【作者】 陈立勇；

【导师】 秦成勇；

【作者基本信息】 山东大学 ， 消化系疾病， 2010， 博士

【摘要】 背景：随着经济的迅速发展和膳食结构的改变,我国大肠癌发病率呈逐年增长的趋势,已成为威胁我国人民身心健康的一个重要疾病。通过膳食中存在的天然抗癌成分来预防癌症是最有效的方法。抗性淀粉(resistant starch, RS)与大肠癌的关系已引起国内外学者的广泛关注。RS主要存在于淀粉含量高的食物中,如谷类、豆类、薯类、根茎蔬菜类、青香蕉等。目前,国内有关抗性淀粉预防大肠癌的动物实验研究十分有限,尤其是抗性淀粉对大肠癌癌前病变的研究未见报道。目的：1通过对食物原料、加工后的熟食和精细加工的食物中RS含量测定结果比较,探讨食物来源和烹调加工对食物中RS含量的影响。2提供中国居民经常摄入的淀粉含量高的食物中的RS含量。3通过膳食调查所得的食物消费数据以及食物中RS含量测定的结果,计算每人每日RS摄入量,报告山东居民和河南居民平均RS摄入量(g/per capita/day)。4提供中国居民膳食RS的主要食物来源,为从饮食上指导和预防大肠癌的发生提供最基础的科学依据。5以氧化偶氮甲烷(azoxymethane, AOM)诱导的Wistar大鼠发生大肠肿瘤为动物模型,通过观察大鼠的生长试验、小肠运动试验以及代谢试验,进一步探讨其可能的防癌机制；同时通过测定大肠癌的早期标志物异性隐窝病灶(aberrant crypt foci, ACF)数目,肿瘤早期生物标记物增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)和核仁组成区嗜银蛋白颗粒(argyrophilic nucleolar organizer regions, AgNORs)以及分析凋亡基因Bcl-2和Bax蛋白,研究不同剂量的抗性淀粉对大肠癌的癌前病变的作用影响,从而为大肠癌早期癌前病变的诊断和治疗提供依据,并对于指导我国居民膳食和预防大肠癌的发生提供依据。方法：1抗性淀粉含量测定：将中国食物营养成分分析检测中心提供的酶-直接法和体外模拟胃肠道酶解法进行改良,用体外模拟胃肠道酶解法对人们经常食用的淀粉含量高的食物或食物原料烹调加工前后直接测定RS含量。2食物摄入资料的获取：为了估计居民平均每日RS摄入量,采用膳食调查,以整群随机抽样的方法随机选取山东省济南市和莱芜市、河南省郑州市和商丘市城市和农村居民,采用24h食物摄入记录法连续记录3d食物摄入,以获得食物摄取资料。为获取其它地区的食物消费信息,收集已有的文献资料上的数据。3中国居民抗性淀粉摄入量估计：根据膳食调查所获得的各种食物消费数据及各种食物RS含量测定结果,计算每人每日RS摄入量,对其它地区的摄入量采用文献报道的食物摄入数据进行粗略估计。4抗性淀粉对AOM诱导的大鼠肠癌肠道功能的影响：选用雄性Wistar大鼠,按体重随机分为5组：阴性对照组、阳性对照组、低剂量组、中剂量组和高剂量组,每组10只。用化学致癌剂[氧化偶氮甲烷(azoxymethane, AOM)]诱导大鼠发生大肠肿瘤,对照组及RS各剂量组分别用普通饲料和含7.6%、15.2%、22.8%RS的饲料喂养。分别于实验第5周末和第7周末通过排便实验和小肠运动实验,研究不同剂量的抗性淀粉对大鼠肠癌肠道功能的影响。测定粪便排出量、粪便pH及肠道转运时间,进行组间比较。同时通过观察大鼠的生长实验,研究RS对其体重的影响。5抗性淀粉对AOM诱导的大鼠癌前病变预防作用的研究：50只Wistar雄性大鼠随机平均分为5组(阴性对照组、阳性对照组和低、中、高剂量组),用AOM诱导大鼠发生大肠肿瘤,造模后分别喂食普通饲料和不同剂量的RS(低剂量7.6%、中剂量15.2%、高剂量22.8%)。于首次注射AOM后13周处死各组大鼠,分离结肠,亚甲蓝染色后镜下计数大肠瘤前损伤—异性隐窝病灶(aberrant cryptfoci,ACF)的数目；并同时用免疫组化法测量其它肿瘤早期生物标记物,如增殖细胞核抗原(PCNA)、核仁组成区嗜银蛋白颗粒(AgNORs)以及凋亡基因Bcl-2和Bax蛋白,进行组间比较,研究条件下抗性淀粉对氧化偶氮甲烷诱发的大鼠大肠癌前病变的作用。结果：1食物中抗性淀粉含量：不同种类食物的RS含量差异较大,以薯类、根茎类蔬菜含量较高,其次为豆类,谷类含量较低。所有生食物中,土豆含量很高；纯化的土豆淀粉、木薯淀粉含量最高,其次为香芋。熟食中,以冷藏的烤土豆含量最高,其次为地瓜粉条、绿豆粉丝、地瓜粉皮等。同种食物经过不同烹调方式的处理后,RS含量普遍降低。油炸和烘烤的食物较一般蒸煮的食物RS含量高。精细加工后的食品中凉皮的RS比例最高,其次是蛋糕、桃酥、米线、薯片、怪味胡豆、麻花。2中国居民抗性淀粉摄入量：中国山东居民RS摄入量平均每人为14.9±9.1g／d,男性为16.2±8.8g/d,女性为14.2±9.3g／d,城市14.1±7.5g/d,农村15.9±10.7g/d,摄入量范围为2.9～49.0g／d；河南居民RS摄入量平均为17.1±6.5g／d,其中男性18.0g±7.8／d,女性15.4±8.8g／d,农村17.9±9.6g／d,城市16.1±5.3g/d,摄入量范围为4.1～63.2g／d。不同个体摄入量差异很大。中国居民RS平均日摄入量为9.7g/d。3中国居民膳食抗性淀粉的主要来源：最主要来源为面制品、米制品和淀粉产品类,薯类和豆类也是RS的主要来源,其次为玉米制品和其它谷类、坚果种子类,蔬菜和香蕉占很小比例。4大鼠体重变化：实验期间(1～15周),各组大鼠生长良好,体重有增长趋势。RS各剂量组大鼠增重较普通饲料组低,且有随着RS浓度的增加而增重较少的趋势。其中高剂量组体重明显低于阳性对照组(P<0.05),差异有统计学意义。5大鼠排便习惯：RS各剂量组大鼠排便量增加、粪便含水量增加,肠道转运时间缩短,中、高剂量组有更显著的效果。6大鼠肠道pH：RS各剂量组粪便pH较对照组剂量依赖性减低,且粪便pH较阳性对照组相比,差异均有统计学意义(P<0.05)。7大鼠结肠ACF计数：阳性对照组及RS各剂量组均发现了AOM诱发的ACF,而阴性对照组则没有ACF形成。与阳性对照组相比,摄食抗性淀粉显著抑制了ACF的发生(P<0.01)。RS各剂量组的异性隐窝总数(aberrant crypt, AC)也较普通饲料组明显减少(P<0.01),呈剂量依赖性降低。8大肠粘膜组织PCNA标记指数和AgNORs颗粒数目：阳性对照组的PCNA标记指数和每核AgNORs颗粒数目显著高于阴性对照组。与阳性对照组比,RS各剂量组的PCNA标记指数和AgNORs颗粒数均有所降低。高剂量组有更显著的效果。9大肠粘膜组织Bcl-2蛋白和Bax蛋白表达：Bcl-2和Bax蛋白在阴性对照组均低表达,与阳性对照组比,RS各剂量组抑制了Bcl-2蛋白的表达,并呈剂量依赖性降低。而Bax的表达显著增加。结论：1体外模拟胃肠道酶解法较好的模拟了食物进入消化道内的代谢情况,是目前国外研究中普遍采用的方法,不仅可用于测定RS含量较多的食物,也适用于RS含量低于1%的样品,测定结果有较好的重现性,费用低,分析效率高。2不同植物来源食物中的RS含量和组成差异较大；加工方式是影响RS含量的另一个因素,粗加工有利于保护谷类食品中的RS不被破坏；同种食物经过不同烹调方式的处理后,含量会明显降低；油炸、烘烤比普通的蒸煮食物含量要高；对比谷类加工食品可以发现粥、谷类饮品的RS较低：此外,熟食物经过一段时间的放置,RS含量一般会增加。3中国居民RS平均摄入量为9.7g/d。此摄入水平与国外报道值比较,高于欧美等国而低于非洲,这可能是由于中国人摄入的谷类食物较多,也可能是中国人大肠癌发病率低于欧洲人的一个原因。4我国居民以谷类为主食,是RS的最主要来源,薯类是膳食RS的最佳来源。建议人们提高RS摄入量可由增加薯类、豆类和谷粒产品的摄入。5抗性淀粉可控制大鼠的体重增长,因此对于肥胖者可能是一种有效的天然减肥食品。6抗性淀粉可缩短肠道转运时间、增加粪便排出量及粪便含水量,从而影响大鼠排便习惯,利于有毒物质及时排出；降低肠腔pH,维持正常的酸性环境。所有这些作用表明RS对肠道内环境健康起到有利的调节作用。7 ACF可以作为一个有价值的中间终点代替癌发生率来研究对大肠癌的化学预防剂。8 PCNA、AgNORs颗粒及凋亡基因等这些生物标记物的应用,为大肠癌早期癌前病变的诊断和治疗提供依据,并对于指导我国居民膳食和预防大肠癌的发生提供最基础的科学依据。可用于临床早期诊断。9癌前病变模型具有周期短、投资少、方法简单可靠的优点,可供其它实验借鉴。更多还原

