CCR3-shRNA promotes apoptosis and inhibits chemotaxis and degranulation of mouse mast cells
CCR3-shRNA promotes apoptosis and inhibits chemotaxis and degranulation of mouse mast cells
复制标题
CCR3-shRNA促进小鼠肥大细胞凋亡并抑制趋化和脱颗粒
DOI：
10.3892/etm.2020.8737
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发表时间：
2020-08-01
影响因子：
2.7
通讯作者：
Wu, Shuhong
中科院分区：
文献类型：
--
作者：
Peng, Haisen;Liao, Bing;Wu, Shuhong
肥大细胞是变应性鼻炎的主要效应细胞。本研究旨在探讨C-C趋化因子受体3（CCR 3）对小鼠系膜细胞增殖、凋亡、趋化和活化脱颗粒的影响。体外培养小鼠骨髓来源的MCs，纯化后用甲苯胺蓝染色和流式细胞仪进行鉴定。构建3种不同的CCR 3-短发夹（shRNA）慢病毒载体，转染MCs，采用逆转录-定量PCR和Western blotting检测CCR 3 mRNA和蛋白表达水平。分别用CCK-8法和流式细胞术检测MCs的增殖和凋亡。通过Transwell分析评估MC趋化性，并使用流式细胞术进行定量。使用ELISA检测MC脱粒的活化。结果表明，MCs得到了适当的分离，并鉴定了CCR 3-shRNA 2在测试的三种shRNAs中具有较高的敲除效果。转染96 h后，CCK-8和流式细胞仪检测结果显示，CCR 3-shRNA 2抑制MC增殖，促进MC凋亡。Transwell实验结果表明，CCR 3-shRNA 2抑制MC的趋化性，而ELISA结果表明，CCR 3-shRNA 2抑制MC的脱颗粒。结论：CCR 3-shRNA 2能有效下调小鼠MC中CCR 3 mRNA和蛋白的表达水平。此外，CCR 3-shRNA 2可促进小鼠MC凋亡，抑制MC增殖、趋化和脱颗粒，提示CCR 3-shRNA 2可作为治疗变应性鼻炎的一种治疗工具。
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 in vitro, 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.