| Background and Objectives: Rheumatoid arthritis (RA) is an autoimmunedisease characterized by joint synovitis and the disease incidence is about 0.4％allover whole world. Nowadays, its etiology is still not clear, and there are not specifictherapeutic measures. The current diagnosis mainly depends on clinical symptoms andauxiliary examinations due to lack of sensitive and specific laboratory indexes atearly stage. Most of patients with a final diagnosis of RA have irreversible jointdamage. It is recognized widely that synovial hyperplasia and angiogenesis are the keyfactors resulting in cartilage and bone destruction. So it is pretty important to establishan animal model which has the identical or similar pathomechanism in the role ofresearches of RA.At present, in China, the major animal models are inducibility inflammationmodels [for instance: collagen induced arthritis (CIA) and adjuvant arthritis(AA)]andspontaneous inflammation model (Campylobacter Jejuni mice), which simulationmodel mainly in terms of hi-syndromes such as wind, cold, humidity, hot, kidneydeficiency, etc, but fail to combine closely with disorder, the synovial hyperplasia andcartilage invasion can not be isolated, because these pathological changes are totallydrived by immune and inflammatory responses. They still have a long-distance to the actual pathomechanism of RA. Therefore, since the middle 90s of last century, muchattention has been quickly paid to new fields studies. In these researches, RA synovialmembrane was transplanted into immunodeficient animals to bulid animal-HuRAgchimera model. Such kind of model is widely used in exploring the pathomechanismof RA, especially in screening new drugs or medications for purpose of inhibitingsynovial hyperplasia and cartilage invasion. However, in China, the generation of thiskind of animal model is just on its first step. Immunodeficiency animals include nudemice, mice with severe combined immune deficiency (SCID) of T cells and Bcells, NOD/SCID mice and BNX mice with immune deficiency of T cells, B cells ornatural killer cells. Currently, SCID is widely used. As for whether NOD/SCID micecan be used as a carrier of RA synovial grafts or not, no report is found.The chimera model of NOD/SCID mice-human rheumatiod arthritis grafts whichwas established in this experiment aims at formation of the new autoimmunity reactionwhich was provoked by RA synovial membrane in NOD/SCID mice, and smulation ofhuman RA animal model outside the human body, meanwhile, researching of indexeswhich are significant in the process of the pathogenesis of RA. It provides a new animalmodel for an in-depth study of RA, especially for preventing synovial hyperplasia andcartilage erosion in terms of Chinese medicine.Methods:(1) Model production The NOD/SCID mice were radomized into twogroups, with 10 mice in each group, of either sex(gender). The RA group synovial:under general anesthesia (0.2ml/100g),the mice were operated in the back with alongitudinal incision(0.8～1.2cm).The prepared normal cartilage slice(0.5～0.8cm)was planted under the skin of the back, then, the synovial tissue from RA patient wastransplanted on the slice. The skin was sutured. Carry out routine disinfection onincision after operation and add 2ml of norfloxacin antibiosis solution (8mg/ml) into200ml water drinking bottle. Dermal sutures out one week later.The second group isused as control group for experiment. The RA group synovial: after implanting intocartilage,place synovium of Osteoarthritis patients on cartilage and suturation.After operation, mice in experiment after operation should be fed with standard animal feed in the same SPF grade environment as before operation, with correspondentintake amount for each day and without limit on water drinking.Keep room tempera-ture of 22±2℃and humidity of 60％. Padding should be exchanged once a week.(2) Hematology detection(TNF-a):Collect blood from mice by eyeball remov-ing method 10 weeks later and centrifugate at 3000 r/min for 20 min.Serum should bepreserve in low-temperature environment.After dissolving 125I-TNF, antibody andstandard with buffer in liquid reagent box,accurately and respectively absorb 100μlstandard and sample with concentrations (0.3, 0.9, 2.7, 8.1 and 24.3 ng/ml) into 5 mlcentrifuge tube.Additional set T tube (total counter tube) and NSB tube (nonspecificbinding tube).Add 100μl marked antigen to T tube and add 100μl marked antigen and100μl water for injection. And add 100μl antigen and 100μl antibody to the othertube. Mix well and mark corresponding No.of experimental mice.Culture in 4℃cons-tant temperature cabinet. Take it out 24 hours later, expect that separation agent is notadded to T tube,respectively add 500μl separation agent to the other tubes.Mix welland stand still at room temperature for 15 min before carrying out centrifugation at3500 r/min for 20 min.Discard supematant and detect radioactive counting of sampleswith pre programmed procedure on radio-immunityγcounter.