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首页> 外文期刊>Neurochemistry International: The International Journal for the Rapid Publication of Critical Reviews, Preliminary and Original Research Communications in Neurochemistry >Adeno-associated virus mediated gene transfer into primary rat brain neuronal and glial cultures: enhancement with the pH-sensitive surfactant dodecyl 2-(1'-imidazolyl) propionate.
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Adeno-associated virus mediated gene transfer into primary rat brain neuronal and glial cultures: enhancement with the pH-sensitive surfactant dodecyl 2-(1'-imidazolyl) propionate.

机译：腺相关病毒介导的基因转移到原代大鼠大脑神经元和神经胶质培养物中：用pH敏感的表面活性剂十二烷基2-（1'-咪唑基）丙酸酯增强。

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摘要

This study evaluated the effects of a novel, pH-sensitive surfactant, dodecyl 2-(1'-imidazolyl) propionate (DIP), on cationic lipid mediated transfection in primary rat brain neuronal and glial cultures. The cationic lipid complex DOTAP/DOPE (1, 2-dioleoyl-3-trimethylammonium propionate and dioleoyl phosphatidylethanolamine, respectively) was added over a range of concentrations (0-120 microg/ml) with DNA concentration kept constant (1.6 microg/ml). The neuron-specific enolase (NSE) and cytomegalovirus (CMV) promoters were found to drive green fluorescent protein (GFP) expression in neuron-enriched and glial cultures, respectively, using adeno-associated virus (AAV) derived constructs. NSE-driven GFP expression was not observed in glial cultures. Addition of DOTAP/DOPE increased transfection efficiency over a wide range of lipid concentrations (5-50 microg/ml) keeping DNA concentration constant (1.6 microg/ml). Addition of DIP to the lipid/DNA complex increased maximum transfection efficiencies in glial and neuronal cultures 2-3-fold. Transfection efficiencies were at their maximum with a similar total lipid concentration (50 &mgr;g/ml) in both cell-types in the presence of DIP. Neuronal cultures were more sensitive than glia to the toxic actions of DOTAP/DOPE, with or without DIP. These results indicate that AAV-mediated gene-transfer to neurons and glia can be facilitated by addition of a pH-sensitive surfactant to cationic liposome/DNA complexes and that endosomal escape could be a limiting factor in transgene expression.
机译：这项研究评估了新型的pH敏感表面活性剂2-（1'-咪唑基）丙酸十二烷基酯（DIP）对原代大鼠脑神经元和神经胶质细胞培养物中阳离子脂质介导的转染的影响。加入阳离子脂质复合物DOTAP / DOPE（分别为1,2-2-油酰基-3-三甲基丙酸铵和二油酰基磷脂酰乙醇胺），并在一定范围的浓度（0-120微克/毫升）下添加DNA浓度恒定（1.6微克/毫升） 。发现神经元特异性烯醇化酶（NSE）和巨细胞病毒（CMV）启动子使用腺相关病毒（AAV）衍生的构建体分别驱动富含神经元的神经胶质和胶质培养物中的绿色荧光蛋白（GFP）表达。在胶质细胞培养物中未观察到NSE驱动的GFP表达。加入DOTAP / DOPE可在多种脂质浓度（5-50 microg / ml）范围内提高转染效率，从而保持DNA浓度恒定（1.6 microg / ml）。在脂质/ DNA复合物中添加DIP可将神经胶质和神经元培养物中的最大转染效率提高2至3倍。在存在DIP的两种细胞类型中，总脂质浓度（50 mg / ml）相似时，转染效率达到最大值。有或没有DIP时，神经元培养物比神经胶质细胞对DOTAP / DOPE的毒性作用更敏感。这些结果表明，可以通过向阳离子脂质体/ DNA复合物中添加pH敏感表面活性剂来促进AAV介导的基因向神经元和神经胶质的转移，并且内体逃逸可能是转基因表达的限制因素。

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