重组腺病毒载体在小鼠海马组织内的转导特征及其细胞亲嗜性  

Gene transfer characteristics and cell tropism of recombinant adenovirus in the hippocampus of adult mouse

作　　者：颜荣[1] 张琳[1] 杨新宇[2] 刘振林[1] 

机构地区：[1]天津市第五中心医院神经外科,300450 [2]天津医科大学总医院神经外科,天津市神经病学研究所,天津300052

出　　处：《中华神经医学杂志》2014年第1期12-16,共5页Chinese Journal of Neuromedicine

基　　金：天津市科技计划项目（10JCYBJC25700）;天津市卫生局科技基金（10KG210）;天津市滨海新区塘沽科技发展资金项目（2012MS05-04）

摘　　要：目的探讨由鼠巨细胞病毒立早启动子（mCMV）介导的血清5型重组腺病毒（rAd5）载体在小鼠海马组织内的转导特征及其对不同细胞亲嗜性的差异。方法取健康成年雄性C57BL／6小鼠42只，立体定向注射一定体积和滴度的rAd5载体，以增强型绿色荧光蛋白（EGFP）为报告基因，于注射后第1、3、7、14、30、45、60天取脑行序列冰冻切片，用荧光显微镜观察EGFP在海马组织内的表达情况。同时采用免疫荧光双重标记法观察EG卵在不同类型细胞中的表达情况。结果rAd5载体所携带的EGFP在注射后第1天已开始表达。在第3-45天均处在表达高峰，在注射后第60天，EGFP的表达量已明显减少。在海马组织内，rAd5载体介导的EGFP报告基因主要在齿状回颗粒细胞下层（SGZ）表达，在颗粒细胞层（GCL）表达很少，在海马CA1～CA3区锥体细胞层中无表达。免疫荧光双重标记显示，rAd5很少转导神经元，对SGz的神经前体细胞则有很强的转导效率。结论rAd5载体可以快速高效并长时程介导目的基因的表达，且对小鼠海马SGZ神经前体细胞具有高度亲嗜性。Objective To explore the gene transfer characteristics and different cell tropism of the recombinant adenovirus serotype 5 （rAd5） driven by murine cytomegalovirus （mCMV） immediate-early promoter in the hippocampus of adult mouse, and provide a basis for adenovirus-mediated transgene research in the hippocampus of mouse in the future. Methods Forty-two male healthy adult C57BL/6 mice were injected with the titre and volume matched rAd5 vectors which contained enhanced green fluorescent protein （EGFP） sequences as a reporter gene. Then animals （n=6 for each point） were killed 1, 3, 7, 14, 30, 45, 60 days after injection. The coronal cryosections of brains were processed, and EGFP expression was observed with fluorescence microscopy. Double-label immunofluorescence method was used to observe the EGFP expression in different types of cells to identify the cell tropism in the hippocampus. Results The expression of EGFP carried by rAd5 vectors was detected on the first day after the transfection, with peak expression between days 3 and 45, while EGFP activity decreased sharply 60 days aider transfection. EGFP highly expressed in the dentate subgranular zone （SGZ） of the hippocampus but rarely in the granule cell layer （GCL） of the dentate gyrus. Moreover, no EGFP was detected in the pyramidal cell layers of CA1 to CA3 area. The counterstaining showed that rAd5 mainly transduced neuronal precursor cells in the SGZ of the hippocampus and rarely transduced neurons in the GCL. Conclusion The rAd5-mediated gene transfer is rapid and efficient and can keep for a long time in suitable titre; moreover, rAd5 has a highly specific tropism with the neuronal precursor cells in the SGZ of the hippocampus in mice.

关 键 词：重组腺病毒 增强型绿色荧光蛋白 海马 颗粒细胞下层 亲嗜性 

分 类 号：R741[医药卫生—神经病学与精神病学]