(3) Histopathology analysis: Model mice were killed by neck vertebrae disloat-ion method and strip plan with tissue scissors and scalpel. Fix with 4％paraformal-dehyde for 8 hours and carry out decalcification with ethylenediamine tetraaceticacid(EDTA). Then carry out dehydration with alcohol gradient and clarify withxylene.Embed with paraffine.Cut into slices of 4μm thickness and paste on silicifiedglass piece. Dry it, ready for use. Pathologic detection index and procedure:①HEstaining: after dewaxing, dehydration of alcohol gradient, water washing, HE dye,dehydration and mounting, carry out histological observation under micros cope,and estimate for synovial hyperplasia,cartilage invasion and cartilaged degradation②Detection of VEGF hybridization in situ method: slice sheet after dewaxing isfinished after procedures such as after fixing,prehybridization, hybridis ation of mark-ed probe,washing, containning,adding antibody, DAB coloration and redye mounting by hematoxylin.Observe specific positive expression under microscope.③Experimentoperation is finished after procedures of detection of apoptosis with Terminal deoxynu-cleotidy transferase-mediated dUTP nick end labeling(TUNEL) method: After digestedfor 30 minutes with trypsase at 37℃, based on dewaxing and adding enzyme markedantibody, enzyme labelled antibody should be incubated for 60 minutes at 37℃, DABcoloration and redye mounting by hematoxylin and so on step. The professional imageanalysis software,Image Pro plus 6.0 was used to analyse the difference of the meanintegrated optical density(MIOD) and mean value of stained (AREA) in the twogroups.(4) Statistics analysis: Statistics analysis should be carried out on experimentaldata with SPSS13.0 software package, with statistics results denoted in form of(?)±s.model mice blood serum of TNF-a contents and semiquantitative analysis datain T test,synovium hyperplastic degree,cartilage corrosion degree and cartilage degra-dation degree analyzing in 2 independent sample test,P=0.05 for significant difference.Rusults:(1) General state of mouse model: There should be no typical graft-versus-hostdisease(GVHD) behavior such as hand touching skin bone sense, bad mobility andsparse capill for NOD/SCID mice during experiment period.Survival rate for 10weeks of model mice under SPF environment is 100％.(2) Statistical analysis results for TNF-a: There are obviously difference inserum amount of TNF-a between for the RA synovial group and the OA group (0.80±0.06 vs 0.70±0.03),which has significant difference(P＜0.001).(3) Histopathology indicated that:①HE dye: there is only a little amount ofsynovium cell hyperplastic and inflammatory cell for OA synovium group undermicroscope, implanted synovium tissue is mainly replaced by strip material formedfrom fibroplasia,without manifest corrosion and injury of cartilage.A great amount ofsynovial cell hyperplastic and biochemical center can be obviously seen to be formedin RA synovial group.Histological interstitial substance in disease part becomesloosen.It becomes granulation with unsharp boundary or block strong acidophic red dye substance without structure.Edge of cartilage is obviously damaged by corrosionof vs tissue.The OA synovial group and the RA group synovial hyperlasia (1.50±0.51vs 2.56±1.15),cartilage invasion (1.15±0.51 vs 2.42±1.29) and cartilage degradation(1.50±0.51 vs 2.44±1.04),two sets have significant difference(P=0.040).②VEGF:sp-ecific positive expression of VEGF mRNA of cytolymph is very rare on slice for OAvs group under microscope.And specific behavior to exhibit brown yellow by cytoly-mph expressing VEGF mRNA can be clearly seen on slice of RA synovial group.③Apoptosis: It can be obviously seen for OA synovium group that cell nucleus isdyed to brown yellow granulation under microscope,which is cell apoptosis under.positive expression with specificity is not easy found for RA synovial group. Semiqu-antitative analysis of the RA synovial group and the OA synovial: (MIOD:959.23±80.89 vs 168.37±12.87),(Area:1890.51±159.14 vs 180.21±8.25), they have significantdifference (P＜0.001).Conclusions:It can establish the chimera model of NOD/SCID mice-human rheumatoidarthritis grafts successfully through NOD/SCID mice with severe combined immuno-Deficiency. It can be seen from the relative detection result of the model,afterNOD/SCID mice had been done a tissue transplantation and had been raised tenweeks,tansplanted human RA synovial membranes continue to maintain the ability ofHyperplasia and erosion.Furthermore,they continue to highly express TNF-a andVEGF, however, apoptosises are obviously inhibited.The model have SimulatedPathological process from RA synovial cells hyperplasia to cartilage damage in theautoimmune state outside human body.The establishment of human animal model laysa good foundation for further research on RA research and has wide clinical applicationvalue.
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