慢病毒载体介导的RNAi对小鼠外周血CD34^+细胞CCR5表达的影响

Effects of lentiviral vector-mediated RNAi on expression of CCR5 in CD34^+ cells in peripheral blood of mice

目的构建含趋化因子受体5(CCR5)-短发夹状RNA(shRNA)的重组慢病毒,观察其对小鼠外周血CD34+造血干细胞(CD34+细胞)CCR5 mRNA表达的影响。方法使用Ambion RNAi靶序列及参考相关文献设计并确定RNA干扰(RNAi)靶序列。将靶序列转入克隆载体pBShH1扩增,再转入含增强型绿色荧光蛋白(EGFP)的FG12慢病毒载体,酶切、测序鉴定。包装CCR5-shRNA慢病毒并测定滴度,转染小鼠外周血CD34+细胞,检测转染效率。将CCR5-shRNA慢病毒转染的CD34+细胞回输小鼠体内,1周后采用Real-Time PCR检测细胞CCR5 mRNA的表达。结果相关鉴定结果显示:目的片段插入了FG12载体,并带入pBSHH1上的H1 RNA polymeraseⅢ启动子,CCR5-shRNA慢病毒载体构建完成;病毒感染滴度为5×107 TU/mL;CCR5-shRNA慢病毒对小鼠外周血CD34+细胞的转染效率为97.9%;Real-Time PCR检测结果显示:转染CCR5-shRNA慢病毒的CD34+细胞回输后,小鼠外周血CD34+细胞CCR5 mRNA表达显著下降。结论成功制备高滴度的CCR5-shRNA重组慢病毒,该病毒在体内能有效下调小鼠外周血CD34+细胞CCR5 mRNA的表达,为治疗器官移植后排斥反应的研究奠定了基础。

Objective To construct the recombinant lentivirus carrying chemokine receptor 5(CCR5)-short hairpin RNA(shRNA),and investigate its effects on the expression of CCR5 mRNA of CD34+ hematopoietic stem cells(CD34+ cells) in peripheral blood of mice. MethodsRNA interference(RNAi) target sequence was designed by Ambion RNAi target sequence and references.The target sequence was amplified after transduction into plasmid pBSHH1,and was transducted into FG12 lentiviral vector containing enhanced green fluorescent protein(EGFP).The recombinant lentivirus of CCR5-shRNA was packaged,and the virus titer was determined.The recombinant lentivirus was transducted into CD34+ hematopoietic stem cells in peripheral blood of mice,and the transduction efficiency was measured.Then,CD34+ cells transfected with CCR5-shRNA lentivirus were injected into mice,and the expression of CCR5 mRNA was detected by Real-Time PCR after one week. ResultsThe lentivirus was verified to carry both RNAi target sequence and H1 RNA polymerase III gene.CCR5-shRNA lentiviral vector was successfully constructed.The lentiviral infection titer was 5×107 TU/mL.The efficiency of CCR5-shRNA lentivirus in transfection of CD34+ hematopoietic stem cells in peripheral blood of mice was 97.9%.Real-Time PCR revealed that the expression of CCR5 mRNA in CD34+ cells in peripheral blood of mice significantly decreased after CD34+ cells transfected with CCR5-shRNA lentivirus were injected into mice. ConclusionThe recombinant CCR5-shRNA lentivirus of high titer is successfully constructed,which effectively reduces the expression of CCR5 mRNA in CD34+ cells in peripheral blood of mice and lays a foundation for the treatment of rejection after organ transplantation.