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目的探讨家蝇3龄幼虫抗菌肽对K562细胞膜的作用。方法通过针刺感染大肠埃希菌诱导家蝇3龄幼虫大量表达抗菌肽,然后经过研磨、离心、固相萃取、反相高效液相色谱分离纯化家蝇3龄幼虫抗菌肽,采用四甲基偶氮噻唑蓝比色法和光镜观察法筛选对K562有抑制作用的抗菌肽;用不同质量浓度的峰5、峰8处理K562细胞,采用台盼蓝拒染法测定细胞存活率和生长曲线的变化;用光学显微镜和荧光显微镜检测细胞膜形态的变化;用荧光分光光度计检测细胞膜荧光素的渗漏;用激光共聚焦显微镜观察细胞膜的损伤程度。结果低质量浓度(<50μg/ml)对细胞存活率无明显影响,细胞整体形态变化不大,细胞荧光素渗漏较少[(18.95±0.05)～(22.49±0.68)],细胞损伤甚微;高质量浓度(≥50μg/ml)使细胞存活率明显下降,细胞形态变化逐渐增大,细胞荧光素渗漏较多[(62.77±4.08)～(70.81±0.18)],甚至大分子荧光素DextranFITC可以通过。结论家蝇3龄幼虫抗菌肽对K562细胞膜的作用存在质量浓度阈值,低质量浓度时可引起膜通透性增加,高质量浓度时可引起细胞膜严重损伤。家蝇幼虫抗菌肽可能通过直接杀伤作用抑制K562细胞生长,其作用机制首先是通过对膜作用,然后可能进一步对膜作用从而使膜上孔洞不断扩大或者进入细胞后经过其他途径抑制K562细胞生长。
Objective To investigate the effect of antibacterial peptides on the membrane of K562 cells in housefly 3rd instar larvae. Methods The 3rd instar larvae of Housefly were induced by acupuncture to express large quantities of antibacterial peptides. Then the antibacterial peptides of 3rd instar larvae of Musca domestica were isolated and purified by grinding, centrifugation, solid-phase extraction and reverse-phase high performance liquid chromatography. Tetramethyl Azo-thiazolyl blue colorimetric method and light microscopy were used to screen antimicrobial peptides with inhibitory effect on K562 cells. K562 cells were treated with different concentrations of peak 5 and peak 8, and cell viability and growth curve were determined by trypan blue exclusion Changes of cell morphology were observed by light microscope and fluorescence microscope. Fluorescence spectrophotometer was used to detect the leakage of cell membrane fluorescein. The damage degree of cell membrane was observed by laser scanning confocal microscopy. Results Low concentration (<50μg / ml) had no significant effect on the cell viability, with little change in overall cell morphology, less fluorescein leakage [(18.95 ± 0.05) ~ (22.49 ± 0.68)], ; High-quality concentration (≥50μg / ml) significantly decreased cell viability, increased cell morphological changes, and increased leakage of fluorescein (62.77 ± 4.08 ~ 70.81 ± 0.18), even macromolecular fluorescein DextranFITC can be passed. Conclusion The antibacterial peptides of 3rd instar larvae of Musca domestica had a threshold of mass concentration on the membrane of K562 cells. Low mass concentration could increase the permeability of the membrane and cause serious cell membrane damage in high concentration. Musca domestica larvae antibacterial peptides may inhibit the growth of K562 cells through direct killing. The mechanism of action is that the membrane may act on the membrane first and then may further affect the membrane, thereby further expanding the membrane pores or inhibiting the growth of K562 cells after entering the cells.