【作者】 王永恒；

【导师】 耿美玉；

【作者基本信息】 中国海洋大学 ， 海洋生物， 2010， 博士

【摘要】 乳腺癌为发生于乳房的恶性肿瘤,是危害妇女健康的主要恶性肿瘤。目前世界范围内,乳腺癌的发病率已由女性恶性肿瘤中的第2位跃居到首位,其死亡率高达40%以上,其对人类健康的严重危害已引起了世界卫生组织和医疗界人士的高度重视。我国虽是乳腺癌的低发地区,但其发病率亦正逐年上升。与西方发达国家相比,我国乳腺癌的发病年龄高峰提早了10-15年,即：从30岁以后发病率开始上升,发病年龄高峰在40-49岁,以中年人居多。这个时期的妇女正处于最佳工作年龄段。乳腺癌发病率的增加和发病年龄的年轻化,无疑对我们整个社会及个体家庭产生了巨大负面影响。探索有效治疗乳腺癌的药物,是当今医药界的研究热点和紧迫任务。现代医学研究发现：突变型p53基因参与乳腺癌的发生；乳腺癌中p21表达与p53基因相关；p21可能间接调控cyclinD1、cyclinE表达。而正、负性调控因子p21、cyclinD1、cyclinE基因表达异常及cyclinDl基因异常是乳腺癌早期频发的分子事件。上述事件参与乳腺癌的发生发展,证实了周期失控是乳腺癌发生的最重要分子机制。中医学对乳腺癌早有描述。中医将乳腺癌多称为“乳岩”,也有称为“乳石痈”、“石榴翻花发”、“乳栗”等。凡结块如石、溃后状似岩洞者称曰“岩”,而患生于乳房者称为“乳岩”。隋代巢元方《诸病源候论》所载乳石痈“结核如石”即是对本病的早期记录。“乳岩”之名,最早见于宋、金年代。中医认为乳腺癌的主要病因和发病机制在于：肝气郁结、冲任失调、毒热蕴结、气血两虚。肝气郁结,肝经失于疏泄,气血壅滞,乳络不畅,则乳房结块；先天不足,或多产房劳,肝肾亏虚,冲任失养,致乳络不荣,则乳房肿块质硬；气火痰热,结聚肝胃二经,经脉瘀滞,化生乳岩,毒热蕴结,致肿块破溃,则浸淫秽臭；乳岩日久,气血耗伤,则贫血消瘦,疼痛难忍,五脏俱衰。目前乳腺癌的治疗以手术为主,配合放、化疗等综合疗法,但放、化疗所带来的毒副反应非常明显,致综合治疗效果不能令人满意。而采用中药对症复方,不仅能不同程度地缓解中晚期乳腺癌患者的临床症状,而且可以明显减少乳腺癌患者术后并发症,减轻乳腺癌放、化疗所致的毒副反应。其机理在于中医从整体出发,强调调整机体阴阳、气血、脏腑功能的平衡,内治和外治相结合,根据不同的临床证候辨证论治。这对增进乳腺癌患者术后的体质恢复,改善患者的生存质量,提高生存率,降低复发率,具有重要的临床意义和广泛的应用前景。中药是我国重要的资源宝库。依据中医理论,基于中药复方多组分物质基础的联合应用,组合近10万复方应用临床预防和治疗各种疾病,已有两千五百多年的历史。采用中药复方对乳腺癌进行联合治疗,可能成为我们预防和治疗乳腺癌的独特优势。虽然以往中医药治疗乳腺癌的研究报道很多,但大多仅局限于一般的临床观察,或个案验例,或单方验方。不仅在辨证分型上有较多的争议、在临床观察的设计上缺乏系统性和前瞻性对照,而且对于中药治疗乳腺癌的作用机理尚缺乏比较深入的实验研究,对中药复方的有效成分尚不明了,这大大限制了其推广应用和走出国门。因此,利用现代科学技术手段,结合多学科研究方法,探索中药复方的作用机制和有效成分,已成为中药复方科学使用的必要前提。消岩液是依据中医传统理论组建的中药复方,在临床进行了为期十年基于病症的疗效观察,表明了其能够明显增加乳腺癌病人的五年存活率。为了进一步阐明中药复方消岩液的有效成分、探明其具体作用机制,本文利用现代生物技术、蛋白质组学技术及现代药理学技术,结合现代医学对乳腺癌的认识,对该中药复方的作用机制和分子靶点进行了深入系统的研究。本课题中,我们选用依据中医学的辨证施治理论,参考民间验方进行科学筛选后得到中药复方,且在临床进行了为期十年基于病症的疗效观察,表明其确能明显增加乳腺癌病人五年存活率的消岩液。在体内,我们通过接种乳腺癌细胞4T07,建立了小鼠移植瘤模型,结果发现消岩液不仅能明显控制小鼠原位乳腺癌的体积,抑制其瘤重,而且可以明显抑制癌细胞接种引起的小鼠脾脏指数的增加。上述结果提示,消岩液对乳腺癌具有明显的治疗作用。在体外,采用血清药理学方法,我们观察了消岩液对乳腺癌细胞株及其他肿瘤细胞株增殖、细胞周期的抑制作用。采用SRB法,MTT法,流式细胞术,westernblot等多种技术,结果发现,消岩液不仅可以明显抑制乳腺癌细胞的增殖,同时对其他肿瘤细胞株细胞均有一定的抑制作用。进一步研究发现,消岩液可以明显抑制细胞周期调控因子的激活,具有一定的微管抑制作用。细胞水平的酶谱分析发现,消岩液还能够抑制EGFR的活化,细胞划痕实验和transwell小室实验表明消岩液对细胞的迁移和侵袭也具有一定的抑制作用。正是通过上述综合作用,中药复方消岩液得以最终发挥其抗乳腺癌和其他癌症的作用。本文首次通过集成的技术体系,从多个角度,利用多种方法,比较系统地揭示了中药复方消岩液抗乳腺癌的分子机制,为乳腺癌的综合药物治疗开辟了新途径。本项研究不仅提高了中药复方消岩液的应用开发价值,而且为中医药治疗癌症的可信性提供了重要理论支持。更多还原

