| 李文文,曲妍佳,肖君华,李 凯,周宇荀.MicroRNA-29b过表达慢病毒载体的构建及其生物学特性的研究[J].现代生物医学进展英文版,2017,17(25):4807-4811. |
| MicroRNA-29b过表达慢病毒载体的构建及其生物学特性的研究 |
| Construction of MicroRNA-29b Overexpression Lentiviral Vector and Study on Its Biological Characteristics |
| Received:December 09, 2016 Revised:December 29, 2016 |
| DOI：10.13241/j.cnki.pmb.2017.25.002 |
| 中文关键词: microRNA-29b 慢病毒颗粒 GT1-7细胞 靶基因 性发育相关基因 |
| 英文关键词: microRNA-29b Lentivirus GT1-7 Target gene Sexual development related genes |
| 基金项目:上海市科学技术委员会资助项目（15DZ1940802） |
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| 中文摘要: |
| 摘要 目的：构建针对小鼠microRNA-29b过表达的慢病毒载体，研究其在小鼠神经元GT1-7细胞系中的生物学特性。方法：化学合成两条寡聚核苷酸单链，通过搭桥互补延伸成DNA双链，形成miR-29b的前体结构，将酶切后的慢病毒载体FUGW通过同源重组的方法与miR-29b的前体结构进行连接，构建相应microRNA-29b 过表达慢病毒载体，并包装成病毒颗粒后转染小鼠神经元细胞系GT1-7，通过博来霉素药物筛选获得稳转株，RT-PCR检测相关基因在mRNA转录水平上表达量情况。结果：测序图谱证实重组慢病毒表达质粒f-F-miR-29b构建成功，GT1-7细胞稳转株中，miR-29b的表达量与对照组相比提高了约28倍，其靶基因DCX，Vdac1，Pten的表达量有所抑制，性发育相关基因LH-β，kiss-1，Inshulin，IGF-I，GPR54，GnRH，leptin-R没有明显变化。结论：利用慢病毒筛选的方法，成功在小鼠神经元GT1-7细胞中获得microRNA-29b过表达稳转株，为以后microRNA-29b的生物学特性的研究奠定了基础。 |
| 英文摘要: |
| ABSTRACT Objective: To construct a lentiviral vector overexpression of micrRNA-29b and investigate the biological characteris- tics in mouse neuronal cell lines GT1-7. Methods: We chemically synthesized two oligonucleotide single-stranded, complete the comple- mentary by bridging extension into DNA double-stranded to form miR-29b precursor structure. The restriction enzyme digested vector plasmid FUGW was ligated to the precursor structure of miR-29b by homologous recombination to construct the corresponding lentiviral vector of microRNA-29b overexpression, and the stable cells were obtained in the mouse neuronal cell line GT1-7 by bleomycin drug screening. RT-PCR was used to detect the expression level of related genes at mRNA transcription level, Results: The recombinant lentiviral expression plasmid f-F-miR-29b was successfully constructed, and the expression level was about 30 times higher than that of the control group. The expressions of DCX, Vdac1 and pten were inhibited, have no changes in sex developmental related genes LH-β, kiss-1, Inshulin, IGF-I, GPR54, GnRH and leptin-R. Conclusion: Using the method of lentivirus screening, the microRNA-29b overex- pressing stably transformed cells was successfully obtained in mouse neuron GT1-7 cells, which laid a foundation for the study of biolog- ical characteristics of microRNA-29b. |
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