腺病毒介导脑源性神经营养因子基因转移对大鼠创伤性脑损伤后诱导型一氧化氮合酶表达及细胞凋亡的影响

Effect of recombinant adenovirus-mediated brain derived neurotrophic factor gene transfer on induced nitric oxide synthase expression and apoptosis after traumatic brain injury in rats

目的 :研究重组腺病毒介导的脑源性神经营养因子 (BDNF)基因转移对创伤性脑损伤 (TBI)后诱导型一氧化氮合酶 (i NOS)表达及细胞凋亡的影响。方法 :将 4μl重组腺病毒载体注入承受单侧大脑皮质重锤打击大鼠的海马 ,对照组注射病毒缓冲液。伤后 3小时及 1、3、7和 14日 ,利用免疫组织化学单标和双标染色、原位杂交 /组织化学染色及 DNA原位末端标记等方法 ,检测大鼠伤侧大脑皮质和海马各区 i NOS、BDNF及凋亡相关信号表达的改变。结果 :与对照组相比 ,各损伤组大鼠大脑皮质及海马各区 i NOS阳性细胞于伤后 3小时开始显著增多 ,3日达高峰。多数 i NOS阳性细胞同时呈现凋亡相关蛋白阳性反应或分子生物学标记方法(TUNEL )阳性反应 ,但很少同时表达 BDNF m RNA。注射病毒载体组伤后 3日和 7日 ,海马 CA1区和海马齿状回门区 (DH)表达 i NOS、凋亡相关蛋白的细胞及凋亡细胞均显著减少 (P均 <0 .0 1) ,而表达 BDNF m RNA的神经元显著增多。结论 :TBI诱导海马细胞表达 i NOS及诱导海马细胞凋亡 ;腺病毒介导的 BDNF基因转移通过下调 i NOS表达 ,增加 BDNF表达及减少细胞凋亡而保护海马神经元。

Objective:To investigate the effects of recombinant adenovirusmediated brain derived neurotrophic factor (BDNF) gene transfer on the expression of induced nitric oxide synthase (iNOS) and cellular apoptosis after traumatic brain injury (TBI) in rats.Methods:Adult Wistar rats sustained a weightdrop impact on the right cerebral cortex,and then 4 μl of purified recombinant adenovirus vector (RAV) or virus buffer was injected into the right hippocampus.Immunohistochemistry,in situ hybridization as well as terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) were used to determine the expression of iNOS,BDNF,and apoptosisassociated signals in the hippocampus and cerebral cortex area at 3 hours,1,3,7 and 14 days after the injury.Results:Compared to control,iNOS positive cells in the cortex and hippocampus in injured groups were significantly increased at 3 hours post injury,and peaking at 3 days.Most of iNOSlike cells simultaneously showed signals either of apoptotic associated proteins or of TUNEL,but few of BDNF.In hippocampal CA1 and hilar dentate area in RAV group on 3 and 7 days after TBI,the expression of iNOS, apoptoticassociated proteins and TUNEL were markedly decreased (all P <0 01),and BDNF significantly increased compared with that of other groups.Conclusions:These evidences suggest that TBI can upregulate the expression of iNOS and induce hippocampal cell apoptosis,and that RAVmediated BDNF gene transfer might protect hippocampal neurons through downregulating the expression of iNOS and inhibiting neuron apoptosis.