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目的构建过表达miR-145的慢病毒载体,建立稳定过表达miR-145的鼻咽癌细胞株。方法构建慢病毒表达质粒pLVTHM/miR-145;293FT细胞进行慢病毒包装,生产的慢病毒感染鼻咽癌细胞株CNE1和CNE2,经流式细胞仪筛选后,建立稳定表达miR-145的鼻咽癌细胞株;最后应用荧光定量PCR检测病毒感染后鼻咽癌细胞株CNE1和CNE2中miR-145的表达水平。结果荧光定量PCR结果和测序验证pLVTHM/miR-145重组质粒构建成功;包装生产的慢病毒感染鼻咽癌细胞CNE1和CNE2后,与阴性对照组比较,其表达水平分别升高4000倍和4500倍。结论成功构建了pLVTHM/miR-145慢病毒重组质粒,建立稳定表达miR-145的鼻咽癌细胞株CNE1和CNE2,为研究miR-145在鼻咽癌发生发展过程中的基因调控和相应的作用机制提供了有用的细胞模型。
Objective To construct lentiviral vector overexpressing miR-145 and establish a cell line of nasopharyngeal carcinoma stably overexpressing miR-145. Methods The lentiviral expression plasmid pLVTHM / miR-145 was constructed. 293FT cells were infected with lentivirus to infect nasopharyngeal carcinoma cell lines CNE1 and CNE2. After screening by flow cytometry, the nasopharyngeal carcinoma cells stably expressing miR-145 Finally, the expression level of miR-145 in nasopharyngeal carcinoma cell lines CNE1 and CNE2 after viral infection was detected by fluorescence quantitative PCR. Results The recombinant plasmid pLVTHM / miR-145 was successfully constructed by fluorescence quantitative PCR and sequencing. Compared with the negative control group, the packaged recombinant lentivirus was infected by CNE1 and CNE2 lentivirus and the expression levels were increased 4000 times and 4500 times . Conclusion The recombinant plasmid pLVTHM / miR-145 was successfully constructed and CNE1 and CNE2 cell lines stably expressing miR-145 were established. To study the gene regulation and its role of miR-145 in nasopharyngeal carcinoma Mechanisms provide useful cell models.