钩藤叶水提物对TH-GFP转基因斑马鱼甲基苯丙胺依赖的影响

Effect of Uncaria macrophylla leaf aqueous extract on methamphetamine dependence in TH-GFP transgenic zebrafish

目的培育TH-GFP转基因斑马鱼幼鱼,建立转基因斑马鱼幼鱼甲基苯丙胺依赖模型,观察钩藤叶水提物对模型幼鱼的影响。方法收集健康斑马鱼受精卵,向卵黄囊中注射TH-GFP质粒,构建TH-GFP转基因斑马鱼幼鱼。取转基因斑马鱼幼鱼,随机放入含有60,6,0. 6,0. 06 mg·L-1甲基苯丙胺溶液中,3d后,将斑马鱼幼鱼固定,然后放入液态的低熔点琼脂糖中,用荧光体视显微镜观察其头部的荧光变化。取转基因斑马鱼幼鱼,随机分为空白组、对照组(甲基苯丙胺0. 6 mg·L-1)、高剂量实验组(钩藤叶水提物330 mg·mL-1)和低剂量实验组(钩藤叶水提物33 mg·mL-1)。除空白组外,将各组斑马鱼幼鱼放入0. 6 mg·L-1的甲基苯丙胺溶液中自由游动。48 h后,将高、低剂量实验组斑马鱼幼鱼放入相应浓度的钩藤叶水提物中,甲基苯丙胺模型组放入鱼用生理盐水中,自由游动。24 h后,将各组斑马鱼幼鱼固定,放入液态的低熔点琼脂糖中,用激光扫描共聚焦显微镜观察其荧光变化。结果与空白组相比,显微注射THGFP质粒后的斑马鱼幼鱼头部出现明显荧光。在绿色荧光下,与空白组斑马鱼幼鱼相比,对照组斑马鱼幼鱼头部荧光变化不明显,其余各组斑马鱼幼鱼头部荧光变化明显。将各组斑马鱼幼鱼放入相应浓度的钩藤叶水提物中24 h后,在共聚焦显微镜下观察,与空白组相比,对照组斑马鱼幼鱼头部荧光明显增强。与对照组相比,钩藤叶水提物组斑马鱼幼鱼头部荧光有所减弱。结论 TH-GFP转基因斑马鱼可以用于观察斑马鱼甲基苯丙胺依赖变化。0. 6 mg·L-1的甲基苯丙胺溶液可以使TH-GFP转基因斑马鱼头部荧光发生明显增强,而钩藤叶水提物可以降低这种荧光的改变。

Objective To cultivate TH-GFP transgenic zebrafish juveniles and establish a methamphetamine-dependent model of transgenic zebrafish juveniles,and to observe the effect of water extract from Uncaria leaves on the model juveniles.Methods Fertilized eggs of healthy zebrafish were collected and TH-GFP plasmid was injected into yolk sac to construct TH-GFP transgenic juvenile zebrafish.Transgenic zebrafish juveniles were randomly put into methamphetamine solution containing 60,6,0.6,0.06 mg·L-1.After 3 d,zebrafish juveniles were fixed and then put into liquid agarose with low melting point.The fluorescence changes of their heads were observed by fluorescence stereomicroscope.The juvenile genetically modified zebrafish were randomly divided into blank group,control group(methamphetamine 0.6 mg·L-1),and high dose(Uncaria leaves water extract 330 mg·mL-1),low dose(33 mg·mL-1)experimental groups.Except for blank group,the juvenile zebrafish of each group were placed in 0.6 mg·L-1 methamphetamine solution to swim freely.48 h later,the juvenile zebrafish of high and low dose experimental group were put into the corresponding concentration of water extract of Uncaria leaves,and control group was put into the fish physiological saline and swimmed freely.After 24 h,zebrafish juveniles were fixed in liquid agarose with low melting point,and their fluorescence changes were observed by laser scanning confocal microscopy.Results Compared with blank group zebrafish juvenile,the fluorescence of the head of the juvenile zebrafish after microinjection of TH-GFP plasmid was obvious.Under green fluorescence,compared with the blank group,the fluorescence of the head of the juvenile zebrafish in control group did not change significantly,while the fluorescence of the head of the juvenile zebrafish in other groups changed significantly.The fluorescence of the head of juvenile zebrafish in control group significantly increased compared with that in the blank group after 24 h of putting the juvenile zebrafish in the corresponding concentration of water extract from Uncaria leaves.The results showed that the fluorescence of the head of juvenile zebrafish in the control group was significantly higher than that in the blank group.Compared with the control group of the methamphetamine(0.6 mg·L-1),the head fluorescence of juvenile zebrafish in the water extract group of Uncaria leaves decreased.Conclusion TH-GFP transgenic zebrafish can be used to observe the changes of methamphetamine dependence in zebrafish.The fluorescence of TH-GFP transgenic zebrafish head could be enhanced by 0.6 mg·L-1 methamphetamine solution,while the water extract of Uncaria leaves could reduce the fluorescence change.