Effect of P2X7 R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7 / 中国病理生理杂志

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 P2X7 R gene in the RAW264.7 cells was achieved by re-combinant shRNA plasmid targeting murine P2X7 R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X7 R silencing in G418 resistant cells was verified by immunofluorescent micros-copy and real-time PCR, respectively.The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation as-say.The cell cycle distribution and apoptosis were evaluated by flow cytometry.RESULTS:The expression of P2X7 R at mRNA and protein levels was down-regulated by 80% in shP2X7 R group compared with negative control ( NC) plasmid transfection.In addition, P2X7 R-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 P2X7 R expression was successfully es-tablished.P2X7 R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macro-phages.