靶向miR-126慢病毒表达载体的构建及意义

Construction of a lentiviral vectors of antisense oligonucleotide targeting on the miR-126 and its signficance

目的:检测微小RNA-126(microRNA-126,miR-126)在CD4+CD25+调节性T细胞(regulatory T cells,Tregs)中的表达水平,同时构建基于慢病毒的miR-126反义寡核苷酸序列(antisense oligonucleotides,ASOs)表达载体。方法:实时定量PCR特异性探针法检测miR-126在Tregs中的表达水平;针对miR-126序列,设计合成其ASOs序列,退火后连接至经AgeⅠ酶和EcoRⅠ酶双酶切的pGCsil-LV-GFP载体上,连接产物转化DH5α感受态细胞,对经PCR鉴定为阳性的载体(命名为pGCsil-miR-126-ASOs)进行测序分析;将构建成功的pGCsil-miR-126-ASOs表达质粒和pHelper 1.0、pHelper 2.0质粒共转染293T细胞,浓缩病毒颗粒并测定所获病毒滴度;将制备好的病毒颗粒感染经TGF-β体外诱导培养的小鼠CD4+CD62L+初始T细胞,72 h后用流式细胞仪检测其Foxp3的表达变化。结果:实时定量PCR结果显示miR-126在CD4+CD25+Tregs中的表达明显高于CD4+CD25-T细胞(P<0.01);测序结果证明成功构建pGCsil-miR-126-ASOs重组质粒载体,包装并获得高浓度的慢病毒颗粒,病毒滴度为9×108TU/mL;重组的病毒能明显抑制Tregs的外周诱导(P<0.05)。结论:成功构建miR-126 ASOs的慢病毒表达载体,并获得高浓度的病毒颗粒。

Objective： To evaluate the expression of miR-126 in human peripheral blood CD4＋ CD25 ＋ regulatory T cells （ Tregs）, construct a lentiviral vector of antisense oligonucleotides （ASOs） a- gainst miR-126. Methods： The expression level of miR-126 in Tregs and CD4 ＋ CD25 - T cells was determined by real-time PCR respectively. The ASOs against miR-126 were synthesized and inserted into the pGCsiI-LV-GFP plasmid and constructed the pGCsil-miR-126-ASOs plasmid which was indentified by RT-PCR and DNA sequencing. Additional, pHelper 1.0, pHelper 2.0 and pGCsil-miR-126-ASOs vectors were cotransfected into 293T cells by LipofectamineTM 2000. After 48 h cultrue, the supernatant was harvested and the titer of pGCsil-miR-126-ASOs lentivirus was determined by limiting dilution analysis. Finally, CD4 ＋ CD62L＋ naive T cells were infected with pGCsil-miR-126-ASOs lentivirus at MOI （multiplicity of infection） = 100 and cultured in the presence of anti-CD3 antibody and anti-CD28 antibody plus IL-2 and TGF-β for another 72 h, the expression of Foxp3 was analyzed by flow cytometry. Results ： The expression level of miR-126 in Tregs was significantly higher than CD4 ＋ CD25 - T cells （ P 〈 0. 01 ）. Furthermore, the miR-126 ASOs inhibited the induction of Tregs in vitro （P 〈 0. 05 ）. Conclusion： The lentiviral vector of miR-126 ASOs was constructed successfully and laid the foundation for the further research on the miR-126 ASOs in regulation of Treg functions.