Constitution And Application Of Human Tumor Necrosis Factor α Transgenic Arthritis Mice

【Objective】:Human tumor necrosis factor alpha(hTNFα) plays a key role in the course of stimulation and amplification of inflammatory in rheumatoid arthritis(RA),which is an important drug target in the treatment of RA. When drugs block its activity, RA disease can effectively alleviate. This paper constructed the hTNFα transgenic mice and to evaluate the clinical symptoms through screening clinical features 、 pathological characteristics and cytokine detection, on this basis, the application of the model of pre clinical(AT132) and the sale(Adalimumab and Infliximab) of the hTNF alpha monoclonal antibody drugs and synthetic drugs(sinomenine hydrochloride) to carry out the experiment evaluation, for the application of hTNF model in alpha transgenic mice arthritis arthritis drug screening to provide experimental basis.【Method】:Using recombinant hTNFα/β globin genetically modified carrier, hTNFα transgenic mice were obtain.By regularly the weight and arthritis clinical symptoms of hTNFα mice were evaluated; Pathological changes of the of joint tissues and organs of hTNFα mice were observed; The expression of hTNFα of mice’s joint tissues and organs were decteded by immunohistochemical; In mice serum and synovial joints, hTNFα、IL-1α、IL-1β、IL-2、IL-4、IL-5、IL-6、IL-12、IL-15、IL-17、mTNFα、IFNγ and GM-CSF were quantitative analysis by flow cytometry. Finaliy, transgenic mouse model with clinical characteristics of rheumatoid arthritis hTNFα were selected.Application of the model, through clinical observation, pathological detection andhTNFα quantitative analysis, pharmacodynamic evaluation experiment of the preclinical(AT132) and commercial hTNFα drug(Adalimumab and Infliximab) and chemical synthetic drug(sinomenine hydrochloride) were carried out. Providing erimental basis of hTNFα arthritis in mice model which are applied to screening arthritis drug.【Results】:1. Establish hTNFαarthritis mice model. By PCR identification,hTNFα positive mice showed arthritis symptoms. Weight: the FVB mice, during observation period, in 4 ~ 18 weeks after birth,hTNFα transgenic mice weight grow slowly. Clinical symptoms: along with the age growth, hTNFα transgenic mice showed joint swelling deformation, grip strength decline, the overall flexibility decreases, joints stiff, activity damaged severely.Pathological characteristics: hTNFα transgenic mice pathological changes of rheumatoid arthritis were observed in the knee, ankle joint and wrist joint. the mainly pathological changes show inflammatory cells infiltration, synovial hyperplasia, pannus and appearance of osteoclasts, synovial cells fibrosis, eventually lead to the destruction of the cartilage and bone, and other tissues and organs did not. HTNFα immunohistochemical localization: hTNFα in joint synovial tissue was abnormally high expressed. It can also be found in other parts of the tissues and organs hTNFα positive reaction. Cytokines detection:compared with WT mice, in hTNFα transgenic mice articular synovial, hTNFαexpress abnormally elevated(p < 0.01).The expression of hTNFα、IL-1α、IL-1β、IL-2、IL-4、IL-5、IL-6、IL-12、IL-15、IL-17、TNFα、IFNγ and GM-CSF in serum and synovial joints alpha, did not see significant difference.2. The application of hTNF α transgenic mice model. AT132: when hTNF αtransgenic mice were treat by anti-hTNF αdrug Α T132(30 mg/kg) at 10 weeks, arthritis symptoms of hTNFα transgenic mice were inhibited and the expression of hTNFαin articular synovial tissues significantly reduce. The evaluation of AT132concentration-response relationship found when the dose of 1.25 mg/kg and 0.625 mg/kg can still show good clinical treatment result, when the dose to reduce to 0.312 mg/kg and0.156 mg/kg, AT132 treatment is invalid. The experimental results show that the effective dose of hTNFα mice AT132 is 0.625 mg/kg. Adalimumab: different concentration anti-hTNFαdrug Adalimumab(12 mg/kg, 6 mg/kg, 3 mg/kg) treat hTNFαtransgenic arthritis mice, in 8 weeks, the symptoms of TNF α arthritis in mice were suppressed and expression of TNF α in articular synovial tissues significantly reduce, which is effective.Infliximab: different concentration anti-hTNF α drug-resistant Infliximab(60mg/kg, 30 mg/kg, and 15 mg/kg) treat hTNFαtransgenic arthritis mice, in 8 weeks, the symptoms of TNF α arthritis in mice were suppressed and expression of TNF α in articular synovial tissues significantly reduce, which is effective. Sinomenine hydrochloride:medication hTNF α transgenic arthritis mice, 4 weeks hTNF α arthritis symptoms of mice was not suppressed, expression of TNFαin articular synovial tissues is no significant difference, which is ineffective【Conclusion】:1. hTNFα transgenic arthritis mice model was established successfully, and get the important phenotypic index of hTNF α transgenic arthritis mice in weight, clinical manifestation, pathological characteristics and cytokine levels,which laid the foundation for the model application.2. By using the model, pharmacodynamics and concentration-response relationship of preclinical drug AT132 were evaluated, and found that AT132(30 mg/kg) were treated to hTNF α transgenic arthritis in mice,which is effective, and the minimum effective concentration is 0.625 mg/kg.3. By using the model, hTNFα drug(Adalimumab and Infliximab) and non- hTNαdrug(sinomenine hydrochloride) were treated to hTNF α transgenic arthritis mice and evaluate the pharmacodynamics were evaluated, hTNF α drug(Adalimumab andHuman tumor necrosis factor(hTNF α) transgenic animal of rheumatoid arthritis models; AT132; Infliximab;Adalimumab; Sinomenine;Infliximab) is effective and non- hTNFα drug is ineffective.