【作者】 常欣峰；

【导师】 韩立民；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2014， 博士

【摘要】 原发性肝癌是一种常见的严重危害人类健康的恶性肿瘤。目前其总体疗效不容乐观,患者的生活质量差,存活率低,故探索行之有效的治疗方法,延长患者的生存期是一个亟待解决的问题。鳖甲煎丸是医圣张仲景所创,以活血、补益、散结为主。既有攻伐之效,亦含补益之功。研究表明,鳖甲煎丸具有较好的抑制肝癌生长,防止肝癌转移的作用,但其具体抗癌机制仍不甚明确。本课题分别应用体内动物模型和体外细胞模型对鳖甲煎丸抗肝癌的作用和机制进行研究,为充分开发利用祖国中医药资源治疗肝癌提供实验依据。目的：通过在体和离体实验,观察鳖甲煎丸对H22荷瘤小鼠和人肝癌细胞Bel-7402的影响,从促进肿瘤细胞凋亡及抑制肿瘤新生血管形成这两方面来初步探讨鳖甲煎丸抗肝癌作用的可能机制,为临床进一步应用该药物治疗肝癌提供实验依据。方法：系统学习古今文献,从病名、病因、病机、辨证分型、治疗等方面阐述祖国医学对原发性肝癌的认识及鳖甲煎丸的临床应用和实验研究进展；运用血清药理学、形态学、流式细胞术、MTT法、Western blot等多种实验方法,将在体和离体实验相结合,从整体水平、细胞水平分子水平等不同角度研究鳖甲煎丸对肝癌生长的抑制作用,探讨其作用机制。结果：1.文献研究：肝癌的发生是人体内、外因素共同作用的结果,“虚”、“瘀’“热”是其主因,多在正气虚弱基础上,气滞血瘀、痰湿互结、癌毒湿热蕴结于肝而成。鳖甲煎丸以活血、补益、散结为主。既有攻伐之效,亦含补益之功。临床被用来治疗肝癌,取得了良好的效果。2.在体实验：鳖甲煎丸对H22荷瘤小鼠的影响(1)鳖甲煎丸各剂量组的瘤重均显著降低,提示鳖甲煎丸有明显的抑瘤效果。(2)鳖甲煎丸中、高剂量组脾脏指数与模型组比较显著升高；各剂量组胸腺指数比较无明显差异。提示鳖甲煎丸可以提高荷瘤小鼠的免疫功能,抑制肿瘤生长。(3)流式细胞术结果显示,与模型对照组比较,鳖甲煎丸各剂量组可使H22肝癌小鼠外周血中CD4+含量显著增加,CD8+含量显著下降,且升高了CD4+/CD8+比值。提示鳖甲煎丸可以通过增强机体免疫力,发挥抑瘤作用。(4)凋亡蛋白检测结果表明：鳖甲煎丸各剂量组可降低肿瘤组织Bcl-2蛋白表达,增加Bax蛋白表达,降低Bcl-2/Bax比值,同时可抑制p-ERK合成,促进p-JNK合成。提示鳖甲煎丸可阻滞ERK1蛋白磷酸化,促进JNK蛋白磷酸化,进而降低抑调亡蛋白Bcl-2表达,提高促凋亡蛋白Bax表达,调整Bcl-2/Bax比值来达到促进肿瘤凋亡的目的,且具有一定的量效关系。(5)鳖甲煎丸各剂量组肿瘤组织中VEGF、Delta-4和NICD蛋白表达均显著降低。提示鳖甲煎丸可通过降低VEGF、Delta-4和NICD蛋白表达,抑制肿瘤新生血管的形成,进而抑制肿瘤生长。3.离体实验：鳖甲煎丸含药血清对人肝癌细胞Bel-7402的影响(1)倒置显微镜下观察可见,用药组细胞形态发生改变,部分细胞变小变圆,胞浆量减少,浆内可见大量透明颗粒,细胞成单个生长状态,贴壁能力下降,脱落增多,部分细胞呈悬浮生长。(2)MTT实验结果表明,不同剂量的鳖甲煎丸含药血清对Bel-7402细胞生长均有一定的抑制作用,且随着药物剂量的增加,作用时间的延长抑制作用亦逐渐增强。提示鳖甲煎丸含药血清有抑制肝癌细胞Bel-7402增值的作用。(3)Annexin V/PI双染色流式细胞术检测结果表明,随着鳖甲煎丸剂量的增加,细胞凋亡比例亦相应增加。提示鳖甲煎丸含药血清可以促进Bel-7402细胞凋亡。(4)Western blot检测结果显示：鳖甲煎丸各剂量组均可抑制(?)p-ERK生成,促进JNK磷酸化,同时上调Bax,降低Bcl-2,提示鳖甲煎丸通过阻滞ERK1/2信号通路,激活JNK信号通路,从而起到抑瘤作用。(5)Western blot检测结果显示：在VEGF、Delta-4表达上,鳖甲煎丸各剂量组与正常血清组比较均显著减弱;而在NICD表达上,中、高剂量鳖甲煎丸含药血清组NICD表达与正常血清组比较降低明显。提示鳖甲煎丸可以通过阻断VEGF-D114-Notch信号通路转导,抑制肿瘤血管生成而发挥抑瘤作用。结论：鳖甲煎丸可有效抑制肝癌细胞生长,增强机体免疫功能,改善生存质量。其抑瘤机制可能与以下信号通路有关：(1)抑制ERK1的活化,阻断ERK1/2信号通路,从而促进肿瘤细胞凋亡；(2)促进JNK磷酸化,激活JNK信号通路,从而抑制Bcl-2、提高Bax的表达,促进肿瘤细胞凋亡;(3)阻断VEGF-D114-Notch信号通路,抑制肿瘤血管生成,减少肿瘤血液供应,最终控制肿瘤生长。更多还原

