铁调素绿色荧光蛋白转基因对斑马鱼成骨代谢的影响  

Research on osteogenesis metabolism of transgenic zebrafish by hepcidin green fluorescent protein

作　　者：姜宇[1] 李斌 朱国兴[1] 徐又佳[3] 

机构地区：[1]南京医科大学附属无锡市第二人民医院骨科,214000 [2]宜兴市善卷骨科医院 [3]苏州大学附属第二医院骨科

出　　处：《中华医学杂志》2016年第13期1053-1057,共5页National Medical Journal of China

基　　金：国家自然科学基金（81273090,81572179）;江苏省重点专项（BL2014044）;苏州市临床重点专项（LCZX201305）;无锡市科技局项目（CSE31N1506）

摘　　要：目的探讨铁调素抑制对于成骨代谢影响的机制。方法以受精后0．5h斑马鱼的受精卵予以构建好的铁调素基因绿色荧光蛋白（GFP）的质粒进行显微注射受精卵，连续120h每间隔6h观察铁调素在斑马鱼中的表达。在同等情况下对同时注射ATG吗啉环反义寡核苷酸和绿色荧光蛋白质粒后的幼鱼于第4天进行铁染色，与野生型比较观察铁含量变化。于第9天分别进行茜素红染色，比较硬骨的形态差异。对注射ATG吗啉环反义寡核苷酸和GFP后的幼鱼于第4天提取的cDNA采用实时荧光定量聚合酶链反应（FQ-PCR）法检测相关成骨代谢的基因表达，比较和绿色荧光蛋白组的成骨下游基因的差异。结果对注射铁调素基因绿色荧光蛋白转基因的120h的观察中发现铁调素基因72h在斑马鱼中开始表达，96h时最强烈，ATG吗啉环反义寡核苷酸后第4天斑马鱼进行铁染色，与GFP相比色素沉着明显变深，对注射ATG吗啉环反义寡核苷酸后第9天斑马鱼进行茜素红染色与绿色荧光蛋白组染色相比，硬骨茜素红染色变浅且肋骨及尾鳍未发育，注射ATG吗啉环反义寡核苷酸后第4天的斑马鱼做定量FQ—PCR进行检测相关成骨代谢的基因表达量与GFP组斑马鱼成骨基因表达相比均下调。结论铁调素绿色荧光蛋白转基因斑马鱼在体内表达强烈，而抑制铁调素的表达造成斑马鱼体内铁蓄积，使得斑马鱼的成骨基因降低，造成骨发育迟缓，骨量减低。Objective To investigate the effect of hepcidin downregulation on the osteogenesis metabolism and involved mechanism based on the construction of hepcidin green fluorescent protein （GFP） in the transgenic zebrafish. Methods The eggs of zebrafish were transfected by hepcidin GFP plasmid by microinjection in half an hour after fertilization. The expression of hepcidin in the zebrafish was checked every 6 hours for 120 hours. Iron staining change was done in the larvae of zebrafish transfected by ATG morpholine and GFP plasmid compared with wild type in g days in the same conditions. Alizarin red staining of bone and cartilage was performed in 9 days and the expression of bone metabolic cDNA differences was analysed with real-time PCR on 4th day. Results Hepcidin expressed begin on 72 hours and strongest in 96 hours on GFP transgenic zebrafish. Iron staining was significantly darker than the GFP-control type on 4th day. Alizarin red staining became shallow and ribs and caudal fin did not change furthermore in zebrafish transfected by ATG morpholine. PCR related metabolic gene expression was expressed down in zebrafish transfected by ATG morpholine. Conclusion Hepcidin expressed strongest on GFP transgenic zebrafish on 96 hours. The inhibition of hepcidin caused iron accumulation and lowered the osteogenesis led to retardation and reduction of bone in zebrafish.

关 键 词：成骨细胞 吗琳类 代谢 斑马鱼 铁调素绿色荧光蛋白 

分 类 号：R336[医药卫生—人体生理学]