Krüppel样因子4基因干扰对RAW264.7细胞凋亡和吞噬的影响

Effect of Krüppel like factor 4 gene silencing on apoptosis and phagocytosis of murine RAW264.7 macrophages

目的应用RNA干扰技术沉默小鼠RAW264.7巨噬细胞Krüppel样因子4(KLF4)基因,建立稳定干扰细胞株,观察其对细胞增殖、凋亡和吞噬活性的影响。方法针对小鼠KLF4基因设计合成重组KLF4 shRNA质粒,脂质体法将重组质粒转染至RAW264.7细胞,经G418筛选后获得稳定表达细胞株。Western blot法检测细胞中KLF4蛋白的表达;CCK-8法检测细胞增殖活性;AnnexinV-FITC/PI双染色法检测细胞的凋亡情况;荧光素标记的大肠杆菌结合流式细胞仪检测细胞的吞噬活性。结果 KLF4 shRNA能明显抑制RAW264.7细胞的KLF4蛋白的表达,抑制率76%以上;从第3天开始,shKLF4组细胞的生长速度明显低于NC组(转染阴性质粒pGPU6/GFP/Neo-NC)(P<0.05);WT组(野生型RAW264.7)、NC组和shKLF4组的细胞凋亡率分别为:1.73%、6.85%和12.76%,shKLF4组明显高于NC组(P<0.05);各组细胞的平均荧光强度分别为:WT组(122.0±2.80)、NC组(48.97±5.69)和shKLF4组(80.10±4.61),与NC组相比,shKLF4组的细胞吞噬活性明显增高(P<0.05)。结论成功构建了稳定干扰KLF4基因表达的RAW264.7巨噬细胞株,KLF4下调能够明显抑制RAW264.7细胞增殖,促进细胞凋亡,增强细胞的吞噬活性。

Objective To investigate the effect of down-regulation of KrOppel like factor 4 （KLF4） by RNA interference on proliferation, apoptosis and phagocytosis in murine RAW264.7 macrophage cell line. Methods Stable KLF4 silencing in RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine KLF4 gene via liposome mediated transfection, followed by G418 selection. The efficacy of KLF4 silencing in G418 resistant cells was verified by fluorescent microcopy and Western blotting, respectively. Proliferative activity was analyzed by CCK-8 assay. Apoptosis and phagocytosis were evaluated by annexinV-FITC/PI staining and flow cytometry with assistant use of fluorescein-labeled E. coil K-J2 particles, respectively. Results KLF4 protein expression was significantly down-regulated by the recombinant shRNA plasmid as compared with negative control （NC） plasmid transfection, inhibition rate being over 76%. From the third day, KLF4-sUencing cells exhibited lower proliferative activity as compared with NC RAW264.7 cells （ P 〈 0.05）. In resting state, apoptosis rate in wide type, NC and KLF4-silencing cells were 1.73%, 6.85% and 12.76%, respectively, and KLF4-silencing resulted in a statistical difference in apoptosis as compared with NC cells （P 〈 0.05）. Finally, phagocytic capability in wide type, NC and KLF4-silencing cells revealed by the mean fluorescence intensity of engulfed FITC-labeled E. co/i particles were 122.0 ＋ 2.80, 48.97 ＋5.69 and 80.10 ~4.61, respectively, and compared with NC cells, KLF4-silencing cells had a significant increase in phagocytosis （P 〈 0.05）. Conclusion KLF4 gene silencing inhibits RAW264.7 macrophage proliferation, increase apoptosis and enhance phagocytic function in resting state, which provides a novel tool for revealing the role of KLF4 in macrophage immunity.