超声空化脂质体微气泡促反义寡核苷酸转染的体外实验研究

Ultrasound Activation of Liposome Microbubbles Enhances the Transfer of Antisense Oligonucleotides in Vitro

目的:探讨超声空化脂质体微气泡转染反义寡核苷酸HA2 741的有效性与安全性。材料和方法:12孔板培养人乳腺癌细胞并分为:(1)单纯超声照射;(2 )单纯造影剂;(3 )单纯HA2 741;(4 )超声照射+HA2 741;(5 )造影剂+HA2 741;(6)造影剂+HA2 741+超声照射;(7)脂质体+HA2 741;(8)脂质体+HA2 741+超声照射。声波发射强度-18.0～0dB ,照射时间5～2 40s ,给予实验刺激后,荧光显微镜观察HA2 741的转染率及细胞的活性,免疫组化检测HER -2蛋白的表达。结果:第(6)组HA2 741转染率显著高于其余各组,约94.6% ,造影剂浓度2 %、5 %时转染效率最高,提高造影剂浓度,转染效率无改变,但细胞活性显著下降。声波发射强度-3 .0～-6.0dB ,照射时间3 0～60s ,细胞转染效率最高。乳腺癌细胞的HER -2蛋白表达下调,其程度与HA2 741转染率呈正相关。结论:超声空化微气泡造影剂显著增加了基因的转染,安全、简便具有良好的靶向性。

Purpose: To explore the efficiency and safety of transferring of antisense oligonucleotides by ultrasound activation of liposome microbubbles. Materials and Methods: Cultured human breast cancer cells in 12 holes plate were grouped to receive the following eight treatment separately. (1)ultrasound irradiation alone; (2)contrast agent alone; (3)HA2741 alone; (4)ultrasound irradiation and HA2741; (5)contrast agent and HA2741; (6) contrast agent and HA2741 and ultrasound irradiation; (7)liposome and HA2741; (8)liposome and HA2741and ultrasound irradiation. The ultrasound machine was set sound wave intensity of -18.0dB～0dB and exposure time of 5～240s. After experimental treatment, the fluorescence microscope was used to observe the transferring rate of HA2741 and cell activity after PI staining. And immuno-histochemical staining was used to detect the expression of HER-2 protein. Results: The highest transfection rate of HA2741 is 94.6% in group 6 at 2 % and 5% concentration of contrast agent. The increased contrast agent would lead to decreased cellular activity. The transefection efficiency is highest with emission intensity of -3.0dB～-6.0dB and exposure time of 30～60s. The HER-2 expression of breast cancer cells decreased in positive correlation with HA2741 transfection. Conclusion: Ultrasound activation of microbubbles contrast agent can markedly increase the transfection of genes and it has good target orientation with its safety and simplicity.