抗CⅡTA核糖核酸酶P抑制Daudi细胞表面MHC-Ⅱ类抗原的表达  

Inhibitory Effects of Anti-CⅡTA RNase P on Major Histocompatibility Complex ClassⅡ Antigens' Expression in Daudi Cells

作　　者：何飞[1] 吴书林[1] 孙明[2] 郭荣[3] 

机构地区：[1]广东省人民医院心内科,广东省心血管病研究所,广州510080 [2]中南大学湘雅医院心内科,长沙410078 [3]广东省人民医院血液科,广州510080

出　　处：《中国生物化学与分子生物学报》2007年第5期405-409,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：广东省医学科学技术研究基金资助项目(No.A2005017);广东省人民医院科学技术研究基金资助项目(No.Y2004087);广东省自然科学基金资助项目(No.04003959;No.06020896);国家自然科学基金资助项目(No.30571771)~~

摘　　要：探讨抗MHC-Ⅱ类分子转录激活因子(CⅡTA)的核糖核酸酶P对Daudi细胞表面MHC-Ⅱ类分子表达的抑制作用.M1-RNA是核糖核酸酶P的催化活性单位.以pTK117质粒为模板,PCR扩增带有抗CⅡTA第452及629位点的引导序列的M1-RNA(M1-452-GS及M1-629-GS),再分别插入pUC19载体(pUC19-M1-452-GS和pUC19-M1-629-GS).从Raji细胞中克隆CⅡTA基因DNA片段(114～800)后插入pGEM-7zf(+)质粒.将重组M1-RNA与靶基因的mRNA进行细胞外共孵育,显示仅pUC19-M1-629-GS可特异性地切割靶基因mRNA.再将M1-629-GS克隆入psNAV载体(pA629)并稳定转染Daudi细胞株,RT-PCR检测其CⅡTA的mRNA水平,流式细胞术检测其HLA-DR、DP、DQ抗原表达.与对照组比较,M1-629-GS阳性Daudi细胞的CⅡTAmRNA含量减少90.19%(P<0·05),其HLA-DR、DP、DQ抗原表达分别降低91.97%、90.19%、92.36%(P<0·05).研究表明,抗CⅡTA的核糖核酸酶P可通过抑制CⅡTA的转录而降低Daudi细胞表面的MHC-Ⅱ类分子的表达.The inhibitory effect of anti-major histocompatibility complex class Ⅱ （MHC- Ⅱ ） transactivator（C Ⅱ TA） Rnase P on expression of MHC- Ⅱ was investigated in Daudi cells. M1-RNA is the catalytic unit of Rnase P. M1-RNAs with different guide sequences （GS） recognizing C Ⅱ TA gene at 452 or 629 site （M1-452-GS, M1-629-GS） were obtained by PCR amplification from pTK117 plasmid. These two M1-RNAs were cloned into pUC19 vector （named pUC19-M1-452-GS and pUCI9-MI-629-GS）. DNA fragment （114 - 800） in C Ⅱ TA was obtained from Raji cell via RT-PCR and was inserted into pGEM-7zf（ ＋ ） plasmid. The recombinant M1- RNAs and their target mRNA were mixed and incubated in cell-free conditions. It showed that pUC 19-M1-629-GS could exclusively cleave the target mRNA only. M1-629-GS was also cloned into the psNAV vector （named pA629）. Daudi cells were stably transfected with pA629 and the level of C Ⅱ TA mRNA was measured by RT- PCR. The expressions of classical MHC-Ⅱ （HLA-DR, -DP, -DQ） were analyzed by flow cytometry. Compared with the control group, the content of C Ⅱ TA mRNA in pA629-positive Daudi cells decreased 90.19 % （P 〈 0.05） while the expression of HLA-DR, HLA-DP and HLA-DQ on pA629-positive Daudi cells decreased 91.97%, 90.19% and 92.36%, respectively （ P 〈 0.05） .The results demonstrates that anti-C Ⅱ TA RNase P could decrease MHC-Ⅱ expression in Daudi cells through inhibiting C ⅡTA transcription.

关 键 词：MHC-Ⅱ类分子转录激活因子(CⅡTA) 核糖核酸酶P(M1-RNA) 移植免疫 

分 类 号：R593.2[医药卫生—内科学]