CAJ | 학술논문

目的观察诱导型一氧化氮合酶(iNOS)反义基因重组腺相关病毒载体(rAAV-AsiNOS)对急性完全性脊髓缺血的神经保护作用.方法采用腹主动脉阻断法建立脊髓缺血模型,应用下传神经源性运动诱发电位对模型进行评估,苏木素-伊红(HE)切片观察缺血对脊髓前角运动神经元的影响,半定量逆转录-聚合酶链反应(RT-PCR)检测单纯缺血各组G24、G48、G72和G120及rAAV-AsiNOS导入各组RG24、RG48、RG72和RG120,iNOS、p38MAPK和Caspase-3 mRNA在脊髓缺血后不同时间点的表达水平.结果重组病毒载体rAAV-AsiNOS导入各组与缺血各组比较下传神经源性运动诱发电位,重组病毒组均可以部分恢复,正常脊髓前角运动神经元病理损害程度减轻;RT-PCR检测iNOS,p38MAPK和Caspase-3 mRNA在脊髓缺血中后期不同时间点的表达水平不一致,3种基因在缺血24 h组(G24)和病毒转入组缺血24 h(RG24)相对值分别为0.45和0.25、0.56和0.27、0.50和0.24;在G120和RG120相对值分别为0.53和0.33、0.64和0.25、0.54和0.25,重组病毒载体各组与同时间点缺血组比较均明显减少,差异均有统计学意义(P＜0.05).结论 iNOS、p38MAPK和Caspase-3可能共同参与急性脊髓完全性缺血损伤过程;通过抑制iNOS表达,抑制了p38MAPK和Caspase-3表达,从而改善脊髓缺血损伤.
목적 관찰유도형일양화담합매(iNOS)반의기인중조선상관병독재체(rAAV-AsiNOS)대급성완전성척수결혈적신경보호작용.방법 채용복주동맥조단법건립척수결혈모형,응용하전신경원성운동유발전위대모형진행평고,소목소-이홍(HE)절편관찰결혈대척수전각운동신경원적영향,반정량역전록-취합매련반응(RT-PCR)검측단순결혈각조G24、G48、G72화G120급rAAV-AsiNOS도입각조RG24、RG48、RG72화RG120,iNOS、p38MAPK화Caspase-3 mRNA재척수결혈후불동시간점적표체수평.결과 중조병독재체rAAV-AsiNOS도입각조여결혈각조비교하전신경원성운동유발전위,중조병독조균가이부분회복,정상척수전각운동신경원병리손해정도감경;RT-PCR검측iNOS,p38MAPK화Caspase-3 mRNA재척수결혈중후기불동시간점적표체수평불일치,3충기인재결혈24 h조(G24)화병독전입조결혈24 h(RG24)상대치분별위0.45화0.25、0.56화0.27、0.50화0.24;재G120화RG120상대치분별위0.53화0.33、0.64화0.25、0.54화0.25,중조병독재체각조여동시간점결혈조비교균명현감소,차이균유통계학의의(P＜0.05).결론 iNOS、p38MAPK화Caspase-3가능공동삼여급성척수완전성결혈손상과정;통과억제iNOS표체,억제료p38MAPK화Caspase-3표체,종이개선척수결혈손상.
Objective To explore the neuroprotective effects of recombinant rAAV-AsiNOS transfection in vitroon spinal cord ischemia.Methods The spinal cord ischemic injury animal model was established by occlusion of the abdominal aorta.The spinal cord ischemic animal models were estimated by desending neurogenic motor evoked potentials,the ultrastructure of spinal cord after ischemic injury was observed under the light microscopy.The mRNA expression levels of iNOS,p38MAPK and Caspase-3 were semi-quantitatively detected and compared by using reverse transcription-polymerase chain reaction (RT-PCR) in G24,G48,G72,G120 groups and RG24,RG48,RG72 and RG120 groups.Results The amplitude pathological section of recombinant rAAV-AsiNOS transfection groups were gradually improvement,RT-PCR showed that there is significant difference between the rAAV-AsiNOS transfection groups and the ischemic groups for the expression of mRNA of iNOS,p38MAPK and Caspase-3.The relative values of three genes both 24 h in ischemic group (G24) and 24 h in ischemia group with viruses (RG24) were 0.45 and 0.25,0.56 and 0.27,0.50 and 0.24;and the relative values of both G120 and RG120 were 0.53 and 0.33,0.64 and 0.25,0.54 and 0.25.The expression of three genes in recombinant viral vectors group is significantly lower than those in the ischemic groups with the same time,and the difference was significant (P＜0.05 ).Conclusion The recombinant rAAV-AsiNOS transfection in vitro spinal cord ischemia can reduce the expression of mRNA of iNOS,p38MAPK and Caspase-3.