慢病毒干扰水通道蛋白4促进脊髓损伤修复与其上调电压依赖性阴离子通道2相关性的研究  

Study on the Promotion of Spinal Cord Injury Repair by the Lentiviral Interference with AQP4 and Its Correlation with the Up-regulation of VDAC2

作　　者：吕芾 张华阳 黄守娟 向轩颐 郑莉 张晓[1] 张奇兰[1] Lv Fu;Zhang Huayang;Huang Shoujuan;Xiang Xuanyi;Zheng Li;Zhang Xiao;Zhang Qilan(Chengdu Medical College,Chengdu 610500,China)

机构地区：[1]成都医学院

出　　处：《成都医学院学报》2019年第6期687-695,共9页Journal of Chengdu Medical College

基　　金：国家级大学生创新创业训练项目(No:201713705040)

摘　　要：目的采用慢病毒干扰水通道蛋白4(AQP4)的表达,观察电压依赖性阴离子通道2(VDAC2)在脊髓损伤后的表达变化,探讨VDAC2的差异性表达对脊髓损伤修复过程的影响。方法将SD大鼠随机分为脊髓钝挫伤(SCC)组、假手术(Sham)组、AQP4慢病毒干扰(AQP4-SH-LV)组和空载(Vector)组。采用Allen′s法制备SCC模型;Gene MANIA生物信息学方法预测AQP4与VDAC2的关联;通过注射包装AQP4siRNA的质粒,构建慢病毒干扰AQP4的SCC模型。BBB评分评价大鼠后肢的运动功能;苏木精-伊红(HE)染色显示脊髓组织损伤状况;实时荧光定量PCR(qRT-PCR)测定VDAC2mRNA表达量;免疫组织化学染色(IHC)检测VDAC2定位及半定量情况。结果1)BBB评分结果显示,脊髓钝挫伤后1dSCC组和Vector组得分明显降低,接近0分。AQP4-SH-LV组得分的下降趋势与Vector组相似,但各时间点评分均高于后者。HE染色结果显示,脊髓顿挫伤后脊髓组织发生水肿,干扰AQP4后水肿程度有所减轻。2)qRT-PCR结果表明,与Sham组相比,SCC组各时间点VDAC2的mRNA转录水平降低(P<0.05);慢病毒干扰AQP4,SCC术后第7天,AQP4-SH-LV组体内VDAC2mRNA转录水平较Vector组上调(P<0.05)。3)免疫组化结果显示,VDAC2免疫阳性产物主要分布于细胞质。SCC组各时间点VDAC2免疫阳性细胞数均低于Sham组(P<0.05);AQP4-SH-LV组各时间点VDAC2免疫阳性细胞数均高于Vector组。4)GeneMANIA结果显示,AQP4和VDAC2两分子间具有特定的生理联系,如共表达、共定位、共享蛋白质结构域等。结论脊髓损伤后,大鼠体内VDAC2的转录水平下降,不利于脊髓组织损伤后的恢复。慢病毒干扰AQP4可上调VDAC2转录水平,并促进脊髓损伤的修复。Objective To observe the expression changes of voltage-dependent anion channel 2(VDAC2)in spinal cord contusion(SCC)by the lentiviral interference with aquaporin 4(AQP4)and explore the effect of the differential expression of VDAC2 on the repair process of spinal cord injury.Methods The adult Sprague-Dawley rats were assigned randomly to the SCC group,sham operation group,AQP4-SH-LV group and empty vector group.The SD rat models with SCC were established by the Allen’s free-fall hammer hitting method.The Gene MANIA Bioinformatic analysis was used to predict the correlation between AQP4 and VDAC2.The SSC models interfered by AQP4 was constructed by injecting the plasmid packaged with AQP4 siRNA.The motor function was evaluated by Basso,Beattie,Bresnahan(BBB)Locomotor Rating Scale,hematoxylin-eosin staining(HE staining)was used to show the status of the spinal cord injury,quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression level of VDAC2 mRNA,and immunohistochemistry(IHC)was used to determine the localization and semi-quantification of VDAC2.Results Firstly,the BBB scores in the SCC group and empty vector group were significantly reduced and they were close to zero.The downtrend of the BBB score in the AQP4-SH-LV group was similar to that in the empty vector group,but the scores of AQP4-SH-LV group were higher than those of the empty vector group at each time point.The results of HE staining showed the edema in spinal cord after SCC and the degree of spinal edema was reduced after the interference of AQP4.Secondly,the results of qRT-PCR revealed that the transcriptional levels of VDAC2 mRNA in the SCC group were significantly lower than those in the sham operation group at each time point(P<0.05),and those in the AQP4-SH-LV group were significantly higher than those in the empty vector group at the 7 th day after the SCC operation by the lentiviral interference with AQP4(P<0.05).Thirdly,the IHC results showed that the immunoreactive positive reactants of VDAC2 were mainly di

关 键 词：脊髓损伤 细胞水肿 VDAC2 AQP4 线粒体 

分 类 号：R361[医药卫生—病理学]