【摘要】 目的:观察流体力学尾静脉注射对绿色荧光蛋白基因器官靶向性的影响,为今后质粒载体的基因治疗和功能研究寻找潜在的靶器官。方法:实验于2005-12/2006-04在江西省分子医学重点实验室完成。选用健康雄性昆明鼠40只,将32只小鼠按随机数字表法分为流体力学注射和常规注射两大组,每大组再分为转染组和对照组两个小组(n=8),并设正常对照组(n=8)。①流体力学转染组将100μg/只绿色荧光蛋白表达质粒溶液2mL在5s内快速注入尾静脉;对照组仅在5s内注入林格氏液2mL。②常规注射组则将2mL林格氏液或绿色荧光蛋白表达质粒溶液在30s左右注入尾静脉。注射结束后24h采集各组小鼠血清检测转氨酶,并采集肝、脾、心、肾、肺和脑组织进行冰冻切片,部分肝组织采用多聚甲醛固定后切片,荧光显微镜下观察。结果:40只小鼠全部进入结果分析,无脱失。①流体力学注射组和常规注射组小鼠血清转氨酶与正常对照组比较差异均无显著性意义(P>0.05)。②常规尾静脉注射引起少数肾小球细胞表达绿色荧光蛋白,而肝、脾、心、肺及脑等组织未见明显绿色荧光蛋白表达。③流体力学注射引起肝内绿色荧光蛋白高水平表达,肝细胞表达率接近45%,其他组织则无绿色荧光蛋白表达。结论:流体力学方法是肝靶向性的活体基因转染方法,绿色荧光蛋白可作为该方法进行目的基因研究的一个可靠和方便的示踪剂。更多还原

【Abstract】 AIM: To investigate the target organ for gene transfection by an intravenous injection of green fluorescent protein (GFP)-expressed plasmid DNA using hydrodynamics-based or regular procedure, so as to search the potential target organ for future gene therapy and function research of plasmid vector.METHODS: The experiment was conducted in the Key Laboratory of Jiangxi Cell Medicine from December 2005 to April 2006. Forty healthy male Kunming mice were selected and randomly divided into hydrodynamics-based injection group, regular injection group and normal control group (n =8), and the former two groups were subdivided into transfection group and control group (n =8). ①2 mL GFP-expressed plasmid (100 μg for each animal) was intravenously injected into the mice of hydrodynamics-based transfection group rapidly in 5 seconds; the control group was only injected with 2 mL Ringer solution in 5 seconds. ②2 mL Ringer solution or GFP-expressed plasmid was injected into the regular injection groups in 30 seconds. Twenty-four hours later, the tissue and blood samples were collected to detect the contents of serum alanine aminotransferase (ALT) and aspartate transaminase (AST). Meanwhile, the expression levels of GFP in liver, spleen, heart, lung, kidney and brain were determined under fluorescent microscope by frozen sections.RESULTS: All 40 mice were involved in the result analysis with no loss. ①Compared with the normal control group, the levels of serum AST of hydrodynamics-based group and regular injection groups had no statistical difference (P > 0.05). ②Regular intravenous injection resulted in GFP expression in a few glomerular cells, while no obvious GFP expression was found in liver, spleen, heart, lung and brain. ③The hydrodynamics-based injection of naked GFP plasmid DNA into mice induced high expression level of GFP gene in liver, and approximately 45% of expression rate was achieved. However, no trace was found in other tissues. CONCLUSION: Hepatic delivery of foreign gene can be accomplished by hydrodynamics-based injection, and GFP is a reliable and convenient tracer agent in this procedure.更多还原