巨噬细胞冠蛋白-1 siRNA对小鼠巨噬细胞吞噬功能的影响

Effect of Coronin-1 siRNA on Phagocytosis of Mouse Macrophages

巨噬细胞冠蛋白-1(Coronin-1)与结核分枝杆菌(Mycobacterium tuberculosis,Mtb)逃逸免疫杀伤有关。该研究探讨了p S-EGFP-SP-Coronin-1si RNA重组质粒靶向抑制巨噬细胞Coronin-1表达后对小鼠巨噬细胞吞噬功能的影响。用该质粒转染小鼠巨噬细胞系RAW264.7,采用RT-PCR法和Western blot检测质粒转染前后细胞Coronin-1的表达水平。以耻垢分枝杆菌(Mycobacterium smegmatis)分别感染转染质粒组细胞和对照组细胞,通过细胞内细菌菌落计数和细胞爬片抗酸染色法评估巨噬细胞转染质粒前后对细菌的吞噬能力;利用流式细胞术检测转染质粒组细胞及对照组细胞吞噬耻垢分枝杆菌后不同时段的细胞凋亡水平。结果显示,该质粒能显著抑制RAW264.7细胞Coronin-1的m RNA水平及蛋白表达水平;感染耻垢分枝杆菌6 h后,转染质粒组细胞内细菌数显著高于对照组细胞(P<0.05);感染耻垢分枝杆菌48 h后,转染质粒组细胞凋亡水平显著高于对照组细胞(P<0.05)。以上结果表明,p S-EGFP-SP-Coronin-1si RNA重组质粒能显著抑制巨噬细胞Coronin-1的表达,并可显著促进巨噬细胞吞噬细菌和凋亡杀菌,为开发靶向巨噬细胞Coronin-1的抗结核基因治疗措施奠定基础。

Coronin-1 of macrophage is associated with the escaping immune killing of Mycobacterium tuberculosis（Mtb）. This study was aimed to investigate the effect of p S-EGFP-SP-Coronin-1 si RNA plasmid on phagocytosis of mouse macrophages. The RAW264.7 cells were transfected with the plasmid, and the expression level of Coronin-1 in macrophages before and after plasmid transfection was detected by RT-PCR and Western blot. After the plasmid transfection group cells and the control group cells were infected with Mycobacterium smegmatis respectively, the ability of phagocytosis of macrophages was evaluated by colony count of intracellular bacteria and acid fast staining of cells on cover slips, and the apoptosis level of the plasmid transfection group cells and the control group cells after phagocytosis of Mycobacterium smegmatis was detected by the flow cytometry. The results showed that the plasmid can significantly inhibit the m RNA level and protein expression of Coronin-1 in RAW264.7 cells. The number of intracellular bacteria in the plasmid transfection group cells was significantly higher than that of the control group cells after infection with Mycobacterium smegmatis for 6 h（P〈0.05）. The apoptosis level in the plasmid transfection group cells were significantly higher than that of the control group cells after infection with Mycobacterium smegmatis for 48 h（P〈0.05）. These results indicated that the p S-EGFP-SPCoronin-1 si RNA plasmid can significantly inhibit the expression of Coronin-1 in macrophages, and significantly promote phagocytosis of bacteria and the bactericidal function through apoptosis of macrophages, which lay a foundation for the development of anti-tuberculosis gene therapy targeting macrophage Coronin-1.