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目的观察经静脉注射基因与微泡声学造影剂,同时经皮超声辐照肝脏方式能否增强小鼠肝脏基因定位转染及其转染效率。方法昆明种小白鼠24只,随机分为4组,每组6只。第1组为单纯质粒转染组:经尾静脉快速注入2ml含20μg乙型肝炎核心抗原(pcDNA3.1/HBV)质粒的生理盐水溶液。第2组为单纯微泡转染组:经尾静脉快速注入2ml含20μg质粒的微泡声学造影剂。第3组为单纯超声转染组:经尾静脉快速注入2ml含20μg质粒的生理盐水溶液,同时采用频率为1MHz、声强为0.5W/cm2的超声波经小白鼠体表肝区辐照1min。第4组为超声与微泡转染组:经尾静脉快速注入2ml含20μg质粒的微泡声学造影剂,同时经小白鼠体表肝区局部采用相同剂量超声辐照1min。7天后处死小鼠,分别取肝、肾、肺、心、脾、骨骼肌组织进行pcDNA3.1/HBV免疫组化检测,记录不同组织每高倍视野(×400)pcDNA3.1/HBV表达阳性细胞数。结果第1、2组肝、肾组织内偶见pcDNA3.1/HBV表达弱阳性细胞,肺、心、脾、骨骼肌组织未见表达阳性细胞。第3组肝组织内可见少量pcDNA3.1/HBV阳性细胞,每高倍视野表达阳性细胞(26.5±3.9)个。肾、肺、心、脾、骨骼肌组织未见表达。第4组肝组织内pcDNA3.1/HBV表达最强,每高倍视野表达阳性细胞(84.2±4.4)个,为第3组的约3.2倍。肾、肺、心、脾、骨骼肌组织未见表达。结论静脉注射黏附质粒的微泡声学造影剂同时经体表给予一定强度的超声辐照,能显著增强辐照部位局部组织的基因转染与表达,有望成为一种安全、高效的体内基因定位转染新技术。
Objective To observe whether intravenous injection of gene and microbubble acousto-contrast agent and simultaneous transdermal ultrasound irradiation of the liver can enhance the liver gene transfection and transfection efficiency in mice. Methods 24 Kunming mice were randomly divided into 4 groups with 6 rats in each group. Group 1 was a plasmid-transfected group: 2 ml of normal saline solution containing 20 μg of hepatitis B core antigen (pcDNA3.1 / HBV) plasmid was rapidly injected through tail vein. Group 2 was a simple micro-bubble transfection group: 2 ml of microbubble acousto-contrast agent containing 20 μg of plasmid was rapidly injected via tail vein. Group 3 was a simple ultrasound transfection group: 2ml saline solution containing 20μg plasmids was rapidly injected through the caudal vein, and irradiated by ultrasonic wave of 0.5W / cm2 at frequency of 1MHz for 1min. Group 4 was transfection with ultrasound and microbubbles: 2ml microbubble acoustic contrast medium containing 20μg of plasmid was injected into the caudal vein rapidly, and the same dose of ultrasound was applied to the liver of mice for 1min. After 7 days, the mice were sacrificed and the liver, kidney, lung, heart, spleen and skeletal muscle tissues were harvested for immunohistochemical detection of pcDNA3.1 / HBV, and the positive cells (pcDNA3.1 / HBV positive) number. Results The positive cells of pcDNA3.1 / HBV were weakly expressed in the first and second groups of liver and kidney, but no positive cells were found in lung, heart, spleen and skeletal muscle. A small amount of pcDNA3.1 / HBV positive cells were observed in Group 3 liver tissues, with 26.5 ± 3.9 cells in each high power field. Kidney, lung, heart, spleen, skeletal muscle tissue was not expressed. The expression of pcDNA3.1 / HBV in group 4 was the strongest, and the number of positive cells per high power field was (84.2 ± 4.4), which was about 3.2 times of that in group 3. Kidney, lung, heart, spleen, skeletal muscle tissue was not expressed. Conclusion Intravenous injection of the microbubble-associated acoustic contrast medium can give a certain intensity of ultrasound irradiation on the surface of the body, which can significantly enhance the gene transfection and expression in the local tissues of the irradiation site. It is expected to be a safe and efficient gene localization transfer in vivo Dyeing new technology.