WO2019056404A1 - 脆弱拟杆菌在制备用于治疗和预防肿瘤的药物中的应用 - Google Patents

脆弱拟杆菌在制备用于治疗和预防肿瘤的药物中的应用 Download PDF

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WO2019056404A1
WO2019056404A1 PCT/CN2017/103940 CN2017103940W WO2019056404A1 WO 2019056404 A1 WO2019056404 A1 WO 2019056404A1 CN 2017103940 W CN2017103940 W CN 2017103940W WO 2019056404 A1 WO2019056404 A1 WO 2019056404A1
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bacteroides fragilis
tumor
treating
pharmaceutical composition
bacteroides
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French (fr)
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曾谷城
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中山大学
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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  • the present invention relates to the field of biomedicine, and in particular to the use of Bacteroides fragilis or a composition comprising Bacteroides fragilis for the treatment and/or prevention of tumors.
  • Cancer has become the "first killer" of human beings.
  • the World Health Organization (WHO) published the Global Cancer Report 2014, which is hosted by the International Agency for Research on Cancer (IARC), the official cancer agency of WHO. More than 250 scientists from more than 40 countries participated in the compilation, more than 180 worldwide. The overall situation and trends of the country's 28 cancers were fully described and analyzed. The report predicts that global cancer cases will show rapid growth, from 14 million in 2012 to 19 million in 2025 and 24 million by 2035.
  • the main difficulty in cancer treatment especially in the treatment of advanced tumors in the advanced stage, is the inability to control cancer metastasis.
  • Chemotherapy is currently recognized as the main treatment, and its main purpose is to kill cancer cells in the body.
  • chemotherapeutic drugs kill cancer cells, they also damage normal human cells. Moreover, chemotherapeutic drugs, especially in high-dose applications, can induce genomic disorders, increase heterogeneity and outlier formation, promote drug resistance, lead to more invasive cell formation, and lead to further spread of cancer. Clinical results show that only about 7% of cancers are truly effective against chemotherapy. In recent years, cancer immunotherapy has received increasing attention. On February 4, 2016, the American Society of Clinical Oncology (ASCO) released the 2016 ASCO Annual Report on Cancer Research, and immunotherapy was named the largest advance in cancer research in 2015. As Dr. Julie M. Vose, President of ASCO, said: “Immunotherapy is the most revolutionary breakthrough in the field of cancer.
  • ASCO American Society of Clinical Oncology
  • Probiotics are a kind of active microorganisms beneficial to the host. They are the general term for active beneficial microorganisms that are colonized in the human intestines and reproductive system and can produce exact health effects to improve the balance of the micro-ecology of the host and play a beneficial role.
  • probiotics can settle in the intestinal mucosa after ingesting human and animal body, in the intestine
  • the built-in group and the prevention of adhesion of harmful microorganisms can help maintain the health of humans and animals by maintaining the natural microflora in the intestines and promoting the formation of healthy, growable microbial preparations by biological individuals.
  • More and more researchers are focusing on probiotics and are gradually realizing their powerful therapeutic effects. Many recent efforts on bacterial therapies for cancer have focused on non-pathogenic strains.
  • Bifidobacterium is a non-pathogenic obligate anaerobic bacterium and has been successfully used to target tumors and as a therapeutic carrier, but has not been shown to have an oncolytic effect.
  • Escherichia and Klebsiella pneumoniae have been used in the treatment of intestinal and pulmonary cancer, respectively, as a "site-specific immunomodulator" and have played a more significant role in inhibiting tumor growth.
  • Bacteroides fragilis is an obligate anaerobic bacterium that is negative for Gram stain, rod-shaped, blunt and densely stained at both ends, and has a capsule, no spores, and no motility. And non- enterotoxin-type. Bacteroides fragilis is part of the normal flora of human and animal gut, mainly in the colon. In addition, the respiratory tract and genitourinary tract can also grow. Numerous studies have shown that Bacteroides fragilis has good curative effect on prevention and treatment of acute and chronic enteritis, dysbacteriosis, upper respiratory tract infection and neurosis.
