超声介导微气泡造影剂促反义寡核苷酸转染的有效性研究

目的探讨超声介导脂质体微气泡转染反义寡核苷酸HA-2741的有效性。方法12孔板培养人乳腺癌细胞,分组为:(1)单纯超声照射;(2)单纯造影剂;(3)单纯HA-2741;(4)超声照射 HA-2741;(5)造影剂 HA-2741;(6)造影剂 HA-2741 超声照射;(7)脂质体 HA-2741;(8)脂质体 HA-2741 超声照射。造影剂浓度2%,超声波发射频率1.3MHz,强度-3dB,照射时间30s,予与实验刺激后,流式细胞仪定量分析HA-2741的转染率,RT-PCR的检测乳腺癌细胞的HER-2mRNA水平,免疫细胞化学检测HER-2蛋白的表达。结果造影剂 HA-2741 超声照射组HA-2741的转染率显著高于其余各组,约94.6%,造影剂 HA-2741 超声照射组乳腺癌细胞的HER-2mRNA水平显著降低,HER-2蛋白表达明显减低,其程度与HA-2741转染率呈正相关。结论超声介导微气泡造影剂显著增加基因的转染效率,方法简便,安全,具有良好的靶向性。

Effects of Ultrasound Mediated Microbubbles in the Transfection of Antisense Oligonucleotides

Objective To explore the efficiency of transfection of antisense oligonucleotides HA-2741 by ultrasound mediated liposome microbubbles.Methods Cultured human breast cancer cells in 12 holes plate were divided into the following eight methods of treatment separately.(1)ultrasound irradiation alone,(2)contrast agent alone,(3)HA-2741 alone,(4)ultrasound irradiation and HA-2741,(5)contrast agent and HA-2741,(6)contrast agent and HA-2741 and ultrasound irradiation,(7)liposome and HA-2741,(8)liposome and HA-2741 and ultrasound irradiation.The concentration of contrast agent was 2 %.The emission frequency of the ultrasound probe is 1.3 MHz and sound wave intensity of-3 dB and exposure time of 30 s.After treatment,the flow cytometry was used to dynamically assay the transferring rate of HA-2741.RT-PCR was used to detect the contents of HER-2 mRNA and immuno-histochemical staining was used to detect the expression of HER-2 protein.Results The highest transefection efficiency of HA-2741was 94.6 % in group 6.The contents of HER-2 mRNA and the expression of HER-2 protein of breast cancer cells decreased in positive correlation with HA-2741 transfection.Conclusions Ultrasound mediated microbubbles can be a new technique to markedly increase the gene transfection.It has good targeting property and is safe and easy to use.