脂质体介导C-erbB-2反义寡核苷酸转染小鼠乳腺癌TM40D细胞的效率及毒性

The evaluation of the effects of cationic lipid-mediated uptake of C-erbB-2-specific antisense oligonucleotides and toxicity in TM40D cells

目的探讨阳离子脂质体为载体的反义寡核苷酸(ODNs)转染小鼠乳腺癌TM40D细胞的最佳转染效率及对细胞的毒性作用。方法分别将有/无脂质体介导的反义ODNs转染小鼠乳腺癌TM40D细胞,应用流式细胞仪观察不同时间的反义ODNs的转染效率,荧光显微镜观察反义ODNs在细胞内的分布,全自动生化分析仪检测转染培养上清液的乳酸脱氢酶(LDH)浓度。结果无脂质体介导的反义ODNs转染细胞效率随着时间的延长增加,6h达到63.00%。脂质体介导的反义ODNs转染细胞4h时转染效率达到高峰为72.23%。细胞内荧光强度与细胞转染效率相关。无脂质体介导的FAM标记的反义ODNs分布在细胞浆,脂质体介导的反义ODNs在细胞浆及细胞核内均有分布。各组LDH浓度无统计学差异。结论脂质体介导组反义ODNs转染效率高于无脂质体组,同时转染高峰提前出现。脂质体促进反义ODNs进入细胞核内。短时间内阳离子脂质体及反义ODNs对细胞的毒性不明显。

Objective To determine the optimal effect of cationic lipids on oligonucleotides （ODNs） uptake and their toxicity in mouse breast cancer TM40D cells. Methods TM40D cells were incubated with oligonuclotides with or without liposome. The transfection ratios of antisense ODNs in different time were detected by flow cytometry. ODNs distribution in the cells was observed through fluorescence microscopy. Lactate dehydrogenase in cell-culture mediums was examined. Results The transfection ratio of antisense ODNs without liposome gradually increased with time progression, and reached 63 % at 6 h. The transfection ratio of liposome-mediated delivery of ODNs was from 50. 23 to 72.23 %. The time of the optimal effect was at 4h. Intracellular fluorescence intensity correlated with transfection ratio. In the absence of liposome, fluorescence staining in cytoplasm, not in nucleus, was observed. In presence of liposome, the cells showed fluorescence staining in cytoplasm and nucleus. There was no statistical difference about lactate dehydrogenase concentrations among three groups. Conclusion Cationic lipid as a transfection carrier enhanced uptake of ODNs in TM40D cells and the peak of uptake appeared earlier. Cationic lipid and antisense ODNs had no apparent toxicity to the cells during short contact.