【作者】 许刚；

【导师】 张亚历；

【作者基本信息】 中国人民解放军第一军医大学 ， 内科学， 2003， 博士

【摘要】 北景／目的 姜茶素是由天然植物活性成分姜黄素和儿茶素组成的，这两种成分副作用极小，几乎无毒性作用。研究证实姜黄素、儿茶素单独应用对肿瘤的的发生都有一定的预防作用，但是两者联合应用是否有协同抑制大肠癌发生的作用，目前国内外尚无这方面的研究报道。大肠异常隐窝病灶(aberrant crypt foli，ACF)是比较理想的大肠癌中间生物学评价指标之一，而环氧化酶(Cyclooxygenase-2，COX-2)和过氧化物酶体增殖物激活受体γ(Peroxisome Proliferator-Activated Receptorγ，PPARγ)则是与恶性肿瘤细胞的生长和生存密切相关的两个药物作用的分子靶点。本实验首次将由儿茶素和姜黄素组成的姜茶素应用于二甲基肼(DMH)诱导的大鼠大肠癌的化学预防研究，旨在初步阐明以下几个方面的问题： 1．探讨姜茶素对DMH诱导的大鼠ACF形成及大肠癌的化学预防作用，评价姜茶素对大肠癌的化学预防作用效果。 2．探讨姜茶素对大鼠大肠癌细胞的增殖及凋亡有何影响，从细胞动力学及凋亡方面初步阐明其抑制肿瘤的机理。 3．探讨姜茶素对DMH诱导的大鼠大肠癌变过程中COX-2和PPARγ表达的影响，以期能从分子水平上初步阐明姜黄素和儿茶素联合应用抑制肿瘤的机理。 方法 1．利用DMH诱导大鼠大肠癌模型，实验大鼠分5个组：(1)阴性对照组；(2)阳性对照组(DMH组)；(3)姜黄素预防组(DMH+0.2％姜黄素)；(4)儿茶素预防组(DMH+0.2％儿茶素)；(5)姜茶素预防组(DMH+0.2％姜黄素+0.2％儿茶素)，其中阴性对照组20只，其它四组各30只。观察各预防药物对诱癌后第12W大鼠大肠ACF数量、第32W大鼠大肠癌的发生率、肿瘤抑制率、大鼠体积及分化程度的影响。应用美蓝染色法观察ACF的数量，HE染色法进行形态学观察和病理定性分析。 2．利用细胞增殖核抗原(PCNA)表示细胞增殖活性，利用细胞原位末端标记技术(Tunel)检测细胞凋亡情况，观察姜茶素、儿茶素和姜黄素对大鼠大肠癌细胞增殖和凋亡的影响。 .3.利用免疫组织化学方法、RT一pCR和免疫印迹(Westemblotting)方法分别检测诱癌12w时大鼠大肠粘膜组织和32W时大鼠大肠癌组织COX一2及PPARY表达的变化，并对这些指标的变化进行定性和定量分析。结果 1.姜茶素对DMH诱导的大鼠ACF数量的影响实验第12周时除阴性对照组外，阳性对照组及3个药物预防组均有ACF形成。姜茶素、姜黄素和儿茶素组大鼠大肠ACF的平均数量明显低于阳性对照组 (尸<0.01)。姜茶素组大鼠大肠AcF的平均数量和由3个以上异常隐窝构成的AcF的平均数量低于姜黄素组、儿茶素组(P<0.05)，而姜黄素和儿茶素单独应用组间相比差异无显著性。 2.姜茶素对DMH诱导大鼠大肠癌的影响实验第32周时除阴性对照组外，阳性对照组、姜黄素、儿茶素和姜茶素预防组均诱导出大肠癌，诱癌率分别为81.巧%、61.11%、58.82%和38.89%，3组药物都能降低大鼠大肠癌的发生率，其中姜茶素组大肠癌发生率明显低于阳性对照组(P<。.05)。姜茶素组大肠癌的抑制率(52.15%)高于姜黄素组(24.79%)和儿茶素组(27.61%)(P<0.05)。研究还发现姜茶素组大肠癌的平均数量和平均体积均最低，与阳性对照组相比差异有显著性(尸<0.05)。无论是姜黄素、儿茶素还是姜茶素对大肠癌的分化程度均无明显影响。 3.姜茶素对大肠癌细胞增殖指数、凋亡指数的影响与阳性对照组相比3个药物预防组大鼠大肠癌组织的增殖指数明显降低(尸<0.05)、凋亡指数明显增高(尸<0.05)，其中姜茶素组的作用效果最强。 4.姜茶素对COX一2表达的影响DMH可以诱导12w大鼠大肠粘膜组织和32W大鼠大肠癌组织COX一2 mRNA和蛋白的表达。RT一PCR结果显示:各组均可见COX一2 mRNA的扩增条带，阴性对照组的扩增带明显弱于其它组;诱癌12w时，3个药物预防组都能抑制COX一2 mRNA的表达，与姜黄素、儿茶素组相比，姜茶素组的抑制作用最强(尸<0.05);实验32W时阳性对照组及3个预防药物组大肠癌组织COX一2 mRNA的表达量明显升高，但各组间差异不明显。westem blotring方法检测了COX一2蛋白的表达情况，结果显示:COX一2蛋白的表达与COX一2 mRNA的表达情况基本一致，但阴性对照组无COX一2蛋白的表达。 5.姜茶素对Pp月卿表达的影响免疫组化结果显示:无论是诱癌12w的大肠粘膜组织还是诱癌32w的大肠癌组织均有PPAR丫表达。RT一PcR结果显示:1 Zw时各组大鼠大肠粘膜PPA砌InRNA的表达无显著差异(P>0.OS);实验32W阳性对照组及3个预防药物组大肠癌组织PPARy 1llRNA的表达明显高于阴性对照组(P<0.05)，但阳性对照组与3个预防药物组间差异不明显。westem bfotting结果显示PPARY蛋白的表达与PPA砌mRNA的表达基本一致。结论 1.姜茶素、姜黄素和儿茶素均能抑制大鼠诱癌早期ACF的数量，对DMH诱导的大鼠大肠癌有明显的化学预防作用。姜黄素和儿茶素联合应用对大肠癌的发生有协同预防作用，这种协同预防作用有可能是通过协同抑制ACF的形成实现的。 2.姜茶素、姜黄素和儿茶素能够抑制大鼠大肠癌细胞的增殖，诱导大肠癌细胞的凋亡，其中姜更多还原

