重组杆状病毒载体介导钠碘转运体基因在胰岛细胞中表达的研究

刘帅，潘昱，张一帆.重组杆状病毒载体介导钠碘转运体基因在胰岛细胞中表达的研究[J].上海交通大学学报.2013, 33(3):263-268

Notch 1;急性T淋巴细胞白血病;斑马鱼;双荧光示踪

刘帅，潘昱，张一帆

上海交通大学医学院附属瑞金医院核医学科,上海,200025

2013年

263-268

2,万方

目的 探讨以杆状病毒为载体,钠碘同向转运体(NIS)基因作为报告基因监测胰岛细胞的可行性.方法 通过Bac-to-Bac杆状病毒表达系统制备绿色荧光蛋白(GFP)和NIS基因重组杆状病毒(Bac-GFP和Bac-NIS).分别以不同感染复数(MOI)的Bac-GFP感染大鼠胰岛细胞,荧光显微镜观察胰岛细胞的感染百分比和荧光表达强度；以MOI为40的Bac-GFP感染胰岛细胞,荧光显微镜下观察荧光表达时间及细胞生存状态.以不同MOI的Bac-NIS感染胰岛细胞,测定感染细胞的碘摄取情况；以MOI为40的Bac-NIS感染胰岛细胞,动态观察感染细胞摄碘能力的变化以及高氯酸钠(NaClO4)对细胞摄碘的影响.结果 Bac-GFP可高效感染SD大鼠胰岛细胞,对细胞无毒性作用；感染的胰岛细胞百分比和荧光强度随MOI的提高而上升,细胞感染百分比可高达95％以上(MOI =40),且荧光表达时间可持续2周左右.Bac-NIS感染的胰岛细胞表现出摄碘功能增强和NaClO4抑制摄碘的特性,且感染细胞摄碘的放射性计数随MOI的提高而增加；MOI为40时,Bac-NIS感染细胞摄碘的放射性计数是空白对照的12倍.结论 杆状病毒介导的NIS基因在胰岛细胞中高效表达,有效介导放射性碘摄取,可用对胰岛细胞移植情况的监测. Objective 1'o establish stable germline ICN1(intracellular domain Notchl)transgenic and double tracer 'I' acute lymphoid leckemia, T一ALL zebrafish models by overexpressing human 1CN1 gene in fli-FGFP zebrafish through T lymphocytes specific Rag2 promoter. Methods ICN1 plasmid carrying red fluorescent protein "reporter gene"(Rag2- 1CN 1一FGFP) was constructed and microinjected into zebrafish single一cell embrSros, then the FO(founder) generation was identified by fluorescence screening for establishing the zebrafish stable transgenic germline. The expression of ICN1 gene and protein was determined by fluorescence microscope, RT-PCR and Western blotting, and the phenotype of T-ALL zebrafish models was identified by flow、ytometry and peripheral blood smear. Results Twenty of the 867(2. 3 0I0)mosaic FO zebrafish embryos injected with the constructed vector were identified to be the germline transgenic zebrafish, including 9 males and 11 females. The FO adult fish of sexual maturity was copulated as one一to-one form, then F1 generation was produced. By using general view fluorescence microscope, RT-PCR and Westers blotting, the expression of ICN1 was validated at tl}e general view, mRNA and protein levels. Flow cytometry and peripheral blood smears suggested a significant reduction in the proportion of red blood cells and an increase of lymphocyte ratio in transgenic zebrafish as(ompared to control group. Conclusion Notchl gene induced T一ALL zebrafish models with double tracer system have been successfully constructed, which may provide a research platform for the in-depth discussion on the ICN1 in the promotion of regulatory mechanism of T-ALL and high throughput drug screening ira vivo.。