Effect of P2X7R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7 |
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Authors: | SU Cheng-cheng ZHANG Yi-dan MA Yong-qiang CHEN Xue-fen XIANG Guo-an ZHOU Xin PENG Shou-chun LIN Zhi-chun WEI Lu-qing JI Wen-jie |
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Institution: | 1. Department of Respirology and Critical Care Medicine, Logistics University of Chinese People's Armed Police Forces, Tianjin 300162, China;
2. Tianjin Key Laboratory of Cardiovascular Remodeling and Target Organ Injury Institute of Cardiovascular Disease and Heart Center Tianjin 300162, China;
3. Hebei Medical University, Shijazhuang 050017 China |
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Abstract: | AIM: To establish a cell line of stable silencing of P2X7 receptor (P2X7R) expression through short hairpin RNA (shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line. METHODS: Stable silencing of P2X7R gene in the RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine P2X7R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X7R silencing in G418 resistant cells was verified by immunofluorescent microscopy and real-time PCR, respectively. The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation assay. The cell cycle distribution and apoptosis were evaluated by flow cytometry. RESULTS: The expression of P2X7R at mRNA and protein levels was down-regulated by 80% in sh P2X7R group compared with negative control (NC) plasmid transfection. In addition, P2X7R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05). Compared with NC cells, P2X7R silencing resulted in an increase in the phagocytosis of the cells (P<0.05). CONCLUSION: A cell line RAW264.7 of stable silencing of P2X7R expression was successfully established. P2X7R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macrophages. |
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Keywords: | P2X7 receptor RNA interference RAW264 7 macrophages Cell proliferation Cell phagocytosis |
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