【Abstract】 Breast cancer is a kind of disease threatening women. The increasing number of the breast cancer patients and the high mortality rate have been attracting the attentions of the World Health Organization and doctors. And in China, the patients of the breast cancer are characterized by the younger women that aged forty to fifty, while those are in their best time of working. And this festher exerts high influence on the patient’s family and society. Thus it is a hot pot to look for the effective medicine to treat breast cancer.The modern medicine studies show that the mutant of p53 gene is involved in breast cancer, which is related to the p21 gene. The p21 gene can regulate the expression of cyclin D1 and cyclin E. While the abnormal expression of p21 gene, cyclin D1, and cyclin E are the frequent events in the early time of breast cancer, which suggest the loss control of cell cycle is an important factor in the happening and developing of the breast cancer.Traditional Chinese medicine (TCM) is an important mine and it has had a 2,500 year’s history for TCM to adopt 100,000 different formulae to prevent and treat various diseases. The breast cancer was described and treated long time ago. Even though TCM has its own advantages of treating breast cancer and has been demonstrating good effectiveness in clinical practice, the dim effective constituents of many TCM-based formulae and their unclear action mechanisms have been restricting themselves in spreading. Therefore, it is necessary and possible, with modern technology and combining the multi-suject methods, to elucidate the effective constituents and mechanisms of those TCM-based formulae and thereafter spreading their applications. XiaoYanYe (XYY) is a formula based on traditional Chinese meicine theories and it has been showing good clinical efficacy that can increase the five years survival rate of breast cancer patients in clinical trials. For elucidating the mechanisms of XYY and spreading its applications, modern biotechnology, proteomics and pharmcological technologies have been adopted to explore the mechanisms and molecular targets of XYY.To observe the effects of traditional Chinese medicine XYY on breast cancer beared mice, Balb/C female mice were planted with breast cancer cells 4TO7. After 50 days administration of XXY, the size of the tumor was measured. And results showed not only the size and weight were reduced, but also the increased spleen and lung index were inhibited, which suggest that XXY can inhibit the growth of breast cancer in mice. In in vitro study, serum pharmacologic techniques were adopted, and the proliferation inhibitive effects of XYY on breast cancer cell were observed with SRB, MTT, flow cytometry and westernblot. The results showed that XYY can evidently inhibit both breast cancer cell lines proliferation. The further studies displayed that XYY can influence on the cell cycle. The detailed mechanisms of XYY inhibiting breast cancer cell line proliferation are attributed to the influence of XYY on the cell cycle regulators and microtubes. And the enzyme spectrum analysis on the cell showed that XYY can also control the activation of the VEGFR, which is further confirmed by the cell migration and the cell invasion experiments. All these results suggested XYY is able to inhibit breast cancer..It is first time that the antitumor mechanisms of XYY were elucidated from in vitro, cellular and molecular level through compositive technology systems. This study opens up a good source for the breast cancer treatment and possesses important theoretical value.更多还原