家蝇幼虫抗菌肽对K562细胞膜的质量浓度阈值作用

Mass concentration threshold of Musca domestica larvae antimicrobial peptides in K562 cell membrane

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 Musca domestica larva antimicrobial peptides on the cell membrane of tumor cells K562.Methods Antimicrobial peptides of M.domestica larvae with inhibition activity to K562 were sieved,through some processes that induced by acupuncture with Escherichia coli induction,isolated and purified by trituration,centrifuga1ization,solid phase extraction（SPE） and reversed-phase high-performance liquid chromatography（RP-HPLC）,detected activity by MTT colorimetric method and observed light microscope.After treatment with different concentrations of peaks 5 and 8,the viability,morphological changes,morphological changes,membrane fluorescein leakage and destruction of K562 cells were detected by trypan blue excluding test,optical microscope and fluorescence microscope,spectrofluorophotometer,laser confocal microscope（LSCM）,respectively.Results Low concentrations（50 μg/ml） of M.domestica antimicrobial peptides resulted in intact viability,little change of cellular structure,a little of fluorescein leakage [（18.95±0.05）-（22.49±0.68）] and few destruction in K562 cells;however,high concentrations（≥50 μg/ml） of M.domestica antimicrobial peptides induced lower viability,cellular form change increased gradually,a great deal of fluoresceinleakage [（62.77±4.08）-（70.81±0.18）],even macromolecular fluorescent element Dextran-FITC could leak through.Conclusion There is a concentration threshold of M.domestica larvae antimicrobial peptides on K562 cell membrane.Low concentrations of M.domestica larvae antimicrobial peptides can cause the membrane leakage increasing of cell membrane,high concentrations can lead to severe damage of cell membrane,respectively.The antimicrobial peptides inhibit the growth of K562 cells through directly killing the cells,and the mechanism is that firstly damage the membrane function,then may expand the holes or inhibit the growth by other ways after entering the cells.