Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we ex...Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B(AF488-CTB).This was injected into the gastrocnemius muscle of rats,and it was found that motor,sensory,and sympathetic neurons were labeled in the spinal ventral horn,dorsal root ganglia,and sympathetic chain,respectively.Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle.The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection.It was found that labeled motor and sensory neurons could be observed 12 hours post-injection.The intensity was found to increase over time,and the morphology appeared clear and complete 3-7 days post-injection,with clearly distinguishable motor neuron axons and dendrites.However,14 days after the injection,the quality of the images decreased and the neurons appeared blurred and incomplete.Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features,and the surrounding microglia were also found to be unaltered.Overall,these results imply that the cholera toxin subunit B,whether unconjugated or conjugated with Alexa Fluor,is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.展开更多
将麦芽糖结合蛋白(maltose-binding protein,MBP)、霍乱毒素B亚基(cholera toxin B subunit,CTB)以及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因重组构建MBP-CTB-EGFP并原核表达纯化,利用HEK293T细胞模型探究MBP...将麦芽糖结合蛋白(maltose-binding protein,MBP)、霍乱毒素B亚基(cholera toxin B subunit,CTB)以及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因重组构建MBP-CTB-EGFP并原核表达纯化,利用HEK293T细胞模型探究MBP-CTB-EGFP穿透细胞膜的能力,从而开发新型黏膜佐剂.进一步地,利用原肌球蛋白(tropomyosin,TM)重组融合蛋白MBP-CTB-TM,将其应用于Balb/c小鼠致敏实验,探究融合蛋白的致敏性及其对小鼠食物过敏相关免疫反应的影响,以达到降低过敏原剂量提高建模效率的效果.结果表明:MBP-CTB-EGFP具有作为黏膜佐剂的能力,从而促进外源蛋白进入HEK293T细胞内,且与转染方式相比,携带外源蛋白进入细胞的效率更高.另一方面,利用融合蛋白MBP-CTB-TM免疫小鼠,TM特异性IgE的OD450 nm达到0.4,而天然TM致敏组仅为0.05,此外融合蛋白致敏还导致高水平的TM特异性IgG1、IgG2a产生.本研究表明,融合蛋白MBP-CTB-TM致敏效果更好,有可能达到降低过敏原用量的效果,为后续食物过敏研究提供了有力工具.展开更多
基金supported by the CACMS Innovation Fund,No.CI2021A03407(to WZB)the Project of National Key R&D Program of China,No.2019YFC1709103(to WZB)+1 种基金the National Natural Science Foundation of China,Nos.81774432(to JJC),81774211(to WZB),82004492(to JW),81801561(to DSX)the Fundamental Research Funds for the Central Public Welfare Research Institutes of China,Nos.ZZ13-YQ-068(to JJC),ZZ14-YQ-032(to JW),ZZ14-YQ-034(to DSX).
文摘Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries.However,it is not always clear which tracer should be used to yield optimal results.In this study,we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B(AF488-CTB).This was injected into the gastrocnemius muscle of rats,and it was found that motor,sensory,and sympathetic neurons were labeled in the spinal ventral horn,dorsal root ganglia,and sympathetic chain,respectively.Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle.The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection.It was found that labeled motor and sensory neurons could be observed 12 hours post-injection.The intensity was found to increase over time,and the morphology appeared clear and complete 3-7 days post-injection,with clearly distinguishable motor neuron axons and dendrites.However,14 days after the injection,the quality of the images decreased and the neurons appeared blurred and incomplete.Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features,and the surrounding microglia were also found to be unaltered.Overall,these results imply that the cholera toxin subunit B,whether unconjugated or conjugated with Alexa Fluor,is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.
文摘将麦芽糖结合蛋白(maltose-binding protein,MBP)、霍乱毒素B亚基(cholera toxin B subunit,CTB)以及增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因重组构建MBP-CTB-EGFP并原核表达纯化,利用HEK293T细胞模型探究MBP-CTB-EGFP穿透细胞膜的能力,从而开发新型黏膜佐剂.进一步地,利用原肌球蛋白(tropomyosin,TM)重组融合蛋白MBP-CTB-TM,将其应用于Balb/c小鼠致敏实验,探究融合蛋白的致敏性及其对小鼠食物过敏相关免疫反应的影响,以达到降低过敏原剂量提高建模效率的效果.结果表明:MBP-CTB-EGFP具有作为黏膜佐剂的能力,从而促进外源蛋白进入HEK293T细胞内,且与转染方式相比,携带外源蛋白进入细胞的效率更高.另一方面,利用融合蛋白MBP-CTB-TM免疫小鼠,TM特异性IgE的OD450 nm达到0.4,而天然TM致敏组仅为0.05,此外融合蛋白致敏还导致高水平的TM特异性IgG1、IgG2a产生.本研究表明,融合蛋白MBP-CTB-TM致敏效果更好,有可能达到降低过敏原用量的效果,为后续食物过敏研究提供了有力工具.
