The Research On RNAi Inhibiting Effect To MyD88 Gene Expression In Rat Myeloid Dendritic Cell

Objective:To investigate the feasibility of inhibiting the MyD88 expression in rat myeloid dendritic cell(DC)by shRNA plasmid vector.Methods:In vitro culture of the rat myeloid DC was performed by medium containing necessary cytokines for DC growth and the expression of MHC-Ⅱantigen and OX-62 antigen were assayed by flow cytometry(FCM). Three different shRNA which sequences were specified to MyD88 mRNA were designed and synthesized in vitro and ligated to plasmid pGenesil-1.Trials are divided into control group, GenePorter 3000 group, pHK-shRNA group, pMyD881-shRNA group, pMyD882-shRNA group and pMyD883-shRNA group.DNA sequencing was used to examine whether the recombinant plasmids pMyD88-shRNA were correct. Transfeted DC with GenePorter 3000 and optimized the transfectional parameters. Real-time QRT-PCR was used to detect mRNA transcriptional levels of MyD88 and WesternBlot was applied to detect MyD88 protein level after gene tranfection.Result:Mean 1.0×10'DC per rat acquired in vitro were identified by FCM. DNA sequencing results verified corrent construction of shRNA plasmids. PCR showed that mRNA transcription of MyD88 was obviously inhibited by pMyD88-shRNA posttransfection, while there were no obvious changes and differences between control group, pHK-shRNA control group and GenePorter 3000 group (P<0.01).pMyD881-shRNA group exhibited the strongest inhibiting effect among pMyD88-shRNA groups. Western Blot results showed similar inhibiting effects. pMyD881-shRNA group also showed stronger effects among pMyD88-shRNA groups.Conclusion:In vitro culture of the rat myeloid DC by medium containing necessary cytokines for DC growth is a reliable method to acquire sufficient DC which show typical morphous and high expression levels of MHC-Ⅱand OX-62. OnThe 12th day in cell culture by flow cytometry, the MHC-Ⅱand OX-62 antigen expression was 91.36% and 72.70% respectivly.pMyD88-shRNA groups show significant inhibiting effect of MyD88 gene and its protein expression versus control group,pHK-shRNA group and GenePorter 3000 group. pMyD881-shRNA produces the strongest effect among the pMyD88-shRNA groups and may be used for further study.