基于斑马鱼模型评价柴胡协同索拉非尼抗肝细胞癌的功效

发布时间：2018-04-07 15:35

本文选题：肝细胞癌　切入点：柴胡　出处：《成都中医药大学》2015年博士论文

【摘要】：目的：肝癌是严重危害人类健康的全球性肿瘤,柴胡为疏肝解郁之要药,加之作为经典的归肝经药物,临床上被广泛用于肝癌的治疗。本研究提出柴胡具有靶向性以“修饰”抗肝癌药物,将其运送至肿瘤部位,以提高治疗效果的这一假说,选择TG品系fli1a-EGFP转基因斑马鱼为实验对象,以体视荧光显微镜为主要观察工具,客观、系统而直接地评价柴胡协同索拉非尼抗HCC的功效,进而验证我们提出的假说的可能性,为柴胡在临床上应用于肝癌的治疗提供客观的实验依据。方法：通过体视显微镜(明场)和体视荧光显微镜分别观察22hpf转基因斑马鱼模型胚胎的血管表型来评价柴胡抑制新生血管生成的作用；记录2dpf野生型斑马鱼胚胎暴露于不同浓度梯度柴胡水提物的死亡数,拟合LC50、LC10,估算MNLC,评估柴胡水提物的急性毒性；构建高表达荧光素酶的HepG2稳转株,通过裸鼠验证其成瘤性,通过显微注射将CM-Dil试剂染色的稳转株注射入2dpf的转基因斑马鱼胚胎的卵黄囊内,立即评估人肝癌转基因斑马鱼卵黄囊移植瘤模型是否成功建立；通过体视显微镜和体视荧光显微镜观察,定量分析并评价柴胡协同索拉非尼抑制转基因斑马鱼HCC移植瘤生长的功效。结果：柴胡对转基因斑马鱼新生血管的生成无抑制作用；柴胡水提物致野生型斑马鱼胚胎的死亡率呈明显的浓度依赖关系,柴胡水提物对斑马鱼胚胎72h-LC50为670 u g/ml.72h-LC10为205μg/ml.72h-MNLC为155μg/ml,柴胡水提物对斑马鱼胚胎及幼鱼的急性毒性较轻；确定了人肝癌转基因斑马鱼卵黄囊移植瘤模型被成功建立,柴胡不能增强索拉非尼对转基因斑马鱼人肝癌移植瘤生长的抑制作用。结论：柴胡对转基因斑马鱼新生血管的生成无抑制作用。柴胡对斑马鱼胚胎及幼鱼急性毒性作用较轻。柴胡作为一味经典的归肝经药物,并不能增强索拉非尼对转基因斑马鱼人肝癌移植瘤生长的抑制作用。
[Abstract]:Objective: liver cancer is a global tumor that seriously endangers human health. Bupleurum chinensis is widely used in the treatment of liver cancer.In this study, the hypothesis that Bupleurum chinense has the target of "modified" anti-hepatoma drugs to be transported to the tumor site to improve the therapeutic effect was proposed. The TG strain fli1a-EGFP transgenic zebrafish was selected as the experimental object.With stereoscopic fluorescence microscope as the main observation tool, objective, systematic and direct evaluation of the efficacy of Radix Bupleurum combined with Solafenil in anti-#en0# was carried out, and the possibility of our hypothesis was verified.To provide an objective experimental basis for the clinical application of Bupleurum chinense in the treatment of liver cancer.Methods: the effects of Bupleurum chinense on angiogenesis were evaluated by observing the vascular phenotype of 22hpf transgenic zebrafish embryos by stereoscopic microscope and stereoscopic fluorescence microscope.The death number of 2dpf wild type zebrafish embryos exposed to different concentration gradient water extract of Bupleurum chinensis was recorded, the LC50 LC10 was fitted to estimate the acute toxicity of the aqueous extract of Bupleurum chinense, and the HepG2 stable transformation strain with high luciferase expression was constructed, and its tumorigenicity was verified by nude mice.The stable transformed strain stained with CM-Dil reagent was injected into the yolk sac of transgenic zebrafish embryos with 2dpf by microinjection, and the successful establishment of human liver cancer transgenic zebrafish yolk sac transplantation model was evaluated immediately.The effects of Radix Bupleurum and Solafenil on the growth of transgenic zebrafish HCC xenografts were quantitatively analyzed and evaluated by stereoscopic microscope and stereoscopic fluorescence microscope.Results: Bupleurum chinense had no inhibitory effect on angiogenesis of transgenic zebrafish, and the death rate of wild type zebrafish embryos induced by water extract of Bupleurum chinense was in a dose-dependent manner.The 72h-LC50 of water extract of Bupleurum chinensis to zebrafish embryos was 670u g/ml.72h-LC10 = 205渭 g/ml.72h-MNLC, and the acute toxicity of water extract of Bupleurum chinense to zebrafish embryos and young fish was less acute, the human liver cancer transgenic zebrafish yolk sac transplantation model was established successfully.Bupleurum Bupleurum could not enhance the inhibitory effect of Solafenil on the growth of human liver cancer transplanted with transgenic zebrafish.Conclusion: Bupleurum chinense has no inhibitory effect on angiogenesis of transgenic zebrafish.The acute toxicity of Bupleurum chinense to zebrafish embryo and juvenile was mild.Bupleurum chinensis, as a classic drug, can not enhance the inhibitory effect of Solafenil on the growth of human liver cancer transplanted with transgenic zebrafish.
【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】：R735.7

【相似文献】