WO2022016806A1 - 一种杜氏杆菌在结肠癌预防或治疗中的应用 - Google Patents

一种杜氏杆菌在结肠癌预防或治疗中的应用 Download PDF

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WO2022016806A1
WO2022016806A1 PCT/CN2020/139831 CN2020139831W WO2022016806A1 WO 2022016806 A1 WO2022016806 A1 WO 2022016806A1 CN 2020139831 W CN2020139831 W CN 2020139831W WO 2022016806 A1 WO2022016806 A1 WO 2022016806A1
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application according
dubosiella
colon cancer
newyorkensis
intestinal
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朱升龙
陈永泉
姜旋
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江南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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  • the invention relates to the application of Dunaliella in the prevention or treatment of colon cancer, and belongs to the technical field of biomedicine and microorganisms.
  • Colorectal cancer is a multifactorial disease that involves many links in the process of occurrence and development. Its causative factors include genetic and environmental factors, and living and eating habits are also highly correlated with colorectal cancer.
  • the symptoms of colorectal cancer are not obvious in the early stage, but as the tumor enlarges, patients will experience changes in bowel habits, diarrhea, alternating blood in the stool with local abdominal pain, and constipation. In the advanced stage, symptoms such as anemia and weight loss occur. Studies have shown that "high-fat, high-protein, high-calorie, low-fiber" diet and smoking and drinking habits will greatly increase the risk of disease.
  • gut microbiota In the human gut, especially the colon, there is a complex micro-ecosystem composed of a large number of microorganisms, which is called gut microbiota. The number of bacteria accounts for the vast majority.
  • the normal human gut contains different types and quantities of bacteria, including beneficial bacteria, harmful bacteria and neutral bacteria.
  • the intestinal flora is involved in many basic physiological activities of the human body, such as promoting the development and maturation of the intestinal tract, promoting material metabolism and absorption, synthesizing vitamins, stimulating immune function, resisting the invasion of pathogens, maintaining the intestinal barrier, and degrading cholesterol. Intestinal flora can achieve anti-tumor effects by degrading and removing carcinogenic factors in the body and activating anti-tumor cytokines in the body.
  • gut microbiota dysbiosis is prevalent in patients with colorectal cancer.
  • the structure of intestinal flora changes.
  • Probiotics in the intestinal flora have the effect of stabilizing the abundance of flora and inhibiting inflammatory diseases.
  • Regulating intestinal flora is one of the important means to prevent and treat colorectal cancer. Using these probiotics to maintain intestinal homeostasis can reduce the occurrence of intestinal inflammatory diseases.
  • probiotics in the intestinal tract can form intestinal mucosa in the intestinal tract and prevent the adhesion and invasion of pathogenic bacteria.
  • Foods containing Lactobacillus and Bifidobacterium can be used as prebiotics to repair intestinal epithelial mucosa, inhibit tumor cell proliferation, and have certain preventive and therapeutic effects on colorectal tumors. It can be seen that probiotics play a very important role in the human body's anti-cancer and anti-cancer, but currently there are few anti-cancer and anti-cancer drugs prepared from probiotics on the market.
  • colorectal cancer The occurrence of colorectal cancer (CRC) is affected by both genetic factors and environmental factors. Most patients with colorectal cancer have mutations in the tumor suppressor gene Apc gene. Therefore, with Apc gene mutation, adenomatous polyps can spontaneously form
  • Apc min/+ mouse is a good animal model to study the mechanism of bowel cancer. Bowel carcinogenesis is influenced by both genetic and environmental factors. Although 5-15% of bowel cancers are attributed to genetic factors, the vast majority of bowel cancers are sporadic and occur along the adenoma-adenocarcinoma pathway. The most important environmental factors affecting the occurrence of colorectal cancer include diet, microbial exposure and host immunity.
  • the present invention adopts the intestinal cancer susceptibility gene Apc Min/+ mice were added with high-fat diet to establish a colon cancer mouse model to illustrate the effect of probiotics in the prevention or treatment of colon cancer.
  • Dubosiella As a new genus isolated and identified from biological samples, Dubosiella has shown the effects of regulating metabolism in the body, improving intestinal immunity and promoting the body's resistance to inflammatory diseases, and can affect a variety of life activities of individuals.
