摘要
目的本研究采用靶向表皮生长因子受体(EGFR)的单克隆抗体对磁性纳米基因载体颗粒进行靶向修饰后进行针对肝癌的基因治疗,观察其对肝癌细胞HepG2的基因治疗效果。方法制备靶向EGFR的SPIO磁性纳米基因载体。对肝癌细胞HepG2进行EGFR免疫荧光染色证明其在细胞膜上的表达。以Lipofectamine^(TM) 2000推荐质粒用量的1/20进行分组转染,EGFR-Ab-Poly MAG100基因载体组作为(EP)组;Lipofectamine^(TM) 2000基因载体组作为(LF)组;未转染空白对照细胞作为(NC)组。荧光显微镜观察纳米造影剂的细胞内吞效果。荧光实时定量PCR检测目的基因mRNA表达。western blot法进行蛋白表达鉴定。四甲基偶氮唑盐法检测基因治疗效果。HCCR-2 mRNA、HCCR-2蛋白和Ki67蛋白表达水平数据、A值的比较采用单因素方差分析和t检验。结果免疫荧光法染色观察发现,肝癌HepG2细胞膜高表达EGFR蛋白。磁性纳米基因载体有效链接EGFR抗体。EP组细胞中质粒大量进入HepG2细胞,质粒内吞量大于LF组和NC组。EP组HCCR2 mRNA的相对表达量为(11.25±0.23)%,相对表达量明显低于NC组,差异具有统计学意义(t=23.57,P=0.00)。EP组HCCR2蛋白的相对表达量为(0.13±0.02),明显低于NC组的0.62±0.05,差异具有统计学意义(t=29.31,P=0.00)。EP组Ki67蛋白的相对表达量为(0.22±0.04),明显低于NC组的(0.83±0.07),差异具有统计学意义(t=18.88,P=0.00)。转染24 h、48 h后,EP组的A值明显低于NC组,差异具有统计学意义(t=-22.02,-34.73,P=0.00,0.00)。结论采用EGFR单克隆抗体修饰后的磁性纳米基因载体颗粒,可以实现对肝癌细胞的高效基因治疗,进而实现对肝癌细胞的增殖抑制。
Objective This study used epidermal growth factor receptor （EGFR） monoclonal antibody to deliver magnetic nanoparticles as gene carrier to HepG2 cells. Methods SPIO magnetic nanoparticles with EGFR Ab were prepared. The expression of EGFR in hepatocellular carcinoma cell line HepG2 was demonstrated by immunofluorescence staining. LipofectamineTM 2000 was used to transfect EGFR-Ab-PolyMAG100 gene vector （EP group）, LipofectamineTM 2000 gene vector （LF group） and blank vector （NC group）. The endocytosis effect of contrast agent was observed with a fluorescence microscope. Fluorescent real time quantitative PCR was used to detect target gene mRNA expression. Protein expression was evaluated by western blot. Cell proliferation was detected by MTT method. MRNA HCCR-2, HCCR-2 and Ki67 protein expression levels were compared by ANOVA and t test. Results The expression of EGFR in the HepG2 cell membrane was observed by immunofluorescence staining. Magnetic nanoparticles were effectively linked to EGFR antibody. The relative expression of mRNA HCCR2 in the EP group was （11.25±0.23） %, significantly lower than that of the NC group （t = 23.57, P = 0.00）. The relative expression amount of HCCR2 protein in the EP group was （0.13± 0.02）, which was significantly lower than that of the NC group （0.62±0.05） （t =29.31, P = 0.00）. The relative expression of Ki67 protein in the EP group was （0.22±0.04）, which was significantly lower than that of the NC group （0.83 ±0.07） （t =18.88, P =0.00）. At 24 h and 48 hours after transfection, the A value of the EP group was significantly lower than that of the NC group （t = -22.02, -34.73, P = 0.00, 0.00）. Conclusions In summary, using EGFR monoclonal antibody modified magnetic nanoparticle particles; gene therapy for hepatocellular carcinoma cells can be achieved.
出处
《中华细胞与干细胞杂志（电子版）》
2016年第4期215-220,共6页
Chinese Journal of Cell and Stem Cell（Electronic Edition）
基金
国家自然科学基金(81201918)
广东省科技计划项目(2012B031800099)
关键词
纳米结构
基因疗法
受体
表皮生长因子
肝肿瘤
Nanostructures
Gene therapy
Receptor, Epidermal growth factor
Liver neoplasms