  • the technical problem to be solved by the present invention is to provide a method capable of treating and/or preventing a tumor in view of the above-mentioned drawbacks existing in the current tumor treatment.
  • the present invention provides the use of Bacteroides fragilis for the treatment and/or prevention of tumors.
  • the tumor of the present invention may be various solid tumors such as, but not limited to, murine melanoma tumor B16-F10, human liver cancer BEL7405 and SMMC7721, human prostate cancer PC-3, human breast cancer MCF-7, human fibrosarcoma cell HT1080, mouse. Bladder sarcoma cells MB-49 and solid tumors such as mouse glioma C6.
  • the method of treating and/or preventing a tumor is combined with other methods of treating and/or preventing a tumor.
  • the additional methods of treating and/or preventing a tumor include, but are not limited to, chemotherapy, radiation therapy, gene therapy, surgery, or a combination thereof.
  • therapeutic and prophylactic compositions comprising Bacteroides fragilis.
  • the therapeutic and prophylactic compositions comprise Bacteroides fragilis.
  • the therapeutic and prophylactic compositions do not contain other microbial strains.
  • the Bacteroides fragilis can delay tumor growth.
  • the tumor is a solid tumor.
  • the tumor includes, but is not limited to, melanoma.
  • the Bacteroides fragilis is any one of the following: Viable Bacteroides viable cells; Bacteroides fragilis that have been genetically engineered, engineered or modified, attenuated, chemically treated, physically treated or inactivated; Bacteroides fragilis lysate; and/or Bacteroides fragilis culture supernatant.
  • the present invention also provides the use of Bacteroides fragilis in the preparation of a medicament for treating and/or preventing a tumor, wherein the tumor is melanoma, or about breast, liver, lung, skin Tumors of cancer, kidney, prostate, nervous system or bladder.
  • the present invention provides a pharmaceutical composition for treating and/or preventing a tumor, wherein the pharmaceutical composition comprises a pharmaceutically effective amount of Bacteroides fragilis and pharmaceutically acceptable thereof Carrier. Wherein the Bacteroides fragilis is an active ingredient.
  • the Bacteroides fragilis is any one of the following: Bacteroides fragilis live cells; genetically recombined, engineered or modified, attenuated, chemically treated, physically treated or inactivated Bacteroides fragilis; Bacteroides fragilis lysate; and/or Bacteroides fragilis culture supernatant.
  • the pharmaceutical composition may be any one or more of pharmaceutically acceptable dosage forms including, but not limited to, tablets, capsules, oral solutions or lyophilized powders.
  • the pharmaceutically acceptable carrier is skim milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, gum arabic, calcium phosphate, alginate, Gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil a mixture of one or more.
  • the present invention also provides a food for treating and/or preventing a tumor, wherein the food comprises Bacteroides fragilis.
  • Bacteroides fragilis is any one of the following: Bacteroides fragilis live cells; Bacteroides fragilis after genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation Bacteroides fragilis lysate; and/or Bacteroides fragilis culture supernatant.
  • the present invention also provides a food additive for treating and/or preventing a tumor, wherein the food additive comprises Bacteroides fragilis.
  • the Bacteroides fragilis is any one of the following: a viable cell of Bacteroides fragilis; a fragile gene after genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation Bacillus; Bacteroides fragilis lysate; and/or Bacteroides fragilis culture supernatant.
  • the present invention also provides a health care for treating and/or preventing tumors.
  • the health care product comprises Bacteroides fragilis.
  • the Bacteroides fragilis is any one of the following: a viable cell of Bacteroides fragilis; a fragile gene after genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation Bacillus; Bacteroides fragilis lysate; and/or Bacteroides fragilis culture supernatant.
