Zhu Xin-Hua, Liao Bing, Xu Yi, Liu Ke, Huang Yun, Huang Quan-Long, Liu Yue-Hui
Department of Otolaryngology Head and Neck Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China.
Department of Head and Neck Surgery, The Tumor Hospital of Jiangxi Province, Nanchang, Jiangxi 330006, P.R. China.
Mol Med Rep. 2017 Feb;15(2):696-702. doi: 10.3892/mmr.2016.6085. Epub 2016 Dec 29.
RNA interference has been considered as an effective gene silencing method in basic and preclinical investigations. The aims of the present study were to construct a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the murine CC chemokine receptor 3 (mCCR3), and to investigate its effects on the proliferation and apoptosis of mouse eosinophils. A recombinant lentiviral vector expressing four fragments of mouse CCR3 shRNA (pLVX‑mCCR3‑1+2+3+4‑shRNA) was constructed using subcloning techniques. This novel lentivirus was then packaged into 293T cells by co‑transduction with plasmids, including Baculo p35, pCMV R8.2 and VSV. The interference effects of the vector were verified using polymerase chain reaction (PCR) and western blot analyses. The effects of the interference on the proliferation and apoptosis of mouse eosinophils were investigated using 3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium and terminal deoxynucleotidyl transferase dUTP nick end labeling methods, respectively. The results of the PCR and western blot analyses confirmed that the novel recombinant vector, pLVX‑mCCR3‑1+2+3+4‑shRNA, had high efficiency in inhibiting the mRNA and protein expression levels of mCCR3 in mouse eosinophils. The downregulation of mCCR3 significantly inhibited proliferation of the eosinophils. Furthermore, the present study found that the downregulation of mCCR3 significantly promoted apoptosis of the eosinophils. Therefore, the downregulation of mCCR3 led to the inhibition of proliferation and induction of apoptosis in mouse eosinophils. The predominant characteristics of allergic rhinitis are eosinophil infiltration and release of inflammatory mediators, which appear in a variety of clinical manifestations. The results of the present study indicate that mCCR3 silencing may serve as a putative approach for the treatment of allergic rhinitis.
在基础研究和临床前研究中，RNA干扰已被视为一种有效的基因沉默方法。本研究旨在构建一种表达靶向小鼠CC趋化因子受体3（mCCR3）的短发夹RNA（shRNA）的慢病毒载体，并研究其对小鼠嗜酸性粒细胞增殖和凋亡的影响。采用亚克隆技术构建了表达小鼠CCR3 shRNA四个片段的重组慢病毒载体（pLVX‑mCCR3‑1+2+3+4‑shRNA）。然后通过与包括杆状病毒p35、pCMV R8.2和VSV在内的质粒共转导，将这种新型慢病毒包装到293T细胞中。使用聚合酶链反应（PCR）和蛋白质印迹分析验证载体的干扰效果。分别使用3‑（4,5‑二甲基噻唑‑2‑基）‑5‑（3‑羧基甲氧基苯基）‑2‑（4‑磺基苯基）‑2H‑四唑和末端脱氧核苷酸转移酶dUTP缺口末端标记法研究干扰对小鼠嗜酸性粒细胞增殖和凋亡的影响。PCR和蛋白质印迹分析结果证实，新型重组载体pLVX‑mCCR3‑1+2+3+4‑shRNA在抑制小鼠嗜酸性粒细胞中mCCR3的mRNA和蛋白质表达水平方面具有高效性。mCCR3的下调显著抑制了嗜酸性粒细胞的增殖。此外，本研究发现mCCR3的下调显著促进了嗜酸性粒细胞的凋亡。因此，mCCR3的下调导致小鼠嗜酸性粒细胞增殖受到抑制并诱导其凋亡。过敏性鼻炎的主要特征是嗜酸性粒细胞浸润和炎症介质释放，表现为多种临床表现。本研究结果表明，沉默mCCR3可能是一种治疗过敏性鼻炎的潜在方法。
