超声联合脂质体微气泡介导反义寡核苷酸转染的实验研究

发布时间：2018-01-02 20:13

本文关键词：超声联合脂质体微气泡介导反义寡核苷酸转染的实验研究　出处：《中国人民解放军军医进修学院》2006年博士论文　论文类型：学位论文

【摘要】：目的:研究超声介导脂质体微气泡造影剂转染反义寡核苷酸的有效性与安全性,探讨超声联合微气泡造影剂增强转染的机制。材料和方法: 1、体外实验:12孔板培养人乳腺癌细胞并分组为:① 单纯超声照射;② 单纯造影剂;③ 单纯反义寡核苷酸HA2741(HA2741);④ 超声照射+HA2741;⑤ 造影剂+HA2741;⑥ 造影剂+HA2741+超声照射;⑦脂质体+HA2741;⑧脂质体+HA2741+超声照射。超声仪探头发射频率1.3MHz,声波发射强度-18.0～0dB,照射时间5～240s,微气泡浓度0.5～30%,反义寡核苷酸HA2741浓度为12～200nmol/L,给予实验刺激后,荧光显微镜动态观察5′-FAM修饰的HA2741的转染率,碘化丙啶PI染色后观察细胞的活性,采用RT-PCR的方法检测HER-2 mRNA的水平,免疫细胞化学检测HER-2蛋白的表达,原子力显微镜(AFM)观察细胞膜形态的改变。 2、体内实验:健康大鼠分为:① 对照组;② 单纯反义寡核苷酸cy5ODN组;③ 造影剂+cy50DN组;④ 超声+cy5ODN组;⑤ 超声+造影剂+cy5ODN组。改变造影剂浓度与超声照射时间,予与实验刺激后,激光共聚焦显微镜检测荧光表达强度,RT-PCR的方法检测CTGF的mRNA的水平,Westem Blot检测CTGF蛋白表达水平,检测血清中GPT、GOT、BUN、Cr水平,以评价该方法的负作用。结果: 1、体外实验:造影剂+HA2741+超声照射组HA2741的转染迅速,转染率显著高于其余各组,约94.6%,是超声照射+HA2741组的22.5倍,造影剂+HA2741组的30.5倍,脂质体+HA2741组的8.3倍;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,细胞活性显著下降。声波发射强度-3.0～6.0dB,照射时间30～60s,细胞转染效率最高。乳腺癌细胞的HER-2 mRNA的水平降低,HER-2蛋白表达下调,其程度与HA2741转染率呈负相关。单纯超声照射组及造影
[Abstract]:Objective: To investigate the efficacy and safety of ultrasound mediated liposome microbubbles in transfection of antisense oligonucleotides, and to explore the mechanism of ultrasound combined with microbubble contrast agent in enhancing transfection.
Materials and methods:
1, in vitro experiments: 12 pore plate cultured human breast cancer cells are grouped as follows: simple ultrasound irradiation; the contrast agent alone; the simple HA2741 antisense oligonucleotide (HA2741); the ultrasound contrast agent +HA2741 +HA2741; 5; the contrast agent +HA2741+ ultrasonic irradiation; the liposome and liposome +HA2741+ + HA2741; ultrasound irradiation. Ultrasound probe frequency 1.3MHz, acoustic emission intensity of -18.0 ~ 0dB, irradiation time was 5 ~ 240s, the micro bubble concentration of 0.5 ~ 30%, HA2741 antisense oligonucleotide concentration is 12 ~ 200nmol/L, experimental stimulation, fluorescence microscope and dynamic modification of HA2741 '-FAM 5 transfection rate, cell viability was iodide propidium PI staining, RT-PCR assay was used to measure the HER-2 levels of mRNA, HER-2 protein expression was detected by immunocytochemistry, atomic force microscopy (AFM) to observe the morphological changes of the cell membrane.
2 in vivo experiment: healthy rats were divided into the control group; the simple antisense oligonucleotide of cy5ODN group; the contrast group +cy50DN and group +cy5ODN; ultrasound; ultrasound contrast agent + +cy5ODN group. The change of contrast agent concentration and ultrasonic irradiation time, to experiment and stimulation, the fluorescence confocal microscopy expression strength, RT-PCR method to detect the CTGF mRNA level, the expression level of CTGF protein was detected by Westem Blot, serum GPT, GOT, BUN, Cr, with a negative effect evaluation of the method.
Result:
1, in vitro: transfection of contrast agent +HA2741+ ultrasound irradiation group HA2741 rapidly, the transfection rate was significantly higher than the other groups, about 94.6%, is 22.5 times of ultrasonic irradiation in +HA2741 group, 30.5 times of contrast agent in +HA2741 group, 8.3 times of liposome group +HA2741; contrast agent concentration of 2%, 5% of the highest transfection efficiency, increase the contrast agent concentration, transfection efficiency, cell activity decreased significantly. The acoustic emission intensity of -3.0 ~ 6.0dB, irradiation time was 30 ~ 60s, the highest transfection efficiency. Breast cancer cells HER-2 reduced levels of mRNA, HER-2 protein expression, the degree and the transfection rate of HA2741 was negatively related to simple ultrasound irradiation group and contrast.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346

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