【作者】 周京旭；

【导师】 张振书；

【作者基本信息】 第一军医大学 ， 内科学消化， 2001， 博士

【摘要】 背景/目的： 人类许多肿瘤的形成和发展是一个长期的多阶段的过程，化学预防可作用于其中的任何阶段。从低风险的正常人群到因环境、生活方式、基因易感性和癌前病变等因素造成的中等风险人群，再到曾患过肿瘤而易再发肿瘤的高风险人群，都可以采用适当的化学预防措施降低肿瘤的发病率和病死率。因而，人们希望能找到安全、高效的肿瘤化学预防药物。食物来源的天然药物因其具备较低的毒副作用、较高的安全性和较高的依从性，可被各类人群尤其是低风险人群长期服用，而倍受关注。姜黄素是天然植物姜黄的主要活性成分，生活中可作为调料和食物染色剂。姜黄素的药理作用广泛，具有抗炎、抗氧化、降血脂等作用。近年来，其对肿瘤的化学预防作用受到重视，具有抗癌谱广，毒副作用小等特点。尽管已对姜黄素的作用机制进行了初步阐明，但对其作用的分子靶点依然不清。基因芯片能同时检测几千个甚至上万个基因的表达，使系统的研究姜黄素作用的分子机制成为可能。本研究旨在探讨以下问题：1）观察姜黄根粉对亚硝胺诱导的实验性大鼠肝癌的化学预防作用，评价姜黄素 对肝癌的化学预防效果；2）寻找姜黄素对肝癌预防作用评价的中间生物指标，并利用中间生物指标建立 姜黄素预防效果的早期判别函数；3）利用基因芯片技术探讨姜黄素对肝癌化学防护作用的分子机理，分析姜黄素 98级博士学位论义 可能作用的基因功能组。方 法：1）利用二乙基亚硝胺和 Nitrosomorpholine诱导 Wistar大鼠原发性肝癌模型。 预防组A于诱癌前7天喂食含2％姜黄根粉饲料，预防组B于诱癌24周后加用 2％姜黄根粉饲料。分别于 16、20。24周宰杀部分大鼠，观察大鼠肿瘤发生情 况。32周时，宰杀所有大鼠，统计各组大鼠的肿瘤发生率、淋巴结转移率和 肺转移率。HE染色进行形态学观察和病理定性分析；2）利用细胞增殖核抗原和细胞原位末端标记技术分别检测各组肝组织的细胞增 殖指数和细胞凋亡指数，并用这两个指标作为中间生物标记评价姜黄素对肝 癌的化学预防作用，建立以它们为参数的判别函数。3）分别取诱癌早期（20周）和诱癌晚期（32周）组织和对应的姜黄素化学预防 组织，进行基因芯片检测。用异硫氰酸肌一步法抽提组织RNA，比色检测纯度， 电泳检测RNA的完整性，用oligodT亲和柱过柱纯化，得到纯mRNA。逆转录 为 CDNA，分别用荧光染料CY3和 CYS标记成探针，与大鼠基因芯片杂交，42 OC孵育15l7hr，洗片，扫描，进行结果分析。结 果：1）大鼠经 32周二乙基亚硝胺和 Nitrosomorpholine处理，肝癌的发生率在诱癌 组为100％（12／12），肿瘤在肝脏形成多发癌灶（22．3土6．0个），最多达31 个，同时肺转移率为75．0％（9／12），淋巴结转移率为66．7％（S／12）。而姜黄素 早期预防组 A肝癌的发生率仅为 16．7见 肝癌结节明显小而少，平均为 2．0土 1．4，未发现肺转移和淋巴结转移；而在诱癌的中晚期枯4周）给予姜黄素，肝 癌的发生率为100％，肝癌结节达9．8士2．2，肺转移率和淋巴结转移率均为 16．7叭2／l人形态学上，诱癌早期，诱癌组呈典型肝硬化表现，形成假小叶。 而预防组则仅见少量气泡样变性和点状坏死：诱癌晚期，诱癌组可见肿瘤形南方医院全军消化内科研究所 第5贞 98级博士学位论文 成，而预防组仅见轻度肝硬化形成。同时，诱癌组可见许多肺转移结节，镜 下见肿瘤细胞呈灶性生长；而预防组则少见。2）与诱癌组相比，预防组增殖指数在 16，20，24和 32周均较同期诱癌组明显 降低而凋亡指数明显增高，差异显著。提示姜黄素对肝癌的化学预防作用与 抑制增殖和诱导凋亡有关。利用增殖指数和凋亡指数为参数，建立起判别函 数，以期在早期判定姜黄素的预防效果。3）应用基因芯片检测了诱癌早期和诱癌晚期姜黄素可能的作用靶点，发现在早 期调节 217个基因的表达，而晚期调节 106个基因的表达，与多个功能基因 组有关。结 论：1）姜黄素对亚硝胺诱发的大鼠肝癌具有明显的化学预防作用，其早期化学预防 作用良好，明显降低肝癌的发生率和多结节性，降低肿瘤自发转移率；晚期 给予姜黄素也有一定的防护作用；能延缓疾病进展，降低肝癌的转移率。2）细胞增殖指数和细胞凋亡指数可作为评价姜黄素化学预防作用的中间生物指 标，可在诱癌的早中期反映姜黄素的化学预防效果，对今后的临床实践有借 鉴作用。3）姜黄素对肝癌的化学预防作用与调节肝脏代谢功能，抑制增殖，诱导凋亡， 抗细胞骨架，增强机体兔疫力和桔抗亚硝胺的基因调节作用有关。更多还原

