摘要
目的:研究基因转染后高表达吲哚胺2,3-双加氧酶(IDO)的树突状细胞(DC)对T细胞增殖的抑制作用,以探讨其在器官移植中排斥反应的作用。方法:采用逆转录-聚合酶链式反应(RT-PCR)从腹膜炎小鼠的小肠组织中获取IDO全长cDNA片段,将其克隆至真核表达质粒pTracer中,构建真核表达质粒IDO-pTracer,随后用LipofectamineTM2000介导转染小鼠脾脏DC,分为未转染组、空载体转染组、IDO基因转染组和IDO基因转染并添加1-甲基色氨酸(1-MT)组,以混合淋巴细胞反应的方法检测转染后DC对同种异系T细胞增殖的作用。结果:克隆的小鼠IDO基因全长cDNA大小约1.3kb,无碱基突变,以构建的重组真核表达质粒IDO-pTracer转染DC后,混合淋巴细胞反应结果显示IDO基因转染组的刺激指数(SI)低于未转染组、空载体转染组及IDO基因转染并添加1-MT组,差异均有统计学意义(P<0.05)。结论:成功构建了IDO基因的真核表达载体IDO-pTracer,且IDO基因转染DC具有抑制异系T细胞增殖的作用。
Objective： To demonstrate the effect of indoleamine 2,3-dioxygenase （IDO） transfected dendritic cells （DCs） in preventing the rejection of organ transplantation. Methods： IDO cDNA was obtained by RT-PCR technique from small bowl tissue of a peritonitis model mouse and was inserted into eukaryotic expression vector pTracer to construct the recombinant expression plasmid IDO-pTracer. The recombinant plasmid was transfected into mouse splenic DCs by LipofectamineTM 2000. The transfected DCs were co-cultured with allogeneic lymphocytes. Results： IDO-pTracer was successfully generated and verified by sequencing. And the result of mixed lymphocyte reaction （MLR） displayed the stimulation index （SI） was significantly lower in the transfected DC group than those of non-transfected group and 1-methyl-tryptophan （1-MT） addition group. Conclusion： The eukaryotic expression vector IDO-pTracer is constructed successfully, and the transfected DCs can inhibit Tcell proliferation which can be prevented by 1-MT.
出处
《天津医药》
CAS
北大核心
2010年第6期496-498,547,共4页
Tianjin Medical Journal
基金
天津市重中之重科学基金资助项目(项目编号:05YFGDSF02600)