  • the present invention provides the application of Dubosiella newyorkensis or a probiotic preparation containing Dubosiella in preparing a product for preventing, alleviating or improving colon cancer.
  • the Dunaliella is NYU-BL-A4, which is published in the patent with the publication number of US2018125900A1.
  • the probiotic preparation is used to improve the intestinal immunity of the body and inhibit the proliferation activity of intestinal cancer cells.
  • the product is a probiotic formulation that can be used as a dietary supplement and therapeutic agent.
  • the probiotic preparation in addition to Dunaliella, contains other excipients, including but not limited to excipients or food additives; the content of the Dunaliella in the probiotic preparation It is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 10 cfu/mL or 1.0 ⁇ 10 7 to 1.0 ⁇ 10 10 cfu/g.
  • the product includes a medicament or a pharmaceutical composition for use in at least one of (a) to (d):
  • the content of the Dunaliella in the medicine or the pharmaceutical composition is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 10 cfu/mL or 1.0 ⁇ 10 7 to 1.0 ⁇ 10 10 cfu/g.
  • the inflammatory factors are Tnf- ⁇ , Cox2, IL-6, and interleukin-1 ⁇ .
  • the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field.
  • the product includes but is not limited to food, health product drink, enteral nutrition preparation, dietary supplement, veterinary medicine or feed additive.
  • the food, health product drink, enteral nutrition preparation, dietary supplement, veterinary drug or feed additive further contains the technical field that those skilled in the art appropriately select ingredients according to the dosage form or purpose of use A common ingredient that can be used with other ingredients.
  • the conventional adjuvants include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.
  • Dubosiella newyorkensis NYU-BL-A4 in the present invention has a close relationship with colon cancer, it can reduce the expression of the verification factor related to colon cancer at the molecular level, and then can inhibit the proliferation of colon cancer cells, Decrease organ weight and number of colon adenomas in colon cancer patients.
  • the performance of this strain of inhibiting or reducing the expression level or proliferation ability of colon cancer-related factors, coupled with its probiotic ability, can be used to prepare drugs that alleviate the occurrence of intestinal inflammatory factors and inhibit the proliferation of related cells. It is beneficial to regulate intestinal immunity and maintain intestinal health, and has broad market prospects.
  • Fig. 1 is a graph showing the effect of Dubosiella newyorkensis NYU-BL-A4 on the organ weight of colon cancer mice.
  • Figure 2 is a graph showing the effect of Dubosiella newyorkensis NYU-BL-A4 on the number of colonic adenomas.
  • Figure 3 is a graph showing the effect of Dubosiella newyorkensis NYU-BL-A4 on colon cancer cell proliferation.
  • Fig. 4 is a graph showing the effect of Dubosiella newyorkensis NYU-BL-A4 on the expression of inflammatory factors induced by colon cancer.
  • Mouse C57BL/6J-ApcMin/Nju (Apc min/+ ) was purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • the high-fat feed was purchased from Nantong Trofee Biological Co., Ltd. (TP23300), which was a high-fat purified feed with a fat energy supply ratio of 60%.
  • Reverse transcription cDNA synthesis use Takara's reverse transcription kit (Cat. No.: RR036A).
  • Example 1 Dubosiella newyorkensis NYU-BL-A4 reduces organ weight in mice with colon cancer
  • the lower viable bacterial pellet was mixed with anaerobic PBS in a strict anaerobic environment to prepare a bacterial solution with a concentration of 3.0 ⁇ 10 9 cfu/mL for animal and cell experiments. Bacterial liquid is now prepared before the mouse to ensure the activity of the strain. The strains were stored in a -80°C long-term refrigerator.
  • the animal experiment method in this example was to select 30 4-week-old male Apc min/+ mice with a body weight of about 18-20 g. All mice were raised in the Experimental Animal Center of Jiangnan University according to the standard operating procedures for raising SPF mice, and they were allowed to eat irradiated sterilized feed and water ad libitum. During the feeding period, the food and water intake, activity level, fur gloss and fecal properties of each mouse were monitored daily, and the mice were weighed and recorded weekly.