  • the invention establishes a mouse melanoma model by a transplant tumor research method, and detects and identifies the action of Bacteroides fragilis in a mouse melanoma model, and the invention proves that the Bacteroides fragilis can significantly inhibit melanoma in vitro. Survival, and can effectively inhibit the growth of transplanted tumors in mice, suggesting that Bacteroides fragilis has important development and application value in the clinical treatment of tumors.
  • Fig. 1 is a schematic diagram showing the experimental procedure for detecting the therapeutic effect of Bacteroides fragilis and inactivated Bacteroides fragilis on mouse melanoma.
  • Figure 2 shows the inhibitory effect of Bacteroides fragilis and B. fragilis inactivated on human transplanted tumors in mice.
  • Figure 3 shows the inhibition of Bacteroides fragilis and inactivated Bacteroides fragilis on the growth of human transplanted tumors in mice.
  • Figure 4 is a comparison of the size of mouse melanoma after treatment with Bacteroides fragilis.
  • Bacteroides fragilis for use in the treatment and/or prevention of tumors of the present invention or the pharmaceutical compositions, foods, nutraceuticals and food additives containing the Bacteroides fragilis of the present invention are applied to a subject, It can be applied to the indications described above and exhibits the functions described above, all of which have been tested within the scope of the invention, hereinafter, merely for illustration, only one of which is described in the examples In part, however, it should not be construed as limiting the invention.
  • the Bacteroides fragilis referred to in the present invention includes, but is not limited to, any one of the Bacteroides fragilis viable cells, the inactivated Bacteroides fragilis, the Bacteroides fragilis lysate, and/or the Bacteroides fragilis culture supernatant.
  • the tumor is a solid tumor.
  • the tumor includes, but is not limited to, melanoma.
  • the pharmaceutical composition for antitumor provided by the present invention comprises a pharmaceutically effective amount of Bacteroides fragilis.
  • the "pharmaceutically effective dose” is referred to as 10 6 - 10 10 CFU, preferably 10 9 CFU.
  • the Bacteroides fragilis includes any one of the following: Bacteroides fragilis live cells; genetically recombined, engineered or modified, attenuated, chemically treated, physically treated or inactivated Bacteroides fragilis; Bacteroides fragilis lysate; / or Bacteroides fragilis culture supernatant.
  • the pharmaceutical compositions include, but are not limited to, tablets, capsules, oral solutions or lyophilized powders.
  • the pharmaceutically acceptable carrier includes, but is not limited to, skim milk, lactose, glucose, sucrose, sorbitol, mannose, trehalose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, One or more of fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.
  • the Bacteroides fragilis of the present invention can also be made into foods, health care products or food additives and the like.
  • the food, health care product or food additive comprises Bacteroides fragilis, genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation of Bacteroides fragilis, Bacteroides fragilis lysate and/or fragile Any one of Bacteroides culture supernatants.
  • These foods, health supplements or food additives can be used to treat and/or prevent tumors.
  • Step 1 Take a freeze-dried strain of Bacteroides fragilis, add 200 ⁇ L of TSB medium, reconstitute, absorb 20 bases, draw blood plates, and anaerobic tank gas control system after pumping in biochemical incubator 37 dishes, anaerobic culture for 48 hours;
  • Step 2 Pick up the monoclonal colonies into 10 mL TSB medium, 37, and anaerobic culture for 12 h;
  • Step 3 Take 1 bottle of 500mL TSB medium, access 1% (v/v) strain, 37, anaerobic culture for 48h;
  • Step 4 The bacteria solution was centrifuged at 6000 rpm for 10 min. Wash twice with physiological saline, and finally reconstitute the bacterial sludge with physiological saline for use and count the viable bacteria.
  • Figure 1 is a schematic diagram showing the experimental procedure for the detection and prevention of Bacteroides fragilis and inactivation of Bacteroides fragilis on tumors (melanoma) in mice.
  • the culture method of Bacteroides fragilis is the same as in Example 1.
