腺病毒载体介导血管内皮生长因子165基因治疗新生大鼠缺氧缺血性脑损伤

目的 研究腺病毒介导血管内皮生长因子(VEGF)165基因转移对新生大鼠缺氧缺血性脑损伤(HIBD)的治疗作用.方法 采用细菌内同源重组技术构建腺病毒重组载体Ad-VEGF.7日龄SD大鼠随机分成3组:假手术组(20只)、HIBD(20只)、Ad-VEGF移植组(Ad-VEGF组,20只),Ad-VEGF移植组在HIBD后3 d于大鼠左侧感觉运动皮层区(坐标AP:+0.3mm,ML:-2 mm,DV:-1.5 mm)立体定位注射2μl重组体腺病毒悬液.移植后7 d采用免疫组织化学法检测鼠脑VEGF蛋白;采用原位缺口末端标记法(TUNEL法)检测鼠脑神经元凋亡情况;大鼠3～4周龄时采用T迷宫觅食实验及足错误、姿势反射、肢体放置实验对其学习记忆、空间能力和感觉运动功能等行为学进行评估;采用尼氏染色法检测鼠脑海马CAl区和皮层单位面积内神经元数目.结果 免疫组织化学法结果 显示皮层与海马Ad-VEGF组VEGF阳性细胞数均明显多于HIBD组(68.09±3.37比24.65±3.37,68.37±3.17比25.14±1.86,均P<0.05).TUNEL结果 显示Ad-VEGF组鼠脑神经元凋亡数目明显低于HIBD组(151.4±21.7比264.4±16.3,P<0.05);行为学检测结果 显示Ad-VEGF组的各项行为学测试成绩均较HIBD组有明显改善(均P<0.05),尼氏染色结果 显示Ad-VEGF组鼠脑海马CAl区和皮层单位面积内神经元数日明显多于HIBD组(70.6±2.3比55.3±2.1,95.1±2.8比70.1±2.7,均P<0.05).结论 腺病毒载体介导的VEGF165基因治疗可增加VEGF蛋白的表达,减少脑细胞凋亡、改善脑损伤后的远期行为学功能,减轻缺氧缺血后的脑损伤,具有神经保护作用.

Adenovirus-mediated vascular endothelial growth factor165 gene therapy in treatment of hypoxic-ischemic brain damage: experiment with rats

Objective To investigate the therapeutical effect of adenovirus-mediated vascular endothelial growth factor (VEGF)165 gene transplantation in treatment of hypoxic-ischemic brain damage. Methods Recombinant vector of adenovirus-mediated VEGF165 gene (Ad-VEGF) was constructed by bacterial homologous recombination technology. Sixty 7-day-old Sprague-Dawley rats were randomly divided into 3 equal groups: hypoxic-ischemic brain damage (HIBD) group undergoing ligation of the left common carotid artery and inhalation of 8% oxygen for 2 hours, Ad-VEGF group undergoing injection of Ad-VEGF into the left sensorimotor cortex with the help of stereo-positioner 3 days after hypoxia-ischemia ( HI), and sham operation group. Seven days after transplantation, 5 rats from each group were killed with their left brains taken out. The VEGF protein expression was detected by immunohistocbemistry, and the neuron apoptosis was detected by terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL). Since the age of 28 days T-maze foraging test was conducted. At the age of 34 days, sensorimotor tests were performed. After the behavioral tests all the rats were killed. The number of neurons in the CA1 region of the hippocampus and cerebral cortex was detected by Nissl's staining. Results Immunohistochemistry showed that the density levels of VEGF positive cells in cerebral cortex and hippocampus of the Ad-VEGF group were (68.09 ±3.37) and (68.37±3.17) respectively, beth significantly higher than those of the HIBD group [ (24.65±3.14) and (25.14±1.86) respectively, both P < 0.05 ]. TUNEL showed that the number of apoptotic neurons of the Ad-VEGF group was ( 151.4 ± 21.7), significantly lower than that of the HIBD group [ (264.4 ±16.3), P <0. 05]. Behavioral tests showed the percentages of accuracy on day 4 of the Ad-VEGF and sham operation groups were both significantly higher than that of the HIBD group (both P <0.01 ). Nissl's staining showed that the numbers of neurons per unit area in the hippocampal CAI area and cortex of the Ad-VEGF group were (70.6±2.3) and (95.1±2.8) respectively, both significantly higher than those of the HI group [ (55.3 ± 2. 1 ) and (70.1 ± 2.7) respectively, both P < 0.05 ]. Conclusion Adenovirns vector-mediated VEGF gene therapy increases the VEGF protein expression, decreases neuron apeptosis, improves the long-term behavioral function after brain damage, and reduces hypoxic ischemic brain injury, thus possessing neuroprotective effects.