两种抑制MHCⅡ类分子表达方法的比较  

Appraise for two methods suppressing the MHC class Ⅱ molecules expression

作　　者：欧启水[1] 林琪[1] 林琳[2] 黄立东[2] 陆佩华[2] 周光炎[2] 

机构地区：[1]福建医科大学基因诊断研究室福建医科大学附属第一医院检验科,福州350005 [2]上海第二医科大学免疫学研究所,上海200025

出　　处：《中国免疫学杂志》2006年第1期75-77,81,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金项目资助(399934302;30000157)上海市科技发展基金福建省自然科研基金(2000A014)福建省教委青年科研基金(99A055)资助

摘　　要：目的:评价cⅡTA(MHC class Ⅱ transactivator,CⅡTA)基因突变体和反义RNA对MHCⅡ类分子的抑制效率和胞内稳定性,为改造MHC分子奠定基础。方法:用PCR、酶切及连接技术,构建不含起始密码子的pcDNA3mCⅡTA2,含起始密码子的pcDNA3mCⅡTA3突变体及CⅡTA基因的反义RNA-pcDNA3/CⅡTA反。用脂质体转染法,将突变体、反义RNA及空载体pcDNA3转入PIEC细胞和L23细胞中。持续四个月,流式细胞术观察它们对PIEC/L23细胞株MHCⅡ类(SLA-DR)分子的诱导性和组成性表达的影响。结果:转染pcDNA3、pcDNA3mCⅡTA2后,PIEC/L23细胞SLA-DR的表达没有受到抑制,转染pcDNA3mCⅡTA3后,9/20(45．0％)的PIEC细胞、10/20(50．0％)的L23细胞SLA-DR的表达受到明显抑制,抑制率高达 90％以上;转染pcDNA3mCⅡTA反后,7/15(46．7％)PIEC细胞、5/15(33．3％)L23细胞SLA-DR的表达受到明显抑制,抑制率高达85％以上。持续检测4个月,转染pcDNA3mCⅡTA3的PIEC/L23细胞SLA-DR的表达受抑率均在90％以上。转染反义 RNA的PIEC/L23细胞在第65/45天,SLA-DR表达抑制率降至10％以下。结论:构建成功的CⅡTA突变体和反义RNA均能有效抑制SLA-DR表达,但CⅡTA突变体稳定存在于胞内的时间长,构建突变体是利用CⅡTA抑制MHCⅡ类分子的好方法。Objective：To appraise the efficiency and stability of the mutant and anti-sense RNA of CⅡTA gene. Methods;Restrictive enzymes digestion, PCR and synthesized oligonueleotide strands were used to construct three mutants including pcDNA3mCⅡTA2, pcDNA3mCⅡTA3 and anti-sense RNA of CⅡTA. All these reconstructed plasmid were transfected into PIEC and I23 cell lines by lipofectamine. Flow cytometry were used to determine the changes of the MHC class Ⅱ molecules for 4 months. Results： pcDNA3mCⅡTA3 and anti-sense RNA of C ⅡTA could suppress significantly the expressions of SLA-DR molecules in PIEC and I23 cell, while pcDNA3 empty vector and pcDNA3mC ⅡTA2 had no effect on the MHC class Ⅱ expression. The suppression of pcDNA3mC ⅡTA3 for PIEC/L23 cells would persist for 4 months, while that of anti-sense RNA of CⅡTA persist for 45～65 days. Conclusion：The mutants pcDNA3mCⅡTA3 and anti-sense RNA of C Ⅱ TA can suppress the expression of SLA-DR molecules, the mutants pcDNA3mCⅡTA3 is more stable than anti-sense RNA of C Ⅱ TA. For suppression of the MHC class Ⅱ molecules, the mutant pcDNA3mC Ⅱ TA3 is better than the anti-sense RNA of C Ⅱ TA.

关 键 词：CⅡTA SLA 突变体 反义RNA 

分 类 号：R392.12[医药卫生—免疫学]