  • Step 1 Take a freeze-preserved strain, add 200 freeze-preserved bacteria culture medium, reconstitute, draw 20 base, blood plate, and anaerobic tank gas control system after pumping in the biochemical incubator at 37 degrees Anaerobic culture for 48 hours;
  • Step 2 Pick up the monoclonal colonies into 10 mL TSB medium, 37-base, anaerobic culture for 12 h;
  • Step 3 Take 1 bottle of 500mL TSB medium, access 1% (v/v) strain, 37, anaerobic culture for 48h;
  • Step 4 The bacteria solution was centrifuged, centrifuged with a centrifuge, and centrifuged at 6000 rpm for 10 minutes.
  • the inactivated bacterial solution was obtained by heating in a 70 degree water bath for 30 minutes.
  • Bacteroides fragilis culture broth was treated by sonication using a sonicator, and it was broken for 2 seconds, stopped for 5 seconds, and continued for 20 minutes to obtain the Bacteroides fragilis lysate.
  • the culture solution of Bacteroides fragilis was centrifuged by a centrifuge, and the supernatant was cultured at 6000 rpm for 10 min to obtain a culture supernatant of Bacteroides fragilis.
  • mice C57BL/6 mice were 36 in 3 to 4 weeks, and they were in good mental state and purchased from the Experimental Animal Center of Sun Yat-sen University. The mice were randomly divided into 3 groups, 12 in each group. The 3 groups were control group, live bacteria gavage group and inactivated bacteria gavage group.
  • mice Three groups of mice were intragastrically administered with 109 CFU of Bacteroides fragilis, inactivated Bacteroides fragilis and controls, and were intragastrically administered for 1 week. Body weight was measured every day. Subsequently, mouse tumor (melanoma) cells B16 were grown to log phase, cells were digested with TE, neutralized in medium, cells were collected by centrifugation, and washed twice with DPBS to remove residual serum, and