溴化乙锭诱导的短暂脱髓鞘增强PRV的逆行转导效率 *

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。

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

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.