  • mice After 7 days of acclimatization, 30 mice were randomly divided into 3 groups, 10 mice in each group, divided into:
  • the Dubosiella intervention group (Dubosiella) consuming a high-fat diet, the mice were fed a high-fat diet and water ad libitum, and the mice received 0.2 mL of live Dubosiella solution 3.0 ⁇ 10 by gavage three times a week. 9 cfu/mL;
  • HFD High-fat diet group
  • mice After 12 weeks of feeding, fresh feces of each mouse were collected on the 12th week of the experiment, the mice were sacrificed by cervical dislocation after collecting feces, and the liver, spleen and spleen were isolated and weighed.
  • the distal small intestine and colon were partially embedded in paraffin and partially frozen in liquid nitrogen.
  • the colon is a single segment, and the small intestine is divided into three segments.
  • the intestinal canal is cut and flattened along the long axis. The number and size of intestinal adenomas in each segment are observed and recorded under a dissecting microscope.
  • the distal small intestine and colon were rolled up and fixed along the horizontal axis, placed in 10% formalin, embedded in paraffin as soon as possible, and the remaining part was frozen in liquid nitrogen.
  • Example 2 Dubosiella newyorkensis NYU-BL-A4 can significantly reduce the number of colonic adenomas
  • mice The grouping, modeling and treatment methods of experimental mice were the same as those in Example 1.
  • Example 3 Dubosiella newyorkensis NYU-BL-A4 significantly inhibits colon cancer cell proliferation
  • the cell experimental treatment method was the same as that in Example 1.
  • two colorectal cancer cells human colon adenocarcinoma cell line Caco-2 and human colon cancer cell line HCT116, were used, and the cells were treated with a medium containing Dunaliella, and the treatment conditions included different time gradients and concentration gradients.
  • Caco-2 culture method use MEM complete medium containing 20% fetal bovine serum FBS, 1% non-essential amino acids and 1% penicillin streptomycin, and culture cells at 37°C and 5% CO 2 ;
  • HCT116 culture method DMEM medium containing 5% fetal bovine serum, 1% penicillin streptomycin was used to culture cells at 37°C and 5% CO 2 .
  • the new medium was replaced every other day.
  • the Caco-2 cells were passaged at a ratio of 1:2 (the cells were seeded into two cell flasks for culture), and the HCT116 cells were passaged at a ratio of 1:3 (the cells were cultured in 1:3). were inoculated into 3 cell flasks for culture).
  • Discard the old culture medium during subculture add sterile PBS (phosphate buffered saline) to wash it twice, then add 0.25% (0.25g/100mL) trypsin to digest the suspended adherent cells, and inoculate an appropriate amount of cells into the culture Flasks continue to incubate or proceed to plating.
  • sterile PBS phosphate buffered saline
  • WST-8 can be reduced to orange-yellow formazan by dehydrogenase in mitochondria.
  • the shade of color is linearly related to the number of cells, and the more cells proliferate, the darker the color.
  • the Cell Counting Kit-8 (CCK-8 kit) of Shanghai Biyuntian Company was used to detect the proliferation of colorectal cancer cells. The operations were as follows:
  • the size of the absorbance OD 450 can reflect the cell proliferation.
  • the human colon adenocarcinoma cell line Caco-2 and the human colon cancer cell line HCT116 were cultured in a system containing Dunaliella, and Dunaliella could The growth of two intestinal cancer cell lines was significantly inhibited in a clear time-dependent manner.
  • RNA from intestinal adenoma tissue Take intestinal adenomas (2-4 polyp tissue blocks) from each group of APC Min+ mice and put them into 1.5 mL EP tubes without RNase, add 1 mL of Trizol to each tube, and add steel beads with high Fully grind with a flux tissue grinder, let stand for 10 min at room temperature, add 200 ⁇ L of chloroform, invert and mix for 30 s, and let stand for 5 min at room temperature. Centrifuge at 4°C for 15 min at 12000 rpm. Pipette the upper aqueous phase containing total RNA into a new RNase-free EP tube.