the cells were resuspended in DPBS. The cells were counted and 10 7 cells were injected subcutaneously into each mouse. The mice were continuously treated with intragastric administration. One week later, the tumor-bearing mice were sacrificed, and the subcutaneous tumor masses were dissected to measure the tumor size.
  • melanoma mouse tumor (melanoma) cells B16 were grown to log phase, cells were digested with TE, neutralized in medium, cells were collected by centrifugation, and washed twice with DPBS to remove residual serum, and the

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Abstract

提供一种脆弱拟杆菌以及包含所述脆弱拟杆菌的治疗性或预防性组合物在治疗和/或预防肿瘤中的应用。还提供了脆弱拟杆菌以及包含所述脆弱拟杆菌在制备用于治疗和/或预防肿瘤的药物中的用途，可以显著抑制肿瘤的生长。还提供了脆弱拟杆菌在制备用于治疗和/或预防肿瘤的药物组合物、食品、保健品及食品添加剂中的应用。

Description

脆弱拟杆菌在制备用于治疗和预防肿瘤的药物中的应用 技术领域
本发明涉及生物医药领域，具体涉及使用脆弱拟杆菌或包含使用脆弱拟杆菌的组合物治疗和/或预防肿瘤的应用。
背景技术
癌症已成为人类的“第一杀手”。世界卫生组织(WHO)发表的《全球癌症报告2014》由世卫组织下属的官方癌症机构国际癌症研究中心(IARC)负责，来自40多个国家的250多位科学家参与编撰，对全球180多个国家的28种癌症的总体情况和流行趋势进行了全面的描述和分析。报告预测全球癌症病例将呈现迅猛增长态势，由2012年的1400万人，逐年递增至2025年的1900万人，到2035年将达到2400万人。癌症治疗尤其是中晚期的进展性肿瘤治疗的主要困难是无法控制癌症转移。化疗是目前公认的主要治疗手段，其主要目的是杀灭体内的癌细胞。但是化疗药物在杀死癌细胞的同时，也会损伤人体正常细胞。而且，化疗药，尤其大剂量应用时，可诱导基因组混乱，增加异质性和离群者形成，促进药物抵抗，导致更多侵袭性细胞形成，导致癌症的进一步扩散。临床结果显示真正对化疗有效的癌症只有大约7％。近年来，癌症的免疫治疗愈来愈受到重视。2016年2月4日美国临床肿瘤学学会(ASCO)发布了《2016年ASCO癌症研究进展年报》，免疫治疗被评为2015年癌症研究的最大进展。正如ASCO主席Julie M.Vose医生所说：“免疫治疗是癌症领域最具革命性的突破，这种新疗法不仅改善了患者的生活，也为未来的研究指明了方向”。当前肿瘤免疫疗法将成为继手术、放疗和化疗之后的第四种癌症主要治疗方法，研究安全、廉价、高效和低副作用的癌症免疫药物是当前全世界的研究热点。
细菌在癌症治疗中的应用可以追溯至十九世纪晚期，甚至存在更早的细菌在治疗癌症中的效力的报告。益生菌是一类对宿主有益的活性微生物，是定植于人体肠道、生殖系统内，能产生确切健康功效从而改善宿主微生态平衡、发挥有益作用的活性有益微生物的总称，其具有缓解乳糖不耐症、调整肠道微生态、增强自身免疫力等多种功效，益生菌在摄入人和动物体内后，它们能够在肠粘膜上定居，在肠道 内建群并且防止有害微生物在其上粘附，能通过保持肠内自然微生物区系，促进生物个体形成健康的可生长的微生物制剂，帮助维持人和动物的健康。目前越来越多的研究人员聚焦于益生菌，逐渐意识到它们强大的治疗功效。许多最近的关于癌症的细菌疗法的工作已经聚焦于非致病菌株。双歧杆菌属是非致病的专性厌氧菌，且已经被成功地用于靶向肿瘤和用作治疗载体，但是没有表明具有溶瘤作用。近年来，有研究将大肠杆菌Escherichia、肺炎杆菌分别被用于肠和肺病癌肿，作为“部位特异性免疫调节剂”并起到了较明显的抑制肿瘤生长的效果。
