表达VEGF的重组腺相关病毒对大鼠创伤性脊髓损伤的保护作用及其机制研究

目的探讨表达VEGF的重组腺相关病毒(recombinant adeno-associated virus,rAAV)对大鼠创伤性脊髓损伤(spinal cord injury,SCI)的保护作用及其机制。方法取144只成年雄性SD大鼠(体重200～250 g)随机分为4组,每组36只。假手术组(A组)仅手术暴露脊髓。模型对照组(B组)、rAAV-绿色荧光蛋白(green fluorescentprotein,GFP)组(C组)、rAAV-hVEGF165-GFP组(D组)制备创伤性SCI大鼠模型,术后即刻分别予以20μL生理盐水、rAAV-GFP病毒、rAAV-hVEGF165-GFP病毒脊髓注射治疗。术后3、7 d采用BBB评分观察大鼠后肢运动功能变化;术后7 d通过脊髓组织尼氏小体染色观察脊髓组织病理学变化,脊髓组织神经元透射电镜检测及TUNEL凋亡细胞染色观察脊髓神经元凋亡,Western blot检测水通道蛋白4(aquaporin 4,AQP-4)表达;术后1、3、5、7 d ELISA法检测VEGF165蛋白表达。结果 BBB评分显示术后3、7 d D组神经功能较B、C组显著改善(P<0.05)。尼氏小体染色显示术后7 d D组脊髓组织结构破坏明显轻于B、C组(P<0.05)。ELISA检测显示D组VEGF165蛋白表达呈低剂量缓释表达,术后3、5、7 d,D组VEGF165蛋白表达均高于其他3组(P<0.05)。透射电镜检测及TUNEL凋亡细胞染色结果显示术后7 d D组脊髓神经元凋亡较B、C组显著减少,凋亡率显著低于B、C组(P<0.05)。Western blot结果显示术后7 d D组脊髓组织AQP-4蛋白表达明显较B、C组减少(P<0.05)。结论表达VEGF的rAAV通过抗神经元凋亡及减轻脊髓水肿对大鼠创伤性SCI起保护作用。

Neuroprotective effects of recombinant adeno-associated virus expressing vascular endothelial growth factor on rat traumatic spinal cord injury and its mechanism

Objective To investigate the neuroprotective effects of recombinant adeno-associated virus(rAAV) expressing vascular endothelial growth factor(VEGF) on traumatic spinal cord injury(SCI) of rat and its mechanisms.Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups,and each group contained 36 rats.The rats in sham group(group A) received dorsal laminectomy without SCI and microinjection,the rats in model control group(group B),rAAV-green fluorescent protein(GFP) group(group C),and rAAV-hVEGF165-GFP group(group D) received dorsal laminectomy with SCI and injection of 20 μL saline,rAAV-GFP viruses,or rAAV-hVEGF165-GFP viruses,respectively.At 3 and 7 days after operation,Basso-Beattie-Bresnahan(BBB) score was used to evaluate the neurologic function.At 7 days after operation,Nissl’s body staining was used to evaluate the histopathological changes;apoptosis was con rmed by transmission electron microscope examination and TUNEL staining;the expression of aquaporin 4(AQP-4) was detected by Western blot assay.At 1,3,5,and 7 days,ELISA assay was used to detect the VEGF165protein expression.Results According to BBB scores,the neurologic function in group D was signi cantly better than those in groups B and C at 3 and 7 days after operation(P < 0.05).Nissl’s body staining showed that tissue damage in group D was signi cantly milder than those in groups B and C at 7 days after operation(P < 0.05).ELISA results showed that VEGF165protein expression was slowly-released in low dose in group D,and the expression in group D was signi cantly higher than that in groups A,B,and C at 3,5,and 7 days after operation(P < 0.05).The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was signi cantly lower than that in groups B and C at 7 days after operation(P < 0.05).The results of Western blot showed that AQP-4 expression in group D was signi cantly decreased when compared with that in groups B and C at 7 days after operation(P < 0.05).Conclusion The rAAV expressing VEGF has neuroprotective e ects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.