铁蓄积对斑马鱼幼鱼骨发育的影响

Influence of iron accumulation on bone development of zebrafish larvae

目的通过对斑马鱼受精卵进行高铁环境干预,观察铁积蓄对斑马鱼幼鱼骨代谢的影响,探讨铁蓄积对于骨代谢的影响。方法不同浓度的枸橼酸铁铵(ferric ammonium citrate,FAC)(25、50、100、200 mg/L)干预野生斑马鱼交配,收集鱼卵置于4个含相应FAC浓度的水环境中,每24小时予以更换含同样浓度FAC的水1次,同时设不含FAC为对照组,观察斑马鱼在不同铁环境下生存的情况。于第4天采用实时荧光定量聚合酶链反应(fluorescent quantification-polymerase chain reaction,FQ-PCR)法检测相关成骨代谢的基因表达,比较和对照组基因组的成骨下游基因的差异,并测量幼鱼的铁含量及铁染色,观察铁含量变化;于第9天进行茜素红染色,比较与对照组的硬骨形态差异。结果不同浓度FAC处理后,斑马鱼骨矿化面积成剂量依赖性降低,其中200 mg/L FAC组与对照组对比T值为40.567(P<0.01);FQ-PCT结果显示:相关早期表达成骨代谢的基因表达量与对照组斑马鱼成骨基因表达相比显著下调,差异有统计学意义(P<0.05);铁染色结果显示:FAC组斑马鱼色素沉着变深,与对照组相比,100和200 mg/L两组的幼鱼铁含量升高[(21.5±3.4)μg/L vs.(12.5±3.6)μg/L,(22.8±2.9)μg/L vs.(12.5±3.6)μg/L],差异有统计学意义(P<0.05);茜素红染色结果显示:与对照组相比,FAC组硬骨染色变浅且肋骨及尾鳍未发育。结论高铁环境下造成斑马鱼体内铁蓄积,使斑马鱼成骨基因降低,造成骨发育迟缓,矿化障碍。

Objective To examine the effect of iron accumulation on bone metabolism in zebraiish larvae by creating high iron environment. Methods Different concentrations of fen＇it ammonium citrate （FAC） （25, 50, 100, 200 mg/L） were used to interfere with copulation of zebrafish. Water without FAC was used as a control. Eggs were collected and placed into water containing different FAC. We changed water containing the same concentration of FAC every 24 h in order to study the survival of zebrafish. The expression of osteogenesis metabolic gene was measured by Fluorescent Quantification- Polymerase Chain Reaction （FQ-PCR） on day 4 to explore the differences of the downstream genes between those in experimental groups and control groups. Quantify iron content was also conducted by iron staining. Alizarin red staining was carried out to compare bony shape of experimental groups with control. Results The mineralization area was reduced in a dose-dependent-manner after FAC treatment at the concentration 200 mg/L and T value was 40. 567 compared to the control group （ P〈0. 0001 ）. Iron staining results showed that pigmentation of zebrafishs became darker in FACgroup than that in control group. Iron content of zebrafishs in 100 mg/L and 200 mg/L groups significantly increased compared with control group [ （21.5±3.4） μg/Lvs （12.5±3.6） μg/L, （22.8±2.9） μg/Lvs. （12.5±3.6） μg/L （P〈0.05） 1. Alizarin red staining re-sults showed that bony shape became shallow and, while and caudal fin were not developed. FQ-PCR results demonstrated that expression intensity of early distribution of metabolic genewere downregulated compared with control group. Conclusion High iron environment caused iron accumulation in zebrafish body, as a result of lowering the expression intensity of zebrafish osteogenesis genes and causing retardation of bone development as well as disorders of abnormal bone mineralization.