摘要
目的研究腺病毒介导的血管内皮生长因子(VEGF)165基因转移对新生大鼠缺氧缺血性脑损伤(HIBD)的神经保护作用。方法采用细菌内同源重组技术构建Ad-VEGF腺病毒重组载体。7日龄Sprague-Dawley大鼠随机分成4组,假手术组(n=20)、HIBD组(n=25)、病毒缓冲液移植组(Buffer组,n=20),Ad-VEGF移植组(Ad-VEGF组,n=25)。使用Rice法制成HIBD模型,Ad-VEGF移植组和Buffer组在HIBD后3d于大鼠左侧感觉运动皮层区分别立体定位注射2 μL重组体腺病毒悬液或病毒缓冲液;移植后7d采用RT-PCR法检测鼠脑VEGF165 mRNA的表达;采用原位缺口末端标记法(TUNEL法)检测鼠脑皮质神经元凋亡情况;采用免疫组织化学法分别检测VEGF蛋白表达及CD34表达计数大脑皮质微血管密度;30日龄时采用放射型迷宫觅水试验进行行为学测试,35日龄采用苏木精-伊红染色观察鼠脑组织病理学。结果Ad-VEGF组VEGF165基因表达较HIBD组及Buffer组明显增高(P<0.05);Ad-VEGF组脑细胞凋亡数目较HIBD组及Buffer组减少(P<0.05);Ad-VEGF组平均大脑皮质微血管数及VEGF蛋白表达较HIBD组及Buffer组明显增多(P<0.05);Ad-VEGF组行为学测试成绩较HIBD组及Buffer组改善(P<0.05);Ad-VEGF组鼠脑皮层神经元变性坏死较HIBD组及Buffer组减轻。结论腺病毒载体介导的VEGF165基因转移可增加新生鼠脑组织VEGF165 mRNA及VEGF蛋白的表达,减少脑细胞凋亡、增加新生脑血管形成,减轻缺氧缺血性脑损伤,改善远期学习记忆功能。
Objective To investigate the protective effects of adenovirus-mediated vascular endothelial growth factor （Ad-VEGF） 165 gene transfer against hypoxic-ischemic brain damage （HIBD） in neonatal rats. Methods Ad-VEGF recombinant adenovirus was constructed by bacterial homologous recombination technology. Seven-clay-old Sprague-Dawley rats were randomly assigned to 4 groups ： sham-operated （ n = 20 ）, HIBD （ n = 25 ）, buffer-treated （ n = 20 ）, and Ad- VEGF-treated （ n = 25 ）. The HIBD model was prepared by permanent occlusion of left common carotid artery, followed by exposure to 8 % oxygen for 2 hrs. In the Ad-VEGF-treated and the Buffer-treated groups, 2 μL recombinant adenovirus suspension or buffer was injected into the left sensorimotor cortex of the rat brain 3 days after HIBD. Seven days after transplantation, VEGF165 mRNA expression was detected using RT-PCR. Neuronal apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling （TUNEL）. CD34 and VEGF protein were detected using immunobistocbemistry. Microvascular density in the cerebral cortex was measured based on CD34 positive cells. A radial ann maze test was performed from 30 postnatal days to evaluate long-term learning and memory functions. At 35 postnatal days, the rats were sacrificed for cerebral histological examinations by bematoxylin and eosin. Results The expression of VEGFI65 mRNA increased in the Ad-VEGF-treated group more than in the untreated HIBD and the buffer-treated groups （ P 〈 0.05 ）. The number of apeptotic neurons was less in the Ad-VEGF-treated group compared with that in the untreated HIBD and the buffer-treated groups （P 〈 0.05 ）. Microvascular density and VEGF positive cells increased in the Ad-VEGF-treated group compared with that in the untreated HIBD and the buffer-treated groups （P 〈 0. 05 ）. In the radial arm maze test, the Ad-VEGF-treated group had more improved achievements than the HIBD and the buffer groups （ P 〈 0.05 ）. Neuronal degeneration and necrosis were lessened in the Ad-VEGF-treated group compared with the HIBD and the buffer groups. Conclusions Ad-VEGF gene transfer can increase the expression of VEGF mRNA and VEGF protein, decrease neuronal apoptosis, and increase angiopoiesis in the brain. This attenuates brain damage and improves long-term learning and memory functions in neonatal rats after HIBD.
出处
《中国当代儿科杂志》
CAS
CSCD
2008年第6期737-742,共6页
Chinese Journal of Contemporary Pediatrics
基金
国家自然科学基金资助项目(30500541)
关键词
缺氧缺血性脑损伤
血管内皮生长因子
基因转移
新生大鼠
Hypoxie-ischemic brain damage
Vascular endothelial growth factor
Gene transfer
Neonatal rats
作者简介
张珊珊，女，硕士研究生。主攻方向：小儿脑损伤。
[通讯作者]郑湘榕，女，博士，副教授，中南大学湘雅医院儿科，邮编：410008。
