抑制小鼠低氧诱导因子-1α基因的小干扰RNA腺病毒载体的构建和功能鉴定

Construction and functional identification of siRNA adenoviral vector targeting HIF-1α in mouse

目的构建针对小鼠低氧诱导因子-1α（hypoxia-inducible transcription factor-1α，HIF-1α）基因的小干扰RNA（small interference RNA，siRNA）腺病毒载体AdHIF-1α-shRNA也GFP，鉴定AdHIF-1α-shRNA-FGFP在培养的小鼠胚胎神经干细胞中对HIF-1α表达的影响，为研究HIF-1α信号途径在神经干细胞生物学中的作用提供基础。方法采用生物信息学方法设计合成针对HIF-1α的siRNA靶DNA序列，通过DNA重组技术构建小鼠HIF-1α-shRNA-EGFP腺病毒载体，PCR和测序鉴定。体外转染培养的小鼠前脑皮质神经干细胞，流式细胞仪检测转染效率，对AdHIF-1α-shRNA-EGFP转染的细胞进行低氧处理，双重免疫荧光检测小鼠胚胎神经干细胞中HIF-1α表达，逆转录-聚合酶链反应（RT-PCR）和Westernblot定量HIF-1α基因和蛋白水平。结果所克隆的序列和已知序列一致；AdHIF-1α-shRNA-EGFP可高效转染小鼠前脑皮质神经干细胞；RT-PCR和Westernblot结果显示，AdHIF-1α-shRNA-EGFP转染小鼠前脑皮质神经干细胞后，低氧条件下小鼠前脑皮质神经千细胞中HIF-1α的基因和蛋白表达分别降低52．05％和67．27％。结论构建的AdHIF-1α-shRNA也GFP能够成功抑制低氧条件下小鼠前脑皮质神经干细胞中HIF-1α的表达。

Objective To construct an adenoviral vector targeting hypoxic inducible factor-1α （HIF-1α） gene in mouse and to identify the effect of the adenoviral vector, AdHIF-1α hRNA-EGFP, on the expression of HIF-1α in cultured mouse embryonic neural stem cells, and provide a basis for investigating the contribution of HIF-1α signaling pathway in neural stem cell biology. Methods The HIF-1α template DNA sequence was designed by using bioinformatics. DNA recombinant technique was used to construct recombinant adenoviral vector, AdHIF-1α-shRNA-EGFP. Polymerase chain reaction （PCR） and sequencing were performed. The neural stem cells from frontal cortex of the mouse were transfected with AdHIF-1α-shRNA-EGFP in vitro. The efficiency of transfection was detected by flow cytometry. The transfected cells were cultured under hypoxia condition. Double immunofluorescent staining of nestin and HIF-α was performed, and the expression of HIF-1α was quantified by reverse transcription -polymerase chain reaction （RT-PCR） and Western blot analysis. Results The sequence cloned into the adenoviral vector was identical with the one synthesized chemically. The neural stem cells were transfected effectively by AdHIF--1α-shRNA-EGFP. The results from RT-PCR and Western blot analysis showed that the expression of HIF-1α gene and protein in the neural stem cells transfected with AdHIF-1α- shRNA-EGFP decreased by 52.05% and 67.27% under hypoxia condition, respectively. Conclusion AdHIF-1α-shRNA-EGFP could effectively inhibit hypoxia -induced expression of HIF-1α gene and protein in the mouse neural stem cells in vitro.