转基因系构建斑马鱼胰岛β细胞发育模型的建立

发布时间：2018-05-26 00:32

本文选题：斑马鱼 + β细胞　；参考：《贵阳中医学院》2008年硕士论文

【摘要】： 目的:本课题旨在利用转基因技术,构建活体的胰岛β细胞绿色荧光可示踪的斑马鱼系,从而为在此基础上进一步破坏胰岛β细胞,建立活体的胰岛β细胞可示踪的,可供高通量药物筛选的糖尿病胰岛β细胞破坏模型作出前期准备。方法:通过转基因技术,PCR技术、分子克隆等方法,建立GFP标记胰岛B细胞的遗传学转基因斑马鱼品系;活体呈像动态示踪胰岛B细胞的起源和成熟;全胚胎原位杂交和基因组PCR证实GFP阳性细胞与内源性胰岛B细胞共定位。结果:1.获得INS部分启动子的克隆和重组质粒INS:GFP,显微镜注射建立生殖系胰岛素转基因斑马鱼。 2.通过生殖系转基因斑马鱼的筛选和荧光观察,24hpf GFP标记的胰腺β细胞群被探测到位于第二、三体节下方中线两侧的单一的椭圆形实体。随着斑马鱼胚胎的发育,胰腺逐渐增大并向右迁移,72hpf可以观察到胰腺β-细胞位于腹中线的右侧。卵黄囊消失后,120hpf明显看到胰腺右偏,同时下移到鱼鳔下方的十二指肠背侧。 3.全基因组PCR证实GFP在基因组中的稳定整合,全胚胎原位杂交说明了活体转基因斑马鱼的GFP细胞为胰岛β-细胞。结论:1.利用转基因技术,构建了胰岛β细胞可示踪斑马鱼系。 2.通过PCR和全胚胎原位杂交的方法证实所建立的胰岛β细胞可示踪斑马鱼系是完全成功的。 3.本研究的成功为进一步建立可供高通量药物筛选的胰岛β细胞破坏斑马鱼疾病模型奠定了研究基础。
[Abstract]:Objective: to construct a living zebrafish line with green fluorescent tracer of islet 尾 cells by using transgenic technology, so as to further destroy the islet 尾 cells and establish in vivo islet 尾 cells traceable. Diabetic islet 尾-cell destruction model for high-throughput drug screening was prepared. Methods: genetic transgenic zebrafish strains labeled with GFP were established by means of transgene technique and molecular cloning, and the origin and maturation of islet B cells were traced in vivo. Whole embryo in situ hybridization and genomic PCR confirmed that GFP positive cells were colocated with endogenous islet B cells. The result is 1: 1. The partial promoter of INS was cloned and the recombinant plasmid ins: GFP was obtained, and the reproductive line insulin transgenic zebrafish was established by microscope injection. 2. By screening and fluorescence observation of transgenic zebrafish, a single elliptical entity located at the middle line of the second and third ganglia was detected by 24hpf GFP labeled pancreatic 尾 cell group. With the development of zebrafish embryos, the pancreas gradually enlarged and migrated to the right of 72hpf. It was observed that the pancreatic 尾-cells were located on the right side of the middle line of abdomen. At 120h after the disappearance of yolk sac, the pancreas could be seen to the right and move down to the dorsal side of the duodenum below the swim bladder. 3. The whole genome PCR confirmed the stable integration of GFP in the genome. The in situ hybridization of whole embryo indicated that the GFP cells of transgenic zebrafish were islet 尾-cells. Conclusion 1. Islet 尾-cells traceable zebrafish lines were constructed by transgenic technique. 2. The establishment of islet 尾 cells was proved to be completely successful by PCR and whole embryo in situ hybridization. 3. The success of this study laid a foundation for the further establishment of islet 尾 -cell destruction model for zebrafish disease.
【学位授予单位】：贵阳中医学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R587.1;R-332

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