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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。
OBJECTIVE: To study the effect of inhibiting the P2X7 receptor (P2X7R) gene expression in murine macrophage RAW264.7 cells by RNA interference and to establish a stable interfering cell line and to observe its effect on cell proliferation and apoptosis. Methods: P2X7R shRNA recombinant plasmids were transfected into RAW264.7 cells by lipofectamine. After G418 selection, stable interfering cell lines were obtained. Cells were divided into the wild-type (WT) group, the negative control (NC) group and the sh P2X7R group. The expression of P2X7R mRNA was detected by Real-time PCR and the expression of P2X7R protein by Western blot. The cell growth activity was detected by CCK-8. The expression of 5-ethynyl-2 ’ -deoxyuridine, Ed U) were added to test the cell proliferation activity; flow cytometry analysis of cell cycle distribution and phagocytosis. Results: P2X7R shRNA significantly inhibited P2X7R mRNA and protein expression in RAW264.7 cells with the inhibition rate above 80%. After 48 h, the growth rate of P2X7R group was significantly higher than that of NC group and WT group (P <0.05), and the proportion of cells in proliferating phase was significantly increased (P <0.05), indicating that down-regulation of P2X7R gene could significantly promote cell proliferation. The cell cycle of P2X7R group changed, the proportion of S phase and G2 / M phase increased obviously, and the proliferation index increased (P <0.05). The phagocytic activity of P2X7R group was significantly higher than that of NC group (P <0.05). Conclusion: The mouse macrophage cell line RAW264.7 stably interfering with the expression of P2X7R gene was successfully constructed and sh P2X7R could significantly promote the proliferation of RAW264.7 cells and change the phagocytic activity of the cells.