UNC5B基因靶向shRNA表达质粒的构建及体外RNA干扰

目的：探讨人UNC5B基因的双链RNA在体外对UNC5B表达的干扰作用及其规律。方法：构建两个能产生UNC5B的发夹状RNA（shRNA）的质粒载体Pgenesil—UNC581及Pgenesil—UNC582作为实验组，并构建无关序列shRNA表达质粒Pgenesil—NC作为阴性对照，将3种质粒分别转染脐静脉内皮细胞（HUVEC）。转染24h后，荧光倒置显微镜下观察质粒转染效率，用G418筛选得到稳定转染的细胞，通过逆转录聚合酶链反应（RT—PCR）检测UNC5B在HUVECs中的表达情况。结果：转染24h后，在荧光显微镜下可见绿色荧光，转染效率约为40％～50％。实验组经G418筛选后稳定转染的细胞UNC5BmRNA均受到抑制。两种UNC5B基因靶向shRNA表达质粒对HUVECs中UNC5B抑制率分别为88％和52％。结论：本研究所构建的UNC5BshRNA表达质粒可在体外高效特异地抑制UNC5B的表达，为进一步实验奠定了基础。

Establishment of UNC5B shRNA and Its Effect on UNC5B Expression in Vitro

Objective: To construct the siRNA plasmids for UNC5B gene, which has been reported to be the only receptor of neural guidance axon Netrin-1 selectively expressed on vascular system and their inhibitory effects on UNC5B expression in vitro. Methods: Plasmids UNC5B shRNA complimentary to the sequence responsible for the function donmain of human UNC5B was constructed. Plasmid expressing irrelevant shRNA with a random combination was used as negative control. The recombinant plasmid vector was transfected into HUVECs with liposome. Cells were observed under fluorescence at 24 h post-transfection. After screened with G418 for three weeks, the stable transfected cells were obtained. RT-PCR was used to investigate the inhibitory effect of the recombinant plasmid on UNC5B expression in vitro. Results: Transfection of UNC5B shRNA was observed under fluorescence at 24 h.The transfection efficiency was about 40%~50%. After screened with G418, the expression of UNC5B in HUVECs at mRNA level was inhibited. The inhibitory efficiency was 88% and 52% respectively. Conclusion: The study demonstrated that the construct of UNC5B shRNA successfully interfered in the UNC5B expression in vitro. It provides a basis for a further investigation of effect of UNC5B.