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

汤维芳;刘菲;陈世彩;陈东辉;李孟;朱敏辉;郑宏良;

1:第二军医大学长海医院耳鼻咽喉头颈外科

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。

关键词

人肝细胞生长因子;慢病毒;喉肌;成肌干细胞

基金项目(Foundation):

国家自然科学基金(81070774/H1302)资助

作者

汤维芳;刘菲;陈世彩;陈东辉;李孟;朱敏辉;郑宏良;

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人肝细胞生长因子慢病毒喉肌成肌干细胞

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汤维芳刘菲陈世彩陈东辉李孟朱敏辉郑宏良