脆弱拟杆菌(Bacteroides fragilis)是一种革兰氏染色阴性、杆状、两端钝圆而浓染，有荚膜、无芽胞、无动力的专性厌氧细菌，其分为产肠毒素型和非产肠毒素型。脆弱拟杆菌作为人及动物肠道正常菌群的一部分，主要存于结肠中，此外，呼吸道胃肠道及泌尿生殖道也可定植生长。众多研究表明，脆弱拟杆菌对防治急慢性肠炎、菌群失调、上呼吸道感染和神经官能症等具有较好疗效。
发明内容
本发明所要解决的技术问题是针对目前肿瘤治疗中所存在的上述缺陷，提供一种能够治疗和/或预防肿瘤的方法。
为了实现上述目的，本发明提供了脆弱拟杆菌(Bacteroides fragilis)在治疗和/或预防肿瘤中的应用。
本发明所述肿瘤可以为各种实体瘤，例如但不限于鼠黑色素肿瘤B16-F10、人肝癌BEL7405及SMMC7721、人前列腺癌PC-3、人乳腺癌MCF-7、人纤维肉瘤细胞HT1080、鼠膀胱肉瘤细胞MB-49及鼠神经胶质瘤C6等实体瘤等。
在某些实施方案中，将所述治疗和/或预防肿瘤的方法与其它治疗和/或预防肿瘤的方法进行组合。在某些实施方案中，所述其它治疗和/或预防肿瘤的方法包括但不限于是化学疗法、放射疗法、基因疗法、外科手术或它们的组合。本文还描述了包含脆弱拟杆菌的治疗性和预防性的组合物。在某些实施方案中，所述治疗性和预防性的组合物含有脆弱拟杆菌。在某些实施方案中，所述治疗性和预防性的组合物不含有其它微生物菌株。在一个方面，所述脆弱拟杆菌可以延缓肿瘤的生长。在一个方面，所述肿瘤是实体瘤。在某些实施方案中，所述肿瘤包括但不限于：黑色素瘤。
根据本发明的一个方面，上述的应用中，所述脆弱拟杆菌为以下中的任意一种： 脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
为了更好地实现上述目的，本发明还提供了脆弱拟杆菌在制备用于治疗和/或预防肿瘤的药物中的用途，其中，所述肿瘤是黑色素瘤、或关于乳房、肝、肺、皮肤、癌、肾、前列腺、神经系统或膀胱的肿瘤。
根据本发明的一个方面，本发明还提供了一种用于治疗和/或预防肿瘤的药物组合物，其中，所述药物组合物包括药学有效剂量的脆弱拟杆菌及其在药学上可接受的载体。其中，所述脆弱拟杆菌为活性成分。
优选地，上述的药物组合物中，所述脆弱拟杆菌为以下中的任意一种：脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
优选地，上述的药物组合物中，所述药物组合物可以是药学上可行的任一种或多种剂型，包括但不限于为片剂、胶囊剂、口服液或冻干粉剂。
优选地，上述的药物组合物中，所述药学上可接受的载体为脱脂奶、乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、海藻糖、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁或矿物油中的一种或多种的混合物。
为了更好地实现上述目的，本发明还提供了一种用于治疗和/或预防肿瘤的食品，其中，所述食品包括脆弱拟杆菌。
优选地，上述食物中，所述脆弱拟杆菌为以下中的任意一种：脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
为了更好地实现上述目的，本发明还提供了一种用于治疗和/或预防肿瘤的食品添加剂，其中，所述食品添加剂包括脆弱拟杆菌。
优选地，上述食品添加剂中，所述脆弱拟杆菌为以下中的任意一种：脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
为了更好地实现上述目的，本发明还提供了一种用于治疗和/或预防肿瘤的保健 品，其中，所述保健品包括脆弱拟杆菌。
优选地，上述保健品中，所述脆弱拟杆菌为以下中的任意一种：脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
本发明以移植性肿瘤研究法建立小鼠黑色素瘤模型，通过在小鼠黑色素瘤模型中对脆弱拟杆菌的作用进行检测和鉴定，本发明通过实验证明，脆弱拟杆菌能够显著抑制体外黑色素瘤的存活，并能有效抑制小鼠体内移植肿瘤的生长，提示脆弱拟杆菌在肿瘤临床治疗中具有重要的开发和应用价值。
附图说明
图1为检测脆弱拟杆菌及灭活脆弱拟杆菌对小鼠黑色素瘤的治疗作用实验流程示意图。
