[重组腺相关病毒表达血管内皮生长因子对大鼠创伤性脊髓损伤的神经保护作用及其机制]

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

作者信息

Qiang Hui, Zhang Chen, Shi Zhibin, Ling Ming

机构信息

Department of Orthopaedics, Shaanxi Provincial People's Hospital (the Third Affiliated Hospital of School of Medicine, Xi'an Jiaotong University), Xi'an Shaanxi, 710068, P.R.China.

出版信息

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Jun;26(6):724-30.

PMID:22792773

Abstract

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 microL 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 confirmed 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 VEGF165 protein expression.

RESULTS

According to BBB scores, the neurologic function in group D was significantly 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 significantly milder than those in groups B and C at 7 days after operation (P < 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly 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 significantly 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 significantly 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 effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.

摘要

目的

探讨表达血管内皮生长因子（VEGF）的重组腺相关病毒（rAAV）对大鼠创伤性脊髓损伤（SCI）的神经保护作用及其机制。

方法

将144只雄性Sprague Dawley大鼠随机分为4组，每组36只。假手术组（A组）大鼠行背部椎板切除术但无脊髓损伤及微量注射；模型对照组（B组）、rAAV-绿色荧光蛋白（GFP）组（C组）和rAAV-hVEGF165-GFP组（D组）大鼠行背部椎板切除术并造成脊髓损伤，分别注射20 μL生理盐水、rAAV-GFP病毒或rAAV-hVEGF165-GFP病毒。术后3天和7天，采用Basso-Beattie-Bresnahan（BBB）评分评估神经功能。术后7天，采用尼氏染色评估组织病理学变化；通过透射电镜检查和TUNEL染色确认细胞凋亡；采用蛋白质印迹法检测水通道蛋白4（AQP-4）的表达。在术后1、3、5和7天，采用酶联免疫吸附测定（ELISA）法检测VEGF165蛋白表达。

结果

根据BBB评分，术后3天和7天，D组神经功能明显优于B组和C组（P<0.05）。尼氏染色显示，术后7天，D组组织损伤明显轻于B组和C组（P<0.05）。ELISA结果显示，D组VEGF165蛋白表达呈低剂量缓慢释放，术后3天、5天和7天，D组表达明显高于A组、B组和C组（P<0.05）。透射电镜和TUNEL染色结果显示，术后7天，D组脊髓神经元凋亡率明显低于B组和C组（P<0.05）。蛋白质印迹结果显示，术后7天，D组AQP-4表达与B组和C组相比明显降低（P<0.05）。