發(fā)布時(shí)間：2018-04-07 15:35

  本文選題：肝細(xì)胞癌　切入點(diǎn)：柴胡　出處：《成都中醫(yī)藥大學(xué)》2015年博士論文

【摘要】：目的：肝癌是嚴(yán)重危害人類健康的全球性腫瘤,柴胡為疏肝解郁之要藥,加之作為經(jīng)典的歸肝經(jīng)藥物,臨床上被廣泛用于肝癌的治療。本研究提出柴胡具有靶向性以“修飾”抗肝癌藥物,將其運(yùn)送至腫瘤部位,以提高治療效果的這一假說,選擇TG品系fli1a-EGFP轉(zhuǎn)基因斑馬魚為實(shí)驗(yàn)對(duì)象,以體視熒光顯微鏡為主要觀察工具,客觀、系統(tǒng)而直接地評(píng)價(jià)柴胡協(xié)同索拉非尼抗HCC的功效,進(jìn)而驗(yàn)證我們提出的假說的可能性,為柴胡在臨床上應(yīng)用于肝癌的治療提供客觀的實(shí)驗(yàn)依據(jù)。方法：通過體視顯微鏡(明場(chǎng))和體視熒光顯微鏡分別觀察22hpf轉(zhuǎn)基因斑馬魚模型胚胎的血管表型來評(píng)價(jià)柴胡抑制新生血管生成的作用；記錄2dpf野生型斑馬魚胚胎暴露于不同濃度梯度柴胡水提物的死亡數(shù),擬合LC50、LC10,估算MNLC,評(píng)估柴胡水提物的急性毒性；構(gòu)建高表達(dá)熒光素酶的HepG2穩(wěn)轉(zhuǎn)株,通過裸鼠驗(yàn)證其成瘤性,通過顯微注射將CM-Dil試劑染色的穩(wěn)轉(zhuǎn)株注射入2dpf的轉(zhuǎn)基因斑馬魚胚胎的卵黃囊內(nèi),立即評(píng)估人肝癌轉(zhuǎn)基因斑馬魚卵黃囊移植瘤模型是否成功建立；通過體視顯微鏡和體視熒光顯微鏡觀察,定量分析并評(píng)價(jià)柴胡協(xié)同索拉非尼抑制轉(zhuǎn)基因斑馬魚HCC移植瘤生長(zhǎng)的功效。結(jié)果：柴胡對(duì)轉(zhuǎn)基因斑馬魚新生血管的生成無抑制作用；柴胡水提物致野生型斑馬魚胚胎的死亡率呈明顯的濃度依賴關(guān)系,柴胡水提物對(duì)斑馬魚胚胎72h-LC50為670 u g/ml.72h-LC10為205μg/ml.72h-MNLC為155μg/ml,柴胡水提物對(duì)斑馬魚胚胎及幼魚的急性毒性較輕；確定了人肝癌轉(zhuǎn)基因斑馬魚卵黃囊移植瘤模型被成功建立,柴胡不能增強(qiáng)索拉非尼對(duì)轉(zhuǎn)基因斑馬魚人肝癌移植瘤生長(zhǎng)的抑制作用。結(jié)論：柴胡對(duì)轉(zhuǎn)基因斑馬魚新生血管的生成無抑制作用。柴胡對(duì)斑馬魚胚胎及幼魚急性毒性作用較輕。柴胡作為一味經(jīng)典的歸肝經(jīng)藥物,并不能增強(qiáng)索拉非尼對(duì)轉(zhuǎn)基因斑馬魚人肝癌移植瘤生長(zhǎng)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.
【學(xué)位授予單位】：成都中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.7

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