重组苦瓜叶蛋白MAP30诱导人食管癌细胞株EC-1.71凋亡的实验研究

Experimental study of recombinant MAP30-induced apoptosis of human esophageal carcinoma EC-1.71 cells

目的探讨用重组苦瓜蛋白MAP30(Momordica anti-HIV protein of 30 ku)诱导人食管癌细胞株EC-1.71细胞凋亡状况。方法 MAP30诱导EC-1.71细胞后,透射电镜观察细胞凋亡的形态特征;JC-1荧光探针检测细胞线粒体膜电位(ΔΨm)变化;ELISA法检测细胞培养上清液中细胞色素c(Cytc)和肿瘤坏死因子-α(TNF-α)的水平;免疫细胞化学法检测细胞ERK1/PERK1,AKT/PAKT和NF-κB表达;荧光微板阅读仪检测细胞caspase-12活性。结果 MAP30诱导可以使EC-1.71细胞固缩,核膜扭曲,核染色质聚集成块并靠近核膜,内质网高度膨胀、扩张;ΔΨm水平下降;Cytc和TNF-α含量增加;胞质中ERK1、AKT表达增强而胞核中PERK1、PAKT表达降低,NF-κB活性降低,caspase-12活性增强。结论 MAP30可以诱导EC-1.71细胞凋亡。

Objective To investigate the effects of recombinant MAP30 （Momordica anti-HIV protein of 30 ku） on apoptosis of human esophageal carcinoma EC-1.71 cells. Methods Following the treatment with MAP30, the uhrastructural and morphologic changes of EC-1.71 cell apoptosis were observed by electron microscope. The level of mitochondria membrane potential （△φm） was displayed by JC-1 fluorescent probe assay. The cytochrome C （Cytc） and TNF-α were determined by ELISA. The expression of ERK1/PERK1, AKT/PAKT and NF-κB protein were showed by immunocytochemical stain. The activity of caspase-12 was measured by fluorometric assay. Results The nuclear fragmentation, chromosome condensation, abnornalities of mitochondrial cristae and shrinkage of the apoptotic cell were visible. The endoplasmic reticulum swelled and expanded. The level of △φm decreased and the contents of Cytc and TNF-α increased in supernatant. The expressions of ERK1 and AKT in cytoplasm increased but PERK1 and PAKT in cell nucleus decreased. NF-KB protein translocated from nucleus to endochylema which was identified by immunocytochemical stain. The increased activity of caspase-12 was showed in time- and dose-dependent manner by fluorometric assay. Conclusion The recombinant MAP30 could induce the EC-1.71 cell apoptosis.