RNAi抑制大鼠骨髓源树突状细胞MHC-Ⅱ表达的研究

The research on RNAi inhibiting effect to MHCⅡ expression in rat myeloid dendritic cell

目的:应用RNAi技术探讨靶向主要组织相容性复合物Ⅱ类抗原反式转录激活因子(CⅡTA)的shRNA质粒载体抑制大鼠骨髓源树突状细胞(DC)表面MHC-Ⅱ类抗原表达的可行性。方法:体外培养大鼠骨髓源DC;流式细胞仪检测DC表面抗原MHC-Ⅱ、OX-62表达情况;体外合成针对CⅡTA mRNA序列特异性CⅡTA shRNA并构建质粒,在阳离子脂质体介导下转染DC;流式细胞仪检测细胞表面抗原MHC-Ⅱ表达水平的变化。结果:(1)经体外培养DC可用于后续反应,体外构建质粒成功;(2)流式细胞仪检测显示CⅡTA-shRNA质粒组中DC转染后MHC-Ⅱ的抗原水平均较对照组明显降低。结论:利用CⅡTA shRNA质粒载体沉默CⅡTA基因从而下调MHC-Ⅱ的表达的策略是可行的,为降低移植排斥反应提供了实验依据。

Objective： To investigate the feasibility of inhibiting the MHC-Ⅱ expression in rat myeloid dendritic cell （DC） by shRNA plasmid vector targeting MHC-Ⅱ transactivator （CⅡ TA）, which might regulate MHC-Ⅱ expression for suppressing immune rejection. Methods： In vitro culture of the rat myeloid DC was performed and the expression of MHC-Ⅱ antigen and OX-62 antigen were assayed by flow cytometry （FCM）. The shRNA of CⅡ TA mRNA were designed and synthesized in vitro and ligated to plasmid pGenesil-1, transfected DC with Lipofectamine2000 and optimized the transfectional parameters. FCM was applied to detect MHC-Ⅱ antigen. Results： （1） DC acquired in vitro was identified by FCM. DNA sequencing results verified corrent construction of shRNA plasmids. （2） FCM results showed similar MHC-Ⅱ antigen inhibiting effects with mRNA transcriptional changes of CⅡ TA and MHC-Ⅱ. Conclusion： The significant correlation between CⅡ TA and MHC-Ⅱ indicates the feasibility to inhibit MHC-Ⅱ expression by RNAi inhibiting CⅡ TA, which could be used to treat the graft rejective reaction.