构建人HCN4基因重组腺病毒载体及其对大鼠骨髓间充质干细胞的感染效率  

Construction of recombinant adenovirus containing human HCN4 gene and its transfection efficiency in rat bone marrow mesenchymal stem cells

作　　者：袁桂仪[1] 伍卫[1] 周淑娴[1] 张玉玲[1] 雷娟[1] 

机构地区：[1]中山大学附属第二医院心血管内科,广东省广州市510120

出　　处：《中国组织工程研究与临床康复》2009年第45期8944-8948,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:超极化激活及环化核苷酸门控阳离子通道(hyperpolarization-activated cyclic nucleotide-gated cation channel,HCN)基因由于具有不增加诱发心律失常的风险、能够接受自主神经系统调节等优势,成为目前最受关注的生物起搏备选基因。目的:构建携带人HCN4基因的重组腺病毒载体,并测定其对大鼠骨髓间充质干细胞的感染效率。设计、时间及地点:细胞-基因学体外实验,于2008-02/09在中山大学附属第二医院林百欣实验中心完成。材料:SD大鼠10只,由中山大学实验动物中心提供。携带目的基因人HCN4cDNA的质粒pcDNA3.1-HCN4、人胚肾293细胞、大肠杆菌DH5α由中山大学附属第二医院林百欣实验中心保存。腺病毒穿梭质粒pShuttle-CMV、骨架质粒pAdxsi购自北京诺赛基因组研究中心有限公司。方法:质粒pcDNA3.1-HCN4用HindⅢ+XbaⅠ双酶切后回收HCN4片段,亚克隆至pShuttle-CMV中,得到重组穿梭质粒;I-CeuⅠ+I-SceⅠ双酶切处理pShuttle-CMV-HCN4,回收CMV-HCN4片段,亚克隆至腺病毒骨架载体pAdxsi,得到重组腺病毒质粒;重组腺病毒质粒酶切线性化后,应用脂质体法转染293细胞进行包装扩增,得到重组腺病毒AdHCN4;应用AdHCN4转染大鼠骨髓间充质干细胞。主要观察指标:重组腺病毒质粒载体的鉴定,重组腺病毒的鉴定及滴度测定,重组腺病毒的感染效率。结果:构建的重组穿梭质粒pShuttle-CMV-HCN4用HindⅢ+XhoⅠ双酶切,得到大小为3600bp(HCN4)和5100bp(pShuttle-CMV)两个片段,DNA测序结果证实人HCN4基因的全长序列已正确插入到pShuttle-CMV穿梭质粒中;重组腺病毒质粒pAdxsi-CMV-HCN4用XhoⅠ酶切得到7个片段,而作为对照的空腺病毒质粒只得到6个片段;重组腺病毒质粒在293细胞中包装后产生的重组腺病毒对293细胞有致病作用;重组腺病毒AdHCN4PCR鉴定可见657bp的阳性扩增条带;经多次重复感染后,病毒滴度检测达2.5×1011PFU/mL。成功转染AdHCN4的大鼠骨髓�BACKGROUND： The hyperpolarization-activated cyclic nucleotide-gated cation channel （HCN） gene is not increase the risk of induced cardiac arrhythmia, and can accept the modulation function of autonomic nervous system. Therefore, it is the first candidate gene for biological pacemakers. OBJECTIVE： To construct recombinant adenovirus vector containing human HCN4 gene and evaluate its transfection efficiency in rat bone marrow mesenchymal stem cells （MSCs）. DESIGN, TIME AND SETTING： The in vitro cytology-gene experiment was performed at the Lin Bai-xin Experimental Center, Second Hospital of Sun Yat-sen University from February to September 2008. MATERIALS： Ten SD rats were supplied by the experimental animal center of Sun Yat-sen University. Plasmid pcDNA3.1-HCN4 containing target gene human HCN4, human embryo kidney 293 cells and Escherichia coli DH5a were preserved by our experimental center; Shuttle plasmid pShuttle-CMV containing green fluorescent protein gene and adenoviral backbone plasmid pAdxsi were bought from SinoGenoMax Co., Ltd. METHODS： HCN4 cDNA segment was liberated from the cloning vector of pcDNA3.1-HCN4 via Hind Ⅲ＋Xba Ⅰ, and subcloned into pShuttle-CMV, which was digested by I-Ceu Ⅰ ＋I-Sce Ⅰ double enzyme and subcloned into adenoviral plasmid to form recombinant adenovirus plasmid. Recombinant adenovirus plasmid was transfected into 293 cell lines by liposome, and the recombinant adenovirus AdHCN4 was packaged and transfected into rat MSCs. MAIN OUTCOME MEASURES： The identification of recombinant adenovirus plasmid vector; identification of recombinant adenovirus and its titration test; the transfection efficiency of recombinant adenovirus. RESULTS： Cloned sequence about 3.6kbp was obtained by Hind Ⅲ＋Xho Ⅰ digestion after HCN4 cDNA segment was cloned into pShuttle-CMV. DNA sequencing results indicated that the clone location was correct. Recombinant adenovirus plasmid was cut into seven fragments while empty vector gained only six fragments after

关 键 词：HCN4 腺病毒载体 骨髓间充质干细胞 基因转染 

分 类 号：R394.2[医药卫生—医学遗传学]