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目的血管生成与很多疾病有关。目前对于信号传递的下游的代谢了解甚少,探寻与血管相关的新的生物学机制对于疾病的防治意义重大。斑马鱼血管生成评价模型是目前公认的评价血管生成作用的优良模型,能够把血管生成独立出来研究,与移植瘤等模型不同,无须考虑肿瘤等其他因素的干扰。同时借鉴分子生物学科中靶点研究的思路和方法,建立“双向验证”策略,进行血管生成相关的代谢型生物标志物进行研究。方法采用转基因斑马鱼Tg(vegfr2:GFP)幼鱼作为血管生成研究模型,在斑马鱼发育至24h时,采用靶点抑制剂PTK787和中药丹红注射液进行双向处理,观察丹红注射液(DHI)对PTK787造成损伤的逆转作用,用Image J软件对节间血管(ISVs)进行定量分析,作为血管生成评价指标;同步处理后的斑马鱼每20条为一个样本,进行组织破碎、提取、去蛋白后,进行衍生化处理,然后用GC-MS联用仪进行分析,每组6个重复;采用Simca-P软件进行代谢组模式判别分析。结果在本实验条件下,30~60μL·m L-1的丹红注射液对PTK787造成的斑马鱼节间血管生成的损伤具有明显逆转作用。代谢轮廓分析结果显示,正常组、PTK787处理组、丹红注射液处理在散点图中呈现了明显的损伤和逆转趋势。进一步的分析发现,PTK787处理斑马鱼后,酪氨酸、次黄嘌呤、苯丙氨酸、天冬氨酸、谷氨酰胺、磷酸水平明显升高,肌醇明显降低,而丹红注射液处理后,发现这些代谢物呈现了显著逆转。代谢网络分析显示,这些代谢物可能与缬氨酸-亮氨酸-异亮氨酸生物合成途径、苯丙氨酸-酪氨酸-色氨酸合成途径、苯丙氨酸代谢、甘氨酸-天冬氨酸-谷氨酰胺代谢、磷酸肌醇代谢途径有关。结论本研究利用斑马鱼模型和代谢组学技术对血管生成相关的下游代谢信号进行了探讨,并建立了双向处理法,快速筛选发现了一些相关的代谢物,与常规的药物处理进行模式判别,准确率更高,对利用斑马鱼模型研究疾病相关的代谢途径提供了新的研究方法。
The purpose of angiogenesis and many diseases. At present, little is known about the downstream metabolism of signaling, and exploring new biological mechanisms related to blood vessels is of great significance for the prevention and treatment of diseases. The zebrafish angiogenesis evaluation model is currently recognized as an excellent model for evaluating angiogenic effects and is capable of studying angiogenesis independently of other models such as xenografts without the interference of other factors such as tumors. At the same time, drawing on the ideas and methods of target research in molecular biology, we established a “two-way validation” strategy to study the metabolic biomarkers related to angiogenesis. Methods The transgenic zebrafish Tg (vegfr2: GFP) juvenile fish was used as a model for angiogenesis. When the zebrafish developed to 24h, the target inhibitor PTK787 and Chinese traditional medicine Danhong injection were used for bidirectional treatment. The effects of Danhong injection (DHI ) On PTK787 damage caused by the reversal of the use of Image J software for quantitative analysis of internus blood vessels (ISVs) as an indicator of angiogenesis; synchronized zebrafish every 20 as a sample of tissue disruption, extraction, go After derivatization, the proteins were analyzed by GC-MS with 6 replicates in each group. Simca-P software was used for discriminant analysis of metabolome patterns. Results Under the experimental conditions, Danhong injection of 30 ~ 60 μL · m L-1 could significantly reverse the damage of zebrafish intervertebral vascularization induced by PTK787. Metabolic profile analysis showed that the normal group, PTK787 treatment group, Danhong injection treatment in the scatter plot showed obvious damage and reversal trend. Further analysis showed that tyrosine, hypoxanthine, phenylalanine, aspartic acid, glutamine, and phosphoric acid levels were significantly increased and inositol significantly decreased after PTK787 treatment of zebrafish, while Danhong injection treatment After these metabolites were found to show a significant reversal. Metabolic network analysis showed that these metabolites may be related to the valine-leucine-isoleucine biosynthesis pathway, the phenylalanine-tyrosine-tryptophan pathway, the phenylalanine metabolism, the glycine-day Aspartate - glutamine metabolism, phosphoinositide metabolic pathway. Conclusion In this study, we used the zebrafish model and metabonomics techniques to explore the downstream metabolic signals related to angiogenesis and established a two-way treatment method. Some related metabolites were found by rapid screening, and were compared with the conventional drug treatment for pattern discrimination. The accuracy rate is higher, which provides a new research method for the study of disease-related metabolic pathways by zebrafish model.