【摘要】 目的构建携带针对人miR-100-3p抑制序列的慢病毒载体,建立稳定表达的该载体的小鼠胚胎成纤维(MEF)细胞。方法设计并合成特定的miR-100-3p抑制序列克隆入载体h U6-MCS-Ubiquitin-EGFP-IRES-puromycin构建重组载体,将该载体与p Helper1.0载体和p Helper2.0载体三质粒共感染293T细胞得到所需的病毒液,最后感染MEF细胞,并通过嘌呤霉素筛选出稳定感染的细胞株。检测MEF细胞中绿色荧光蛋白(GFP)的表达和hsa-miR-100-3p在MEF细胞中的表达水平,验证慢病毒在MEF细胞中的抑制表达效果。结果测序结果证明成功构建了重组质粒,并成功的包装成慢病毒,所得病毒液感染MEF细胞并筛选出稳定细胞株。流式分析提示,带有绿色荧光蛋白的目的重组质粒在小鼠胚胎成纤维细胞细胞中的表达(显著抑制miR-100表达)达到了97.4%和95.1%,PCR进一步验证这一结果。结论 hsa-MiR-100-3p抑制序列慢病毒沉默载体及抑制miR-100-3p表达的MEF细胞可被成功构建,为后续miR-100的功能研究奠定基础。更多还原

【Abstract】 Objective To establish the mouse embryonic fibroblast( MEF) cells lines with stable expression of MiR-100-3p inhibitor,which lays a foundation for its function research of the role of miR-100 in the interaction of environmental pollutants and Src homology 2 domain tyrosine phosphatases( SHP-2) gene mutation. Methods Designed and synthesized a particular DNA fragment as hsa-MiR-100-3p inhibitor,which was then cloned into the h U6-MCS-Ubiquitin-EGFP-IRES-puromycin vector. This vector was then co-transfected with lentiviral packaging vectors p Helper1. 0 and p Helper2. 0 into 293 T to produce lentiviral particles. Finally,MEF cells were infected with the above-mentioned lentiviral particles and the stable cell lines expressing hsa-miR-100-3p inhibitor were selected by puromycin. The expression of green fluorescent protein( GFP) and the expression of miR-100 were detected which indicated its inhibition effect of lentiviral vector in MEF cells. Results The construction of the recombinant plasmid was confirmed by sequencing. The subsequent infection confirmed that the packaging of lentiviruses was successfully. MEF cells successfully infected with hsa-miR-100-3p inhibitor. Flow cytometry indicatesthe expression of green fluorescent protein( significantly inhibition the expression of miR-100) has reached 97. 4%and 95. 1%,as well as proved by RT-PCR. Conclusion We successfully constructed the has-miR-100-3p lentiviral silencing vector,and established the stable inhibition cell lines which lay a foundation for its function research of miR-100.更多还原