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目的:构建miRNA的慢病毒表达载体,使其在原代培养的大鼠胚胎大脑皮层神经细胞中稳定表达。方法:将该干扰片段构建入慢病毒载体(pL KD-Ubc-eG FP-U6-shRNA)的U6启动子下游,该载体可以实现在干扰小片段RNA的同时表达绿色荧光蛋白EGFP基因,便于观察载体工作状态。通过脂质体转染法转染原代培养的大鼠胚胎大脑皮层神经细胞,观察转染的原代培养的大鼠胚胎大脑皮层神经细胞的形态及表达情况。结果:针对筛选的miRNA构建的慢病毒载体,经过DNA测序结果和琼脂糖凝胶电泳鉴定成功构建了筛选的miRNA的慢病毒表达载体,重组质粒转染原代培养的大鼠胚胎大脑皮层神经细胞,经24 h后在荧光倒置显微镜下可以观察到部分细胞带有绿色荧光,筛选的miRNA构建的慢病毒载体在原代培养的大鼠胚胎大脑皮层神经细胞中稳定表达。结论:筛选的miRNA的慢病毒表达载体构建成功,并稳定的转染到原代培养的大鼠胚胎大脑皮层神经细胞中,为miRNA对神经细胞存活功能的研究提供了平台。
OBJECTIVE: To construct a lentiviral expression vector of miRNA and make it stably expressed in primary cultured rat cerebral cortex neurons. Methods: The interference fragment was constructed into the downstream of the U6 promoter of lentiviral vector (pL KD-Ubc-eG FP-U6-shRNA), which can express the green fluorescent protein EGFP gene while interfering with the small fragment of RNA for observation Carrier work status. Primary cultured rat embryo cerebral cortex nerve cells were transfected by lipofectamine to observe the morphology and expression of neuronal cells in primary cultured rat embryonic cortex. Results: The lentiviral vector constructed by the selected miRNA was identified by DNA sequencing and agarose gel electrophoresis. The lentiviral expression vector of the selected miRNA was successfully constructed, and the recombinant plasmid was transfected into primary cultured rat embryonic cerebral cortex neurons After 24 h, some cells were observed under fluorescent inverted microscope with green fluorescence. The lentiviral vector constructed by the selected miRNA was stably expressed in primary cultured rat cerebral cortex neurons. CONCLUSION: The lentiviral expression vector of selected miRNA was successfully constructed and stably transfected into primary cultured rat cerebral cortex neurons, which provided a platform for miRNAs to study the survival of neurons.