斑马鱼中骨形态发生蛋白2a敲降对骨代谢的影响

Effects on osteoblasts by bone morphgenetic protein 2a knoched down in zebrafish

目的通过显微注射吗啉环反义寡核苷酸(morpholino oligo,MO),使斑马鱼骨形态发生蛋白2a (bone morphgenetic protein 2a,BMP2a)基因敲降,观察BMP2a基因表达抑制后对斑马鱼骨代谢的影响。方法以受精后半小时斑马鱼受精卵为模型,将MO显微注射到受精卵(20个,对照组相同处理),对注射MO后及野生型(wild type,WT)的幼鱼于第9天分别进行茜素红染色,比较硬骨的形态差异,同时运用Image J软件对染色体积进行统计,比较差异。对注射MO后的幼鱼于第4天提取的c DNA采用实时荧光定量PCR(QPCR)方法检测Runx2a、Runx2b、Sp7、Alp骨形成相关基因和WT基因组的成骨下游基因表达的差异。对注射MO后第4天的斑马鱼予以成骨代表性的两个基因Runx2a进行原位杂交,比较WT幼鱼相关成骨基因表达部位的差异和信号强弱。结果与WT染色相比,注射抑制BMP2a的MO后第9天斑马鱼硬骨茜素红染色变浅,体积更小(MO vs. WT:3 276±153 vs. 4 768±231,P<0. 05)。QPCR检测显示,注射MO后第4天的斑马鱼成骨代谢基因表达量与WT斑马鱼对应基因相比均下调(Runx2a:0. 348±0. 002 vs. 1. 007±0. 032;Runx2b:0. 525±0. 021 vs. 1. 013±0. 021; Sp7:0. 483±0. 026 vs. 1. 124±0. 173; Alp:0. 136±0. 012 vs. 1. 233±0. 237; P<0. 05)。就Runx2a原位杂交而言,注射MO后第4天斑马鱼信号表达弱于同期WT斑马鱼。结论通过基因敲降技术使得斑马鱼BMP2下调,直观地观察到斑马鱼的骨发育迟缓,运用分子手段发现其基因下调造成一系列的成骨基因降低,用原位半定量方法靶向定位到斑马鱼蝶骨上,基因下调后造成斑马鱼的蝶骨信号消失。

Objective Bone morphgenetic protein 2a(BMP2a) was knocked down by the microinjection of morpholino oligo (MO) in zebrafish embryo. To observe the influence on bone metabolism after expression inhibitory of BMP2a in zebrafish. Methods We treated half an hour post-fertilization zebrafish with MO microinjection. At the 9th day, Alizarin Red staining was performed for measuring vertebral bodies. The expression of Runx2a, Runx2b, Sp7, Alp cDNA differences were analysed with real-time PCR on the 4th day. Compared to the wild type of juvenile fish, Runx2a in situ hybridzation was associated to part of the distribution of different gene expressions. Image J software was used to make statistical analysis on area of dyeing and to compare the differences. Results Alizarin red staining was shallow compared with wild type in zebrafish. Volume comparison was statistically significant(MO vs. WT: 3 276±153 vs. 4 768 ±231, P <0.05). Quantitative PCR related (Runx2a:0.348±0.002 vs. 1.007±0.032;Runx2b:0.525±0.021 vs. 1.013±0.021;Sp7:0.483±0.026 vs. 1.124±0.173;Alp:0.136±0.012 vs. 1.233±0.237;P <0.05). Expression of runx2a in situ hybridization signal was weaker than the wild zebrafish. Conclusions Through the gene knockout technology, expression of bone-formation genes of zebrafish have been discovered reduced. Zebrafish sphenoid bone disappear with semi-quantitative targeted positioning.