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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 silence the KLF4 gene of murine RAW264.7 macrophages by RNA interference and establish a stable interfering cell line to observe the effect of KLF4 on proliferation, apoptosis and phagocytosis. Methods Recombinant KLF4 shRNA plasmid was designed and synthesized based on the mouse KLF4 gene. The recombinant plasmids were transfected into RAW264.7 cells by lipofectamine. The stable cell line was obtained after screening by G418. The expression of KLF4 protein in cells was detected by Western blot, the cell proliferation activity was detected by CCK-8 assay, the apoptosis was detected by Annexin V-FITC / PI double staining, the phagocytosis of cells was detected by fluorescein-labeled E.coli combined with flow cytometry active. Results KLF4 shRNA significantly inhibited the expression of KLF4 protein in RAW264.7 cells with the inhibitory rate of over 76%. From day 3, the growth of shKLF4 group was significantly lower than that of NC group (transfected with negative plasmid pGPU6 / GFP / Neo- NC group and shKLF4 group were 1.73%, 6.85% and 12.76%, respectively, which were significantly higher in shKLF4 group than in NC group (P <0.05) 0.05). The average fluorescence intensity of each group was 122.0 ± 2.80 in WT group, 48.97 ± 5.69 in NC group and 80.10 ± 4.61 in shKLF4 group. Compared with NC group, the phagocytic activity of shKLF4 group was obvious Increased (P <0.05). Conclusion The RAW264.7 macrophage cell line that stably interferes with the expression of KLF4 gene was successfully constructed. The down-regulation of KLF4 can significantly inhibit the proliferation of RAW264.7 cells, promote the apoptosis and enhance the phagocytic activity of RAW264.7 cells.