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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 newly constructed lentiviral vector expressing short hairpin RNA (shRNA) directed against vesicular glutamate transporter 2 (VGLUT2) can effectively infect the spinal cord of rats On the other hand, cultured neurons and identified its specific interference effect on VGLUT2 gene in cultured neurons, which provided a powerful tool to further study the function of VGLUT2 in the whole animal spinal cord. METHODS: Two pairs of screened oligonucleotides that were complementary to each other and targeted to specific shRNA sequences and one negative control, which encode two sites of rat VGLUT2 sequence, were cloned into pGCSIL-GFP plasmid I and EcoRI double digestion) vector, after digestion, sequencing and identification and transfected 293T cells, packaging virus particles. Then, the selected primary lentivirus was infected in vitro and isolated from spinal cord neurons of embryonic rat. Primary cultured neurons were divided into normal group, negative control group, VGLUT2-shRNA-1 group and VGLUT2-shRNA-2 group. The expression of GFP was observed under a fluorescence microscope. At the same time, the expression of VGLUT2 protein in each group was detected by Westernt blot. Results: The results of immunofluorescence showed that GFP fluorescence was observed in primary cultured neurons of VGLUT2-shRNA-1 group and VGLUT2-shRNA-2 group, indicating that these neurons have been efficiently transfected by lentiviral vectors However, the expression of VGLUT2 in VGLUT2 shRNA-1 group and VGLUT2 shRNA-2 group was significantly lower than that in normal group and negative control group. Western blot results showed that the expression of VGLUT2 protein in VGLUT2-shRNA-1 group and VGLUT2-shRNA-2 group was significantly decreased compared with the normal control group and negative control group, accounting for 62.42 ± 1.12% and 66.07 ± 1.33% (P <0.05). However, there was no significant difference between VGLUT2-shRNA-1 group and VGLUT2-shRNA-2 group (P> 0.05) ). CONCLUSION: The recombinant lentiviral vectors of VGLUT2-shRNA-1 and VGLUT2-shRNA-2 can effectively infect primary cultured spinal cord neurons and down-regulate the expression of VGLUT2 in neurons.