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目的总结制作小鼠视神经完全截断性动物模型作为视神经再生研究的经验和体会。方法将雄性Bcl-2高表达转基因小鼠(Bcl-2 transgenic mice)和受GFAP启动子控制表达疱疹病毒-胸苷激酶转基因雌性小鼠(GFAP-TK)交配产生的4只8~12周成年小鼠(20~30g),Bcl-2/GFAP-TK双转基因小鼠作为实验组,同周龄4只Bcl-2转基因小鼠作为对照组。其中Bcl-2/GFAP-TK双转基因小鼠皮下植入缓释泵,连续7d释放更昔洛韦(GCV)100mg.kg-1.d-1以选择性地去除视神经损伤后激活的星形胶质细胞。更昔洛韦缓释泵植入术后2d在两组动物中制作右侧单眼标准完全性视神经钳夹损伤模型,视神经钳夹10d后获取组织标本。采用免疫荧光染色特异性检测再生轴突纤维并进行定量分析;结合罗丹明的霍乱毒素B亚单位(CTB-R)或增强表达绿色荧光蛋白的复制缺陷型腺相关病毒(AAV-EGFP)用作顺行性标记物以显示再生轴突是否到达大脑靶器官。结果在Bcl-2/GFAP-TK双转基因小鼠中存在免疫荧光阳性的再生视神经轴突,再生轴突计数为71.99±24.04,并可见生长锥(growth cone)样结构,但是再生轴突纤维未能延伸达到大脑靶器官。在对照组Bcl-2转基因小鼠中未见明显再生迹象。结论小鼠视神经完全截断性动物模型可用于视神经病变的再生研究。
OBJECTIVE: To summarize the experience and experience of making a completely truncated animal model of optic nerve in mice as a study of optic nerve regeneration. Methods Four 8-12-week-old adulthood born from the mating of Bcl-2 transgenic mice and GFAP-TK transfected female mice expressing GFAP promoter (GFAP-TK) Mice (20 ~ 30g), Bcl-2 / GFAP-TK double transgenic mice as experimental group, the same age 4 Bcl-2 transgenic mice as control group. The Bcl-2 / GFAP-TK double transgenic mice were subcutaneously implanted with sustained-release pump and ganciclovir (GCV) 100 mg.kg-1.d-1 was released for 7 days to selectively remove the activated star after optic nerve injury Glial cells. Two days after ganciclovir sustained-release pump implantation, the right monocular standard complete optic nerve clamp injury model was made in two groups of animals. Tissue specimens were obtained after the optic nerve was clamped for 10 days. Regenerated axonal fibers were detected specifically by immunofluorescence staining and quantified; combined with cholera toxin B subunit of rhodamine (CTB-R) or replication-defective adeno-associated virus (AAV-EGFP) expressing enhanced green fluorescent protein Antegrade markers show whether regenerative axons reach the target organ of the brain. Results There were immunofluorescence positive regenerated axons in Bcl-2 / GFAP-TK double transgenic mice. The axonal regeneration count was 71.99 ± 24.04 and the growth cone-like structure was observed. However, the regenerated axon fibers Can reach the target organ of the brain. There was no obvious sign of regeneration in control Bcl-2 transgenic mice. Conclusion The mouse model of complete truncation of the optic nerve can be used for the regeneration of optic neuropathy.