Zhu X H, Peng H S, Jiang Y L, Wu S H, Tang S Y, Liu Y H
Department of Otorhinolaryngology Head and Neck Surgery, Second Affiliated Hospital of Nanchang University, Nanchang, 330006, China.
Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2019 Jul;33(7):628-634. doi: 10.13201/j.issn.1001-1781.2019.07.013.
The aim of this study is to screen the targeting chemokine receptor 3-RNA interference (CCR3-RNAi) lentiviral expression vector, infect mouse mast cells,observe the expression of this gene in mast cells and the interference efficiency of the virus vector.The pathogenesis of allergic rhinitis lays the foundation.Three pairs of CCR3-shRNA sequences were constructed,and three pairs of double-stranded shRNA oligo were inserted into shRNA lentiviral vectors to construct three shRNA lentiviral recombinant plasmids.The recombinant vector and virus-packed auxiliary plasmids were co-transfected into 293T cells to obtain lentiviral plasmids.The lentiviral plasmids were then transfected into mouse bone marrow-derived mast cells in vitro and purified. The expression level of CCR3 mRNA in mast cells was verified by qRT-PCR,and the expression level of CCR3 protein in mast cells was detected by Western Blot. It was confirmed by sequencing that the lentiviral vector of CCR3 shRNA was successfully constructed, transfected into 293T cells and packaged with virus. Finally the high purity PDSO19-PL-CCR3 lentiviral plasmid was obtained with a virus titer of 3.7×10⁸TU/ml.The lentiviral plasmid was used to infect mouse mast cells.RT-PCR and Western Blot detection assay showed that CCR3shRNA reduced the expression of CCR3 gene in mouse mast cells at the level of mRNA and protein. The CCR3 gene RNAi lentivirus expression vector was successfully constructed.It was found that it downregulated the expression level of CCR3 gene mRNA and protein in mouse mast cells,which laid the foundation for further research on its role in the pathogenesis of allergic rhinitis.
本研究旨在筛选靶向趋化因子受体3-RNA干扰（CCR3-RNAi）慢病毒表达载体，感染小鼠肥大细胞，观察该基因在肥大细胞中的表达及病毒载体的干扰效率，为变应性鼻炎的发病机制奠定基础。构建了三对CCR3-shRNA序列，并将三对双链shRNA寡核苷酸插入shRNA慢病毒载体中，构建了三种shRNA慢病毒重组质粒。将重组载体与病毒包装辅助质粒共转染至293T细胞中，获得慢病毒质粒。然后将慢病毒质粒体外转染至小鼠骨髓来源的肥大细胞中并进行纯化。通过qRT-PCR验证肥大细胞中CCR3 mRNA的表达水平，通过蛋白质免疫印迹法检测肥大细胞中CCR3蛋白的表达水平。经测序证实成功构建了CCR3 shRNA慢病毒载体，并转染至293T细胞中并进行病毒包装。最终获得了高纯度的PDSO19-PL-CCR3慢病毒质粒，病毒滴度为3.7×10⁸TU/ml。用该慢病毒质粒感染小鼠肥大细胞。RT-PCR和蛋白质免疫印迹检测分析表明，CCR3shRNA在mRNA和蛋白质水平上降低了小鼠肥大细胞中CCR3基因的表达。成功构建了CCR3基因RNAi慢病毒表达载体，发现其下调了小鼠肥大细胞中CCR3基因mRNA和蛋白质的表达水平，为进一步研究其在变应性鼻炎发病机制中的作用奠定了基础。
