| 赵保 苏国军 张淑霞 吴峰 苏陈 叶晶亮 韩瑞章 郑林丰.miR-217慢病毒表达载体及稳转胶质瘤细胞系的构建[J].,2015,15(14):2625-2628 |
| 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 ofStable Transfected Glioma Cell Lines Expressed MiR-217 |
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| DOI： |
| 中文关键词: miR-217 胶质瘤细胞 慢病毒 基因表达 |
| 英文关键词: MiR-217 Glioma cell Lentivirus Gene expression |
| 基金项目: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 |
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| 中文摘要: |
| 目的：构建miR-217 慢病毒表达载体并建立稳定表达miR-217 的胶质瘤细胞系，为深入研究miR-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miR-217 前体序列，设计并合成引物。采用PCR的方法扩增含miR-217 前
体的目的片段，酶切后连接至慢病毒表达载体pLVX-EGFP-Puro。将带有miR-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miR-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miR-217 的胶质瘤细胞系。结果：克隆
的miR-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，
miR-217 的表达水平明显升高（P<0.01），表明miR-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miR-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miR-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。 |
| 英文摘要: |
| Objective:To construct a miR-217 lentiviral expression vector and establish a stable transfected glioma cell lines that
overexpressed miR-217, which founded a platformto 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 transfected 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. |
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