利用七氯对斑马鱼多巴胺神经元毒性影响构建帕金森病模型

Establishment of Parkinson’s disease model by the toxicity effect of heptachlor on dopamine neuron in zebrafish

目的:利用七氯对斑马鱼多巴胺神经元的影响,建立斑马鱼帕金森病(PD)模型。方法:将斑马鱼鱼卵分为正常对照组(不予干预),750μmol/L 6-羟基多巴胺(6-OHDA)组和不同浓度七氯组(1 ng/mL、10 ng/mL、100 ng/mL、1000 ng/mL、10000 ng/mL),分别恒温孵育,每24 h观察胚胎发育情况并记录直到受精后120 hpf(h post fertilization,hpf),在120 hpf做行为学测定,然后实时荧光定量PCR(RT-qPCR)测定多巴胺通路酪氨酸羟化酶(TH2)的相对表达量。结果:与正常对照组比较,6-OHDA组幼体出现发育迟缓、脊柱弯曲,运动距离缩短(P<0.05),TH2基因表达降低(P<0.05);与正常对照组比较,1~100 ng/mL七氯组幼体发育无明显变化,但运动距离缩短(P<0.05),TH2基因表达降低(P<0.05),1000~10000 ng/mL七氯组出现色素沉着分布和脊柱弯曲,而且运动距离更短(P<0.05)、TH2基因表达更低(P<0.05),变化具有浓度依赖性;与6-OHDA组比较,1000~10000 ng/mL七氯组运动距离更短,TH2基因表达更低,其中10000 ng/mL七氯组运动距离缩短和TH2基因表达降低有统计学差异(P<0.05)。结论:传统PD模型造模药物6-OHDA和1000~10000 ng/mL七氯都可通过影响多巴胺神经元功能成功建立斑马鱼PD模型,其中10000 ng/mL七氯的造模效果比6-OHDA更显著。

Objective:To establish the model of Parkinson’s disease(PD)by using the effect of heptachlor on dopamine neuronal toxicity in zebrafish.Methods:Zebrafish eggs were divided into normal control group(without intervention),750μmol/L 6-hydroxydopamine(6-OHDA)group and different concentrations of heptachlor group(1 ng/mL,10 ng/mL,100 ng/mL,1,000 ng/mL,10,000 ng/mL).The embryonic development was observed every 24 h and recorded until 120 h post fertilization(hpf)after fertilization.Then the relative expression of tyrosine hydroxylase(TH2)in dopamine pathway was measured by real-time quantitative PCR.Results:Compared with the normal control group,the larvae of 6-OHDA group showed developmental retardation,rachiocamposis,shorter distance of movement(P<0.05)and lower expression of TH2 gene(P<0.05).Compared with the normal control group,the larval development of 1-100 ng/mL heptachlor groups had no significant change,but the movement distance was shorter(P<0.05)and the expression of TH2 gene was decreased(P<0.05).1,000 and 10,000 ng/mL heptachlor groups showed pigmentation distribution and rachiocamposis,and the movement distance was shorter(P<0.05)and the expression of TH2 gene was lower(P<0.05),showing concentration dependent.Compared with 6-OHDA group,1,000 and 10,000 ng/mL heptachlor group had shorter exercise distance and lower TH2 gene expression.Among them,10,000 ng/mL heptachlor groups had shorter exercise distance and significant lower TH2 gene expression(P<0.05).Conclusions:Both traditional PD modeling drugs 6-OHDA,1,000 and 10,000 ng/mL heptachlor can successfully establish zebrafish PD model by affecting the function of dopamine neurons,and the modeling effect of 10,000 ng/mL heptachlor is more significant than that of 6-OHDA.