鱼藤素对斑马鱼胚胎中细胞周期蛋白D1基因下调的作用：整胚原位杂交检测

摘要

BACKGROUND:In the early development of zebrafish embryos, cels divide and proliferate rapidly, but low concentration of deguelin can delay the development of zebrafish embryos. OBJECTIVE:To observe the effect of different concentrations of deguelin on cyclin D1 gene expression in zebrafish embryos. METHODS:Though normal fertilization, zebrafish embryos that incubated to the 2-cel stage (about 0.75 hour after fertilization) and shield stage (6 hours after fertilization) were colected and put into 12-wel plates treated with 100, 200, 400 nmol/L deguelin at 28.5℃in an incubator til the shield period and 24 hours after fertilization, respectively. Simultaneously embroys treated with 1% dimethyl sulfoxide solution were as a control group, cultured in the same conditions. Cyclin D1 RNA probes were prepared for the whole mountin situhybridization, observing staining by an upright fluorescent microscope camera to detect the effect of deguelin on cyclin D1 expression in zebrafish embryos. RESULTS AND CONCLUSION:Deguelin showed significant negative regulation on the expression of cyclin D1 gene in zebrafish embryos. Cyclin D1 expressed in outsourcing cels in embryos of shield stage, and a significant reduction in the expression of cyclin D1 came up with the increasing concentrations of deguelin. In the 400 nmol/L deguelin treatment group, there was nearly no expression of cyclin D1. Cyclin D1 expressed in the brain, central nervous system, immature eye, somites, trunk, and tail of embryos at 24 hours after fertilization, and reduced significantly in the 100 nmol/L deguelin treatment group, especialy in the proliferative area. In the 200 and 400 nmol/L treatment groups, the embryonic development slowed down signficantly, and cyclin D1 gene mainly expressed in the dorsal ectoderm cels.%背景：斑马鱼胚胎发育早期，细胞分裂增殖快速，低浓度的鱼藤素可以延缓斑马鱼胚胎发育。目的：观察鱼藤素对斑马鱼胚胎中细胞周期蛋白D1基因的表达影响。　　方法：收集正常受精后发育至2-cel期(受精后约0.75 h)以及shield期(受精后6 h)斑马鱼胚胎于12孔板，分别用100，200，400 nmol/L鱼藤素在28.5℃的培养箱中避光处理至shield期及受精后24 h。同时用1%的二甲基亚砜溶液作为对照组，于相同条件下培养。后收集对照组及实验组胚胎，制备细胞周期蛋白D1 RNA探针进行整胚原位杂交，于正置荧光显微镜下拍照观察染色情况以检测鱼藤素对胚胎中细胞周期蛋白D1基因的表达影响。　　结果与结论：鱼藤素对斑马鱼胚胎中细胞周期蛋白D1基因的表达有明显的负调控作用。Shield期对照组胚胎细胞周期蛋白D1在外包细胞中泛表达，随着给药浓度的增加，细胞周期蛋白D1的表达显著减少，400 nmol/L鱼藤素处理组几乎不表达。受精后24 h的胚胎中细胞周期蛋白D1在脑部、中枢神经系统、发育未成熟的眼部、体节、躯干、尾部等有表达，100 nmol/L鱼藤素处理组表达明显下调，在增殖性区域几乎不表达。200，400 nmol/L处理组胚胎发育明显延缓，细胞周期蛋白D1主要表达于背侧外胚层细胞。