摘要
目的 探讨抗主要组织相容性复合物(MHC)Ⅱ类分子转录激活因子的锤头状核酶对Raji细胞表面经典MHCⅡ类抗原表达的抑制作用。方法 设计并克隆针对MHCⅡ类分子转录激活因子(CⅡTA)第134、218、464位点的3 个核酶片段(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,成为pIRES2 EGFP Rz464(pRz464),将pRz464稳定转染Raji细胞株,流式细胞术检测Raji细胞表面MHCⅡ类抗原(HLA DR、DP、DQ)的表达,并用逆转录聚合酶链反应分析Raji细胞CⅡTA mRNA的表达。结果 转染pRz464 的Raji细胞表面HLA DR、DP、DQ抗原表达抑制率分别为95.93 %、94.14 %及98.32 %,同时其CⅡTA mRNA的表达显著下调(P<0.05)。结论 Rz464可抑制Raji细胞表面MHCⅡ类抗原的表达。
Objective To investigate the inhibitive effect of hammerhead ribozyme against class Ⅱ major histocompatibility complex (MHCⅡ) transactivator (CⅡTA) on the expression of MHCⅡ on Raji cells.Method Hammerhead ribozyme against CⅡTA recognizing at 134, 218 and 464 sites (Rz134, Rz218, Rz464 respectively) and CⅡTA target RNA were constructed, then cloned into the pGEM-T vector respectively. The recombinant ribozymes and their CⅡTA target RNA were incu -bated in cell-free conditions. It showed that only Rz464 could cleave target RNA exclusively, then it was cloned into the pIRES2-EGFP vector for intracellular analyses (pIRES2-EGFP-Rz464, pRz464). Stable transfectants of Raji cell by pRz464 were detected for classical MHCⅡ (HLA-DR, -DP, -DQ) expression by flow cytometry, and the mRNA of CⅡTA was detected by RT-PCR.Results The expression of HLA-DR, -DP, -DQ on pRz464 positive Raji cells were almost totally inhibited with the inhibitive rate being 95.93 % , 94.14 % , 98.32 % respectively. The expression of CⅡTA mRNA was significantly down-regulated simultaneously ( P < 0.05 ).Conclusion Rz464 can inhibit the expression of CⅡTA and thus the family of MHCⅡmolecules regulated by CⅡTA.
出处
《中华器官移植杂志》
CAS
CSCD
北大核心
2005年第3期160-163,共4页
Chinese Journal of Organ Transplantation
基金
上海市科技发展基金项目资助(0143nm068)
上海市科技发展基金/重大项目子课题项目资助(00DJ140018)