【Abstract】 Background:Because of the rapid developing in economy and changing food habits, there was an increasing trend yearly at colorectal cancer (CRC) incidence in our country. It is an effectively preventive method to tumor by ingesting natural food that has anticancer effect. At present, the relationship of resistant starch (RS) and colon cancer is being paid close attention at home and abroad. A number of food, such as cereals, legumes, tubers, green banana have been shown to contain various amounts of RS. Few animal experiments is reported about RS preventing colorectal cancer, especially concerning the effect of RS on the precancerous leisions of colorectal cancer.Objective:1. To study the effect of processing to RS content by comparing the results of RS content in raw food materials and cooked food products.2. To report the RS contents of typical starchy foods used frequently by Chinese population.3. Based on the RS values measured in the present study and food consumption data obtained from the dietary survey, RS intake (g/per capita/day) was estimated by individual.4. The key sources of RS in Chinese foods were also identified. This will supply the basic science information for the study of dietary guidance and colon cancer prevention.5. We use the model of AOM-induced colorectal cancer in Wistar rat. This study was designed to determine the potential inhibitory influence of RS at different doses through the observation of the rat growth test, the small intestine movement test and metabolic test. And it was also designed to investigate the influence of a premalignant lesion by testing the number of ACF, one of the early landmarks of colorectal cancer, and some other biomarkers, such as PCNA, AgNORs, Bcl-2 and Bax. The results will provide evidence for early diagnosis and treatment of colorectal cancer, and be of great importance for guiding Chinese diets and early preventing colorectal cancer.Methods:1. Determining of resistant starch:to collect the typical starchy foods (raw material and cooked food) used frequently by Chinese population and to determine RS content of before and after processing by an in vitro method that mimicked gastrointestinal conditions.2. Dietary survey:in order to estimate the daily RS intake in the Chinese population, food consumption data from a dietary survey was used. Population was Choosed randomly from city and country (Shandong, Henan, China) by the method of cluster sampling. All subjects were asked to complete an estimated dietary record for 3 consecutive days of food items and liquids each individual consumed.3. Estimating the Chinese intake of RS:based on the RS values measured in the present study and food consumption data (g/per capita/day) obtained from the dietary survey, RS intake was estimated by individual. The main sources of RS in foods were also explained. RS intake was also divided according to the food from which it originated (i.e. tuber, legume or cereals). In addition, data reported from references were used for estamiting the intake of different area.4. Effects of resistant starch on intestine function of AOM-induced colorectal cancer in ratsMale Wistar rats were randomly divided into 5 groups:negative control group, positive control group and three different resistant starch (RS) added groups with the ratio of 7.6%,15.2% and 22.8% (n= 10 each group). For inducing colonic tumorigenesis, rats received chemical carcinogen (AOM) injections by intraperitoneal once a week for two consecutive weeks. Animals in different RS added groups received the corresponding doses of RS while rats in the positive control group and the negative control group received ordinary feed during the whole study. This study was designed to determine the protective effect on intestine of RS at different doses through the observation of the small intestine movement test and