| Objective To investigate the effect of RNAi to DC surface antigen expression and the mechanism of DC modified by shRNA introducing T lymphocyte anergy. Methods Mice myeloid DC was cultured in selective medium containing necessary cytokines for DC growth in vitro. shRNA which sequence was specified to CD40 mRNA was synthesized in vitro and linked with plasmid vector then transfected into DC. The expression levels of CD40 mRNA and surface antigen CD40, MHCII, CD11c were assayed by real time quantitative PCR and flowcytometry before and after shRNA transfection. The effect to DC stimulating the proliferation of T lymphocyte resulting from silencing effect mediated by shRNA was observed through MLC. IL-12 mRNA levels in MLC culture reaction system were determined by real time quantitative PCR. Results About 1.5-2x10~7 myeloid DCs may be harvested from each mouse through cell culture in vitro. After siRNA transfected into DC, the levels of CD40 mRNA are significantly lower companying with reducing of the antigen CD40 from 49.2% to 16.7%, the inhibition of CD40 mRNA is 49.7% from real time quantitative PCR data, but no changing in antigen MHC II, CD11c. The result of MLC demonstrates that the stimulating index to T lymphocyte of DC knocked down by shRNA significantly decreased accompanied by considerably lower IL-12mRNA inhibitor about 44.9% (p<0.01). Conclusion Large quantity of myeloidDC with typically histological configuration and high levels of MHCII and CD11c may be obtained through vitro culture in selective medium which may activate naive T lymphocyte to generate immune response. Transfecting shRNA targeting to CD40 mRNA with Lipofectamine 2000 into DC may efficiently and specifically inhibit CD40 expression. The activation to allogenic T lymphocyte and IL-12 synthesis as well as secretion of DC knocked down by shRNA becomes weaker and lower which might result in immune deviation in Th cells and introduce T lymphocyte anergy.
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