铁调素对敲降斑马鱼中抑制成骨代谢的影响

Hepcidin knocked down to study the metabolism of osteoblast in zebrafish

目的通过对斑马鱼受精卵进行显微注射剪接吗啉环反义寡核苷酸，使其铁调素基因敲降，观察铁调素表达抑制后对斑马鱼骨代谢的影响，探讨铁调素抑制对于骨代谢影响的机制。方法以受精后0．5h斑马鱼的受精卵为模型，予以不同浓度的剪接吗啉环反义寡核苷酸显微注射受精卵，将注射后的幼鱼于第4天进行提取RNA，反转录成cDNA，进行鉴定后得到适当浓度。在浓度为25mg／L的剪接吗啉环反义寡核苷酸注射条件下，对注射剪接吗啉环反义寡核苷酸后的幼鱼于第4天进行铁染色，与野生型比较观察铁含量变化，对注射剪接吗啉环反义寡核苷酸后的幼鱼于第9天分别进行茜素红和阿可辛蓝染色，与野生型比较硬骨和软骨的形态差异；对注射剪接吗啉环反义寡核苷酸后的幼鱼于第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，比较和野生型基因组成骨下游基因的差异。对注射剪接吗啉环反义寡核苷酸后第4天斑马鱼予以成骨代表性的两个基因Runx2a和sp7进行原位杂交，比较野生型的幼鱼相关成骨基因表达部位的差异和信号强弱。结果对注射剪接吗啉环反义寡核苷酸后第4天斑马鱼进行铁染色，与野生型染色比较色素沉着明显变深，对注射剪接吗啉环反义寡核苷酸后第9天斑马鱼进行茜素红和阿尔辛蓝染色分别与野生型染色比较，硬骨茜素红染色变浅且肋骨及尾鳍未发育，软骨阿尔辛蓝染色未有明显改变，注射剪接吗啉环反义寡核苷酸后第4天的斑马鱼行FQ-PCR进行检测相关成骨代谢的基因表达量与野生型斑马鱼成骨基因表达比较均下调，尤其以Runx2a和sp7最为显著，野生型分别为1．00790±0．01117和1．12450±0．17607降至处理后的0．25639±0．01607和0．33147±0．07436。对于注射剪接吗啉环反义寡核苷酸后第4天斑马鱼进行Runx2a和sp7原位杂交的信号表达弱于同期野生型的斑马鱼。结论敲降斑马鱼的铁调素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 Hepcidin was knocked down by the microinjection of oligo morpholine in zebrafish embryo, the influence of bone metabolism was observed after expression inhibitory of hepcidin in zebrafish. The mechanism of hepcidin knocked down effecting on bone metabolism is explored. Methods We treat half an hour post - fertilization zebrafish with different concentrations of oligo morpholine microin- jection. Based on identifion of the cDNA effect. In the concentration of 25 PPM of oligo morpholine, At 4 dph, change of iron content after the injection of larvae is observed compared with wild type, At 9 dph, Alizarin red staining was performed for measuring vertebral bodies and the expression of bone metabolic cDNA differences was analysed with real -time fluorescent quantitative polymerase chain reaction （ FQ - PCR） on 4 dph. Runx2a and sp7 in situ hybridization compared with the wild type of juvenile fish was associated part of the distribution of different gene expression. Results （ 1 ） At 4 dph, iron stain pigmentation was significantly darker than the wild type; （2） Alizarin red staining becomes shallow and ribs and caudal fin was not developed, al sheen blue staining did not change obviously ; （ 3 ） FQ - PCR related metabolic gene expression was expressed down, especially Runx2a and sp7 in the most significant, the wild type was 1. 007 90 ±0. 011 17 and 1. 124 50 ±0. 176 07 was compared with O. 256 39 ±0. 016 07 and 0. 0160 72 ±0. 074 36 after treatment. Conclusion Hepcidin knocked down in zebrafish represent iron accumulation in the body, reducing bone gene expression, bone loss and retarded bone development.