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目的:构建含hsa-miR-128-1和hsa-miR-128-2前体的慢病毒表达载体,建立能够稳定表达目的基因的人胶质瘤U87细胞株。研究hsa-miR-128对胶质瘤细胞增殖的影响。方法:扩增hsa-miR-128前体片段,克隆入Pentr3c载体,利用LR clonaseⅡ酶将目的片段转接于Plenti6.3质粒,包装病毒,以改构的Plenti6.3-mCherry载体包装的病毒为对照,感染人胶质瘤U87细胞,通过Blasticidin筛选出能够稳定表达抗生素抗性的细胞株。荧光显微镜观察mCherry的荧光表达,实时荧光定量检测miR-128的表达量,流式细胞仪检测细胞周期,MTT检测细胞增殖。结果:成功构建重组慢病毒载体Plenti6.3-miR-128-1和Plenti6.3-miR-128-2,经Blasticidin筛选细胞株、mCherry荧光表达和实时荧光定量PCR验证,成功包装出慢病毒。病毒滴度测定在1.1×107-8.3×107TU/mL之间,细胞周期和MTT结果提示miR-128-1和miR-128-2高表达的U87细胞株均表现增殖抑制。结论:成功构建了分别含有两种hsa-miR-128前体的慢病毒表达载体,筛选出稳定表达hsa-miR-128的U87细胞株。miR-128-1和miR-128-2均抑制胶质瘤U87细胞增殖。
OBJECTIVE: To construct a lentiviral vector containing hsa-miR-128-1 and hsa-miR-128-2 precursors and establish a human glioma U87 cell line which can stably express the target gene. To investigate the effect of hsa-miR-128 on the proliferation of glioma cells. Methods: The precursor fragment of hsa-miR-128 was amplified and cloned into Pentr3c vector. The target fragment was transferred to Plenti6.3 plasmid using LR clonase Ⅱ enzyme. The virus was packaged and the virus packaged with the modified Plenti6.3-mCherry vector was In contrast, human glioma U87 cells were infected and cell lines stably expressing antibiotic resistance were selected by Blasticidin. The fluorescence of mCherry was observed by fluorescence microscopy. The expression of miR-128 was detected by real-time fluorescence quantitative analysis. The cell cycle was detected by flow cytometry. The proliferation of mCherry was detected by MTT assay. Results: The recombinant lentiviral vector Plenti6.3-miR-128-1 and Plenti6.3-miR-128-2 were successfully constructed. The lentivirus was successfully packaged by Blasticidin screening, mCherry fluorescence and real-time PCR. The virus titer was determined between 1.1 × 107-8.3 × 107TU / mL. The cell cycle and MTT results showed that the proliferation of miR-128-1 and miR-128-2 highly expressed U87 cell lines all showed inhibition. Conclusion: The lentiviral vector containing two hsa-miR-128 precursors was successfully constructed, and the U87 cell line stably expressing hsa-miR-128 was screened out. Both miR-128-1 and miR-128-2 inhibited glioma U87 cell proliferation.