构建Ad-XIAP感染原代培养老年性聋模型小鼠皮层神经细胞的实验研究

Transfection of primary culture cortical neurons with recombinant adenovirus carrying the X-linked inhibitor of the apoptosis protein gene

目的构建携带绿色荧光(GFP)的X染色体连锁凋亡抑制蛋白基因的重组腺病毒载体(Ad-X IAP)感染体外原代培养老年性聋小鼠模型(C57BL/6J)皮层神经细胞,为后续体内基因治疗实验提供体外研究基础。方法体外培养C57BL/6J小鼠皮层神经细胞,-βTubu lin鉴定其纯度;利用Ad-Easy载体系统通过细菌内同源重组法构建携带X IAP基因的腺病毒表达载体,筛选鉴定并线性化后获得重组腺病毒载体,TC ID50法测定其滴度后转染体外原代培养的皮层神经细胞,观察其感染效率并用RT-PCR检测细胞内X IAP-mRNA表达量。结果 PCR及酶切鉴定穿梭质粒含有X IAP基因,经筛选和鉴定的Ad-X IAP感染皮层神经细胞72h后观察到细胞携带绿色荧光最强,此时X IAP-mRNA表达量最高。结论成功构建了携带X IAP基因的高滴度重组腺病毒载体,滴度高、皮层神经细胞感染率大于90%,为后续试验提供了体外研究基础。

Objective To construct the green fluorescence protein（GFP） labeled recombinant adenovirus vector carrying the XIAP gene and transfect it into cortical nerve cells.Methods A method of primary culture cortical neurons of the C57BL/6J mouse was developed and an experiment cell model of the neurons was established.Morphological changes of neuron cells were observed by a light microscope.Double immuno-staining of β-Tubulin and DAPI were applied to assess the culture purity.The XIAP gene recombinant adenovirus vector was constructed by the homologous recombination.Ad-XIAP was transfected into cortical nerve cells after testing the virus titer determined by tissue culture infectious dose 50（TCID50）.Results The target gene XIAP amplified by PCR was identified by DNA sequencing.The adenoviral vector could be examined 24 hours after transfection,and the green fluorescence intensity became greatest at about 72 hours.Conclusion The recombinant adenovirus vector containing XIAP was successfully constructed and a highly efficient virus was obtained which could efficiently transfect cortical nerve cells and this laid a foundation for following investigations.