| Objective:1. To study probability of in experimental autoimmune myasthenia gravis with rat-derived 97-116(Rα97-116) peptide of the acetylcholine receptor( AchR) a-subunit induced and provide a simple and available animal models for basic researches of MG..2. To explore immunity role of CD28:CTLA4/B7 costimulatory molecules by studying their expressions on peripheral blood cells in experimental autoimmune myasthenia gravis with rat-derived 97-116(Rα97-116) peptide of the acetylcholine receptor a-subunit induced.3. To study effect of nasal tolerance with Rα97-116(V108A) on the clinical manifestation and the immunity function of experimental autoimmune myasthenia gravis.Methods:Female Lewis rats(6-8 weeks) were immunized subcutaneously at several spots with the synthetic peptide Rα97-116 in complete Freund's adjuvan(tCFA) and boosted on day 30 and 60 with the same peptide in incomplete Freund's adjuvant(IFA) to iduced rat models of EAMG in experiment group, or only with phosphate buffer saline(PBS) in control group. Clinical manifestation was evaluated by measurement of body weight and Lennon clinical score. Disease was further confirmed by 3,5 Hz repetitive nerve stimulation (RNS) for positive decremental respnse and ELISA assay for serum AchR-Ab titers.The 5th day after the third immunization, the expressions of CD28,CTLA4, B7-1, B7-2 on the surface of peripheral blood cells, lymphocytes and monocytes were exaimed by flow cytometry. The 8th day after the rats were immunized thrice with rat-derived 97-116 peptide of the AChRα-subunit(Rα97-116), 22 EAMG model Lewis were determined by measurement of body weight and clinical score, repetitive nerve stimulation(RNS) and ELISA assay for serum AchR-Ab titers and were divided randomly into tolerance group and control group. They were respectively immunized with alanine replacement of valine at position 108 petide(V108A) corresponding to Rα97-116 sequence? Rα97-116(V108A) and PBS buffer solution for 10 days by the route of nasal mucous. Then the body weight and Lennon score of two group Lewis rats were measured,serum anti-AChR antibody were tested by ELISA, the expression level of CD28,CTLA4,B7-1 and B7-2 were determined by flow cytometry at different time points.Results:1. Signs of EAMG occurred in 75% of Lewis rats in the EAMG group ,which was confirmed by the Lennon clinical score>1 grade, the positive decremental response D5 >10% of 3,5 Hz RNS and the ratio >2.1 of the serum AchR-Ab titers to the control group. The achievement ratio of EAMG model was 75%.2. Compared with the control group, the expression levels of CD28,CTLA4,B7-1, B7-2 on the surface of peripheral blood cells were increased in the EAMG group(p﹤0.05).3. In the EAMG group , the expressions of CD28,CTLA4 were maily of lymphocytes(p﹤0.05,0.01).Although the expressions of CD28,CTLA4 were increased in monocyte, their expressions were not significantly changed in monocytes when compared to the control group(p﹥0.05). Compared with the control group, the expressions of B7-1, B7-2 were increased in lymphocytes and monocytes(p﹤0.01,0.05). 4. The 20th day after Lewis rats received nasal tolerance with Rα97-116(V108A) , the body weight of rats in the treatment group were increased significantly compared with the control group(p﹤0.05).5. The 10th Lewis rats received nasal tolerance with Rα97-116(V108A), compared with the control group ,the changes of the mean clinical score of rats in the treatment group decreased (p﹤0.05).6. Compared with the control group , the amount of anti-AChR IgG in serum in the treatment group decreased significantly (p﹤0.05) after the 16th day nasal tolerance with Rα97-116(V108A). 7. In the treatment group , the expressions of CD28,CTLA4,B7-1, B7-2 on the surfaceof peripheral blood cells decreased remarkably compared with the control group (p≤0.01) after the 10th day nasal tolerance with Rα97-116(V108A).Conclusions:1. Immunization with the synthetic Rα97-116 peptide can induce EAMG in Lewis rats .The achievement ratio of EAMG model was 75% . This animal models builds a base for basic researches of MG.2. The expressions of CD28:CTLA4/B7 costimulatory molecules was abnormal in the rats with EAMG. The abnormal of CD28:CTLA4/B7 costimulatory pathway reflect the immune state of EAMG, which may have close relation with the occurrence and development of MG.3. Nasal mucous tolerance with Rα97-116(V108A) could ameliorate muscular weakness and Lab indexes in EAMG rats involved in suppressed T cell activation and B cellular immunity function,which may provide a new concept for the therapy of MG.
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