CAJ | 학술논문

目的研究大鼠脑外伤后自噬是否被激活并探讨其在脑外伤后神经细胞损伤和修复中的作用.方法建立大鼠定量脑外伤模型,于脑外伤后不同时间点处死动物并取脑;应用透射电镜检测脑组织自噬双层膜结构以及次级溶酶体的形成情况:应用自噬标记抗体LC3B和Beclin-1对脑外伤后不同时间点的脑组织进行免疫荧光和Westernblot检测;LC3和caspase-3或Beclin 1和Fluoro-Jade双标记检测.结果脑外伤后1 h在损伤区周围即检测到双层膜结构,并且一直持续到脑外伤后32天.脑外伤后1 h,脑组织中LC3和Beclin-1表达增加,损伤后3天内阳性细胞以神经元为主,之后阳性胶质细胞增加,第8天达到高峰,并可持续至脑外伤后32天仍维持高表达.大多数阳性细胞分布在损伤区周围(包括海马)而不是损伤区.此外,脑外伤后24小时以前,在损伤区周围不是所有的LC3阳性细胞都与caspase.3阳性细胞重叠.同样脑外伤后6 h至48 h,Beclin 1阳性海马神经元与Fluoro-Jade染色不重叠.结论脑外伤后自噬被激活,在损伤后早期保护损伤区周围神经细胞免于凋亡和退行性变,并对神经细胞损伤与修复发挥长期作用.
목적 연구대서뇌외상후자서시부피격활병탐토기재뇌외상후신경세포손상화수복중적작용.방법 건립대서정량뇌외상모형,우뇌외상후불동시간점처사동물병취뇌;응용투사전경검측뇌조직자서쌍층막결구이급차급용매체적형성정황:응용자서표기항체LC3B화Beclin-1대뇌외상후불동시간점적뇌조직진행면역형광화Westernblot검측;LC3화caspase-3혹Beclin 1화Fluoro-Jade쌍표기검측.결과 뇌외상후1 h재손상구주위즉검측도쌍층막결구,병차일직지속도뇌외상후32천.뇌외상후1 h,뇌조직중LC3화Beclin-1표체증가,손상후3천내양성세포이신경원위주,지후양성효질세포증가,제8천체도고봉,병가지속지뇌외상후32천잉유지고표체.대다수양성세포분포재손상구주위(포괄해마)이불시손상구.차외,뇌외상후24소시이전,재손상구주위불시소유적LC3양성세포도여caspase.3양성세포중첩.동양뇌외상후6 h지48 h,Beclin 1양성해마신경원여Fluoro-Jade염색불중첩.결론 뇌외상후자서피격활,재손상후조기보호손상구주위신경세포면우조망화퇴행성변,병대신경세포손상여수복발휘장기작용.
Objective To investigate changes of autophagy after traumatic brain injury (TBI) and its possible role.Methods Rat TBI model was established by controlled cortical injury system.Autophagic double membrane structure was detected by transmission electronic microscope.Microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1 were also used to investigate the activation of autophagy post-TBI.Double labeling with LC3 and caspase-3,or Beclin 1 and Fluoro-Jade,to show the relationship between autophagy and apoptosis or neuron degeneration after TBI.Results An increase of autophagic double membrane structure was observed in early stage (1 h),and the increase lasted for at least 32 d post-TBI.LC3 and Beclin 1 proteins also began to elevate at 1 h time point post-TBI in neurons,3 d later in astrocytes,and peaked at about 8 d post-TBI.In both cell types,LC3 and Beclin 1 maintained at a high level until 32 d post-TBI.Most LC3 and Beclin 1 positive cells were near the side (including hippocampus),but not in the core of the injury.In addition,in the periphery of the injury site,not all caspase-3 positive (+) cells merged with LC3 (+) cells post-TBI;In hippocampal area,almost all Beclin 1 (+) neurons did not merge with Fluoro-Jade (+) neurons from 1 h to 48 h post-TBI.Conclusion Autophagy is activated and might protect neurons from degeneration at early stage post-TBI and play a continuous role afterwards in eliminating aberrant cell components.