目的 由损伤的周围神经靶向性导入腺病毒介导的 L ac Z基因 (Ad L ac Z)至脊髓 ,动态观察病毒载体逆行输送到脊髓前角运动神经元、脊髓后根神经节 (dorsal root ganglia,DRGs)感觉神经元及基因产物顺行标记周围神经的全过程和特点。 ...目的 由损伤的周围神经靶向性导入腺病毒介导的 L ac Z基因 (Ad L ac Z)至脊髓 ,动态观察病毒载体逆行输送到脊髓前角运动神经元、脊髓后根神经节 (dorsal root ganglia,DRGs)感觉神经元及基因产物顺行标记周围神经的全过程和特点。 方法 分别将 Ad L ac Z转染大鼠正中神经和胫神经近断端 ,然后以 10 - 0无创线吻合神经。在转染后 9周内的 2 4个不同时间点取出正中神经组的 C5～ T1 脊髓节段、DRGs连同臂丛 ,胫神经组的 L2 ～ L6 脊髓节段、DRGs连同骶丛。将脊髓和 DRGs的 5 0 μm横切片行 X- gal染色和免疫组织化学染色 ,臂丛和骶丛的整个标本分别行 X- gal染色。计数阳性脊髓前角运动神经元、DRGs神经元及周围神经轴突数 ,研究转基因表达在脊髓前角运动神经元、DRGs神经元及正中神经、胫神经的最早时间、高峰时间和持续时间。 结果 L ac Z基因能特异性、高效表达在损伤周围神经的感觉和运动神经元。转基因在脊髓和周围神经的表达严格限于感染神经的同侧。正中神经组标本各部位的转基因表达均早于胫神经组。胫神经组被标记的运动神经元和感觉神经元数均高于正中神经组。表达持续的时间在运动神经元最短 ,然后是感觉神经元 ,在周围神经持续时间最长。在同一组内 ,转基因表达在 DRGs神经元最早 。展开更多
In order to determine the most optimal host cell line,inoculation dose and the time of harvest,we studied the growth pattern and cytopathic effect (CPE) of the mutants of Pseudorabies virus (PRV) Ea strain,gG -/LacZ +...In order to determine the most optimal host cell line,inoculation dose and the time of harvest,we studied the growth pattern and cytopathic effect (CPE) of the mutants of Pseudorabies virus (PRV) Ea strain,gG -/LacZ +,in different cell lines.A clone virus of gG -/LacZ +,the titer of which could be up to 10 -8.0 /0.1 ml,after purification four times by plaques,was obtained.The clone virus was inoculated into different cell lines,BHK-21,IBRS-2,MDBK and PK-15,respectively.Infected BHK-21 could be induced CPE at 12 hours p.i..However,The CPE of MDBK appeared after 24 hours p.i..In terms of the growth pattern,the titer of IBRS-2 could reach the peak level at 40 hours p.i. and the peak titer was up to 10 -8.0 /0.1 ml,which was maintained until 64 hours p.i..In contrast,the peak of BHK-21 was only 10 -6.5 /0.1 ml during the 96 hours p.i.. We also studied the titer of IBRS-2 when it was inoculated with different dose virus from 0.001 PFU/cell to 100PFU/cell.It was discovered that the titer could reached over 10 -7.5 /0.1 ml when inoculated with 0.1PFU/cell,0.01PFU/cell after 30,48 hours p.i.,respectively.These data indicate that IBRS-2 is the most optimal cell and that the titer could reach the peak when inoculated with 0.01PFU/cell and harvested at 40-50 hours p.i..展开更多
文摘In order to determine the most optimal host cell line,inoculation dose and the time of harvest,we studied the growth pattern and cytopathic effect (CPE) of the mutants of Pseudorabies virus (PRV) Ea strain,gG -/LacZ +,in different cell lines.A clone virus of gG -/LacZ +,the titer of which could be up to 10 -8.0 /0.1 ml,after purification four times by plaques,was obtained.The clone virus was inoculated into different cell lines,BHK-21,IBRS-2,MDBK and PK-15,respectively.Infected BHK-21 could be induced CPE at 12 hours p.i..However,The CPE of MDBK appeared after 24 hours p.i..In terms of the growth pattern,the titer of IBRS-2 could reach the peak level at 40 hours p.i. and the peak titer was up to 10 -8.0 /0.1 ml,which was maintained until 64 hours p.i..In contrast,the peak of BHK-21 was only 10 -6.5 /0.1 ml during the 96 hours p.i.. We also studied the titer of IBRS-2 when it was inoculated with different dose virus from 0.001 PFU/cell to 100PFU/cell.It was discovered that the titer could reached over 10 -7.5 /0.1 ml when inoculated with 0.1PFU/cell,0.01PFU/cell after 30,48 hours p.i.,respectively.These data indicate that IBRS-2 is the most optimal cell and that the titer could reach the peak when inoculated with 0.01PFU/cell and harvested at 40-50 hours p.i..
