The Research On RNAi Inhibiting Effect To MHC Ⅱ Expression In Rat Myeloid Dendritic Cell

The Research on RNAi Inhibiting Effect to MHCⅡExpressionin Rat Myeloid Dendritic CellObjective: To investigate the feasibility of inhibiting the MHC-Ⅱexpression in ratmyeloid dendritic cell (DC) by shRNA plasmid vector targeting MHC-Ⅱtransactivator (CⅡTA), which might regulate MHC-Ⅱexpression for suppressingimmune rejection.Methods: In vitro culture of the rat myeloid DC was performed by selective mediumcontaining necessary cytokines for DC growth and the expression of MHC-Ⅱantigenand OX-62 antigen were assayed by flow cytometry (FCM). Three different shRNAwhich sequences were specified to CⅡTA mRNA were designed and synthesized invitro and ligated to plasmid pGenesil-1. DNA sequencing was used to examinewhether the recombinant plasmids pCⅡTA-shRNA were correct. Transfeted DC withLipofectamine2000 and optimized the transfectional parameters. Real-time QRT-PCRwas used to detect mRNA transcriptional levels of CⅡTA and MHC-Ⅱand FCM wasapplied to detect MHC-Ⅱantigen level 48 hours after gene tranfection.Result: Mean 1.0×10~7 DC per rat acquired in vitro were identified by FCM. DNAsequencing results verified corrent construction of shRNA plasmids. PCR showed thatboth mRNA transcription of CⅡTA and MHC-Ⅱwere obviously inhibited bypCⅡTA-shRNA posttransfection, while there were no obvious changes anddifferences between control group, pHK-shRNA control group and Lipofectaminegroup (P＜0.01). pCⅡTA1-shRNA group exhibited the strongest inhibiting effectamong pCⅡTA-shRNA groups. FCM results showed similar MHC-Ⅱantigeninhibiting effects with mRNA transcriptional changes of CⅡTA andMHC-Ⅱ(pCⅡTA-shRNA groups vs control groups and Lipofectamine group, P＜ 0.01)pCⅡTA1-shRNA group also showed stronger effects than other groups.There were obvious correlation between MHC-Ⅱantigen and mRNA transcriptionalchanges of CⅡTA and MHC-Ⅱby Spearman correlation statistics analysis.Condusion: In vitro culture of the rat myetoid DC by selective medium is a reliablemethod to acquire sufficient DC which show typical morphous and high expressionlevels of MHC-Ⅱand OX62. It proves an effective pathway to transfect rat DC withLipofectamine2000. pCⅡTA-shRNA groups show significant inhibiting effect ofCⅡTA and MHC-Ⅱexpression versus control group, pHK-shRNA group andLipofectamine group, pCⅡTA1-shRNA produces the strongest effect among thepCⅡTA-shRNA groups and maybe used for further study. The significant correlationbetween CⅡTA and MHC-Ⅱindicates the feasibility to inhibit MHC-Ⅱexpressionby RNAi inhibiting CⅡTA.