修饰CⅡTA基因抑制异种器官移植中供体猪SLA的表达  

Depression of the expression of swine leukocyte antigen by modification of CⅡTA gene in the xenotransplantation

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

机构地区：[1]上海第二医科大学免疫学研究所

出　　处：《中华微生物学和免疫学杂志》2003年第2期137-142,共6页Chinese Journal of Microbiology and Immunology

基　　金：上海市科技发展基金 ( 99JC14 0 33) ;福建省自然科研基金 ( 2 0 0 0A0 14 );福建省教委青年科研基金资助项目 ( 99A0 5 5 )

摘　　要：目的　构建可抑制猪MHC(SLA)Ⅱ类分子表达的MHCⅡ类分子反式激活因子 (CⅡTA)突变体 ,抑制人CD4+ T细胞对猪细胞株的免疫应答 ,为异种器官的应用研究奠定基础。方法　用PCR、人工合成寡核苷酸、限制性内切酶等技术构建不含起始密码子的pcDNA3mCⅡTA2、含起始密码子的pcDNA3mCⅡTA3突变体。用脂质体转染法将上述 2种突变体及pcDNA3空载体转入猪细胞株PIEC细胞和L2 3细胞。用流式细胞术和RT PCR法观察它们对PIEC/L2 3细胞SLA DR/DQ分子的诱导性和组成性表达的影响。通过混合淋巴细胞反应观察人CD4+ T细胞对转入突变体及空载体后的猪细胞的免疫应答强度的变化。结果　在细胞和分子水平证实pcDNA3mCⅡTA3对PIEC/L2 3细胞SLA DR/DQ的表达起明显抑制作用 ,而pcDNA3mCⅡTA2和pcDNA3空载体无此作用。SLAⅡ类分子受抑制的猪细胞已基本丧失了对人CD4+ T细胞的刺激能力。结论　转染能抑制SLAⅡ类分子表达的突变体使PIEC细胞丧失了对人CD4+ T细胞的刺激能力 ,为异种器官的修饰提供了新的思路。Objective To construct the CⅡTA mutant which could suppress the expression of SLA class Ⅱ molecules and suppress the capability of the porcine cells transfected by the mutant to stimulate human CD4+ T cells. Methods Restriction enzymes, PCR technique and synthesized oligonucleotide strand were used to construct two mutants denominated as pCDNA3mCⅡTA2 and pcDNA3mCⅡTA3. LipofectAMINE was used to transfect all mutants and pcDNA3 plasmid into PIEC/L23 cell lines. RT-PCR and flow cytometry were used to identify the changes of the MHC class Ⅱ inducible/constitutive expression. Fillally, the mixed lymphocyte culture was used to test the capability of PIEC cells of stimulating human CD4+ T cells. Results Mutant pcDNA3mCⅡTA3 suppressed the inducible/constitutive expression of SLA class Ⅱ genes. pcDNA3 vector and pcDNA3mCⅡTA2 in which the initiation codon ATG was not constructed, however, had no effect on the SLA class Ⅱ expression. The PIEC cells transfected by mutant pcDNA3mCⅡTA3 failed to stimulate human CD4+ T cells. Conclusion Human CD4+ T cells failed to response to the PIEC cells transfected by the CⅡTA mutant, the modification of the CⅡTA gene would be useful in xenotransplantation. [

关 键 词：CⅡTA突变体 SLA-DR/DQ 异种器官移植 排斥反应 

分 类 号：R392[医药卫生—免疫学]