血红素加氧酶-1基因过表达对脊髓神经元损伤的保护作用

目的探讨血红素加氧酶-1(HO-1)基因过表达对脊髓神经元损伤的保护作用。方法利用腺相关病毒(AAV)构建HO-1过表达载体,运用免疫荧光标记神经元特异性标志物鉴定原代脊髓神经元,比较正常培养(对照组)、磷酸缓冲盐溶液(PBS)+H2O2(单纯损伤组)、PBS+AAV-EGFP+H2O2(AAV-EGFP组)、PBS+AAV-HO-1+H2O2(AAV-HO-1组)的HO-1、IL-6、TNF-α的蛋白表达与神经凋亡情况。结果HO-1重组腺相关病毒基因测序结果表明,获得的克隆片段和目的基因的DNA序列完全一致。β-tubulin-Ⅲ标记神经元,DAPI用于核定位呈现蓝色,其他细胞不显色。AAV-HO-1组HO-1蛋白表达比对照组高,单纯损伤组、AAV-EGFP组、AAV-HO-1组IL-6蛋白与TNF-α蛋白表达比对照组高,差异有统计学意义(P<0.05),单纯损伤组、AAV-EGFP组IL-6蛋白与TNF-α蛋白表达比AAV-HO-1组高差异有统计学意义(P<0.05)。单纯损伤组与AAV-EGFP组比较细胞凋亡水平差异没有统计学意义(P>0.05)。AAV-HO-1组的细胞凋亡比单纯损伤组和AAV-EGFP组的细胞凋亡比例小,差异有统计学意义(P<0.05)。结论过表达HO-1通过抑制炎症反应减少氧化应激损伤后细胞凋亡,对原代大鼠脊髓神经元氧化应激损伤起保护作用。

Protective effects of heme oxygenase-1 gene overexpression on injured spinal cord neurons

Objective To investigate the protective effects of heme oxygenase-1(HO-1)gene overexpression on injured spinal cord neurons.Methods HO-1 overexpression vector was constructed by using adeno-associated virus(AAV).Primary spinal cord neurons were identified by immunofluorescence labeling.Primary spinal cord neurons were randomly assigned to 4 groups,including normal culture(Control),PBS+H2O2(Vehicle),PBS+AAV-EGFP+H2O2(AAV-EGFP)and PBS+AAV-HO-1+H2O2(AAV-HO-1)groups.Enzyme linked immunosorbent assay(ELISA)was applied to detect the expression of HO-1,IL-6 and TNF-αprotein.The rate of neuronal apoptosis was detected by flow cytometry.Results The sequencing results of HO-1 recombinant adeno-associated virus showed that the DNA sequence of the obtained cloned fragment and the target gene were completely identical.β-tubulin-Ⅲmarked neurons,DAPI was used to dye the nucleus blue,and other cells did not develop color.The expression of HO-1 protein in AAV-HO-1 group was significantly higher than that in control group(P<0.05).The expression of IL-6 and TNF-αprotein in Vehicle,AAV-EGFP and AAV-HO-1 groups was significantly higher than that in control group(P<0.05),the expression of IL-6 and TNF-αprotein in Vehicle and AAV-EGFP groups was significantly higher than that in AAV-HO-1 group(P<0.05).There was no statistically significant difference in the level of apoptosis between the Vehicle group and the AAV-EGFP group(P>0.05).The proportion of apoptosis in the AAV-HO-1 group was significantly lower than that in the Vehicle and AAV-EGFP groups(P<0.05).Conclusion Overexpression of HO-1 protects primary spinal cord neurons from oxidative stress injury due to inhibition of the inflammation and reduction of the neuronal apoptosis.