【Abstract】 Background:Primary liver cancer is a common malignancy and harm human health seriously. At present, to explore effective treatment methods to improve the quality of life and survival of patients is becoming a serious problem, because that the overall efficacy is not optimistic. Bie Jia Jian Wan(BJJW) is created by Zhang Zhongjing, with the effect of activating blood, nourishing, and relieving congestion. Studies have shown that BJJW has better inhibition of liver cancer growth and prevents metastasis of liver cancer, but its exact mechanisms of anti-cancer remain unclear. This project is to study the effect of BJJW and its anti-cancer mechanism by using in vivo animal models and in vitro cell models. It can provide a theoretical basis for using TCM in treatment of liver cancer.Objective:To observe the effect of BJJW in H22tumor-bearing mice and in Bel-7402human hepatoma cells through in vivo and in vitro experiments, and explore the possible anti-cancer mechanisms of BJJW by promoting apoptosis of tumor cells and inhibition of tumor angiogenesis so as to lay the foundation for further clinical application of the drug in treatment of liver cancer.Methods:Systematically study the ancient and modern document, to set forth the understanding of traditional Chinese medicine for primary liver cancer and clinical application and advances in experimental study from the aspects of name of the disease, etiology, pathogenesis, syndromes and treatment. Through different research methods like serum pharmacology, morphology, flow cytometry, MTT method, Western blot and so on, to study the inhibitory effect of BJJW on the growth of liver cancer and investigate its mechanism in vitro and in vivo. Results:1. Literature study:Liver cancer whose main cause is "deficiency of Qi"," blood stasis" and "heat" is the result of interaction of internal and external factors of the human body. Liver cancer basses on’’deficiency of Qi" and results from "Qi stagnation and blood stasis","phlegm-dampness cross knot" and "accumulation of wet and hot". BJJW has the effect of activating blood, nourishing, and relieving congestion. It is clinically used to treat liver cancer and has achieved good results.2. In vivo experiments:Effect of BJJW in H22tumor-bearing mice.(1) The weight of the tumor in BJJWL, BJJWM and BJJWH was significantly reduced, which prompts BJJW has a significant inhibitory effect.(2) Compared with the Model, the spleen index of BJJWM and BJJWH were significantly increased while the thymus index of BJJWL, BJJWM and BJJWH were not significantly different. It shows that BJJW could improve immune function in tumor-bearing mice and inhibit the growth of tumor.(3) Flow cytometry showed compared with Model the content of CD4+and the ratio of CD4+/CD8+in BJJWL, BJJWM and BJJWH were significantly reduced while the content of CD8+was increased. It intimates that BJJW could enhance immunity to inhibit tumor.(4) Western bolt showed BJJWL, BJJWM, and BJJWH reduced the expression of Bcl-2, increased the expression of Bax and decreased the ratio of Bcl-2/Bax. Besides, BJJW could inhibit the synthesis of p-ERK and promote the synthesis of p-JNK. This prompts that BJJW could block phosphorylation of ERK1protein and promote phosphorylation of JNK protein, so as to reduce the expression of anti-apoptosis protein Bcl-2, enhance the expression of apoptosis protein Bax and adjust the ratio of Bcl-2/Bax to achieve the aim of promoting apoptosis of tumor, and it has a certain dose-effect relationship. (5) Western bolt showed BJJWL, BJJWM, and BJJWH reduced the expression of VEGF, Delta-4and NICD. It implies that BJJW could inhibit tumor growth by reducing the expression of VEGF, Delta-4and NICD and inhibiting the formation of tumor angiogenesis.3. In vitro experiments:Effect of serum containing BJJW in Bel-7402human hepatoma cells.(1) Under an inverted microscope, cell morphology changed, some cells became smaller and round, the amount of cytoplasm reduced, pulp was visible within a large number of transparent particles, growth of cells was into a single state, adherent ability decreased, shedding increased and some cells were grown in suspension.(2) MTT showed different doses of serum containing BJJW had a certain inhibition on the growth of Bel-7402cells and as the dosage increased, inhibition increased. It implies serum containing BJJW has a certain inhibition on the growth of Bel-7402cells.(4) Annexin V/PI double staining flow cytometry results showed with the dose of BJJW increased, the proportion of apoptosis increased. It hints serum containing BJJW could promote the apoptosis of Bel-7402cells.(5) Western blot showed BJJW in different concentration group could inhibit the generation of p-ERK, promote phosphorylation of JNK, raise Bax and reduce Bcl-2. It indicates that BJJW may inhibit tumor growth by blocking ERK1/2signal pathway, activating JNK signal pathway.(6) Western blot showed compared with Control the expression of VEGF and Delta4in BJJWL, BJJWM, and BJJWH were significantly reduced, and the expression of NICD in BJJWM and BJJWH were significantly reduced. It hints BJJW may inhibit tumor by blocking the VEGF-D114-Notch signal transduction pathway.Conclusions:Combination of in vivo and in vitro experimental results, we came to the conclusion:BJJW can effectively inhibit the growth of liver cancer, enhance immune function and improve quality of life. The inhibition mechanism may be related to signal transduction as follows:(1) Blocking ERK1/2signal transduction, inhibiting the activation of ERK1, thereby promoting apoptosis of tumor cells.(2) Activating JNK signal transduction, promoting JNK phosphorylation, indirectly inhibiting the activity of Bcl-2, and promoting tumor cell apoptosis.(3) Blocking VEGF-D114-Notch signal transduction, inhibiting tumor angiogenesis and reducing tumor blood supply, ultimately to control tumor growth.更多还原