红花、桃仁、怀牛膝水提物抑制转基因斑马鱼模型新生血管生成的效应研究

发布时间：2018-06-13 19:52

本文选题：肝细胞癌 + 活血化瘀中药　；参考：《成都中医药大学》2016年硕士论文

【摘要】：目的：本实验选用临床上被广泛用于恶性肿瘤治疗的红花、桃仁、怀牛膝等具有活血化瘀功效的中药,基于TG品系flila-EGFP转基因斑马鱼生物模型来筛选其中具有抑制肿瘤新生血管生成效用的中药,客观、系统地评价红花、桃仁、怀牛膝等具有活血化瘀功效的中药在临床上应用于治疗恶性肿瘤的可靠性和安全性,为临床使用活血化瘀中药治疗恶性肿瘤提供客观的实验依据。方法：选用TG品系的flila-EGFP转基因斑马鱼为生物模型,每个样品处理30只斑马鱼胚胎,在12孔板(Falcon)中实验。将工作浓度为200μg/ml的红花、桃仁、怀牛膝水提取物和阳性对照药物-PTK787 (5 μ g/ml)分别处理受精后22小时(22hpf)的斑马鱼胚胎直至48hpf。完整的连接背主动脉(DA)和背长轴血管(DLAV)之间的一段血管,即体节间血管(ISV),是衡量斑马鱼血管新生(angiogenesis)的重要指标。药物处理实验结束后,在体视显微镜(明场)和体视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(ISV)生成的影响。同时,观察和统计各个样品诱导的斑马鱼发育毒性和死亡数。结果：各个样品均未出现药物诱导的斑马鱼发育毒性和死亡数事件。体视显微镜(明场)和体视荧光显微镜下完整的ISVs表型观察显示：空白对照组斑马鱼胚胎的ISVs的表达完整、清晰；与空白对照组相比较,阳性对照组采用瓦他拉尼干预的斑马鱼胚胎的ISV及DLAV的荧光表达缺失；红花、桃仁、怀牛膝水提物组斑马鱼胚胎的ISVs的荧光表达都有不同程度的变细、荧光强度减弱,或分布紊乱；其中,怀牛膝、红花水提物组斑马鱼胚胎ISVs的荧光表达变细、强度减弱较为明显,且分布紊乱,怀牛膝水提物组尤为突出。结论：瓦他拉尼能完全抑制斑马鱼胚胎的ISVs及DLAV;临床治疗肿瘤常用活血化瘀中药红花、桃仁、怀牛膝对斑马鱼胚胎的ISVs有不同程度的抑制作用；活血化瘀中药红花、桃仁、怀牛膝对转基因斑马鱼新生血管生成存在抑制效用,从而抑制肿瘤生长及转移；活血化瘀中药怀牛膝、红花、桃仁抑制肿瘤新生血管生成效应有差异,其中,怀牛膝、红花作用明显,且安全、可靠,无致畸、致死事件；推测肿瘤活血化瘀疗法可以抑制肿瘤血管生成,从而抑制肿瘤生长及转移。
[Abstract]:Objective: to select traditional Chinese medicine which is widely used in the treatment of malignant tumor, such as safflower, peach kernel and Achyranthes bidentata, which has the effect of promoting blood circulation and removing blood stasis. Based on the TG flila-EGFP transgenic zebrafish biological model to screen the Chinese medicine which can inhibit tumor angiogenesis, objective, systematic evaluation of safflower, peach kernel, The traditional Chinese medicine with the effect of activating blood circulation and removing blood stasis is applied to the reliability and safety of malignant tumor in clinic, which provides an objective experimental basis for the clinical use of traditional Chinese medicine for promoting blood circulation and removing stasis in the treatment of malignant tumor. Methods: Flila-EGFP transgenic zebrafish from TG strain was used as a biological model. 30 zebrafish embryos were treated with each sample. Zebrafish embryos with working concentration of 200 渭 g/ml of safflower, peach kernel, Achyranthes bidentata and positive control drug -PTK787 5 渭 g / ml were treated for 22 hours after fertilization until 48hpf. The complete connection between the dorsal aorta (DAA) and the dorsal long axis vessel (DLAV) is an important indicator of angiogenesis in zebrafish. After the drug treatment, the phenotype of blood vessels was observed and photographed under stereoscopic microscope and stereoscopic fluorescence microscope, and the phase was 48hpf. to analyze the effect of each sample on the formation of ISV in zebrafish. At the same time, the developmental toxicity and death number of zebrafish induced by each sample were observed and counted. Results: there were no developmental toxicity and death events induced by drugs in all samples. The complete phenotypic observation of ISVs under stereoscopic microscope and stereoscopic fluorescence microscope showed that the expression of ISVs in zebrafish embryos in blank control group was complete and clear, and compared with that in blank control group. In the positive control group, the fluorescence expression of ISV and DLAV was absent in zebrafish embryos treated with Vataranil, and the fluorescence expression of ISVs of zebrafish embryos in safflower, peach kernel and Achyranthes bidentata water extract groups were all attenuated in varying degrees. The fluorescence expression of ISVs in zebrafish embryos became thinner, the intensity weakened obviously, and the distribution was disordered, especially in the water extract group of Achyranthes bidentata. Conclusion: Vataranil can completely inhibit the ISVs and DLAV of zebrafish embryos, the traditional Chinese medicine safflower, peach kernel and Achyranthes bidentata can inhibit the ISVs of zebrafish embryos in different degrees, and the traditional Chinese medicine safflower and peach kernel for activating blood circulation and removing stasis can inhibit the ISVs of zebrafish embryos in different degrees. The inhibitory effect of Achyranthes bidentata on the angiogenesis of transgenic zebrafish, thus inhibiting tumor growth and metastasis, is different from that of traditional Chinese medicine for promoting blood circulation and removing blood stasis, such as Achyranthes bidentata, safflower and peach kernel, among which, Achyranthes bidentata. The effect of safflower is obvious, safe, reliable, no teratogenicity, lethal event. It is inferred that tumor blood circulation and blood stasis therapy can inhibit tumor angiogenesis, thus inhibit tumor growth and metastasis.
【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285

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