中文摘要：

目的：研究表达载体介导的反义RNA对人巨噬细胞移动抑制因子（MIF）表达的抑制作用。方法：用亚克隆技术构建可转录MIF反义RNA的真核表达载体pcDNA3-antiMIF。用lipofectamine2000分别将pcDNA3、pcDNA3-antiMIF转染可表达MIF的HEK293（293-MIF）细胞，用Real—time定量PCR鉴定MIF mRNA表达水平。将pcDNA3-antiMIF转化人脐静脉血管内皮细胞（HUVECs），建立可表达MIF反义RNA的HUVECs（HUVECs—antiMIF）细胞。将MIF的真核表达载体pSecTag—MIF转染HUVECs—antiMIF，用Real—time定量PCR鉴定MIF mRNA的表达水平。结果：正确构建了MIF反义RNA的表达载体pcDNA3-antiMIF。MIF反义RNA对293-MIF细胞中MIF表达的抑制水平达32％（P〈0．05）。建立稳定表达MIF反义RNA的HUVECs—antiMIF细胞株。HUVECs—antiMIF中MIF的表达受到抑制，表达水平降低40％（P〈0．05）。结论：表达载体介导的反义RNA能有效地抑制MIF的表达，建立了稳定表达MIF反义RNA的HUVECs。

英文摘要：

AIM ： To study the potential of using antisense RNA mediated by expression vector to suppress MIF expression. METHODS： MIF gene was sub - cloned into plasmid poDNA3 to construct MIF antisense RNA expression vector, poDNA3 -antiMIF, which was identified by restriction enzyme digestion and DNA sequencing. By using lipofectamine 2000, plasmid pcDNA3 and pcDNA3 -antiMIF were transfeeted into MIF expression cells, 293 -MIF, separately. 60 h later, the 293 - MIF cells were collected and used to determine the MIF mRNA expression by real - time quantitive PCR. Plasmid poDNA3 -antiMIF was transformed into HUVECs, named HUVECs -antiMIF, to express MIF antisense RNA. HUVECs -antiMIF was screened by sulfate G418 and identified by PCR and RT- PCR analysis. Then the MIF expression vector, pSecTag- MIF, was transfected into HUVECs -antiMIF, and the MIF mRNA expression in HUVECs -antiMIF was determined by quantitative PCR. RESULTS： Restriction enzyme digestion and DNA sequencing analysis showed that MIF antisense RNA expression vector, poDNA3 -antiMIF, was constructed correctly. The results of quantitative PCR showed that MIF mRNA expression was suppressed by MIF antisense RNA at level about 32% （ P 〈 0. 05 ） in 293 - MIF cells. The HUVECs -antiMIF, which expressed MIF antisense RNA, was obtained and identified by PCR and RT - PCR assay. The results of quantitative PCR revealed that MIF mRNA expression was also d.own - regulated by about 40% （ P 〈 0. 05） in HUVECs -antiMIF cells. CONCLUSION： MIF expression was suppressed efficiently by MIF antisense RNA mediated by expression vector, and the HUVECs -antiMIF was established to express MIF antisense RNA.