中文摘要：

目的构建含小鼠Hes1基因的重组腺病毒（rAd）并观察其在小鼠海马组织中的表达情况．为进一步研究Hes1基因在成年小鼠神经再生中的作用奠定基础。方法利用限制酶对pEGFP—miles1质粒和pDC316质粒进行酶切，将获得的miles1目的基因片段和pDC316质粒酶切产物回收,在T4DNA连接酶作用下连接，形成pDC316-mHes1穿梭质粒，并用PCR法及EcoRI＋HindⅢ双酶切法对pDC316-miles1进行鉴定。利用AdMax包装系统，穿梭质粒pDC316-mHes1与骨架质粒pBHGlox_E1,3Cre共转染293细胞，同源重组产生复制缺陷型腺病毒Ad5-mHes1，其报告病毒是包含高强度绿色荧光蛋白（EGFP）的腺病毒Ad5-EGFP。向成年C57BL／6小鼠海马中立体定向注射Ad5-miles1及Ad5-EGFP，Western blotting检测注射后7d Hes1蛋白在海马中的表达情况，荧光显微镜观察EGFP在海马中的表达情况。结果用PCR法及EcoR I＋HindⅢ双酶切法对pDC316-miles1鉴定的实验结果与预期结果相符，经测序后其序列与miles1 CDS序列一致。注射后7d．Hesl蛋白在PBS注射组和Ad5-mHes1注射组海马组织中均有表达，其Hesl蛋白与GAPDH的比值分别为0．363±0．053和0．705±0．128，差异有统计学意义（P〈0．05）。荧光显微镜下在海马齿状回颗粒细胞层中观察到Ad5-EGFP表达的绿色荧光。结论本研究成功构建了Ad5-miles1表达载体系统，并在C57BL／6成年小鼠海马组织中表达了Hes1基因。

英文摘要：

Objective To construct an adenoviral vector containing mouse Hesl gene, observe its expression in the hippocampus of adult mice and build a basis for further investigation of Hes1 gene in adult neurogenesis. Methods The restriction endonuclease was used to digest plasmid pEGFP-mHes1 and pDC316, and then, the products were recovered and connected by T4 DNA ligase and the shuttle plasmid pDC316-mHes1 was constructed which was identified by the method of PCR and EcoRI＋Hind Ⅲ digestion. After that, the shuttle plasmid pDC316-mHes1 was cotransfected into 293 cells with the adenovirus skeleton plasmid pBHGlox_E1,3Cre to obtain the produced replication defective recombinant adenovirus Ad5-mHes1. Then, the recombinant adenovims could be further amplified and purified. The report recombinant adenovimses were Ad5-EGFP containing enhanced green fluorescent protein （EGFP）. Then, Ad5-mHes1 and Ad5-EGFP were stereotactic injected into the hippocampus of the adult C57BL/6 mice and their expressions in the hippocampus were detected. Western blotting was used to detect the Hesl protein level 7 d after the injection. Fluorescent microscopy was used to observe the expression of EGFP in the hippocampus. Results The experimental results of identification by the methods of PCR and EcoRI＋Hind Ⅲ digestion were in accordance with the anticipated results, and the sequences were also the same with mHeslCDS sequences; Hesl gene was expressed in the hippocampus of both the PBS injection group and the Ad5-mHesl injection group 7 d after the injection, and the expression of Hesl/GAPDH in Ad5-mHes1 injection hippocampus （0.705 ±0.128） was statistically different as compared with that in PBS injection group （0.363±0.053, P〈0.05）. Ad5-EGFP strongly expressed in the granular cell layer and subgranular zone （SGZ） of dentate gyrus. Conclusion The adenoviral vector of mouse Hesl gene is successfully established and Hesl gene is expressed in the hippocampus of C57BL/6 adult mice.