Tumor necrosis factor alpha(TNF-?) is one of the key proinflammatory factors in driving and attending chronic inflammatory process in rheumatoid arthritis(RA).TNF-? acts on different kinds of cell in synovial membrane,such as synoviocytes,macrophages,osteoclasts and chondrocytes,which can produce metalloproteinase,collagenase,stromelysin and so on,further induce pannus formation,joint inflammation,bone erosion and cartilage degradation. The TNF inhibitors have been proved by a lot of clinical trials to be an important new group of agents that significantly improve symptoms and signs,induce remission,reduce objectively measured damage in chronic inflammatory conditions,such as RA.There are three kinds of TNF inhibitors: Etanercept,Infliximab and Adalimumab.Etanercept is a fusion protein of human IgG and two p75 TNF receptors.Infliximab is an IgG1 monoclonal antibody(a chimera of human constant and mouse variable regions).Adalimumab is a humanized IgG1 monoclonal antibody with fully human constant and variable regions.The side effects include injection site infusion reactions,infection,lymphoproliferative disease,demyelinating disease,and SLE-like syndromes.

Objective:To construct retroveral vectors,containing the expression sequence of vIL-10 and to transfect rabbit synoviocyte in vitro with recombinant retrovirus and detect the expression of target genes.Methods:①The primers with restrictive enzyme were designed spot and/amplify target gene by PCR from plasmid including vIL-10 gene was amplified.②The retroviral vector pLXSN of target gene was cloned,and identify the aquired plasmid by sequencing.③Co-transfecting the packaging cell GP-293 with constructed retroviral vector and assistant plasmid pVSVG by calcium phosphate-DNA co-precipitation.The medium containing recombinant virus was collected and titer of virus was determined.④Rabbit synoviocytes was transfected with acquired virus in vitro.Detect the protein expression by cell immunohistochemistry.Results:①The recombinant retrovirus rRV-vIL-10 was successfully constructed.The viral titer reached 5?106 cfu/ml.②vIL-10 gene were transduced into rabbit synoviocytes by recombinant retrovirus in vitro.The protein expression of genes could be detected by cell immunohistochemistry.Conclusion:①The recombinant retrovirus rRV-vIL-10 was successfully constructed.②vIL-10 gene were transduced successfully into the rabbit synoviocytes by retroviral vector in vitro.The transduced synoviocytes can express vIL-10 protein.

Objective:To establish a local ex-vivo gene transfer method to treat RA through animal experiments in vivo and in vitro using retrovirus(rV) as a vector which carrying rRV-vIL-10 target gene.Methods:①The rabbit RA models were induced by the rabbit synovial fibroblast cell line which could continuously expreesed hIL-1?. ②In vivo, the rabbit synovial fibroblast cell line was transduced with rRV-vIL-10, then adding G418 to pick out the rRV-vIL-10 positive clon and infecting the rabbit synovium through intra-articular injection. ③RT-PCR, IHC methods were performed to prove the success of gene transfer to the rabbit synovium and expressed the target protein. ④The relative cytokins changes were detected by ELISA before and after gene therapy and evaluated treatment efficacy of rRV-vIL-10.Results:①rRV-vIL-10 was a effective vector which could transfect to the rabbit synovium in vivo through RT-PCR and IHC methods. ②Intra-articular local gene therapy could effectly reduce the synovium inflammation level of rabbit joints and expressed mRNA and vIL-10 protein. The level of cytokin such as IL-1? was decline.Conclusion:Retrovirus-mediated transgene of vIL-10 is successfully transfected to the rabbit synovium ex-vivo and can reduce arthritis inflammation levels of the IL-1? induced arthritis.

Objective To study the analgesic effects of combination of fortanodyn and verapamil. Methods Used the model of the acetic acid induction writhing in mice and the method of hot-plate in mice, then observe the action of the mice after treatment with combination of fortanodyn and verapamil. Results Low dose Verapamil com-bined AP237 (40 mg/kg, 80 mg/kg)could enhance the threshold of pain of the mouse ache response obviously, and postponed the time which the mouse ache appeared (comparing using simply AP237 P <0.05 with controlled group P <0. 01) ;Low dose Verapamil combined AP237 (20 mg/kg,40 mg/kg) had the obvious inhibitory action in the body turning times of the mouse. Conclusion Verapamil can strengthen the analgesia function of AP237.

Objective To investigate influence of Panax notoginseng saponins ( PNS) on Cyclooxygenase-2 (COX-2),Prostaglandin E2(PGE-2)and Phospholipases A2(PLA-2)in patients of acute exacerbation of chronic ob-structive pulmonary disease ( AECOPD) with respiratory failure .Methods One hundred AECOPD patients with re-spiratory failure were divided into the control group (n=50)and PNS group(n=50).The levels of blood serum COX-2,PGE-2 and PLA-2 of patients in the two groups were detected .Results The levels of blood serum COX-2,PGE-2 and PLA-2 of patients in PNS group were lower than those in control group ,the two groups after treatment significantly lower than those before treatment(P<0.05).Conclusion The levels of COX-2,PGE-2 and PLA-2 have influence on respiratory failure.PNS can reduce the COX-2,PGE-2 and PLA-2 concentrations in blood serum of patients ,mean-while it play a prominent role in inhibiting airway inflammatory and obstruction process .

