CⅡTA核酶对肝细胞表面MHCⅡ类分子表达的抑制  

Study on the Inhibitive Effects of CⅡTA Ribozyme on Hepatocytes MHC Ⅱ Molecules Expression

作　　者：金中奎[1] 郭荣[2] 贺强[1] 李宁[1] 陈实[3] 

机构地区：[1]首都医科大学附属北京朝阳医院普外科,100020 [2]广东省人民医院血液科 [3]华中科技大学同济医学院附属同济医院器官移植研究所

出　　处：《天津医药》2005年第4期193-196,共4页Tianjin Medical Journal

基　　金：国家"十五"863课题资助项目(项目编号:2001AA216071)

摘　　要：目的:探讨抗CⅡTA核酶对肝细胞表面MHCⅡ类分子表达的抑制作用。方法:设计并克隆针对CⅡTA第134、218及464位点的核酶(分别为Rz134、Rz218、Rz464)及其相应的CⅡTA靶基因,分别插入pGEM-T载体,进行细胞外切割活性筛选。将切割作用明显的Rz464亚克隆入真核表达载体pIRES2-EGFP(pIRES2-EGFP-Rz464,pRz464)进行细胞内切割分析。pRz464稳定转染人胎肝细胞,流式细胞术检测MHCⅡ(HLA-DR、HLA-DP、HLA-DQ)类抗原表达,RT-PCR分析CⅡTAmRNA水平。结果:pRz464肝细胞表面HLA-DR、HLA-DP、HLA-DQ的诱导型表达分别为(1.01±0.51)%、(4.37±1.28)%和(1.98±0.42)%,与对照组比较分别下调90.65%、89.11%及65.32%;同时CⅡTA的诱导型mRNA含量明显减少(P<0.01)。结论:抗CⅡTA核酶-Rz464降低了CⅡTA的mRNA含量,从而阻止其调控的MHCⅡ类分子的表达。Objective: To study the inhibitive effects of MHC class Ⅱ transactivator CⅡTA ribozyme on hepatocytes MHC Ⅱ molecules expression. Methods: Ribozyme which aimed at CⅡTA 134,218 and 464 sites served as Rz134,Rz218,Rz464 respectively and CⅡTA target gene were designed and cloned. They were inserted in the pGEM-T vector respectively, and were undergone extracellular incision activity screening. Rz464 which had obvious incision effect was sub-cloned into the pIRES2-EGFP vector pIRES2-EGFP-Rz464, pRz464 for intracellular incision analyses. Hepatocytes were stably transfected by pRz464, classical MHC Ⅱ HLA-DR,-DP,-DQ antigen expression were detected by flow cytometer, and mRNA level of CⅡTA was measured by RT-PCR. Results: The expressions of HLA-DR,-DP, and-DQ on pRz464 positive hepatocytes were 1.01 ± 0.51%,4.37 ± 1.28%, and 1.98 ± 0.42% respectively, and were down-modulated by 90.65%, 89.11%, 65.32% respectively compared with that in control group. The mRNA content of CⅡTA decreased significantly P < 0.01. Conclusion: Anti-CⅡTA ribozyme Rz464 decreases mRNA content of CⅡTA and inhibits the expression of MHC Ⅱ molecules regulated by CⅡTA.

关 键 词：CⅡTA 细胞表面 分子表达 核酶 流式细胞术检测 MHCⅡ类分子 RNA含量 真核表达载体 mRNA水平 人胎肝细胞 HLA PCR分析 抑制作用 活性筛选 切割作用 稳定转染 抗原表达 诱导型 靶基因 T载体 细胞外 亚克隆 细胞内 RT- 对照组 

分 类 号：R392.11[医药卫生—免疫学] R692[医药卫生—基础医学]