摘要
健康和长寿是人类生存和发展的永恒主题，而恶性肿瘤是危害人类健康的头号杀手。全世界每年约有1000万新发癌症患者，并且由于人口老龄化、吸烟、饮食习惯和膳食结构的改变和工业化程度的提高，全球癌症的发病数还将以年均3％—5％的速度递增，死亡率也呈逐年上升趋势。
大肠癌是常见的恶性肿瘤，占所有癌症的9％—15％。在许多工业化国家中是第二、三位常见的恶性肿瘤和第二位常见的癌症死亡原因。而在我国，大肠癌年发病率高达35—50/10万，每年大约以2.40％的速度较快增长，是男性第四位的常见肿瘤和第五位常见的癌症死亡原因。
对大肠癌的治疗目前采取的是一种包括手术、放疗、化疗以及生物、免疫治疗等在内的综合治疗，但不管采用哪种手段，大肠癌的总体治愈率仍徘徊在50％左右，长期生存率和生活质量仍不理想。因此，为了进一步促进我国城市经济和社会可持续发展，很有必要适时调整新世纪大肠癌防治策略，寻找其它更经济有效的手段以更好地控制肿瘤，从而实现WHO‘21世纪人人享有卫生保健’的具体目标。
控制癌症通常有两种途径。已延用100多年的经典途径是在临床上出现与局部扩散和转移相关的症状时对疾病进行处理，但往往因为失去根治机会而效果不佳；另一途径是了解突变发生的各种步骤并开发能够在其早期或瘤变前制止或逆转这一过程的化学预防药物。相比而言，前者着重于消除已存在的恶性细胞，而后者则强调消灭疾病于无形之中。无疑，后者对控制癌症是最佳途径也是最根本
浙江大学博士学位论文
的途径凹。肿瘤的发生是一个历时数年（平均 15—20年L复杂的、多阶段渐进
过程，这就为我们干预计划的实施提供了绝好的机会。同时肿瘤是遗传因素和环
境因素相互作用的结果［”］，其中2八以上的肿瘤与吸烟、不合理膳食、环境污染。
职业危害有关，从这一意义上说，肿瘤是一种社会病，社会病的防治更应从预防
着手。因此‘防患于未然’是最合理的癌症防治策略【’‘］。
大肠癌的化学预防由来已久，药物种类繁多，其中最经典的就是非笛体消炎
药（NSAIDS）。许多流行病学和动物实验资料显示非笛体消炎药如阿司匹林具有
降低结直肠癌发生危险的有益作用l”、”］。但现在应用于临床的大多数 NASIDS
存在两个主要问题，一是选择性不高，能同时抑制环氧化酶 1（COX—l）和 COX
一Z，从而带来不必要的副作用；二是要求长时间连续服用【’‘、匕1。因而目前迫切
需要寻找新的高效低毒干预阻断试剂。有些药物己经在这方面显示出较好的前
景，如近来发展的能高度选择性抑制COX一二的选择性非笛体消炎药［““”］；针对
肿瘤发生具体基因的基因预防驯’8］；针对端粒酶的端粒酶抑制剂l’9］；促进恶性
细胞向正常细胞逆转的分化诱导剂【川］；诱导细胞凋亡的凋亡诱导剂【”］；从天然
食物或传统中草药中筛选并分离出来的广谱化学预防剂【n］等。
叶绿酸铜钠（CHL）是天然叶绿素的衍生物，以往在一些老年病和慢性病患
者中使用较多，同时作为一种食品添加剂而被广泛使用，对人体无明显毒副作用
l’‘、纠］。最近的研究显示 CHL对多种诱变剂和致癌物有较强的抗诱变作用口’”刀］，
与肝肿瘤、皮肤肿瘤、大肠肿瘤等多种动物肿瘤的发生呈负相关【‘｝川。其作用机
理目前认为主要涉及以下几方面：1．抗氧化和自由基清除作用［‘’］；2．直接与致癌
物结合形成复合物，影响致癌物的吸收、分布和代谢活化l‘13．影响致癌物引起
的DNA加合物形成口＼4．影响代谢酶的活性，从而影响致癌物代谢及其代谢产
物的降解与排泄［3‘］。然而，CHL发挥化学预防作用的细胞分子生物效应仍然有
待阐明。
基于以上研究背景，本课题以二甲朕（DMH）化学诱导的昆明种小鼠大肠
肿瘤及人肠癌细胞株HT29为研究对象，选择了同大肠癌发生发展密切相关的几
个分子事件：无限制的增殖。凋亡的抑制、端粒酶的表达上调、细胞周期的紊乱
及环氧化酶的高表达为切入点，以体内模型及体外验证的方法探讨CHL对DMH
诱导的小鼠肠肿瘤的预防作用及对HT29细胞增殖、凋亡、端粒酶活性和 COX．2
2
浙江大学博士学位论文
活性等多方面生物学特性的影响。本研究的意义在于明确CHL对大肠肿瘤的化
学预防作用并探讨其可能的机制，为CHL用于肿瘤化学预防提供依据。
第一部分：CHL对DMH诱导的小鼠肠肿瘤的预防作用
将 195只小鼠随机分成 4组，阴性对照组（简称 NS组）10只，每周一次皮
下注射生理盐水，连续20周；阳性对照组（DMH组）50只，按20mg从g剂量每
周皮下注射DMH溶液，连续20周；实验1组（CHL组，诱癌启动阶段给药
组）75只，又按 CHL给药浓度 0刀sing／g、0．ling／g、0．3mg哈分为 3小组，DMH
每周一次诱癌，CHL隔天一次灌胃，连续20周；实验2组（CHLZ组，诱癌启动
后阶段给药组师0只小鼠也按CHL给药浓度分为3小组，DMH诱癌，不同浓度
CHL从第 8周起隔天灌胃
Health and longevity is the eternal subject of existing and developing for human beings. With the developing of economy, the meliorating of medicine condition and the progressing of living standard, malignant tumor becomes one of the main diseases which do harm to health of people. There were about 10 million new cases of cancer in the world every year and about 6 million died from cancer. Furthermore, because of aging, smoking, changing of eating habit and developing of industry, the incidence rate and the death rate will continue increasing.
Colorectal carcinoma was the third common malignancy and the second leading cause of cancer death in many developed countries. In China, the incidence of the colorectal cancer reaches to 35 to 50 cases per 1 million people each year and increases 2.40% every year, colorectal carcinoma ranks 4th common cancer and is 5th cause of cancer death.
