摘要
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 the M1-RNA, the catalytic activity unit of ribonuclease P（RNAase P）, from MHC classs Ⅱ transactivator（CllTA） on the expression of MHC class Ⅱ molecules on HeLa cells, the M1-RNA with guide sequences（GS） recognizing CIITA at 452 and 629 sites（M1-452-GS and M1-629-GS） and the corresponding CIITA-target RNA （114-800） were constructed, and then cloned to vector pUC19 and pGEM-7zf（＋） respectively. The recombinant M1-RNA and their target RNA were incubated in cell-free conditions. It was demonstrated that the M1-629-GS could exclusively cleave the target RNA, furthermore, it was subcloned to vector psNAV for intracellular analysis （psNAV-M1-629-GS, pA629）. Stable transfectants of HeLa cells with pA629 were analyzed for the expressions of classical MHC class Ⅱ molecules（ HLA- DR, -DP, -DQ） by flow cytometry, and the level of CIITA mRNA was measured by RT-PCR. Through the induction with recombinant human interferon-γ（IFN-γ）, the expressions of HLA-DR and -DP on the surface of HeLa cells reduced to 83.03% and 89.91% respectively. Meanwhile, the content of CIITA mRNA with a reduced rate of expression up to 91.12% ±7.97% （P〈0.05）. It is evident from the above observations that the M1-RNA inhibits CIITA, and thus the MHC class Ⅱ molecules regulated by CIITA. This result would provide a new way of approach for the solution of GVHD in hematopoietic stern cell transplantation.
出处
《现代免疫学》
CAS
CSCD
北大核心
2007年第6期453-457,共5页
Current Immunology
基金
国家自然科学基金资助项目(30571771)
广东省人民医院科学技术研究基金资助项目(Y2004087)
广东省医学科学技术研究基金资助项目(A2005017)
广东省自然科学基金资助项目(04003959
06020896)