  • GAPDH-F AGG TCG GTG TGAACG GATTTG (SEQ ID NO.1);
  • GAPDH-R TGTAGA CCA TGTAGT TGA GGT CA (SEQ ID NO.2);
  • Tnf- ⁇ -F CAGGGCGGTGCCTATGTCTC (SEQ ID NO.3);
  • Tnf- ⁇ -R CGATCACCCCGAAGTTCAGTAG (SEQ ID NO. 4);
  • COX-2-F ACGGTCCTGAACGCATTTATG (SEQ ID NO. 5);
  • COX-2-R TTGGCCCCATTTAGCAATCTG (SEQ ID NO. 6).
  • Reaction system (10 ⁇ L system): 5 ⁇ L of 2 ⁇ SYBR GreenIMix, 0.5 ⁇ L of cDNA, 0.5 ⁇ L of upper and lower primers, and 4 ⁇ L of sterile water.
  • the reaction system was added to a 96-well special reaction plate for real-time quantification, and four duplicate wells were set for each sample. Seal the special transparent film cover on the 96-well plate, centrifuge at 1500 rpm for 2 minutes, and place the reaction plate into the BIO RID fluorescence quantitative PCR instrument.
  • Reaction procedure pre-denaturation: 95°C for 10 min; amplification: 95°C for 10s, 60°C for 20s, 72°C for 30s. 45 cycles; melting curve: 95°C for 5s, 65°C for 1min.

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Abstract

提供了一种杜氏杆菌（ Dubosiella newyorkensis）或含有所述杜氏杆菌的益生菌制剂在制备预防、缓解或改善结肠癌的产品方面的应用，所述杜氏杆菌为NYU-BL-A4。

Description

一种杜氏杆菌在结肠癌预防或治疗中的应用 技术领域
本发明涉及一种杜氏杆菌在结肠癌预防或治疗中的应用，属于生物医药及微生物技术领域。
背景技术
结直肠癌是一种多因素诱发的疾病，在发生、发展过程中涉及众多环节。它的致病因素有遗传和环境因素，生活和饮食习惯也与结直肠癌也有很高相关性。结直肠癌早期症状不明显，但随着癌肿的增大患者会出现排便习惯改变、腹泻、便血与局部腹痛、便秘交替等症状，晚期则出现贫血、体重下降等症状。研究表明“高脂肪、高蛋白、高热量、低纤维素”的饮食方式及烟酒习惯会很大程度上增加患病的几率。
在人类的肠道，尤其是结肠中，存在着由大量微生物组成的复杂微生态系统，人们称为肠道菌群。其中细菌的数量占绝大多数。正常人体肠道内含有不同类型、不同数量的细菌，包括有益菌、有害菌和中性菌。肠道菌群参与人体许多基本的生理活动，如促进肠道发育成熟、促进物质代谢与吸收、合成维生素、刺激免疫功能、抵抗病原体的入侵、维护肠道屏障、降解胆固醇等。肠道菌群可通过降解和清除体内的致癌因子，激活体内的抗肿瘤细胞因子等，达到抗肿瘤作用。
