人肝细胞生长因子基因重组慢病毒载体的构建及其在成肌干细胞中的表达  

Construction of Human Hepatocyte Growth Factor Gene Recombinant Lentivirus Vectors and Its Expression in Myoblasts

作　　者：汤维芳[1] 刘菲[1] 陈世彩[1] 陈东辉[1] 李孟[1] 朱敏辉[1] 郑宏良[1] 

机构地区：[1]第二军医大学长海医院耳鼻咽喉头颈外科,上海200433

出　　处：《听力学及言语疾病杂志》2015年第1期45-49,共5页Journal of Audiology and Speech Pathology

基　　金：国家自然科学基金(81070774/H1302)资助

摘　　要：目的构建人肝细胞生长因子(human hepatocyte growth factor,hHGF)基因重组pCMV-G﹠NR-U6-hHGF慢病毒载体,并检测其在成肌干细胞系C2C12细胞中的hHGF表达,为治疗失神经喉肌纤维化的研究提供依据。方法采用RT-PCR法扩增并纯化得到hHGF基因片段,并将其克隆到pCMV-G﹠NR-U6上,再将重组慢病毒载体转化入感受态大肠杆菌DH5α,筛选阳性菌落并行BamHI和HindⅢ双酶切鉴定。对筛选后的重组质粒阳性克隆行PCR鉴定和测序,将表达质粒与包装质粒共转染293T细胞包装hHGF慢病毒,荧光显微镜检测病毒滴度,再进一步转染C2C12细胞(分为空白对照组、空载体组、HGF重组慢病毒过表达组),最后行PCR和WB检测HGF的表达情况。结果 PCR鉴定和测序结果显示所构建的慢病毒载体正确无误,包装后的病毒滴度>1×109 IU/mL;慢病毒过表达组的HGF表达量均较空白对照组、空载体组明显增高,而且72h仍有稳定的表达。结论本实验成功构建了慢病毒载体pCMV-G﹠NR-U6-hHGF,并能够在离体培养的成肌干细胞系C2C12细胞中稳定高效表达HGF,为治疗失神经喉肌等骨骼肌纤维化、提高神经修复效果的实验研究提供了依据。Objective To construct a gene recombinant lentiviral vector pCMV -G -U6 -hHGF and detect its expression in C2C12 myoblast cells .Methods hHGF gene fragments were obtained and purified by RT -PCR method ,and were cloned to pCMV -G＆amp;NR -U6 ,then the restructured lentiviral vector was transformed into e . coli DH5 alpha ,the positive colonies were identified by BamHI and Hind Ⅲ enzyme digestion .The selected positive colonies were tested by PCR and sequencing analysis .The expression plasmids and packing plasmids were co -trans_fected into 293 T cells ,and virus titer was observed under the fluorescence microscope .Furthermore ,transfected C2C12 cells with lenti virus ,and the expression of HGF was detected by PCR and WB methods .ResuIts PCR and sequencing analysis showed that the lentiviral vector was constructed correctly and successfully ,the virus titer was above 1 × 10^9 IU/mL .The results of PCR and WB showed that HGF expression level in the lentiviral vector group was much higher than those of in blank control and negative control groups ,and yet the expression was stable after 72 hours .ConcIusion The lentiviral vector pCMV -G﹠NR -U6 -hHGF has been successfully constructed ,and stable expressed in C2C12 cells .It provides references for experimental study in the fields of the denervated skeletal muscle fibrosis and nerve regeneration treatment .

关 键 词：人肝细胞生长因子 慢病毒 喉肌 成肌干细胞 

分 类 号：R767.62[医药卫生—耳鼻咽喉科]