metabolic test. Fecal pH and fecal output were measured in the 5th weekend and the intestinal transit time measurement was carried out in the7th weekend.Then comparing them among groups. And it was also designed to investigate the influence of weight by testing the rat growth.5. The study of prophylactic effect of resistant starch on precancerous lesions of AOM-induced colorectal cancer in ratsMale Wistar rats were randomly divided into 5 groups:negative control group, positive control group and three different resistant starch (RS) added groups with the ratio of 7.6%,15.2% and 22.8%.For inducing colonic tumorigenesis, rats received chemical carcinogen (AOM) injections by intraperitoneal once a week for two consecutive weeks. Animals in different RS added groups received the corresponding doses of RS while rats in the positive control group and the negative control group received ordinary feed during the whole study. At 13 weeks after the first injection, all of the rats were killed. Colons of rats were removed and the total numbers of ACF as well as the number of crypts per focus were scored using a light microscope at low magnification after staining with 0.5% methylene blue solution. Some other biomarkers (such as PCNA, AgNORs, Bcl-2 and Bax) were also determined by immunohistochemical method and compared among groups to investigate the effect of RS on the precancerous lesion of colon cancer.Results:1. Resistant starch content in food:the contents of RS largely varied with plant source. In general, tubers, tuber vegetables and legumes had more RS than cereals. Among the selected samples of raw material, potato showed high amount of RS. The pure potato starch and cassava starch had the highest RS content. Among the food products, roasted and cooled potato had the highest RS content. The following were sweet potato starch noodle, mung bean starch noodle and Sweet potato starch jelly sheet. The content of RS in cooked foods was lower than raw food material in general. Deep-fried and roasted foods have more RS than general braised foods. The content of RS in cold noodle was the highest in finely processed food.The following were cake, walnut cake, rice noodle, potatochips, Lima bean with special hot sauce and fried dough twist.2. Chinese intake of resistant starch:the daily RS intake of Shandong population was estimated to be 14.9±9.1g, which were 16.2±8.8g/d in male and 14.2±9.3g/d in female. And the value was 14.1±7.5g/d in population who live in the city and 15.9±10.7g/d in population who live in the country. The range of intake was 2.9g～49.0g/d. The daily RS intake of Henan population was estimated to be 17.1±6.5g, which were 18.0±7.8g/d in male and 15.4±8.8g/d in female. And the value was 16.1±5.3g/d in population who live in the city and 17.9±9.6g/d in population who live in the country. The range of intake was 4.1g～63.2g/d. Obviously, RS intake varied considerably among individuals. The average intake of Chinese population was 9.7g/d.3. The key RS sources in the Chinese diet:the key RS sources in the Chinese diet are wheat products, rice products and starch products. Other important sources of RS are tubers and legume products, followed by corn products and other cereals, nuts and seeds. Vegetables and banana contribute fewer amounts of RS to the Chinese diet.4. Body weight alteration of rat:During the whole study, each group of rats grew well and weight was increasing. The body weights of rats in RS groups were lower than control and there was a dose-dependent in lower weight and high RS level. Compared with the positive control group,the body weights of rats in the high dose group was significantly decreased (P＜0.05).5. The fecal excreting habit:Addition of RS to the diet significantly