reports in China there are at least 1 million patients.The pathogenesis of SLE has not yet been fully clarified to date. Generally, the immune dysfunction involved with the genetic quality,sex hormones,environment and the complex interaction of these factors results in the occurrence and persistence of SLE, which is difficult to alleviate. Since the etiopathogenisis are still unkown, SLE is difficult to be cured. However, over the past 20 years, with the rapid development of modem medicine, a means of early diagnosis and treatment increase, the 10-year survival rate of SLE has reached 92 percent, and the prognosis is significantly improved. At the same time, glucocorticoids and immunosuppressants are still the preferred drugs. Usually the treatment of common cases is large doses of glucocorticoid followed by a small dose whenever the condition is better. Stezoid therapy is used for severe cases, and sometime combined with immunsupressant agents (mainly cyclophosphamide and azathioprine) when necessary. The conventional treatment methods enable most patients in stable condition, but with a large quantity of drugs and long time of applicaton, the side effects of drugs can not be ignored, such as inducing or aggravating infection, diabetes, gastrointestinal ulcers, gastrointestinal bleeding or perforation, long-term application of causing hypertension and atherosclerosis, bone fractures and ischemic necrosis, mental disorder, Cushing's syndrome, acne, hirsutism, etc. More than 60 percent of patients died of stezoid side effects, which seriously affect the survival of patients with stage and the quality of life.Therefore, to seek new treatment for SLE has been a hot research both at home and abroad. Mathods such as large dose of immunoglobulin, plasma replacement, and other therapies, although patients's conditions can be temporarily alleviated, can not be widely used because of the high cost and non specificity. New immunosuppressants sincluding B-cell therapy, anti-CD20 monoclonal antibody therapy, T-cell therapy, anti-cytokine treatment, are mostly at the experimental stage, and the exact effect needs to be confirmed. In recent years the treatment effect of hematopoietic stem cell transplantation are encouraging, but there is a high risk and high technology requirements, and some patients can not tolerate the mobilization stage. This therapy is very expensive, and more difficult to promote.SLE is an autoimmune disease in which T and B lymphocyte dysfunction is the main features.It is supposed that the self-reactive T lymphocyte activation and the autoimmune reaction is the central link. People found that in the pathogenesis of SLE, Th cells responding to autoantigen increase. T cell Clones of the lupus rat can provide assistance to B cells, and help them to produce IgG anti-DNA antibodies. About 50% of these T-cell clones have the capacity to identify the body of the T cell epitopes. Th cell clones in both SLE patients and mice identify the bodies epitope of TCRα chain which can combine different TCRβchain, and form a TCRαβtwo peptide chains, identify multiple T cell epitopes to a variety of B-cell cloning, a multiple autoantibodies. The T-cells are repeatedly activated and by antigen, so that the mutation rate increased. The mutant T cells will prompt B cells to produce their own anti-DNA antibodies. These antibodies and their corresponding antigen form immune complexes, which deposit in the glomerular basement membrane, liver, central nervous system, blood vessel wall and other organs and tissues, and then activate the complement, multi-leukocyte and platelet, induce the local inflammatory and vasculitis, vascular occlusion, and finally lead to a variety of clinical disease. If auto-reactive T lymphocyte activation can be suppressed through T cell immune, the development of SLE may be under control.In 1981, Bennun got myelin basic protein (MBP) specific self-reactive T cells from Lewis rats with experimental autoimmune encephalomyelitis (EAE). These cells can induced EAE in normal rats, but if the self-reactive T cells are handled with radiation or mitomycin C before the transfer, the mice is induced the ability of EAE resistance., At that time, the concept of T-cell vaccine was first put forward, in which inactivated or less pathogenic dose of self-reactive T cells or T cell receptor (T cell receptor, TCR) peptide are used as vaccine to trigger regulation of inhibition of specific network, thereby remove self-reactive T cells and control autoimmune diseases. Recent years T-cell vaccines have been extensively studied. In multiple sclerosis, rheumatoid arthritis, post-transplantation of organ and tissues, autoimmune thyroiditis, autoimmune diseases such as viral myocarditis, satisfactory results were obtained in clinical research of treatment for refractory autoimmune