基于大规模人群的群组比较分析，发现肠道菌群结构失调现象在结直肠癌患者中普遍存在。在结直肠癌发生发展过程中，肠道菌群结构均发生变化。研究表明，肠道菌群的变化与结直肠癌的发生、发展密切相关。肠道菌群中的益生菌具有稳定菌群丰度、抑制炎症性疾病的效果。调节肠道菌群是防治结直肠癌的重要手段之一，利用这些益生菌制剂来维护肠道稳态，就可以减少肠道炎症性疾病的发生。肠道内益生菌作为肠道菌群的重要组成部分，可在肠道中形成肠道黏膜，预防致病菌的黏附和侵袭。含有乳酸杆菌和双歧杆菌的食物可以作为益生元修复肠上皮黏膜，抑制肿瘤细胞增殖，对结直肠肿瘤产生一定预防和治疗作用。由此可见益生菌对于人体的防癌抗癌具有十分重要的作用，但是目前市场上从益生菌出发制备的防癌抗癌的药物较少。
因此，有必要进行额外的研究和发掘新的功能菌属以进一步支持益生菌的临床使用，作为结直肠肿瘤的预防性治疗。
发明内容
结直肠癌(colorectal cancer，CRC)的发生受机体遗传因素和环境因素的双重影响，大部 分肠癌患者存在抑癌基因Apc基因的突变.因此，具有Apc基因突变，可自发形成腺瘤性息肉的Apc min/+小鼠是研究肠癌机制的良好动物模型。肠癌发生受遗传因素和环境因素的双重影响，尽管5-15％的肠癌发生归咎于遗传因素，绝大多数的肠癌仍是散发的，沿腺瘤-腺癌途径发生。而影响肠癌发生的最主要环境因素包括饮食，微生物暴露以及宿主免疫。研究发现高脂饮食介导肠道菌群结构失调，具有肠癌易感基因的小鼠在摄入高脂饮食后肠道肿瘤负荷明显增加，基于此，本发明采用具有肠癌易感基因Apc min/+小鼠加高脂饮食建立结肠癌小鼠模型用于阐述益生菌在预防或治疗结肠癌中的效果。
杜氏杆菌属(Dubosiella)作为从生物样本中被分离鉴定的新菌属，并表现出调节体内代谢，提高肠道免疫力和促进机体抵抗炎症疾病等功效，能影响个体多种生命活动。
为解决目前存在的问题，本发明提供了杜氏杆菌(Dubosiella newyorkensis)或含有杜氏杆菌的益生菌制剂在制备预防、缓解或改善结肠癌的产品方面的应用。
在本发明的一种实施方式中，所述杜氏杆菌为NYU-BL-A4，公布于公开号为US2018125900A1的专利中。
在本发明的一种实施方式中，所述益生菌制剂用于提高机体肠道免疫力，抑制肠癌细胞增殖活性。
在本发明的一种实施方式中，所述产品为益生菌制剂，可用作食物补剂和治疗剂。
在本发明的一种实施方式中，所述益生菌制剂中除杜氏杆菌外，含有其他辅料，所述辅料包括但不限于赋形剂或食品添加剂；所述杜氏杆菌在益生菌制剂中的含量为1.0×10 7～1.0×10 10cfu/mL或1.0×10 7～1.0×10 10cfu/g。
在本发明的一种实施方式中，
所述产品包括药物或药物组合物，所述药物或药物组合物用于(a)～(d)至少一方面：
(a)缓解脾脏炎症；
(b)减少结肠腺瘤数；
(c)抑制结肠癌细胞增殖；
(d)调节与结肠癌相关炎症因子表达的。
在本发明的一种实施方式中，所述杜氏杆菌在药物或药物组合物中的含量为1.0×10 7～1.0×10 10cfu/mL或1.0×10 7～1.0×10 10cfu/g。
在本发明的一种实施方式中，所述炎症因子为Tnf-α、Cox2、IL-6、白细胞介素-1β。
在本发明的一种实施方式中，所述药物或药物组合物还包括药学上可接受的赋型剂。
在本发明的一种实施方式中，所述药学上可接受的赋型剂是指任何可用于药学领域的稀 释剂、辅助剂和/或载体。
在本发明的一种实施方式中，所述产品包括但不限于食品、保健品饮品、肠内营养制剂、膳食补充剂、兽药或者饲料添加剂。
在本发明的一种实施方式中，所述食品、保健品饮品、肠内营养制剂、膳食补充剂、兽药或者饲料添加剂中还含有本领域技术人员根据剂型或者使用目的适当选择配料的本技术领域的常用成分，可与其他原料一同使用。
在本发明的一种实施方式中，所述常规辅料包括填充剂、矫味剂、粘合剂、崩解剂、润滑剂、抗酸剂、以及营养强化剂中的一种或多种。
有益效果：
本发明中的杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4与结肠癌有密切的关系，其能从分子水平上降低与结肠癌相关的验证因子的表达量，进而能够抑制结肠癌细胞的增殖、降低结肠癌患者的脏器重量和结肠腺瘤的数量。该菌株所具有的这种抑制或降低结肠癌相关因素表达水平或增殖能力的性能，加上其具有的益生能力，从而能够被应用于制备缓解肠道炎症因子的发生、抑制相关细胞的增殖的产品，从而有利于调节肠道免疫力、维持肠道健康，具有广阔的市场前景。
