鼻粘膜内分泌表达大分子蛋白沿“鼻-脑”通路入脑的研究

The delivery of macromolecular proteins to the brain via "nose-brain" pathway by expressive "NT4-GFP-Ant" recombinant gene in vivo

目的利用分子生物学技术寻求将大分子蛋白类药物沿"鼻-脑"通路长期稳定导入脑内的方法。方法昆明小鼠8只,随机分为两组,每组4只。分别滴鼻给予小鼠已构建成功的PSSHG/NT4-GFP-Ant穿膜肽重组腺相关病毒和对照组PSSHG/GFP重组腺相关病毒,每次每只鼻孔滴入10μl病毒液,每日1次,连续滴鼻5d。10d后处死小鼠,脑组织冰冻切片,共聚焦显微镜观测脑组织内绿色荧光表达情况。结果滴鼻PSSHG/NT4-GFP-Ant重组腺相关病毒小鼠组可在鼻粘膜、嗅球、海马观测到绿色荧光表达,其余脑组织内未见荧光表达。而对照组PSSHG/GFP重组腺相关病毒小鼠组鼻粘膜上可见绿色荧光表达,嗅球、海马、其他脑组织未见荧光表达。结论两种重组腺相关病毒均能感染鼻粘膜细胞,并在鼻粘膜处表达目的蛋白;用腺相关病毒介导体内分泌表达的单纯大分子蛋白不能沿鼻-脑途径入脑;用腺相关病毒介导体内分泌表达经穿膜肽修饰的大分子的报告蛋白可沿鼻-脑途径入脑。

Objective To find a suitable way to deliver macromolecular biological proteins into the brain via nose-brain pathway.Methods The Kunming rats were divided into two groups.One group were given pSSHG/NT4-GFP-Ant AAV at a dose of 10μl per day of each nostril by intranasal administration.The other group were given pSSHG/GFP AAV in the same way.The rats were killed after ten days and frozen slides were made,and then were detected by laser confocal microscope.Results The fluorescence can be observed at the membrana mucosa nasi,olfactory bulb and hippocampus in the group which was given pSSHG/NT4-GFP-Ant by intranasal administration.By contrast,the fluorescence can be observed only at the membrana mucosa nasi in the group which was given pSSHG/GFP.Conclusion The animal experiment confirmed that macromolecular protein modified by cell penetrating peptides can be delivered into brain via ＂nose-brain＂ pathway.