disease treatment. Based on the theory , we try to use the method of T cell vaccine to treat SLE in animal model, analyse laboratory changes, and provide laboratory dates for clinical use. Objective1 .Prepare T-cell vaccine using BXSB mice spleen cells2. Observe 24 hours BXSB rat urine protein, serum level of antibody ANA, Ds-DNA antibody in BXSB mice for a certain period of time before and after T-cell immunity to discuss the effect of T-cell vaccine on BXSB mouse.3. Observe the serum level of IL-18 in BXSB mouse for a certain period of time before and after T-cell immunity, to analysis the mechanism of the he T-cell vaccine.Methods1 .Experimental animals12 of the male BXSB mice are 2.5months old. They were randomly divided into2 groups. One group is consisit of two mice, for T cell vaccine preparation. Another group is cinsist of 10 mice, for observation.2. Isolation of spleen lymphocytes in BXSB miceBXSB rat spleen is removed under aseptic conditions, spleen cells were then made into cell suspension, liquid for lymphocytes isolation is administrated, followed with Hanks solution cleansing 2 times, and last headled with RPMI1640 to reach the cell concentration of 1×10~8/ml. Trypan blue was used to identify the number of viable cells> 95%.3. T cell vaccine Preparation:Add complete culture solution to adjust cell concentration to 2×105/ml. We first put ConA in. Second, culture cells in five percent CO2 37℃sterile box for 48 hours, and then joined mitomycin C to headle cells, finally use RPMI1640 to meet the concentration of 1×10~8/ml for spare.4. T-cell immunity:1×10~7 irradiated T cells were inoculated subcutaneously at 0, one, two week respectively. 5. Inspection item and Detection Methods:Specimen was collected before and1, 2, 4week after the first vaccination. Blood was got by tail cutting, after serum separation, serum level of antibody ANA, Ds-DNA antibody and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA). 24-hour urine was collected in cages for metabolism, and test Maas blue staining was for detecting protein quantitation of 24-hour urine.Results1.24 hours BXSB mice urine protein quantitative difference is significant (F=22.177, P=0.000), there is a downward trend. The quantitative of 1 w, 2w, 4w after the first Immunity is lower than preimmunization. There is no significant difference betweenl w post-immunization and preimmunization (P = 0.519), and the level of 2 w and 4 w immunization was significantly lower than that of the pre and the first immunization (P <0.05).2.Difference of serum level of ANA antibody level in BXSB is significant (F=15.757, P=0.000), there is a downward trend. The quantitative of 1 w, 2w, 4w after the first immunity is lower than preimmunization. There is no significant difference betweenl w post-immunization and preimmunization (P = 0.317), and the level of 2 w and 4 w immunization was significantly lower than that of the pre and the first immunization (P <0.05) but no significant difference between the two groups (P=0.072).3.The serum level of ds-DNA antibody in BXSB mice is significantly different (F= 16.735, P=0.000), there is a downward trend. The quantitative of 1 w, 2w, 4w after the first Immunity is lower than preimmunization. There is no significant difference betweenl w post-immunization and preimmunization (P = 0.569), and the level of 2 w and 4 w immunization was significantly lower than that of the pre- and the first immunization (P <0.05) but no significant difference between the two groups (P=0.056).4.The serum level of IL-18 in BXSB mice is significant different(F=34.583, P=0.000), there is a downward trend. The quantitative of 1 w, 2w, 4w after the first immunity is lower than pre-immunization. There is no significant difference between 1 w post-immunization and pre-immunization (P = 0.130), and the level of 2 w and 4 w immunization was significantly lower than that of the pre and the first immunization (P <0.05).5. In the fourth week after the first immunization, one BXSB mouse died.Conclusion1. The quantitation of 24-hour urine protein and serum level of antibodies in BXSB mice begen to decline after the second immunization, and remain lower level after the third time. We conclude that T cell vaccine can inhibit B cell activation, reducing the formation of autoantibodies and kidney damage, and delaying disease progression in a certain period of time of BXSB mouse.2. The serum level of IL-18 in BXSB mice significantly decreace after T-cell immunity for a period of time. We may conclude that T-cell vaccine plays an important role in the regulation of the immune cells and cytokines.3. T-cell vaccine is to a certain extent effective and safe for treatment of BXSB mouse. This experiment may provide basis for clinical application of T cell vaccine. |
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