内源性VEGF表达对缺血性脑损伤后成年大鼠海马齿状回神经发生影响的实验研究

The effect of endogenous VEGF gene expression on dentate gyrus neurogenesis after cerebral ischemia in adult rats

目的观察缺血性脑损伤后成年大鼠海马内源性VEGF表达对齿状回神经前体细胞增殖水平的影响。方法将携带靶向干扰大鼠VEGF基因shRNA的重组腺相关病毒颗粒通过立体定向给药的方法递送至大鼠右侧海马,术后通过改良的4血管闭塞方法建立成年大鼠缺血性脑损伤动物模型。应用实时荧光定量PCR和免疫组化染色法检测大鼠海马VEGF mRNA的表达水平及齿状回神经前体细胞的增殖水平。结果荧光显微镜下观察重组腺相关病毒颗粒立体定向给药至海马区的大鼠脑片,可见报告基因EGFP的表达。使用实时荧光定量PCR方法,发现rAAV2-EGFP-U6-shRNA(VEGF)立体定向给药大鼠缺血性脑损伤后24h和6d时缺血性脑损伤诱导的大鼠海马VEGF mRNA表达水平反应性升高可被抑制(P<0.05),海马内源性VEGF mRNA表达水平的降低可显著下调缺血性脑损伤后齿状回神经前体细胞增殖水平。结论内源性VEGF基因表达可能是缺血性脑损伤后齿状回神经前体细胞增殖的一个重要启动信号。VEGF是耦联缺血性脑损伤和齿状回神经发生的关键分子。

Objective To investigated the effect of endogenous VEGF gene expression on dentate gyrus neurogenesis after cerebral isehemia in adult rats. Methods The recombinant adeno-associated virus （AAV） vector for expressing short hairpin RNA （shRNA） targeting endogenous VEGF mRNA was delivered into the right hippocampus of adult rats by stereotaxie technique. After operation, these rats were subjected to 4-vessel occlusion （4-VO） cerebral isehemia model. Real Time PCR and immunohistoehemistry were used to detected the endogenous VEGF mRNA and dentate gyrus neurogenesis. Results Under fluorescence microscope,the report gene EGFP could be observed in the brain slice of adult rat that targeted delivery of rAAV2-EGFP-U6- shRNA（VEGF） virus. Compared with isehemia rats, the level of endogenous VEGF mRNA in shRNA-imported ischemia rats significantly decreased at 24h and 6d after ischemia （ P 〈 0.05 ）. Immunohistochemical staining result indicated that the proliferation level of neural precursor cells in right DG of shRNA-imported rats was significantly lower than that in ischemia rats at 6 and 24d after isehemia （P 〈 0.05 ）, but there was no significant difference at 24h after ischemia （ P 〉 0.05 ）. Conclusion The endogenous VEGF gene expression maybe an important start signal related to hippocampus neurogenesis after cerebral ischemia. It is a key molecule coupled cerebral isehemia with dentate gyrus neurogenesis.