大鼠VGLUT2基因shRNA慢病毒载体转染原代培养脊髓神经元对VGLUT2表达的影响  

Effects on expression of VGLUT2 in primary cultured spinal cord neurons transfected shRNA targeting VGLUT2 gene lentiviral vectors in rats

作　　者：方相春[1] 张春奎[1] 唐君[1] 李志红[1] 张婷[1] 李金莲[1] 

机构地区：[1]第四军医大学人体解剖与组织胚胎学教研室暨梁銶琚脑研究中心,西安710032

出　　处：《神经解剖学杂志》2012年第6期537-543,共7页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81070900)

摘　　要：目的:探讨新构建的可以表达针对2型囊泡膜谷氨酸转运体(vesicular glutamate transporter 2,VGLUT2)短发卡RNA(short hairpin RNA,shRNA)的慢病毒载体能否有效地感染大鼠脊髓原代培养神经元,并鉴定其对培养神经元内VGLUT2基因的特异性干扰效果,从而为进一步在整体动物脊髓水平研究VGLUT2的功能提供有力的工具。方法:首先分别将已筛选到的两对互补并靶向作用于编码大鼠VGLUT2序列两个位点的特异性shRNA序列和一个阴性对照的寡核苷酸,克隆到pGCSIL-GFP质粒(经Age I和EcoRI双酶切)载体内,经酶切、测序鉴定及转染293T细胞,包装得到病毒颗粒。然后将筛选出的慢病毒感染体外分离、培养的胚胎大鼠脊髓神经元,将培养的原代神经元分为正常组、阴性对照组、VGLUT2-shRNA-1组和VGLUT2-shRNA-2组,在荧光显微镜下分别观察GFP的表达情况;同时运用Westernt blot检测各组VGLUT2蛋白的表达水平。结果:免疫荧光检测显示,阴性对照组、VGLUT2-shRNA-1组和VGLUT2-shRNA-2组脊髓原代培养神经元均能观察到明显的GFP荧光,表明这些神经元已被慢病毒载体高效转染;但VGLUT2 shRNA-1组和VGLUT2 shRNA-2组神经元VGLUT2的表达量明显低于正常组和阴性对照组。Western blot结果显示,VGLUT2-shRNA-1组和VGLUT2-shRNA-2组的VGLUT2蛋白的表达量与正常组和阴性对照组相比明显有所下降,分别占正常组的62.42±1.12%和66.07±1.33%(P<0.05),但VGLUT2-shRNA-1组和VGLUT2-shRNA-2组之间无显著性差异(P>0.05);阴性对照组与正常组相比亦无明显差异(P>0.05)。结论:VGLUT2-shRNA-1组和VGLUT2-shRNA-2组重组慢病毒载体均能有效地感染原代培养脊髓神经元,并高效下调其神经元内目的基因VGLUT2的表达。Objective： To investigate whether the new recombined lentiviral vectors which express short hairpin RNA （shRNA） targeting the vesicular glutamate transporter 2 （VGLUT2） could efficiently transfeeted the primary cultured rat spinal cord neurons, and detect its specific interference effect on VGLUT2 gene, thus provide a potent tool for further studying the function of VGLUT2 at the level of spinal cord in a whole animal. Methods： Firstly, two specific selected shRNA sequences which target the two sites of VGLUT2 coding sequences and one pair of negative control oligonucleotide sequence were cloned into the pGCSIL-GFP plasmid （ restricted by Age I and EcoRl）. Secondly, after identified by re- striction and sequencing, the pGCSIL-GFP plasmid was transfected into the 293T cells, and the viral panicles were pack-aged. Then, the primary cultured spinal cord neurons of rats were transfected by the lentiviral vectors. The primary cul- tured spinal cord neurons were divided into four groups： normal group, negative control group, VGLUT2-shRNA-1 group, VGLUT2-shRNA-2 group. Finally, the expressional level of the GFP was observed under fluorescence microscope, and the protein quantities of VGLUT2 were tested with Western blot as well. Results： In negative control group, VGLUT2- shRNA-1 group and VGLUT2-shRNA-2 group, green GFP fluorescence could be observed obviously in the primary cul- tured spinal cord neurons, indicating these neurons were effectively transfected by the lentiviral vectors. But the protein quantities of VGLUT2 expression in VGLUT2 shRNA-1 group and VGLUT2-shRNA-2 group were lower than normal group and negative control group. The Western blot examination showed： compared with normal group and negative control group, the expression quantity of VGLUT2 in VGLUT2-shRNA-1 group and VGLUT2-shRNA-2 group were declined sig- nificantly to 62.42 ＋ 1.12% and 66.07 ＋ 1.33% of the normal group, respectively （P 〈 0.05）. However, there is no significant difference between VGLUT2-shRNA-1 and

关 键 词：VGLUT2 RNA干扰 慢病毒载体 脊髓原代培养神经元 大鼠 

分 类 号：R346[医药卫生—基础医学]