腺病毒介导BDNF基因转移对大鼠创伤性脑损伤后iNOS表达及细胞凋亡的影响

Effects of recombinant adenovirus-mediated BDNF gene transfer on iNOS expression and apoptosis after post traumatic brain injury in the rat

研究重组腺病毒介导的脑源性神经营养因子 (brainderivedneurotrophicfactor,BDNF)基因转移对创伤性脑损伤 (TBI)后诱导型一氧化氮合酶 (induciblenitricoxidesynthase,iNOS)表达及细胞凋亡的影响。将重组腺病毒载体 4 μl注入承受单侧大脑皮质重锤打击的大鼠海马 ,对照组注射病毒缓冲液。伤后 3h及 1,3,7,14d利用免疫组化单标 /双标染色 ,原位杂交 /组化染色及DNA末端原位标记等方法 ,检测伤侧大脑皮质和海马各区iN OS、BDNF及凋亡相关信号表达的改变。与对照组相比 ,各损伤组大脑皮质及海马各区iNOS阳性细胞于伤后 3h开始显著增多 ,7d达高峰。多数iNOS阳性细胞同时呈现凋亡相关蛋白阳性反应或TUNEL阳性反应 ,但很少同时表达BDNFmRNA。注射病毒载体组伤后 3,7d ,海马CA1区和DH区表达iNOS、凋亡相关蛋白的细胞及凋亡细胞显著减少 (P均 <0 .0 1) ,而表达BDNFmRNA的神经元显著增多。提示 ,TBI诱导海马细胞表达iNOS及诱导海马细胞凋亡 ;腺病毒介导的BDNF基因转移通过抑制iNOS表达、增加BDNF表达及减少细胞凋亡的机制保护海马神经元。

To investigate the effects of re co mbinant adenovirus mediated brain derived neurotrophic factor (BDNF) gene tran sfer on the expression of induced nitric oxide synthase (iNOS) and cellular apop tosis post traumatic brain injury (TBI)in the rat, adult Wistar rats experience d a weight drop strike on the right cerebral cortex, and then 4 μl of purified recombinant adenovirus vector (RAV) or virus buffer was injected into the right side of hippocampus. Immunohistochemistry, in situ hybridization as well as terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) w ere used to determine the expressions of iNOS, BDNF,and apoptosis associated si gnals in the hippocampus and cerebral cortex area at 3 h, and 1, 3, 7 ,14 d foll owing injury. Compared with the control, iNOS positive cells in the cortex and h ippocampus in each group of injury were significantly increased at 3 h post inju ry, and reached its peak at 7 d. Most of iNOS like cells simultaneously shown s ignals either of apoptotic associated proteins or of TUNEL, but less of BDNF mRN A. In hippocampal CA1 and hilar dentate area in RAV group at 3 d and 7d post TBI ,the expressions of iNOS, apoptotic associated proteins and TUNEL were markedly decreased and BDNF significantly increased compared with that of other groups . These findings suggest that TBI can up regulate the expression of iNOS and in duce hippocampal cell apoptosis, and that RAV mediated BDNF gene transfer might protect hippocampal neurons through down regulating the expression of iNOS and inhibiting cellular apoptosis.