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探讨MHC II类转录激活因子(CIITA)的M1-RNA对细胞表面MHC II类分子表达的抑制。M1-RNA是核糖核酸酶P的催化活性单位,设计并克隆针对CIITA第452、629位点的M1-RNA(分别为M1-452-GS、M1-629-GS)及其相应的CIITA靶基因,分别插入pUC19、pGEM-7zf(+)载体,进行细胞外切割活性筛选。将细胞外切割作用明显的M1-629-GS亚克隆入psNAV载体(psNAV-M1-629-GS,pA629)并稳定转染HeLa细胞株,流式细胞术检测经典的MHC II类抗原(HLA-DR、-DP、-DQ)的表达,RT-PCR检测CIITA的mRNA水平。在重组人γ干扰素诱导下,pA629阳性HeLa细胞株表面HLA-DR、-DP抗原表达分别降低了83.03%及89.91%;同时CIITA的mRNA含量明显减少(P<0.05)。CIITA的M1-RNA抑制了自身mRNA含量,从而阻止其调控的MHC II类分子的表达,为移植物抗宿主病的研究提供了一种新方法。
To investigate the inhibitory effect of M1-RNA of MHC class II transcriptional activator (CIITA) on the expression of MHC class II molecules on the cell surface. M1-RNA is a catalytically active unit of RNase P and M1-RNA (M1-452-GS, M1-629-GS, respectively) and their corresponding CIITA target genes for CIITA positions 452, 629 were designed and cloned , Were inserted into pUC19, pGEM-7zf (+) vector for extracellular cutting activity screening. M1-629-GS with extracellular cleavage was subcloned into psNAV vector (psNAV-M1-629-GS, pA629) and stably transfected into HeLa cell lines. The expression of classical MHC class II antigen (HLA- DR, -DP, -DQ) expression was detected by RT-PCR CIITA mRNA levels. The expression of HLA-DR and -DP on the surface of pA629-positive HeLa cells was decreased by 83.03% and 89.91%, respectively, while the mRNA level of CIITA was significantly decreased (P <0.05). CIITA’s M1-RNA inhibits its own mRNA content, thus preventing its regulation of the expression of MHC class II molecules, providing a new method for the study of graft versus host disease.