附图说明
图1是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4对结肠癌小鼠脏器重量的影响图。
图2是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4对结肠腺瘤数量的影响图。
图3是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4对结肠癌细胞增殖的影响图。
图4是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4对诱发结肠癌的炎症因子表达量的影响图。
具体实施方式
小鼠C57BL/6J-ApcMin/Nju(Apc min/+)，购自上海斯莱克实验动物有限公司。
高脂饲料购于南通特洛飞生物有限公司(TP23300)，为脂肪供能比60％的高脂纯化饲料。
反转录cDNA合成：使用Takara公司的反转录试剂盒(货号：RR036A)。
实施例1：杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4减轻结肠癌小鼠脏器重量
杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4培养、灌胃菌液制备：菌株涂布于补充有5mL/100mL脱纤维绵阳血的布鲁氏琼脂固体培养基，置于厌氧环境下37℃培养72小时； 或者在MTGE肉汤液体培养基中，37℃培养48小时。当进入对数期(OD 600＝0.5)后停止培养。培养基悬液以3000rpm离心5min后，下层活菌沉淀在严格厌氧环境下与无氧PBS混合制备浓度为3.0×10 9cfu/mL的菌液用于动物和细胞实验，每次灌胃小鼠前现配菌液以保证菌株活性。菌株置于-80℃长期冰箱保存。
本例动物实验方法为选取4周龄雄性Apc min/+小鼠30只，体重约18-20g。所有小鼠培养步骤按照SPF级小鼠饲养标准操作规程饲养于江南大学实验动物中心，可自由采食经辐照灭菌的饲料和水。饲养期间，每日关注各小鼠进食饮水量、活跃程度、皮毛光泽及粪便性状，每周称重小鼠并记录。
适应环境7天后，将30只小鼠随机分为3组，每组10只，分为：
(1)摄入高脂饮食的杜氏杆菌干预组(Dubosiella)，小鼠自由采食高脂饮食和水，小鼠每周接受3次灌胃每次0.2mL的杜氏杆菌活菌液3.0×10 9cfu/mL；
(2)单纯摄入高脂饮食组(HFD)：小鼠自由采食高脂饮食和水，每周灌胃0.2mL PBS3次；
(3)摄入普通饮食的完全对照组(ND)：小鼠自由采食普通饮食和水，每次3次灌胃0.2mL PBS缓冲液(pH7.2～7.4)。
饲养12周后，实验第12周时分别收集每只小鼠的新鲜粪便，收集粪便后颈椎脱臼法处死小鼠，分离肝脾和脾脏并称重。将远段小肠和结肠部分行石蜡包埋，部分冻存于液氮中。结肠为单独一段，小肠组织均分为三段，将肠管沿长轴剪开铺平，解剖显微镜下观测并记录各段肠管腺瘤数量及大小。将远段小肠和结肠部分沿横轴卷起固定，置于10％福尔马林，尽快行石蜡包埋，剩余部分冻存于液氮中。
结果如图1a和图1b所示，与高脂对照组相比，杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4干预组能显著减轻高脂诱导脂肪堆积肝脏重量的上升，并能缓解炎症导致的脾脏肿大。
实施例2：杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4能显著减少结肠腺瘤数量
实验小鼠分组、造模及处理方法同实施例1。
结果如图2所示，不同组小鼠在接受不同饮食干预12周后，解剖显微镜下对各组小鼠结肠腺瘤数量进行计数。单纯高脂饮食组结肠腺瘤数目与完全对照组相比显著增加。杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4干预组较单纯高脂饮食组结肠腺瘤数目明显减少，说明菌株干预能有效减少结肠腺瘤数量，维护肠道稳态。
