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Objective: To generate mouse B7-2 gene RNAi lentivirus and study its interference effects on B7-2 expression and T lymphocytes proliferation induced by dendritic cells. Methods: Three sequences specific targeting B7-2 gene and one non-specific sequence were respectively synthesized, and inserted into lentiviral vector, then the recombinant vectors were sequencing. 293 T cells were co-transfected with lentiviral expression plasmid and packaging plasmids to produce recombinant lentivirus which titre was checked according to the expression level of green fluorescent protein ( GFP). Bone marrow cells from C57 BL/6 mice were isolated to differentiate into DCs at the present of GM-CSF, IL-4 and LPS for 48 h, then morphology and phenotypic was identified. DCs were infected by recombinant RNAi lentivirus and then the efficiency of infection and the expression of B7-2 on the surface of DCs were detected by flow cytometry. Effects on the proliferation of T cells were detected by co-culturing with DCs which were infected by B7-2 RNAi lentivirus and murine spleen T cells in vitro. Results: DNA sequencing confirmed that three B7-2 RNAi and one non-specific recombinant lentiviral transfer plasmids were successfully constructed, the titer of recombinant lentivirus was ( 2-4) × 108 TU/ml. The recombinant lentivirus could effectively infect DC and inhibit the expression of B7-2. After the B7-2 recombinant lentivirus infection, the ability of DCs to stimulate the proliferation of T cells decreased obviously ( P＜0. 05). Conclusion: The lentiviral B7-2 gene RNAi vector can effectively silence the expression of B7-2 on the surface of DCs and inhibit the proliferation effect of T cells induced by DCs.
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Objective:To construct lentiviral vector specific for mouse B7-1 RNA interference and study lentivirus-mediated B7-1 gene silencing effects in L929 fibroblast cells.Methods:Three candidate sequences for B7-1 RNAi selected from coding sequence of mouse B7-1 transcription were used to design short hairpin RNA ( shRNA ) templates and then cloned into lentiviral expression plasmid followed with correctness identification of inserted sequence by DNA sequencing.Recombinant lentivirus were prepared by co-transfecting lentiviral expression vector and packaging plasmids into 293T cells.Then the resulting culture supernatant containing infectious lentiviral particles was pooled and centrifuged via ultra-centrifugation.Infectious titer of the preparations was determined by detecting the expression of GFP in 293T cells after transfected by lentivirus.Cultured L929 cells were transfected with lentivirus to deter-mine transduction efficiency and silencing efficacy of B7-1 expression by flow cytometry.Transducted L929 cells were then screened using puromycin to generate stable cell clones followed by flow cytometry analysis of GFP and B7-1 expression.A mixed reaction system consisting of stable B7-1 silencing L929 cells and mouse splenic T cells was used to analyze ability of the established cell line to trigger T cells proliferation.Results: Lentiviral expression vector for mouse B7-1 RNAi was correctly constructed with inserted sequences as designed.Recombinant RNAi lentivirus were prepared with titers ranging (3-5) ×108 TU/ml and efficacy to mediate GFP transgene expression and B7-1 silencing.B7-1 expression and the ability to trigger T cells proliferation of stable L929 cells were suppressed significantly ( P<0.05 ).Conclusion: We generated lentiviral vector specific for mouse B7-1 RNAi with high performance of transduction efficiency as well as B7-1 silencing efficacy and the recombinant RNAi lentivirus can mediated stable B7-1 gene silencing in L929 cells and inhibition of T cells proliferation induced by B7-1/CD28 co-stimulatory signal.
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Objective:To set up a eukaryotic system for high expressing human CD137 and to investigate the role of CD137 and CD137L on signals transduction of cells.Methods:The pCDNA3 plasmid containing full length of human CD137 cDNA sequence(CMV ILA SEN,CIS) and pSV2 dhfr plasmid were cotransfected into dhfr CHO cells by lipoid mediating method. The positive clone was selected with G418. Expression of CD137 on dhfr CHO cells were induced by MTX and detected by RT PCR, immunocytochemistry and flow cytometry.It's activity study was done by method of incorporating 3H TdR.Results:CD137 expressed on the surface of dhfr CHO, expression rate was 96.07%, it's activity study indicated that CD137 increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.Conclusion:dhfr CHO cells that highly express CD137 were established. CD137 can increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.
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Objective To study the expressions of co stimulatory factors CD28 and CTLA 4 on CD3 + T cells in peripheral blood lymphocytes (PBMC) and synovial fluid lymphocytes (SFMC) in patients with rheumatoid arthritis (RA) and the relationship of these molecules with the activity of RA. Methods The lymphocytes were collected from the peripheral blood and synovial fluid in RA patients. The CD3 +CD28 + and CD3 +CTLA 4 + molecules on these cells were measured by dual color fluorescence cytometry. The correlation of the expressions of CD3+CD28+ and CD3 +CTLA 4 + molecules with the activity of RA was statistically analyzed by Spearman. Results ① The level of CD3 +CD28 + in PBMC of RA patients was significantly lower than that of the control group and SFMC ( P