The therapeutic strategy for colorectal cancer is a comprehensive method including surgery, chemotherapy, radiotherapy, biological therapy and so on. But the whole cure rate of colorectal carcinoma was only about 50% in spite of using all these methods and the long-term survival rate and the quality of life were not satisfactied. To accelerate the
maintaining development of economy and society, we should pay more attention to the chemoprevention strategy and look for more economic, more effective methods.
Usually there are two ways to control cancer. The classical one, is treating patients with clinic symptoms which benefited in small part of patients. Another way is blocking the tumourigenesis with the chemoprevention strategy. The former was concentrating on eradicating cells that have already undergone malignant transformation, and the later was focusing on intervening in the process in the cascade of carcinogenic events to prevent the final progression to neoplastic disease. Compared with each other, the later is more promising. Carcinogenesis is a complex and multistage process which needs long time (more than 10-20 years), this situation offers the opportunity to interrupt or reverse tumor development at a still harmless stage. In addition, tumourigenesis involves interaction between genetics and environmental factors, and about 2/3 of tumors are related to environmental factors. To some extent, cancer is a kind of social disease. Preventing social disease should depend on prophylaxis.
The chemoprevention of colorectal carcinoma has long history and a variety of agents have been purported to protect against colon cancer; of these, the non-steroidal anti-inflammatory drugs (NSAIDs) have demonstrated the greatest promise. The epidemiological and experimental evidence indicated that NSAIDs , such as, aspirin, sulindac et al, could reduce the risk of colorectal cancer by up to 50%. However, conventional NSAIDs taken orally inhibit both COX-1 and COX-2 nonselectively at concentrations encountered in the gastrointestinal tract. Thus, any of their beneficial effects on carcinogenesis, or other pathological process would be inseparable from the risk for adverse effects in gastrointestinal tract. So utilization of NSAIDs for
colon-cancer prevention has been hindered by their propensity to cause gastro-intestinal injury. To minimize the incidence of colon cancer, we need badly to seeking of novel high-effective, low-side-effect blocking agents. Some of agents have shown good prospect and got many eyeballs, such as, specif ic inhibitors of COX-2, gene prevention agents, telomerase inhibitors, differentiating inducers, apoptosis inducers and active components from nature products or traditional herbs.
Chlorophyllin (CHL) is the derivative of nature chlorophyll and a mixture of sodium-copper salts that is marketed as an over-the-counter drug for controlling body, fecal, and urinary odor in geriatric and osteomy patients and as an accelerant in wound healing. It is also used extensively as a food additive for coloration. Recently, CHL has been shown to exert profound antimutagenic behavior against a wide range of potential hum
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