实施例3：杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4显著抑制结肠癌细胞增殖
细胞实验处理方法同实施例1。本实施例采用了人结肠腺癌细胞系Caco-2和人结肠癌细 胞系HCT116两种肠癌细胞，用含有杜氏杆菌的培养基处理细胞，处理条件包括不同时间梯度及浓度梯度。
实验细胞培养：
(1)Caco-2培养方法：采用含有20％胎牛血清FBS、1％非必需氨基酸及1％青链霉素的MEM完全培养液，在37℃和5％CO 2条件下培养细胞；
(2)HCT116培养方法：采用含有5％胎牛血清、1％青链霉素的DMEM培养基，在37℃和5％CO 2条件下培养细胞。
隔日更换新的培养液，当细胞汇合程度达70-80％时Caco-2细胞以1:2进行传代(将细胞分别接种至2个细胞瓶内培养)，HCT116以1:3传代(将细胞分别接种至3个细胞瓶内培养)。传代时弃去旧培养液，加入无菌PBS(磷酸缓冲盐溶液)小心清洗2次，弃去后再加入0.25％(0.25g/100mL)胰蛋白酶消化悬浮贴壁细胞，取适量细胞接种到培养瓶继续培养或进行铺板操作。
肠癌细胞铺板及处理：
(1)采用Caco-2及HCT116两种人肠癌细胞系，将细胞汇合程度达70-80％的用胰酶消化并制成细胞悬液；
(2)充分混匀后，取5μL细胞悬液至细胞计数板，于显微镜下计数16个中格所含细胞数目；已知16个中格含有0.1μL细胞悬液，可得悬液细胞总数；
(3)用相应培养基稀释悬液至2000个细胞/100μL，充分混匀后将细胞以每孔100μL种于96孔板(96孔板四周共36个孔不加细胞悬液，每孔加入200μL PBS作为蒸发孔)；
(4)待细胞贴壁完全后，弃去旧培养基，用含有杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4的PBS(磷酸缓冲盐溶液)溶液及空白对照溶液(PBS)处理；同一种处理方式再分别采用不同的处理时间(24h、48h、72h)。
肠癌细胞增殖情况的测定：借助电子耦合剂，WST-8可被线粒体内的脱氢酶还原成橙黄色的formazan。对于同一种细胞，颜色的深浅与细胞数目线性相关，细胞增殖越多则颜色越深。采用上海碧云天公司的Cell Counting Kit-8(CCK-8试剂盒)检测肠癌细胞增殖情况，操作如下：
(1)处理完毕后，弃去旧培养基，每孔加入100μL不含FBS等添加物的基础培养基，再加入10μL CCK8溶液，轻晃平板，促进混匀；
(2)放回细胞培养箱孵育，使用酶标仪在450nm测定吸光度。
结果如图3a和3b所示，吸光度OD 450的大小能反应细胞增殖情况，将人结肠腺癌细胞 系Caco-2和人结肠癌细胞系HCT116在含有杜氏杆菌的体系中进行培养，杜氏杆菌能显著抑制两种肠癌细胞系的生长，并呈明显时间依赖性。
实施例4：杜氏杆菌NYU-BL-A4对诱发结肠癌的炎症因子的调控
肠腺瘤组织总RNA的提取：取各组APC Min+小鼠肠腺瘤(2-4个息肉组织块)放入无RNA酶的1.5mL EP管中，每管加入1mL Trizol，加入钢珠用高通量组织研磨器进行充分研磨，室温静置10min后加入200μL氯仿，颠倒混匀30s，室温静置5min。4℃离心，12000rpm离心15min。吸取上层含总RNA的水相至一新无RNA酶EP管中。在新管中加入1倍体积4℃预冷的异丙醇，室温静置15min。4℃离心，12000rpm离心15min，沉淀RNA，弃掉上清液。用1mL预冷的75％乙醇(DEPC水配置)洗涤沉淀，12000rpm、4℃离心10min，弃掉上清，重复洗涤步骤。室温自然干燥，挥发乙醇，RNA沉淀用DEPC水溶解；利用反转录试剂盒合成cDNA，用于后续实时荧光定量PCR。
SYBR Green法实时荧光定量PCR：
荧光定量PCR引物(上海生工生物工程有限公司合成)：
GAPDH-F：AGG TCG GTG TGAACG GATTTG(SEQ ID NO.1)；
GAPDH-R：TGTAGA CCA TGTAGT TGA GGT CA(SEQ ID NO.2)；
Tnf-α-F：CAGGCGGTGCCTATGTCTC(SEQ ID NO.3)；
