溴化乙锭诱导的短暂脱髓鞘增强PRV的逆行转导效率 <sup>*</sup>

doi:10.13523/j.cb.20191205

中国生物工程杂志

2019, Vol. 39

Issue (12): 35-41 DOI: 10.13523/j.cb.20191205

技术与方法

溴化乙锭诱导的短暂脱髓鞘增强PRV的逆行转导效率 ^*

刘法东¹,段红梅²,郝鹏²,赵文²,高钰丹²,杨朝阳^2,³,李晓光^1,^2,^3,^**(

)

1 北京航空航天大学生物与医学工程学院生物材料与神经再生北京市重点实验室北京 100083
2 首都医科大学神经生物学系北京 100069
3 北京航空航天大学生物医学工程高精尖创新中心生物材料与神经再生北京市国际科技合作基地北京 100083

Ethidium Bromide-Induced Transient Demyelination Increases the Efficiency of Retrograde PRV Transduction

LIU Fa-dong¹,DUAN Hong-mei²,HAO Peng²,ZHAO Wen²,GAO Yu-dan²,YANG Zhao-yang^2,³,LI Xiao-guang^1,^2,^3,^**(

)

1 Beijing Key Laboratory for Biomaterials and Neural Regeneration, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
2 Department of Neurobiology, Capital Medical University, Beijing 100069, China
3 Beijing International Cooperation Bases for Science and Technology on Biomaterials and Neural Regeneration,Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, Beijing 100083, China

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摘要：

目的改进伪狂犬病病毒(Pseudorabies virus,PRV)的注射方法,通过溴化乙锭(ethidium bromide,EB)诱导的短暂脱髓鞘提高PRV的转导效率。方法选用18只正常成年Wistar大鼠,随机分为肌肉组、NS组和EB组(n=6)。肌肉组将2μl滴度为2×10 ⁹的PRV工具病毒注射到胫骨前肌和腓肠肌上;NS组将2μl生理盐水(normal saline,NS)注射到坐骨神经上,1周后相同位置注射2μl滴度为2×10 ⁹的PRV;EB组将2μl 0.1%的EB注射到坐骨神经上,1周后相同位置注射2μl滴度为2×10 ⁹的PRV。大鼠注射PRV 5天后,灌流取材并制作冰冻切片,观察各级神经元的感染情况。结果 EB组大鼠L4-L5脊髓前角神经元和背根神经节(DRG)、T8脊髓中间神经元、C4脊髓中间神经元、延髓、中脑、大脑皮层均有大量神经元被PRV标记。肌肉组和NS组各级神经元仅有少量被PRV标记。结论 EB诱导坐骨神经脱髓鞘后能够显著提高PRV的逆行转导效率。

关键词： 溴化乙锭; 脱髓鞘; 伪狂犬病病毒; 效率

Abstract:

Objective: To improve the efficiency of PRV transduction by ethidium bromide (EB) -induced transient demyelination.Methods: 18 adult Wistar rats were randomly divided into muscle group, NS group and EB group(n=6).In the muscle group, 2μl PRV with a titer of 2×10 ⁹ was injected into the tibial anterior muscle and gastrocnemius muscle. NS group was injected with 2μl normal saline (NS) to the sciatic nerve, and 2μl PRV with a titer of 2×10 ⁹ was injected to the same site one week later. In the EB group, 2μl 0.1% EB was injected into the sciatic nerve, and 2μl PRV with a titer of 2×10 ⁹ was injected into the same position one week later. Five days later, perfusion samples were taken and frozen sections were made to observe the infection of neurons at all levels. Methods: A large number of L4-L5 spinal anterior horn neurons, dorsal root ganglion (DRG)neurons, T8 spinal intermediate neurons, C4 spinal intermediate neurons, medulla oblongata, midbrain and cerebral cortex of rats in EB group were labeled by PRV.A small number of neurons at all levels were labeled by PRV in the muscle group and the NS group.Conclusion: EB- induced sciatic nerve demyelination can enhance the reverse transduction efficiency of PRV.

Key words: Ethidium bromide Demyelination PRV Efficiency

收稿日期: 2019-05-10 出版日期: 2020-01-15

ZTFLH:

Q81

基金资助: * 国家自然科学基金(31730030、31670988、31771053、31650001、31320103903);国家重点研发计划(2017YFC1104001、 2017YFC1104002);北京市科技计划(Z181100001818007);北京市教育委员会2018年度科技计划重点项目(KZ201810025030)

通讯作者: 李晓光 E-mail: lxgchina@sina.com

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引用本文:

刘法东,段红梅,郝鹏,赵文,高钰丹,杨朝阳,李晓光. 溴化乙锭诱导的短暂脱髓鞘增强PRV的逆行转导效率 ^*[J]. 中国生物工程杂志, 2019, 39(12): 35-41.

LIU Fa-dong,DUAN Hong-mei,HAO Peng,ZHAO Wen,GAO Yu-dan,YANG Zhao-yang,LI Xiao-guang. Ethidium Bromide-Induced Transient Demyelination Increases the Efficiency of Retrograde PRV Transduction. China Biotechnology, 2019, 39(12): 35-41.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191205 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I12/35

图1 3组大鼠PRV的注射位点

图2 注射溴化乙锭增强PRV感染运动神经元的效率

图3 注射溴化乙锭增强PRV感染感觉神经元的效率

图4 注射溴化乙锭增强PRV感染T8中间神经元的效率

图5 注射溴化乙锭增强PRV感染C4中间神经元的效率

图6 注射溴化乙锭增强PRV感染脑区核团的效率

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