【摘要】 目的观测PTD4-GFP-VP3融合蛋白诱导人肝癌细胞HepG₂的凋亡效应及其在肿瘤细胞中的定位与其凋亡效应关系的研究。方法构建PTD4-GFP-VP3融合蛋白的原核表达载体,利用人源肝癌细胞株HepG₂,经细胞透膜实验在激光共聚焦显微镜下观察PTD4介导的融合蛋白透膜效应,DAPI染色观测该蛋白在细胞中的定位;利用TUNEL法观察PTIM-GFP-VP3融合蛋白诱导肿瘤细胞的凋亡效应,并利用流式细胞仪检测其凋亡率。结果PTD4-GFP-VP3融合蛋白在孵育细胞0.5h后即可透过细胞膜进入到细胞质中,12h后定位于细胞核并诱导肿瘤细胞凋亡,48h达到最高凋亡效应。结论PTD4能携带GFP-VP3融合蛋白穿透细胞膜,在肿瘤细胞中具有核定位效应,并能诱导肿瘤细胞凋亡,为后期进一步研究VP3的凋亡机制及其抗肿瘤治疗奠定了基础。更多还原

【Abstract】 Objective To observe the location of fusion protein PTD4-GFP-VP3 in human hep- atocarcinoma cell line HepG2,and to study the effect of vp3-induced apoptosis of tumor cells. Methods The prokaryotie expression vector of PTD-GFP-VP3 was constructed.The cytomembrane- penetrating effect and location of fusion protein PTD4-GFP-VP3 in tumor cells killed by DAPI were observed under confocal microscope.The PTD4-GFP-VP3-induced apoptosis of tumor cells in vitro was tested by TUNEL assay and flow cytometer.Results The fusion protein PTD4-GFP-VP3 could penetrate eytomembrane 30 minutes after cuhurey,then the protein moved from cytoplasm to nucle- us,and finally induced apoptosis after 12 hs.The rate of apoptosis was highest at the 48th h.Con- clusion PTD4 can carry fusion protein GPF-VP3 into cells,have the nucleic localization effect and can induce apoptosis of tumor cells.This will provide a basis for the further study of apoptosis and the anti-tumor therapy with VP3.更多还原