Tnf-α-R：CGATCACCCCGAAGTTCAGTAG(SEQ ID NO.4)；
COX-2-F：ACGGTCCTGAACGCATTTATG(SEQ ID NO.5)；
COX-2-R：TTGGCCCCATTTAGCAATCTG(SEQ ID NO.6)。
反应体系(10μL体系)：2×SYBR GreenIMix 5μL，cDNA 0.5μL，上、下引物各0.5μL，无菌水4μL。将反应体系加入96孔实时定量专用反应板，每个样品设置4个复孔。将专用透明薄膜盖在96孔板上封紧，1500rpm离心2分钟后将反应板放入BIO RID荧光定量PCR仪中。
反应程序：预变性：95℃10min；扩增：95℃10s，60℃20s，72℃30s。45个循环；熔解曲线：95℃5s，65℃1min。
计算mRNA水平相对表达量：反应测得各样品荧光强度达到阈值时的Ct值，先用各样品Ct值减去对应处理组的内参基因(GAPDH)的Ct值，得到各样品的△Ct值。对于同一个引物，用各处理组的△Ct值减去对照组的平均△Ct值，得到各样品的△△Ct值。mRNA在各样品间的相对表达量＝2 -△△Ct。
炎症因子的变化与Apcmin+小鼠肠道肿瘤的发生发展密切相关。Apc min+小鼠肠道肿瘤中 炎症反应随着肿瘤的发展和生长而加剧，炎症反应的加剧表明其肿瘤进展更快，恶性程度更高。结果如图4a和4b所示，杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4能显著抑制Apc min+小鼠肠道肿瘤中Tnf-α、Cox2等炎症因子基因表达水平的上调，说明杜氏杆菌有减轻结肠癌发病中炎症的作用。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (12)

  1. 一株杜氏杆菌(Dubosiella newyorkensis)或含有所述杜氏杆菌的益生菌制剂在制备预防、缓解或改善结肠癌的产品方面的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述杜氏杆菌为NYU-BL-A4。
  3. 根据权利要求2所述的应用，其特征在于，所述益生菌制剂用于提高机体肠道免疫力，抑制肠癌细胞增殖活性。
  4. 根据权利要求3所述的应用，其特征在于，所述益生菌制剂中除杜氏杆菌外，还含有辅料，所述辅料包括但不限于赋形剂或食品添加剂。
  5. 根据权利要求4所述的应用，其特征在于，所述杜氏杆菌在益生菌制剂中的含量不低于1.0×10 7cfu/mL或1.0×10 7cfu/g。
  6. 根据权利要求5所述的应用，其特征在于，所述产品包括药物或药物组合物，所述药物或药物组合物用于(a)～(d)至少一方面：
    (a)缓解脾脏炎症；
    (b)减少结肠腺瘤数；
    (c)抑制结肠癌细胞增殖；
    (d)调节与结肠癌相关炎症因子表达的。
  7. 根据权利要求6所述的应用，其特征在于，所述杜氏杆菌在药物或药物组合物中的含量不低于1.0×10 7cfu/mL或1.0×10 7cfu/g。
  8. 根据权利要求7所述的应用，其特征在于，所述炎症因子包括Tnf-α、Cox2、IL-6或白细胞介素-1β。
  9. 根据权利要求8所述的应用，其特征在于，所述药物或药物组合物还包括药学上可接受的赋型剂；所述药学上可接受的赋型剂是指任何可用于药学领域的稀释剂、辅助剂和/或载体。
  10. 根据权利要求9所述的应用，其特征在于，所述产品包括但不限于食品、保健品饮品、肠内营养制剂、膳食补充剂、兽药或者饲料添加剂。
  11. 根据权利要求10所述的应用，其特征在于，所述食品、保健品饮品、肠内营养制剂、膳食补充剂、兽药或者饲料添加剂中还含有常规辅料。
  12. 根据权利要求11所述的应用，其特征在于，所述常规辅料包括填充剂、矫味剂、粘合剂、崩解剂、润滑剂、抗酸剂、以及营养强化剂中的一种或多种。
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