【Abstract】 Backgroud/Objectives: The development of human cancer is a long-time and multiphase process, the scope of chemoprevention encompasses cohorts at all phases of this process梖rom healthy subjects at normal risk, to populations at intermediate risk resulting from environmental and lifestyle factors, genetic predisposition, and precancerous lesions, and then to the patients at high risk for second malignancy. So they expect to get chemopreventive drugs with safety and efficiency. Diet-derived chemopreventive agents are highly interested because of their safety and compliance. Curcumin is dominating active components of Curcuma longa and can be used as spice and edible pigment. Simultaneously, curcumin possess widespread pharmacological effect, such as antiinflammation, antioxidation, anticoagulation et al. Recently, curcumin is attached importance on account of its anticancer properties. Understanding possible molecular targets is very important to elucidate mechanisms for curoumin chemoprevention. Gene chip allows monitoring the expression of thousands or tens of thousands genes simultaneously in one hybridization experiment. Employing this technique, detection of differentially expressed genes regulated by curcumin is greatly facilitated. The thesis aimed to observe the inhibition effects of curcumin on hepatocarcinogenesis induced by nitrosamine on Wistar rats and to evaluate chemopreventive effect of curcumin on hepatocarcingenesis, and to investigate mechanisms for chemoprevention by curcumin with possible molecular targets by gene chip. It is focused on the questions as follows. 1) To observe the inhibition effects of diet containing 2% turmeric powder on hepatocarcinogenesis induced by nitrosamine on Wistar rats. 2) To investigate the intermediate biomarker that evaluates the chemopreventive effects of curcumin on hepatocarcinogenesis in order to be used in clinical research. 3) To discover the possible molecular targets by gene chip in order to elucidate the mechanisms of chemoprevention by curcumin. Methods 1) Wistar rats were given a single i.p. injection of 40mg/kg body weight N-diethylnitrosamine(DEN) at age 6 weeks. After 4 weeks, rats received l2Oppm N-nitrosomorpholine in drinking water for 28 weeks. The curcumin A group started eating 2% turmeric powder-containing diet 7 days before DEN injection until execution. The rats in curcumin B group were given 2% turmeric powder-containing diet until hepatocellular carcinoma had been induced at 24th week. Then, the rats in experimental group and curcumin A group were sacrificed at 16th, 20th and 24th week to examine the development of hepatocellular carcinoma. At the 32nd week, all rats were executed to compare the curcumin group with the experimental group on incidence, multiplicity and metastasis of hepatocarcinoma. The changes of hepatic tissues in morphology were observed through hematoxylin-eosin (HE) staining.. 2) Proliferation index displayed by PCNA through immunohistochemistry and apoptotic index revealed by TUNEL are used to evaluate the chemopreveritive effects on curcumin for every stage of hepatocarcinogenesis induced by nitrosamine, and to set up a discriminant function to develop discriminant analysis in order to determine the preventive effects using intermedial biomarker at early-middle stage. 3) The hepatic tissue specimens come from更多还原