Peng Haisen, Liao Bing, Zhu Xinhua, Liu Yuehui, Jiang Yinli, Wu Shuhong
Department of Otolaryngology Head and Neck Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China.
Exp Ther Med. 2020 Aug;20(2):1030-1038. doi: 10.3892/etm.2020.8737. Epub 2020 May 12.
Mast cells (MCs) are the major effector cells of allergic rhinitis (AR). The present study aimed to investigate the effects of C-C chemokine receptor type 3 (CCR3) on the proliferation, apoptosis, chemotaxis and activated degranulation of mouse MCs. Mouse bone marrow-derived MCs were cultured , purified and identified using toluidine blue staining and flow cytometry. Three different CCR3-short hairpin (shRNA) lentiviral vectors were constructed and transfected into MCs, and the mRNA and protein expression levels of CCR3 were assessed by reverse transcription-quantitative PCR and western blotting. Proliferation and apoptosis of the MCs were measured using Cell Counting kit-8 (CCK-8) assays and flow cytometry, respectively. MC chemotaxis was assessed by Transwell assay and quantified using flow cytometry. The activation of MC degranulation was examined using ELISAs. The results demonstrated that MCs were appropriately isolated, and identified that CCR3-shRNA2 presented the higher knockdown effect among the three shRNAs tested. Following 96 h of transfection, the results of CCK-8 and flow cytometry assays demonstrated that CCR3-shRNA2 inhibited MC proliferation and promoted MC apoptosis. The results from the Transwell assay indicated that CCR3-shRNA2 restrained MC chemotaxis, whereas ELISA results demonstrated that CCR3-shRNA2 suppressed MC degranulation. In conclusion, CCR3-shRNA2 effectively downregulated CCR3 mRNA and protein expression levels in mouse MCs. In addition, CCR3-shRNA2 promoted MC apoptosis and suppressed the proliferation, chemotaxis and degranulation of mouse MCs, suggesting that CCR3-shRNA2 may serve as a therapeutic tool for the treatment of allergic rhinitis.
肥大细胞（MCs）是变应性鼻炎（AR）的主要效应细胞。本研究旨在探讨C-C趋化因子受体3（CCR3）对小鼠MCs增殖、凋亡、趋化性和活化脱颗粒的影响。使用甲苯胺蓝染色和流式细胞术对小鼠骨髓来源的MCs进行培养、纯化和鉴定。构建三种不同的CCR3短发夹（shRNA）慢病毒载体并转染至MCs中，通过逆转录-定量PCR和蛋白质印迹法评估CCR3的mRNA和蛋白质表达水平。分别使用细胞计数试剂盒-8（CCK-8）检测法和流式细胞术测量MCs的增殖和凋亡。通过Transwell检测法评估MC趋化性，并使用流式细胞术进行定量。使用酶联免疫吸附测定法检测MC脱颗粒的激活情况。结果表明，MCs被成功分离，并且发现在所测试的三种shRNAs中，CCR3-shRNA2具有更高的敲低效果。转染96小时后，CCK-8检测法和流式细胞术检测结果表明，CCR3-shRNA2抑制MC增殖并促进MC凋亡。Transwell检测法结果表明，CCR3-shRNA2抑制MC趋化性，而酶联免疫吸附测定法结果表明，CCR3-shRNA2抑制MC脱颗粒。总之，CCR3-shRNA2有效下调小鼠MCs中CCR3的mRNA和蛋白质表达水平。此外，CCR3-shRNA2促进MC凋亡并抑制小鼠MCs的增殖、趋化性和脱颗粒，提示CCR3-shRNA2可能作为治疗变应性鼻炎的一种治疗工具。
