摘要
目的探究氟暴露对斑马鱼幼鱼骨骼发育的影响,并探索其可能的分子机制。方法选择受精后6 h(6 hpf)野生型斑马鱼胚胎,采用氟化钠[NaF,对照组(0 mg/L NaF)、低氟组(25 mg/L NaF)和高氟组(100 mg/L NaF)]暴露至9 d。使用氟离子选择电极法检测斑马鱼幼鱼的整体氟含量,观察并统计斑马鱼幼鱼的死亡及发育情况,采用茜素红染色和阿尔新蓝染色分别分析斑马鱼幼鱼的骨矿化和软骨形成情况,实时荧光定量PCR法分析sry相关高迁移率组盒9(Sox9)的同源基因Sox9a、骨保护素(OPG)和核因子κB受体活化因子配体(RANKL)的表达情况。结果与对照组[(0.12±0.01)μg/140条]比较,低氟组[(0.28±0.03)μg/140条]和高氟组[(0.64±0.10)μg/140条]斑马鱼幼鱼整体氟含量均较高,差异均有统计学意义(P均<0.05)。与对照组比较,高氟组斑马鱼幼鱼体长较短、鱼鳔发育缺失率和脊柱弯曲率均较高(P均<0.05);低氟组茜素红染色骨矿化面积、累计光密度(IOD)和矿化椎骨数均较高,阿尔新蓝染色软骨形成面积较低(P均<0.05);高氟组茜素红染色骨矿化面积、IOD和矿化椎骨数均较低,阿尔新蓝染色软骨形成面积较高(P均<0.05)。与对照组比较,低氟组OPG mRNA表达水平和OPG/RANKL比值均较高(P均<0.05);高氟组RANKL mRNA表达水平较高,OPG/RANKL比值较低(P均<0.05)。结论斑马鱼胚胎至幼鱼时期给予短时间的氟暴露即可造成斑马鱼幼鱼骨骼发育异常,该影响可能与软骨内成骨和OPG/RANKL通路相关。
Objective To study the effects of fluoride exposure on skeletal development of zebrafish larvae and its possible molecular mechanisms.Methods Six hours post fertilization(6 hpf)wild-type zebrafish embryos were selected and exposed to sodium fluoride[NaF,control group(0 mg/L NaF),low fluoride group(25 mg/L NaF)and high fluoride group(100 mg/L NaF)]for 9 days.Fluorine ion selective electrode was used to detect the overall fluorine content of zebrafish larvaes,and the death and development of zebrafish larvaes were observed and counted.Bone mineralization and chondrogenesis of the zebrafish larvaes were analyzed by alizarin red staining and alcin blue staining,respectively.The expression levels of sry-related-high-mobilty-group box 9a(Sox9a),osteprotegerin(OPG)and receptor-activator of nuclear factor kappa beta ligant(RANKL)were analyzed by real-time quantitative PCR.Results Compared with control group[(0.12±0.01)μg/140 larvaes],the overall fluorine contents of zebrafish larvaes in low fluoride group[(0.28±0.03)μg/140 larvaes]and high fluoride group[(0.64±0.10)μg/140 larvaes]were significantly higher,and the differences were statistically significant(P<0.05).Compared with control group,zebrafish larvaes in high fluoride group had shorter body length,higher swim bladder loss rate and higher spinal curvature rate(P<0.05).The alizarin red staining area,integrated optical density(IOD)and the number of mineralized vertebrae were higher in low fluoride group,while the alcin blue staining area of cartilage formation was lower(P<0.05).In the high fluoride group,alizarin red staining area,IOD and the number of mineralized vertebrae were lower,while the alcin blue staining area of cartilage formation was higher(P<0.05).Compared with control group,the expression levels of OPG mRNA and OPG/RANKL mRNA in low fluoride group were higher(P<0.05);the expression level of RANKL mRNA was higher in high fluoride group,while the expression level of OPG/RANKL mRNA was lower(P<0.05).Conclusion A short period of fluoride exposure from zebrafish embryo to zebrafish larvae can cause abnormal bone development of zebrafish larvae,which may be related to endochondral osteogenesis and OPG/RANKL pathway.
作者
龚艺
舒莉萍
陈芳萍
郑丹
官志忠
楼迪栋
Gong Yi;Shu Liping;Chen Fangping;Zheng Dan;Guan Zhizhong;Lou Didong(Department of Forensic Pathology,College of Forensic Medicine in Guizhou Medical University,Guiyang 550004,China;Key Laboratory of Adult Stem Cell Translational Research,Chinese Academy of Medical Sciences,Guiyang 550004,China;Guiyang Maternal and Child Health Care Hospital,Guiyang 550004,China;Key Laboratory of Endemic Diseases and Minority Diseases,Ministry of Education,Guiyang 550004,China;Department of Forensic Medicine,Basic Medical College of Guizhou University of Traditional Medicine,Guiyang 550004,China;Key Laboratory of Toxicology of Forensic Traditional Medicine in Guizhou Province,Guiyang 550004,China)
出处
《中华地方病学杂志》
CAS
北大核心
2022年第2期93-99,共7页
Chinese Journal of Endemiology
基金
国家自然科学基金(81760336)。
关键词
氟
斑马鱼
软骨内成骨
骨骼发育
Fluorine
Zebrafish
Endochondral osteogenesis
Skeletal development
