miR-217慢病毒表达载体及稳转胶质瘤细胞系的构建  

Construction of MiR-217 Lentiviral Expression Vector and Establishment of Stable Transfected Glioma Cell Lines Expressed MiR-217

作　　者：赵保[1] 苏国军[1] 张淑霞 吴峰[3] 苏陈[3] 叶晶亮[1] 韩瑞章 郑林丰[4] 

机构地区：[1]解放军第98医院神经外科,浙江湖州313000 [2]湖州市药检所,浙江湖州313000 [3]解放军第98医院心血管内科,浙江湖州313000 [4]上海交通大学附属第一人民医院放射科,上海200080

出　　处：《现代生物医学进展》2015年第14期2625-2628,共4页Progress in Modern Biomedicine

基　　金：国家留学基金项目(201303810388);上海市自然科学基金项目(12ZR1424900);上海交通大学附属第一人民医院"优秀青年人才"基金

摘　　要：目的:构建mi R-217慢病毒表达载体并建立稳定表达mi R-217的胶质瘤细胞系,为深入研究mi R-217在胶质瘤细胞生长及功能中的作用及机制提供条件。方法:利用人基因组中mi R-217前体序列,设计并合成引物。采用PCR的方法扩增含mi R-217前体的目的片段,酶切后连接至慢病毒表达载体p LVX-EGFP-Puro。将带有mi R-217前体的慢病毒载体及辅助质粒用脂质体的方法转染293细胞,24小时后收集上清液测定病毒滴度。利用实时定量PCR检测mi R-217的表达水平,确定慢病毒载体的表达能力。将病毒感染胶质瘤细胞系U251,通过荧光观察和嘌呤霉素(Puromycin)筛选,获得稳定转染mi R-217的胶质瘤细胞系。结果:克隆的mi R-217前体目的片段经PCR检测和测序分析,序列完全正确、无突变。实时定量PCR检测发现慢病毒感染293T细胞后,mi R-217的表达水平明显升高(P<0.01),表明mi R-217慢病毒表达载体构建成功。经嘌呤霉素筛选后,荧光显微镜观察发现细胞均有稳定的绿色荧光表达,并且mi R-217的表达水平显著升高(P<0.01),是对照组的12.5倍。结论:成功构建了mi R-127慢病毒表达载体和稳转胶质瘤细胞系,为后续研究奠定了良好基础。Objective： To construct a miR-217 lentiviral expression vector and establish a stable transfected glioma cell lines that overexpressed miR-217, which founded a platform to study role of miR-217 in growth and the function of glioma cells. Methods： Primers were designed according the human miR-217 precursor sequence. Then miR-217 precursor was amplified by PCR, and loaded to the lentiviral expression vector pLVX-EGFP-Puro. After infection of 293T cell with the lentiviral vector with miR-217 and helper plasmid by liposome, the supernatant were collected and virus titrations were identified. The expression ability of lentiviral vector was determined by detecting miR-217 levels using real-time PCR. The stable transfected glioma cell line with miR-217 was obtained by fluorescence observation and puromycin resistance screening. Results： PCR and sequencing assay showed that the cloned fragment with miR-217 precursor was identified with no mutation; real-time PCR assay showed that miR-217 was markedly increased in 293T cell transfeeted by lentiviral vector with miR-217（P〈0.01）; these results confirmed that the lentiviral vector with miR-217 has been successfully constructed. After puromycin resistance screening, the remaining cells showed stable green fluorescence, and miR-217 was increased significantly （P〈0.01）, with 12.5-fold to control. Conclusion： We have successfully constructed the miR-127 lentiviral vector and established a stable transfected glioma cell lines, these can pave the road for the further studies.

关 键 词：MI R-217 胶质瘤细胞 慢病毒 基因表达 

分 类 号：Q78[生物学—分子生物学] R739.4[医药卫生—肿瘤]