半乳糖受体介导的c-myc反义寡核苷酸对人肝癌细胞的靶向投递作用

背景与目的:c-myc反义寡核苷酸(antisenseoligodeoxynucleotide,ASODN)能抑制肝癌细胞的增殖,脂质体和腺病毒介导的c-mycASODN投递虽能显著抑制肝癌细胞的增殖,抑制荷肝癌小鼠肿瘤的生长,但这种投递缺乏靶向性;受体介导的药物投递具有高效性和靶向性的特点,已被用于基因及ASODN的靶向投递。本实验以半乳糖(galactose,Gal)-聚乙烯亚胺(polyethyleneimine,PEI)作为载体,介导c-mycASODN作用于人肝癌细胞Bel-7402,探讨c-mycASODN对Bel-7402细胞的靶向投递作用。方法:用流式细胞仪检测Bel-7402、U937细胞对荧光标记的Gal-PEI-c-myc-ASODN(简称Gal-PEI-ASODN)的摄取率及细胞内平均荧光强度;相差/荧光显微镜观察荧光标记的Gal-PEI-ASODN及c-myc-ASODN进入Bel-7402、U937、Raji细胞的形态;台盼蓝拒染法检测不同浓度Gal-PEI-ASODN对这三种细胞增殖的影响。结果:荧光标记的Gal-PEI-ASODN或ASODN与相应细胞孵育10min到4h,Gal-PEI-ASODN-Bel-7402细胞的荧光摄取率为88.25%～98.66%,细胞内平均荧光强度为38.64%～111.90%,与单纯c-mycASODN-Bel-7402细胞组、Gal-PEI-ASODN-U937细胞组相比,所有时间点的细胞荧光摄取率、胞内平均荧光强度均显著增高,经Poission分布检验,均有显著性差异(P<0.01)。相差/荧光显微镜观察到

Targeting delivery effect of galactose receptor-mediated c-myc antisense oligonucleotide on human hepatocellular carcinoma cell line Bel-7402

BACKGROUND & OBJECTIVE: Deliveries of c-myc antisense oligonucleotide (ASODN) mediated by liposome or adenvirus could suppress proliferation of human hepatocellular carcinoma cells, and tumor growth of mice hepatoma model. However, these deliveries lack targeting effects. Receptor-mediated drug delivery is being used in gene and ASODN delivery due to its high targeting efficiency. This study was to evaluate targeting delivery effect of c-myc ASODN mediated by galactose-polyethyleneimine (Gal-PEI) on human hepatocellular carcinoma cell line Bel-7402. METHODS: Bel-7402, and lymphoma cell lines, U937 and Raji, were cultured with fluorescence-labeled Gal-PEI-c-myc ASODN, and c-myc ASODN. The uptaking rates of Gal-PEI-c-myc ASODN, and intracellular mean fluorescence intensities of Bel-7402 and U937 cells were tested by flow cytometry. Morphology of Gal-PEI-c-myc ASODN and c-myc ASODN entering Bel-7402, U937, and Raji cells was observed under phase-contrast fluorescence microscope. Effects of Gal-PEI-c-myc ASODN of various concentrations on proliferation of these cells were detected by trypan blue dye method. RESULTS: After cultured for 10 min to 4 h, the uptaking rate, and intracellular mean fluorescence intensity of Gal-PEI-c-myc ASODN cultured Bel-7402 cells (88.25%-98.66%, and 38.61%-111.9%) were higher than those of c-myc ASODN cultured Bel-7402 cells, and Gal-PEI-c-myc ASODN cultured U937 cells, significant differences were detected by Poisson test (P < 0.01). Observed by phase-contrast fluorescence microscope, Gal-PEI-c-myc ASODN entered Bel-7402 cells effectively, but can't enter U937, and Raji cells effectively at the same concentration. C-myc ASODN can't enter Bel-7402 cells effectively at the same ASODN concentration. After incubation with Gal-PEI-c-myc ASODN (containing 0.25-1.25 micromol/L of c-myc ASODN) for 48 h, proliferation of Bel-7402 cells was significantly inhibited (P < 0.01=, but no significant differences were detected in U937, and Raji cells (P >0.05). CONCLUSION: Gal-PEI-c-myc ASODN has high targeting delivery effect on Bel-7402 cells, which enhances the intercellular concentration of c-myc ASODN effectively, but it has no such effects on U937, and Raji cells.