increased fecal output and water content. In the three groups RS fed rats, further increase in fecal output and water content were seen with the concentration of RS. The intestinal transit time was shortened by addition of RS and high-RS group had a more markedly effect.6. Intestinal tract pH:Fecal pH in the three RS added groups were significantly decreased than the positive control group (P＜0.05).7. The number of colonic ACF:ACF were discovered in both of the positive control group and in RS groups. But the negative control was no ACF formation. Compared with the positive control group, the number of ACF and AC in the three RS added groups were both significantly reduced (P<0.01).8. Colonic PCNA-LI and the number of AgNORs:The number of AgNORs and the PCNA-LI in the positive control group were more than the negative control group. Compared with the positive control group, the number of AgNORs and the PCNA-LI in the three RS added groups were both reduced and high-RS group had a more markedly effect.9. Expression of colonic Bcl-2 and Bax protein:There was a low expression of Bcl-2 and Bax in the negative control group. In addition, RS inhibited the expression of Bcl-2 and enhanced the expression of Bax.Conclusion:1. This method mimics physiologic conditions preferably adopted by the overseas laboratory widely these days. Not only is it usable for low-content (＜1%) starchy foods but also for high-content starchy foods, with its good recurrence, low price and high analytic efficiency.2. The contents and pattern of resistant starch in different kinds of food were largely varied. Different processing of food material was another important factor influencing the content of RS. The content of RS in cooked foods decreased obviously. Deep-fried and roasted foods have more RS than general braised foods. It could come to the conclusion that rough processing is propitious to retain high amount of RS.Compared with cereal processed food, the content of RS of porridges and cereal drinks. In addition, the content of RS increased generally in the cooked food after preservation.3. Findings from this study suggest that the estimated intake of RS by Chinese population is approximately 9.7g/d on average. Comparing with the values reported abroad, the level was higher than American and European but lower than African. It might be a factor that CRC incidence was lower in China than European. The estimate of RS intake provides a valuable reference for researchers and food and nutrition professionals. 4. Cereals are the Chinese staple food and the most fundamental source of RS. Tubers are the optimal source of RS. It would be prudent to increase consumption of tubers, legumes and cereal grain products.5. Dietary RS retarded the body weight gain to rat. Therefore it may be an effective natural approach to the treatment of obesity.6. Dietary RS shorten the GTT and increase the fecal output and moisture. These effects on excreting habit might be beneficial for eliminating toxic substrates timely. It also decreased intestinal tract pH. All these findings showed that RS favourably influence the colonic luminal environment.7.ACF may be a valuable middle point instead of cancer incidence for colorectal cancer research of chemical prevention.8.The application of some biomarkers (such as PCNA, AgNORs, Bcl-2 and Bax) will provide evidence for early diagnosis and treatment of colorectal cancer, and be of great importance for guiding Chinese diets and early preventing colorectal cancer. Therefore it can be used for the clinical early diagnosis.9.Pre-cancerous model has many advantages such as shorter period, less investment, simple and reliable method and so on. It can be used for reference by other experiments.更多还原