家蝇3龄幼虫抗肿瘤肽促K562细胞凋亡作用及其机制研究

目的观察家蝇3龄幼虫抗肿瘤肽对K562细胞核和线粒体膜电位及K562细胞凋亡蛋白半胱天冬氨酸蛋白酶-3(caspase-3)的影响,进而探讨家蝇3龄幼虫抗肿瘤肽是否可以影响线粒体膜电位及促进K562细胞凋亡。方法首先采用Hoechst33258荧光标记法,将家蝇3龄幼虫峰5、峰8组分作用于K562细胞,用荧光显微镜检测K562细胞核荧光强度的变化;其次采用荧光探针罗丹明123标记K562细胞,在激光扫描共聚焦显微镜下观察K562细胞中罗丹明123荧光强度,以细胞内荧光强度表示线粒体膜电位大小;最后用caspase-3检测试剂盒和酶标仪测定K562细胞caspase-3的含量。结果采用Hoechst33258荧光标记法,用荧光显微镜观察到家蝇3龄幼虫峰5、峰8组分作用于K562细胞后部分细胞核的荧光强度增强,说明其可以引起K562细胞发生凋亡;用罗丹明123标记,激光共聚焦显微镜观察发现,家蝇3龄幼虫峰5、峰8组分作用组的K562细胞荧光强度值明显低于对照组(t1=21.30,t2=196.23,P<0.05),说明家蝇3龄幼虫抗肿瘤肽可以使K562细胞线粒体膜电位降低;峰5、峰8组分作用组caspase-3的含量明显高于对照组(t1=146.92,t2=189.56,P<0.05),说明3龄幼虫抗肿瘤肽可以促进K562细胞发生凋亡。结论家蝇3龄幼虫抗肿瘤肽峰5、峰8组分对K562细胞核具有损伤作用,可以引起K562细胞发生凋亡,作用机制是通过降低K562细胞线粒体膜电位,激活caspase-3,扰乱其生理功能,促使其凋亡。

The apoptosis-promoting effect of the anti-tumor peptides from Musca domestica larvae on K562 cells and its mechanism

Objective To observe the effect of anti-tumor peptide from Musca domestica larvae on the nucleus,the mitochondrial membrane potential and apoptosis protein cysteinyl aspartic proteinase-3(caspase-3),and apoptosis of K562 cells.Methods Hoechst33258 fluorescent staining was used to observe the fluorescence intensity under a fluorescence microscope.The peak 5 and 8 components from M.domestica larvae were incubated with the K562 cells and the fluorescence intensity of the K562 cell nuclei was detected.The fluorescent probe rhodamine 123 was used to label the cells,which were observed under LSCM(laser scanning confocal microscope,LSCM) for the intracellular fluorescence intensity of the dye that reflected the mitochondrial membrane potential.And caspase-3 immuosorbent test kit and microplate were used to detect the enzyme activity of caspase-3 in K562 cells.Results It was found that the peak 5 and 8 components from M.domestica larvae,after incubation with the K562 cells,increased the fluorescence intensity of some K562 nuclei,suggesting the occurrence of apoptosis of K562 cells.The fluorescence intensity of the K562 cells incubated with the peak 5 and 8 components from M.domestica larvae and labeled with rhodamine 123 was significantly lower than that of the cells in the control group(t1=21.30,t2=196.23,P<0.05),which indicated that the anti-tumor peptide was able to reduce the mitochondrial membrane potential of K562 cells.Also,the content of caspase-3 in the cells in the test group was significantly higher than that in the cells in the control group(t1=146.92,t2=189.56,P<0.05),which suggested that the antitumor peptide could contribute to apoptosis of the K562 cells.Conclusion The anti-tumor peptides in peak 5 and 8 components can damage the cell nucleus,leading to apoptosis of K562 by reducing the mitochondrial membrane potential,activating the caspase-3,and interfering with the physiological function of the cells.