利用斑马鱼模型与代谢组学技术研究瓦他拉尼抑制血管生成的作用及其机制  

Inhibition effect of vatalanib on angiogenesis and its mechanism based on Zebrafish model and metabonomics

作　　者：吴晓敏 董榕 陈善军 刘拥军 杨官娥[3] 韩利文 WU Xiaomin;DONG Rong;CHEN Shanjun;LIU Yongjun;YANG Guane;HAN Liwen(Department of Pharmaceutical Analysis,College of Pharmacy and Pharmaceutical Sciences,Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan 256200,China;Department of Pharmacy,Second People’s Hospital of Jinzhong;Department of Traditional Chinese Medicine,College of Pharmacy,Shanxi Medical University)

机构地区：[1]山东第一医科大学,山东省医学科学院药学与制药科学学院药物分析教研室,济南256200 [2]晋中市第二人民医院药剂科 [3]山西医科大学药学院中药教研室

出　　处：《山西医科大学学报》2021年第12期1545-1550,共6页Journal of Shanxi Medical University

基　　金：山东省自然科学基金资助项目(ZR2019MH037);中央本级重大增减支项目(2060302-1907-09);山东第一医科大学学术提升计划项目(2019LJ003)。

摘　　要：目的利用斑马鱼模型与代谢组学技术研究瓦他拉尼对血管生成的抑制作用,探索血管生成相关的内源性小分子代谢物。方法采用受精后24 h(hour post fertilization,hpf)的Tg(VEGFR2:GFP)型转基因斑马鱼为实验动物,分为对照组、瓦他拉尼组和恢复组。瓦他拉尼组加入瓦他拉尼0.4μg/ml处理至72 hpf。恢复组加入瓦他拉尼0.4μg/ml处理12 h后移除药液,置正常培养液中培养至72 hpf。对照组置正常培养液中培养至72 hpf。用荧光显微镜观察每组斑马鱼胚胎节间血管的生长情况。收集各组斑马鱼,进行组织破碎、提取代谢物、衍生化处理,采用气相色谱质谱联用(gas chromatography-mass spectrometry,GC-MS)进行代谢组学分析。结果瓦他拉尼处理斑马鱼48 h后,斑马鱼节间血管的生长被显著抑制(P<0.01)。先用瓦塔拉尼处理12 h后,在移除药物继续培养至72 hpf的斑马鱼节间血管出现明显恢复。代谢组学结果显示正常对照组、瓦他拉尼组和恢复组在偏最小二乘法-模式判别(PLS-DA)模型中可明显区分,进一步筛选出16种内源性小分子代谢物可能与斑马鱼血管生成相关,包括丙酮酸、缬氨酸、亮氨酸、异亮氨酸、甘油、磷酸、丝氨酸、脯氨酸、葡萄糖、天冬氨酸、苯丙氨酸、丙氨酸、酪氨酸、棕榈酸、硬脂酸和胆固醇。结论瓦他拉尼对斑马鱼的节间血管生成具有显著的抑制作用,其抑制作用可能与影响血管生成过程的内源性小分子代谢物有关。Objective To research the inhibition effect of vatalanib on angiogenesis in Zebrafish model and explore potential endogenic small molecular metabolites related to angiogenesis by metabonomics. Methods A transgenic zebrafish line, Tg(VEGFR2:GFP), at 24 h post fertilization(24 hpf) was used as experimental animal. All healthy zebrafish were divided into three groups: normal control group, vatalanib group and recovery group. In vatalanib group, zebrafish were treated with vatalanib 0.4 μg/ml to 72 hpf. In recovery group, zebrafish were treated firstly with vatalanib 0.4 μg/ml for 12 h and then were treated with water until 72 hpf without vatalanib. Zebrafish in control group were treated with water to 72 hpf. The intersegmental vessels(ISVs) of each zebrafish were observed under fluorescence microscope. Zebrafish in every well were collected for tissue rupture, metabolite extraction and sample derivatization. GC-MS was used for metabolomics analysis. Results The ISVs of zebrafish were significantly inhibited by vatalanib(P<0.01). When zebrafish was firstly treated by vatalanib for 12 h and then incubated to 72 hpf without vatalanib, ISVs of zebrafish were recovered significantly. The metabonomic experimental results showed that normal control group, vatalanib group and recovery group were significantly distinguished by partial least squares pattern discrimination(PLS-DA) model. A total of 16 endogenous small molecular metabolites(pyruvate, valine, leucine, isoleucine, glycerol, phosphoric acid, serine, proline, glucose, aspartic acid, phenylalanine, alanine, tyrosine, palmitic acid, stearic acid, cholesterol) were screened out, and they were related to zebrafish angiogenesis progress. Conclusion Vatalanib has a significant inhibitory effect on angiogenesis in zebrafish, which may be related with disturbing endogenous metabolites in zebrafish.

关 键 词：血管生成 瓦他拉尼 斑马鱼 代谢组学 生物标志物 

分 类 号：R363[医药卫生—病理学]