基于转基因斑马鱼模型的清热解毒类中药抗血管生成筛选及对照研究

发布时间：2018-05-03 22:04

本文选题：斑马鱼 + 清热解毒　；参考：《成都中医药大学》2015年博士论文

【摘要】：课题背景近年来,斑马鱼作为生物研究模型已经成为一个研究热点。斑马鱼早期发育过程中,胚胎透明,可直接观察到各组织器官。并且斑马鱼新生血管简单,观察清晰,在显微镜下就能够清楚观察到节间血管、血流的形态,并且该实验模型被认为具有有高通量和活体实验的特点,对于研究新生血管具有先天的研究优势。抗新生血管生成是肝癌综合治疗的关键环节,而中药抗肿瘤新生血管治疗已经得到部分文献证实。根据文献资料及临床经验,清热解毒类药物是中医治疗肝癌的基础方法。我们在文献复习和专家共识基础上,选择6种清热解毒类中药进行抗新生血管生成的筛选研究,以TG品系的flila-EGFP转基因斑马鱼胚胎为生物模型,利用其绿色荧光蛋白显影特质,在显微镜下观察中药水煎剂成分对斑马鱼体内血管生成的抑制效果。对其中初筛通过确认具有抑制血管生成作用的药物进行进一步的定量对照试验。试验方案选用TG品系的flila-EGFP转基因斑马鱼为动物模型。将新鲜配置好的中药水提取物(工作浓度：200 μg/ml)(分别为：紫花地丁、白花蛇舌草、半边莲、鱼腥草、漏芦、野菊花)和阳性对照药物-PTK787 (5 μg/ml)处理受精后22小时(22hpf)的斑马鱼胚胎直至48hpf。药物处理实验结束后,将各组将各药物浓度处理后的胚胎在体式荧光显微镜(Nikon SMZ-1500体式荧光显微镜)下进行血管表型观察和拍照,拍照时相为48hpf,以分析各个样品对斑马鱼体节间血管(ISVs)生成的影响。在血管筛选实验中,同时对各个样品诱导的斑马鱼发育毒性和死亡也进行观察和统计。初筛试验后,对具有抗血管生成作用的药物进行ISVs定量计数对比和中药药昧共煎液水提物干预效果的对比。主要结果阳性对照组5 μg/ml PTK787能够完全抑制新生血管,空白对照组0.1%DMSO无抑制新生血管作用,野菊花、漏芦水提物可较为明显抑制斑马鱼节间血管,而紫花地丁、白花蛇舌草、半边莲、鱼腥草水提物不具有抑制斑马鱼节间血管的效应。六种中药水提取为均无导致斑马鱼死亡或者致畸现象。定量计数对照研究中,ISVs计数及新生血管抑制率结果相似,均提示野菊花水提物与野菊花漏芦共煎液水提物抑制新生血管生成效应与PTK787相当,并优于漏芦水提物。结论斑马鱼作为新兴筛选新生血管的模型,具有体积小、胚胎透明、繁殖速度快等优势,可以作为以后此方面的研究重点。在当今治疗肿瘤需要求新求变的时代,并且更看重新生血管对于肿瘤生长、转移和浸润的作用,选用具有抗新生血管的中药以达到抗肿瘤的目的成了研究热点。本研究中,野菊花、漏芦水提物显示了较为优异的抗新生血管的作用,但还需要开展进一步研究,对药物的抗新生血管生成作用基因组学、蛋白组学效应等进行深入探索。
[Abstract]:Background zebrafish as a biological research model has become a research hotspot in recent years. During the early development of zebrafish, the embryo is transparent and the tissues and organs can be observed directly. And the zebrafish neovascularization is simple, the observation is clear, under the microscope can clearly observe the internode blood vessel, the blood flow morphology, and this experimental model is believed to have the high throughput and the living body experiment characteristic, For the study of neovascularization has a congenital advantage. Anti-neovascularization is a key link in the comprehensive treatment of liver cancer, and the anti-tumor angiogenesis therapy of traditional Chinese medicine has been confirmed in some literatures. According to the literature and clinical experience, antipyretic and detoxifying drugs are the basic methods of traditional Chinese medicine in the treatment of liver cancer. On the basis of literature review and experts' consensus, six kinds of antipyretic and detoxifying herbs were selected to screen for anti-angiogenesis. The flila-EGFP transgenic zebrafish embryos of TG strain were used as biological model, and their green fluorescent protein (GFP) development characteristics were used. The inhibitory effect of Chinese traditional medicine decoction on the angiogenesis of zebrafish was observed under microscope. Further quantitative controlled trials were carried out to identify the drugs that could inhibit angiogenesis. The flila-EGFP transgenic zebrafish of TG strain was selected as animal model. Fresh Chinese medicine water extracts (working concentration: 200 渭 g / ml) were prepared. The zebrafish embryos were treated 22 hours after fertilization with -PTK787 + 5 渭 g / ml, a wild chrysanthemum, until 48hpf. At the end of the drug treatment experiment, the embryos treated with each drug concentration were observed and photographed under the Nikon SMZ-1500 phantom fluorescence microscope. 48 hpfs were taken to analyze the effect of each sample on ISVsproduction in zebrafish Internode. In the vascular screening experiment, the developmental toxicity and death of zebrafish induced by each sample were also observed and counted. After the primary screening test, the ISVs quantitative count of the drugs with anti-angiogenic effect and the intervention effect of the water extract of traditional Chinese medicine were compared. Results the positive control group (5 渭 g/ml PTK787) could completely inhibit the neovascularization, while the blank control group (0.1%DMSO) had no inhibitory effect on neovascularization. The water extract of Houttuynia cordata did not inhibit the internode blood vessels of zebrafish. No zebrafish death or teratogenicity were found in water extraction of six kinds of traditional Chinese medicine. The results showed that the water extract of Chrysanthemum chrysanthemi and chrysanthemum chrysanthemi had the same inhibitory effect on angiogenesis as PTK787, and it was superior to the water extract of chrysanthemum chrysanthemi. Conclusion zebrafish, as a new model for screening neovascularization, has the advantages of small size, transparent embryo and fast reproduction rate, which can be used as the research focus in the future. In the present age when the treatment of tumor needs to be new and changing, and more attention is paid to the role of neovascularization in tumor growth, metastasis and invasion, the choice of anti-angiogenic Chinese medicine to achieve the purpose of anti-tumor has become a hot research topic. In this study, the aqueous extracts of Chrysanthemum chrysanthemi and Flos chrysanthemi showed excellent anti-angiogenesis effects, but further studies were needed to explore the genomics and proteomics effects of the drugs.
【学位授予单位】：成都中医药大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R-332;R285.5

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