【Abstract】 Background/ObjectivesJiangchasu is composed of curcumin and catechins, both of which are naturally plant active components and have nearly no adverse effects. It has been proved that both curcumin and catechins posses widespread anticancer properties. Now aberrant crypt foli (ACF) has been proposed as a good intermediate biomarker in colorectal cancer. Cyclooxygenase 2 (COX-2) and peroxisome proliferator-activated receptor gamma (PPARy) have emerged as promising candidate molecular targets for colorectal cancer. The thesis aimed to evaluate whether a combination of curcumin and catechins shows synergistic effects for cancer-preventive activity for rat colorectal cancer induced by dimethylhydrazine (DMH). It is focused on questions as follows.1. To observe the inhibition effects of Jiangchasu on the number ofcolon ACF and colorectal cancer induced by DMH on Wistar rats.2. To evaluate the effects of Jiangchasu, curcumin and catechins on the cell proliferation and apoptosis of rat colorectal cancer were investigated, which was helpful to elucidate the anticancer mechanism of Jiangchasu.3. To detect the effects of Jiangchasu, curcumin and catechins on expression of COX-2 and PPARy in DMH induced rat colorectal cancer, hoping to elucidate the chemoprevention mechanisms of Jiangchasu in molecular levels.MethodsI. Wistar rat colorectal cancer was induced by DMH. All rats were randomly divided into five groups: (1) negative group: standardized food. (2) DMH group: standardized food + DMH. (3) curcumin group: standardized food with 0.2% curcumin + DMH. (4) catechins group: standardized food with 0.2% catechins + DMH. (5) Jiangchasu group: standardized food with 0.2% (curcumin and catechins) + DMH. The number of rat colorectal ACFs was observed by methylene blue staining. The incidence, number and size ofcolorectal cancer were accounted. The changes of rat colorectal tissues in morphology were observed through hematoxylin-eosin (HE) staining.2. Proliferation index displayed by PCNA through immunohischemistry and apoptosis index revealed by TUNEL are used to evaluate the chemoprevention effects of curcumin, catechins and Jiangchasu in rat colorectal cancer induced by DMH .3. The expression of COX-2 and PPAR y in tissues of rats colorectal mucosa at 12W and in tissues of rats colorectal cancer at 32W after first DMH administration were examined with immunohistochemical staining, RT-PCR and Western blotting methods.Results1. The effects of Jiangchasu on the number of ACF At 12 weeks after the first administration, rats belong to groups 2-5 developed ACF. The dietary administration of Jiangchasu, curcumin and catechins caused significantly inhibition in the ACF incidence than that of in DMH treatment group (P<0.01). The inhibition effect of Jiangchasu group was the highest among three chemopreventive groups (P<0.05). There was no difference between curcumin and catechins group.2. The effects of Jiangchasu on colorectal cancer At 32 weeks after the first administration, the incidence of the colorectal cancer were 81.15%, 61.11%, 58.82% and 38.89% in DMH, curcumin, catechins and Jiangchasu group respectively. The incidence of colorectal cancer in Jiangchasu group was significantly lower than that of in DMH group (P<0.05). The average numbers and volumes of rumors were significantly decreased in Jiangchasu group than that of in curcumin or cathchins group administration alone. All preventive drugs had no effects on the differentiation of cancer.3. The effects of Jiangchasu on cell proliferation index and apoptosis index Compared with DMH group, proliferation index in preventive group was markedly decreased and apoptosis index was obviously increased. The effects of Jiangchasu group were significantly higher than that of the other groups.4. The effects of Jiangchasu on expression of COX-2 DMH couldinduce the expression of COX-2 in colon mucosa tissues at 12W and in colorectal cancer tissues at 32W. The results of RT-PCR showed更多还原