主要组织相容性复合物Ⅱ类分子转录激活因子核酶的克隆及其活性  

Cloning and biological activities of riboenzymes against major histocompatibility complex class Ⅱ transactivator

作　　者：郭荣[1] 邹萍[1] 杨序春 陆华中[2] 高峰[2] 曹谊林[3] 张敏[1] 范华骅[2] 

机构地区：[1]华中科技大学同济医学院附属协和医院血液病研究所,武汉430022 [2]上海市血液中心血液工程研究室 [3]上海第二医科大学组织工程重点实验室

出　　处：《中华医学杂志》2003年第8期669-672,共4页National Medical Journal of China

基　　金：上海市科技发展基金资助项目 ( 0 14 3nm0 6 8);上海市科技发展基金 /重大项目子课题资助项目 ( 0 0DJ14 0 0 1- 8)

摘　　要：目的　探讨主要组织相容性复合物 (MHC)Ⅱ类分子转录激活因子 (CⅡTA)核酶抑制MHCⅡ类分子的表达。方法　设计并合成针对人类CⅡTA的一组核酶 ,通过体外制备和活性鉴定 ,筛选出活性较高的核酶 Rz46 4。将Rz46 4克隆到真核表达载体pIRES2 EGFP ,简称为pRz46 4,并稳定转染Daudi细胞株 (pRz46 4 D) ,流式细胞术检测经典的MHCⅡ (HLA DR、 DP、 DQ)类抗原表达 ,RT PCR检测CⅡTAmRNA水平。结果　pRz46 4 D与无关核酶组比较 ,HLA DR、 DP、 DQ抗原表达分别降低了 88.4%、83 .5 %、93.4%;同时CⅡTA的mRNA含量明显减少。结论　提示抗CⅡTA锤头状核酶抑制了自身mRNA含量 ,从而阻止了其调控的MHCⅡ类分子的相应表达。Objective To investigate the cloning and biological activities of riboenzymes against major histocompatibility complex (MHC) class Ⅱ transactivator (CⅡTA) so as to explore the feasibility of using hammerhead riboenzyme to create immune tolerance. Methods Three riboenzymes against MHC CⅡTA were synthesized and their activities were evaluated in vitro. Rz464, the riboenzyme with a better digestion effect, was inserted into the vector pIRES2-EGFP (then called pRz464). Human B lymphoma cells of Daudi line were cultured and transfected with pRz464 (then called pRz464-D). Cultured Daudi cells transfected with riboenzyme without the ability against CⅡTA (Rz-D) were used as control group. The expressions of classic MHC CⅡTA (HLA-DR, DP, and DQ ) on the surface of Daudi cells before and after transfection were examined by flow cytometry. Results The expressions of HLA-DR, DP, and DQ on the surface of cultured Daudi cells (Rz-D) were 97.9±0.8%, 94.3±1.1%, and 54.4±3.1% respectively. The expressions of HLA-DR, DP, and DQ were inhibited by 88.4%, 83.5%, and 93.4% respectively on the surface of pRz464-D cells. Conclusion The hammerhead riboenzyme Rz464 inhibits the expression of CⅡTA mRNAs, thus inhibiting the expression of MHC Ⅱ molecules. It may be used in antigen-specific tolerance induction in allo-transplantation.

关 键 词：克隆 活性 基因表达调控 酶学 移植免疫学 基因疗法 RT—PCR 

分 类 号：R392.1[医药卫生—免疫学]