Objectives To construct two retroviral vectors, one containing human interleukin-1 recep-tor antagonist (hIL-1Ra) gene and the other containing human interleukin-10 (hIL-10) gene and to transfect rabbit synoviocytes in vitro and detect the expression level of target genes. Methods RNA from human peripheral blood mononuclear cells were extracted and target genes were amplified by RT-PCR. The target genes were cloned into retroviral vector pLXSN, which was then transducted into GP2-293 cells to produce recombinant retrovirus. Rabbit synoviocytes were transfected and the expression of target genes was detected by RT-PCR, immunohistochemistry and western-blot. Results The retroviral vector containing hIL-1Ra gene or hIL-10 gene was constructed successfully. The hIL-1Ra gene and hIL-10 gene were transduced respectively into rabbit synoviocytes in vitro. The mRNA level of both genes reached peak in 5 days. In positive cell clones, the protein level of hIL-1Ra reached peak within 30 days and maintained at least 60 days; the protein level of hIL-10 maintained at least 40 days. Conclusion The hIL-1Ra gene and hIL-10 gene can be transduced successfully into rabbit synoviocytes by recombinant retrovirus.

IL-17 is the cytokine secreted by the subgroup of CD4+T cells named Th17.The differentiation,proliferation and cytokine secretion of Th17 are regulated by TGF-?,IL-6,IL-15 and IL-23.IL-17 modulates the production and secretion of proinflammative factors,CXCs,affecting the transfer of neutrophil,the activation and the absorption of bone.It suggests that IL-17 also plays an important role in the pathogenesis of autoimmune diseases.

BAFF is an essential ligand essential for survival and differentiation of peripheral B cells. By interacting with three receptors, BAFF can promote B cell maturation and class switching, enhance humoral immunity and T cell co-stimulation. Over-expression of BAFF leads to autoimmune diseases such as systemic lupus erythematosus (SLE) in mouse model. Treating the mice model with BAFF antagonists can slow-down disease progression and enhance survival rate. Moreover, in some SLE patients serum level of BAFF is elevated and correlated with serum anti-dsDNA titer. The preliminary clinical trial of anti-BAFF monoclonal antibody has shown to be safe and effective. BAFF antagonists are promising therapeutic drugs for SLE.

Objective To compare the phenotypes of abnormal CD4+CD25-Foxp3+ T cells with traditional regulatory T cells (CD4+CD25+Foxp3+) in patients with untreated new-onset lupus (UNoL) and investigate their clinical relevance. Methods The expressions of surface markers (CD25, CD127, CCR4, GITR, CTLA-4) and intracellular marker(Foxp3) on the peripheral blood mononuclear cells from twenty-two UNoL patients were analyzed by flow cytometry analysis, and their clinical relevance were assessed. Results There were no significant differences between CD4+CD25-Foxp3+ and CD4+CD25+Foxp3- T cells in the expressions of GITR, CTLA--4 and CCR4 (P>0.05), but they were significantly lower than those of CD4+CD25+ Foxp3+ T cells in UNoL patients (P<0.01). The percentages of CD127low- in CD4+Foxp3+CD25high,CD4+Foxp3+ CD25low and CD4+Foxp3+CD25+ T cells were (93.8±3.5 )%, (93.7±2.3)% and (92.0±2.1)% respectively (P> 0.05), whereas the expressions of Foxp3 on CD4+CD127low- T subpopulations showed significant differences in CD4+CDI27low-CD25high (91.4±2.6)%, CD4+CD127low-CD25low (71.9±3.3)% and CD4+CD127low-CD25- (9.0± 2.2)% T cells(P<0.01 ). The frequency of CD+CCR4+CD25high T cells correlated negatively with SLEDAI (r=-0.695,P<0.001).and it was significantly lower in lupus nephritis patients(1.10±0.17)%compared with SLE patients without nephritis [(1.61±0.23)%,P<0.01]and healthy controls [(1.75±0.10)%,P<0.01], furthermore,the frequency of CD4+CCR4+CD25low-T cells in lupus nephritis was significantly higher than that in healthy controls[(11.5±2.3)%vs (8.0±1.0)%,P<0.01].Conclusion The increased CD4+CD25-Foxp3+ T cells in the Untreated Newonset Lupus(UNoL)patients mimic activated T effector cells.CD4+CD25high-CD127low-T cells can be used to isolate live CD4+CD25highFoxp3+regulatory T cells.CCR4+regulatory T cells may be involved in the pathogenesis of lupus nephritis.

Objective To obtain the soluble single-chain fragment V (ScFv)monoclonal antibodies (McAbs) against the SSA antigen epitopes.Methods Three octapeptides (60 000 SSA antigen residues 482-493 termed as P1 epitope, residues 310-323 termed as P2 epitope and residues 230-241 termed as P3 epitope) were synthesized on the lysine frame.The McAbs were panned by coating the corresponding as targets.The specificity, affinity and gene squences of the positive clones were assessed.Soluble single-chain fragment V antibodies special for SSA antigen epitopes were expressed and then identificated.Results After 5 rounds of panning, reactive scFv clones contained full-length scFv antibodies coding regions were obtained,with sufficient affinity and specificity for respective antigen peptides.The absorbance values at 410 nm of the fusion protein of anti-P1-P3 activity with the corresponding peptides were 1.43 ± 0.23, 0.82 ±0.31 and 0.80 ± 0.25, and there was also statistically significant difference in the cross reactions ( P ＜ 0.01 ).Three clones were successfully expressed and then purified by His-bind resin.The activity in vivo of soluble ScFv antibodies was identified to be positive by the indirect immune-fluorescence assay on Hep-2 cells.Conclusion Souble ScFv McAbs against corresponding SSA antigen peptides with high affinity,specificity and activity in vivo were obtained, which are to be competent enough for epitopes expression on the target organs.