寻常型银屑病T细胞CCR7表达的报告基因分析

Analysis of report gene of human chemokine receptor CCR7 in patients with psoriasis vulgaris

目的构建人外周血T细胞CCR7报告基因载体并初步验证IL-18对其作用的影响。方法提取健康献血员全基因组DNA，克隆包含人CCR7上游启动子的基因序列，酶切、连接形成重组质粒后转化感受态细菌，酶切及测序后构建报告基因质粒，瞬时转染真核细胞COS，荧光素酶报告基因分析。结果pGL3-basic报告基因质粒与空白对照相比可以检测到荧光素酶基因表达0.024±0.008；含有启动子基因的荧光素酶活性明显高于pGL3-basic（P〈0.01）；IL-18作用后报告基因荧光素酶基因的表达明显增加比单独使用pGL3-2233或pGL3-1037质粒DNA转染COS时明显增加（P〈0.01）。结论成功构建包含有人CCR7启动子的报告基因载体，并且初步验证IL-18可以直接激活CCR7基因上游调控区的启动子，激活CCR7基因表达，从基因水平直接找到IL-18对CCR7表达调控的证据。

Objective To construct CCR7 report gene vector of human T cells and verification the effect of IL-18 on the expression of CCR7. Methods The primers were designed to comprise the upper stream promoter region of human CCR7. T cells were separated and genome DNA was extracted. PCR was used to amplify designed sequence. The PCR products were digested by NheI and HindⅢ, and cloned into pET28-a（ ＋ ） plasmid and sequenced, then were subcloned into pGL3-Basic vector and pGL3 vector comprised CCR7 promoter region. CCR7 reporter gene vector was transient transfected into COS cell and luciferase activity was detected. Results 0. 024 ± 0. 008 luciferase activity was detected in pGL3-Basic report gene vector transfection compared with blank control transfection. The luciferase activity in transfection with reporter gene vector comprised of promoter of CCR7 was higher than that in transfection with pGL3-Basic report gene （ P 〈 0. 01 ）. The luciferase activity after stimulated with IL-18 were higher than that in un-stimulated （ P 〈 0. 01 ）. Conclusion The results show that CCR7 expression is significantly up-regnlated by IL-18 and provide direct evidence on that IL-18 can directly regulate CCR7 expression through upper promoter of CCR7.