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目的:构建人源性mir-148a慢病毒过表达载体,并筛选过表达mir-148a稳定细胞株U251、U87、BT325,鉴定mir-148a在稳定细胞系中的表达水平。方法:设计并合成mir-148a上下游引物,PCR扩增mir-148a基因片段,经过酶切后克隆至慢病毒载体上,构建p GC-FU-3FLAG-SV40-EGFP-IRES-puromycin-mir-148a过表达载体,在293T细胞中与p Helper1.0、p Helper2.0包装产生慢病毒,测定mir-148a慢病毒滴度。用构建好的慢病毒感染神经胶质瘤细胞系U251、U87、BT325,用嘌呤霉素筛选,筛选稳定细胞系后q RT-PCR检测mir-148a的表达情况。结果:测序结果证明mir-148a慢病毒载体构建成功并获得了相应的慢病毒。嘌呤霉素筛选后获得稳定表达mir-148a的U251、U87、BT325细胞系,mir-148a表达水平在三种细胞中分别升高167.38倍、7.19倍、11.46倍。结论:成功构建了mir-148a的慢病毒载体,筛选得到了mir-148a过表达稳定胶质瘤细胞系U251、U87、B325。
OBJECTIVE: To construct human miR-148a lentiviral vector and to screen the mir-148a-overexpressing cell line U251, U87 and BT325 to identify the expression of mir-148a in stable cell line. Methods: The mir-148a upstream and downstream primers were designed and synthesized. The mir-148a gene fragment was amplified by PCR and cloned into the lentiviral vector to construct pGF-FU-3FLAG-SV40-EGFP-IRES-puromycin- 148a overexpression vector. Lentivirus was packaged in 293T cells with p Helper 1.0 and p Helper 2.0, and the titer of mir-148a lentivirus was determined. Glioma cell lines U251, U87 and BT325 were infected with the constructed lentivirus, and the cells were screened with puromycin. The stable cell lines were screened by q RT-PCR to detect the expression of mir-148a. Results: The sequencing results showed that mir-148a lentiviral vector was successfully constructed and the corresponding lentivirus was obtained. After puromycin selection, U251, U87 and BT325 cell lines stably expressing mir-148a were obtained, and the expression levels of mir-148a increased by 167.38 times, 7.19 times and 11.46 times respectively in the three kinds of cells. Conclusion: The lentiviral vector of mir-148a was successfully constructed and the stable glioma cell lines U251, U87 and B325 overexpressing mir-148a were obtained.