| Rheumatoid arthritis (RA) is a chronic, inflammatory, tissue specificautoimmune disease. Because RA’s pathogenesis is very complicated, it hasnot been yet fully understood. Research shows that the autoimmune disorderslead to the joints’ continuous attacked by immune system which may be thereason for long-term chronic inflammation. Although it has been confirmedthat a variety of cells and molecules involved in the joint inflammation andtissue damage, but activation of antigen specific T cell has always beenconsidered to be the central link of RA’s occurrence and development. T cellplays a role in the development of RA mainly through the cells’ interactionand secretion of cytokines. CD4~+T cell, as an important component of effect Tcells, participates in all phases of the immune response, plays a key role in theimmune regulation and is the core and the hub of the cell immunity. At present,the more accepted view is that RA is the result of the reaction of antigenpresented cells and CD4~+T cell. In recent years with the further research onCD4~+T cells in the role of RA, it provides a new theoretical and experimentalevidence for deepening the understanding of the mechanisms of pathogenesisof RA, developing new ideas for the treatment of RA, and finding newtherapeutic targets.Based on improved mice model of collagen-induced arthritis in mice(CIA), this study used CIA as the research object. The role of CD4~+T in thedevelopment of RA disease is clarified and the pathogenesis of rheumatoidarthritis is revealed through the variation of the percentage of the CIA micemodel spleen lymphocytes Th1and Th2cells in different period ofdevelopment of the disease in CD4~+T of cells, the dynamical fluctuation ofinflammatory factor in CIA mice model such as IFN-γ and TNF-α in therheumatoid arthritis disease development period. PartⅠImprovement of CIA model in C57BL/6miceObjective:To establish stable C57BL/6CIA mice model, further build RApathogenesis and treatment research system.Methods:1. One hundred C57BL/6mice of clean grade were randomly dividedinto five groups.20of them were taken as the normal control group, the rest80were randomly divided into4groups and each group used different methodto establish CIA model in order to compare the success rates. Commonmethod group:type II collagen emulsified with an equal volume of completeFreund’s adjuvant. Low dose Group, medium dose group and high dose group:type II collagen emulsified with an equal volume of complete Freund’sadjuvant containing2.5mg/ml,5mg/ml,10mg/ml Mycobacterium tuberculosis.2.35days after strengthening the immune CⅡ, the mice were sacrificedby cervical, removed lesions joint fur and superfluous subcutaneous tissue,kept metatarsophalangeal joint and toe articulatory,4%polyformaldehydefixed,10%EDTA decalcified, conventional dehydration, paraffin embedding,slice, HE staining and observed under microscope.3.49days after strengthening the immune CⅡ,Joint imaging observationusing X-ray machine was implemented on ankle and foot joints of the mice forimaging inspection and analysis.4. Spleen general observation and spleen index determination: takespleen of the mice which were sacrificed for gross observation, weighing,calculate the spleen index and compared with normal control group.Results:1. Compare the success rate of mice model and arthritis index (AI) scoreat different time point in the process of the development of mice disease.Differences between Low dose group (Isometric preparation of CⅡ andFCA containing high poison BCG vaccine2.5mg/ml) and medium dosegroup (Isometric preparation of CⅡand FCA containing high poison BCG vaccine5mg/ml) on success rate were no statistically significant. Howeverthe differences between these two groups and the common method group werestatistically significant (P <0.01). Symptoms of arthritis such as redness of thetoe ministry and ankle joints skin gradually appeared3days afterstrengthening the immune mice. Take the Low dose group for example, detectthe mice AI score at the time of3d,7d,10d,12d,14d,21d,28d,35d afterstrengthening the immune respectively. The low dose group of mice AI valuein different time point had significant differences with the normal controlgroup (P <0.01)2. histopathological studyCompared with normal control group, lesions joint bone of the modelgroup mice (35days after strengthening the immune CⅡ.) was destroyed, alarge number of inflammatory cells invaded.3. imaging examinationImaging examination showed there was swelling in lesions joint softtissue, meanwhile joint space was narrow, articular surface was not smooth,periosteum had new bone formation.4. the changes of mice spleen after immune56days after immune mice spleen became much bigger with black color.The spleen index was significantly increased (P <0.01) compared with thenormal control group.Conclusion:1.It can effectively increase the success rate of CIA mice model whenVirginia type complete adjuvant joined with the highly toxic BCG accordingto2.5mg/ml dose. Differences between Low dose group and medium doseone were no statistically significant. The AI score of the low dose mice grouphad significant differences with the normal control group at the time of3d,7d,10d,12d,14d,28d,35d after strengthening the immune respectively (P <0.01).2. In this experiment system, the development of disease in the C57BL/6CIA mice model was observed through the arthritis performance, imaging, pathological histology and pathological changes, etc.PartⅡ Dynamic observation of Spleen lymphocyte balance ofTh1and Th2Objective:Study on the Dynamic Changes of Spleen lymphocyte balance of Th1andTh2.Methods:1．Observe histopathological aspects of C57BL/6mice on7,14,35daysafter strengthening the immune CⅡ through slice and HE staining of the miceknee.2．Measure dynamic spleen index of C57BL/6mice on7,14,35days afterstrengthening the immune CⅡ.3．Detect splenic lymphocyte proliferation of Th1and Th2level ofC57BL/6mice using flow cell detection instrument on7,14,35days afterstrengthening the immune CⅡ.4．Analyze the secretion of IFN-γ and TNF-a of the C57BL/6mice’sblood serum on7,14,35days after strengthening the immune CⅡ.Results:1. histopathological studyLesions joint bone of the model group mice at various time points wasthe joint synovium pannus formation, cartilage and bone erosion, a largenumber of inflammatory cells invaded.2. examine spleen index at certain points.There was no statistical significance between the spleen index of themodel group mice at various time pointsThe spleen index at various time points was significantly increased (P <0.01) compared with the normal control group.3. Spleen lymphocyte differentiation levels of Th1and Th2at certainpoints.The proportion of Th1on7,14,35days after strengthening the immune CⅡ was higher than normal control group(P <0.01).The proportion of Th1on7，14days after strengthening the immune CⅡwas obviously higher thanthat the proportion of Th1on35days after strengthening the immune CⅡ(P <0.01). Levels of Th2at certain points was no significant difference comparedwith the group of normal.4. The secretion fluctuation of IFN-γ and TNF-α of the C57BL/6mice’sblood serum at certain points.The amount of IFN-gamma in serum on7,14,35days afterstrengthening the immune CⅡ was higher than normal control group (P＜0.01).The amount of TNF-a in serum on7days after strengthening theimmune was higher than normal control group (P＜0.01).Conclusion:1.The proportion of Th1on7，14，35days after strengthening the immuneCⅡ was higher than normal control group.Levels of Th2on7,14,35daysafter strengthening the immune CⅡwas no significant difference comparedwith the group of normal.2.TNF-a was significantly higher in early stage. The amount of IFN-gamma in serum on7,14,35days after strengthening the immune CⅡwashigher than normal control group. |
PDF Full Text Request