干扰P2X_7R基因对RAW264.7细胞增殖和吞噬的影响

Effect of P2X_7R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264. 7

目的:应用RNA干扰技术抑制小鼠巨噬细胞RAW264.7细胞P2X7受体(P2X7R)基因的表达,建立稳定干扰细胞株,并观察其对细胞增殖和凋亡的影响。方法:用脂质体法将P2X7R shRNA重组质粒转染至RAW264.7细胞,经G418筛选后获得稳定干扰细胞株。细胞分为野生型(WT)组、阴性对照(NC)组和干扰(sh P2X7R)组。Real-time PCR法检测细胞中P2X7R mRNA的表达,Western blot检测细胞中P2X7R蛋白的表达;CCK-8方法检测细胞生长活性,5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,Ed U)掺入实验检测细胞增殖活性;流式细胞术分析细胞周期的分布和吞噬情况。结果:P2X7R shRNA能明显抑制RAW264.7细胞的P2X7R mRNA和蛋白的表达,抑制率在80%以上。48 h后,sh P2X7R组细胞的生长速度明显高于NC组和WT组(P<0.05),增殖期细胞比例明显升高(P<0.05),说明下调P2X7R基因能明显促进细胞增殖。sh P2X7R组的细胞周期出现改变,S期和G2/M期的比例明显上升,增殖指数增高(P<0.05)。sh P2X7R组的细胞吞噬活性明显高于NC组(P<0.05)。结论:本研究成功构建了稳定干扰P2X7R基因表达的小鼠巨噬细胞株RAW264.7,sh P2X7R能够明显促进RAW264.7细胞的增殖,改变了细胞的吞噬活性。

AIM： To establish a cell line of stable silencing of P2X7 receptor （P2XTR） expression through short hairpin RNA （shRNA）-mediated interference in murine RAW264. 7 macrophages, and to investigate the proliferation and apoptosis in the celt line. METHODS： Stable silencing of P2X7R gene in the RAW264.7 cells was achieved by re- combinant shRNA plasmid targeting murine P2X7R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2XTR silencing in G418 resistant cells was verified by immunoflaorescent 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 P2XTR at mRNA and protein levels was down-regulated by 80% in shP2XTR group compared with negative control （NC） plasmidtransfection. In addition, P2XTR-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 es- tablished. P2XTR gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264. 7 macro- phages.