大鼠海马CA1脑区慢病毒载体注射方法学改进

Improvement of administration method of lentiviral vector in rat CA1 region of hippocampus

目的:采用电极埋管的改进方式进行大鼠海马CA1脑区慢病毒载体注射的方法,并检测此方法的有效性和安全性。方法:45只健康雄性SD大鼠随机分为假手术组(SH)、生理盐水组(NS)和eGFP慢病毒注射组(eGFP),每组15只。所有实验大鼠在脑立体定位仪下定位海马CA1组织,采用电极埋管的改进方式进行慢病毒注射。NS组给予生理盐水(2μL)、eGFP组给予慢病毒(2μL),SH组只实行手术操作。注射病毒7、14和30 d后,分别处死大鼠并进行全脑冰冻切片,荧光显微镜观察eGFP慢病毒注射组的GFP绿色荧光表达水平和脑区分布。同时取相邻冰冻切片进行Nissl染色,检测eGFP慢病毒注射组的海马CA1脑区的损伤程度。结果:eGFP慢病毒注射成年大鼠海马CA1脑区7、14和30 d后,均可以成功检测到GFP在海马CA1脑区的表达,且表达水平和脑区分布均保持稳定,不随注射后切片时间的变化而变化。Nissl染色结果显示,神经元存活数目在eGFP慢病毒注射组与假手术组、生理盐水组比较均无显著性差异(P>0.05)。结论:成功建立了电极埋管的方式向大鼠海马脑区注射慢病毒的方法,并可在海马CA1脑区高水平表达其所携带的目的基因,为进一步的在成年大鼠海马CA1脑区进行基因操作奠定了基础。

Objective To improve the administration method of lentiviral vector in rat CA1 region of hippocampus by electrode pipe installation and detect its validity and security.Methods Fourty-five male healthy SD rats were randomly divided into three groups： sham operation group（SH）,normal saline group（NS）,GFP gene lentiviral vector injection group（eGFP）.The CA1 region was located by stereotaxic instruments and the lentiviral vector was administered by electrode pipe installation.At day 7,14 and 30 after the injection,SD rats were frozen sectioned and the expression of the green fluorescent protein was detected by fluorescence microscope.Nissl staining was used to detect the hippocampus injury.Results The stable expression of the green fluorescent protein was detected in rat CA1 region of hippocampus at day 7,14 and 30 using this method and the number of survival neurons had no significant difference between three groups（P〉0.05）.Conclusion The method of electrode pipe installation to inject lentiviral vector in rat hippocampus is successfully established and the high level expression of fluorescent protein can be detected with no significant injury,which lays the basis for its application in the gene therapy of the neurodamaged diseases.