图2为对脆弱拟杆菌及灭活脆弱拟杆菌对小鼠体内人源移植肿瘤发生抑制作用的检测
图3为对脆弱拟杆菌及灭活脆弱拟杆菌对小鼠体内人源移植肿瘤生长抑制作用的检测。
图4为脆弱拟杆菌治疗后小鼠黑色素瘤大小对比图。
具体实施方式
下面将结合具体实施例对本发明作进一步说明。需要指出的是，由本发明中的用于治疗和/或预防肿瘤的脆弱拟杆菌或含有本发明的脆弱拟杆菌的药物组合物、食品、保健品和食品添加剂在施用于受试者后，都可以应用于上文所述的适应症并展现出上文所述的功能，在本发明范围内的所有剂型均已测试，下文中，仅仅是为说明，只在实施例中描述了其中一小部分，然而不应将其理解为对本发明的限制。
本发明所指的脆弱拟杆菌包括但不限于脆弱拟杆菌活菌体、灭活脆弱拟杆菌、脆弱拟杆菌裂解物和/或脆弱拟杆菌培养上清液中的任意一种。
所述肿瘤是实体瘤。在某些实施方案中，所述肿瘤包括但不限于：黑色素瘤。
本发明还提供的用于抗肿瘤的药物组合物包括药学有效剂量的脆弱拟杆菌。其中，所称的“药学有效剂量”为106-1010CFU，优选为109CFU。所述脆弱拟杆菌包 括以下中的任意一种：脆弱拟杆菌活菌体；基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。所述药物组合物包括但不限于为片剂、胶囊剂、口服液或冻干粉剂。所述药学上可接受的载体包括但不限于为脱脂奶、乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、海藻糖、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁或矿物油中的一种或多种。
本发明的脆弱拟杆菌还可以被制成食品、保健品或食品添加剂等。所述食品、保健品或食品添加剂均含有脆弱拟杆菌活菌体、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌、脆弱拟杆菌裂解物和/或脆弱拟杆菌培养上清液中的任意一种。这些食品、保健品或食品添加剂均可用于治疗和/或预防肿瘤。
实施例1 脆弱拟杆菌培养
培养方法
步骤1：取一支冻干保存脆弱拟杆菌(Bacteroides fragilis)菌种，加入200μL TSB培养基，复溶，吸取20基，血平皿划线，厌氧罐气体控制系统抽气后在生化培养箱中37皿、厌氧培养48h；
步骤2：挑取单克隆菌落接入10mL TSB培养基，37，、厌氧培养12h；
步骤3：取1瓶500mL TSB培养基，接入1％(v/v)菌种，37，、厌氧培养48h；
步骤4：取菌液离心，6000rpm、10min。用生理盐水洗涤2次，最后用生理盐水复溶菌泥备用并进行活菌计数。
实施例2 脆弱拟杆菌对小鼠肿瘤(黑色素瘤)的治疗作用实验
图1为检测脆弱拟杆菌及灭活脆弱拟杆菌对小鼠肿瘤(黑色素瘤)治疗及预防作用实验流程示意图。
1、培养方法
脆弱拟杆菌培养方法同实施例1。
2、样品准备
1)脆弱拟杆菌ZY-312活菌体的制备
步骤1：取一支冻干保存菌种，加入200支冻干保存菌培养基，复溶，吸取20基，血平皿划线，厌氧罐气体控制系统抽气后在生化培养箱中37度、厌氧培养48h；
步骤2：挑取单克隆菌落接入10mL TSB培养基，37基、厌氧培养12h；
步骤3：取1瓶500mL TSB培养基，接入1％(v/v)菌种，37，、厌氧培养48h；
步骤4：取菌液离心，，用离心机进行离心，离心条件6000rpm、10min。
用生理盐水洗涤2次，最后用生理盐水复溶菌泥备用并进行活菌计数。
2)脆弱拟杆菌灭活菌体
在温度70度水浴锅中加热30min获得灭活菌液。
3)脆弱拟杆菌裂解液
脆弱拟杆菌培养菌液，采用超声破碎仪进行超声破碎法处理，破2秒，停5秒，持续20分钟，获得脆弱拟杆菌裂解液。
4)脆弱拟杆菌培养上清液
脆弱拟杆菌培养菌液，用离心机进行离心，离心条件为6000rpm、10min，获得脆弱拟杆菌培养上清液。
3、脆弱拟杆菌对肿瘤防治作用的小鼠实验
实验动物：C57BL/6小鼠3～4周36只，精神状态良好，购自中山大学实验动物中心。将小鼠随机分成3组，每组12只，3组分别为对照组、活菌灌胃组、灭活菌灌胃组
实验过程和结果：
对3组小鼠分别灌胃109CFU的脆弱拟杆菌、灭活脆弱拟杆菌及对照，连续灌胃1周，每天测定体重。随后待小鼠肿瘤(黑色素瘤)细胞B16生长到对数期，用TE消化细胞，培养基中和，离心收集细胞，并用DPBS洗涤两次，除去残留血清，用DPBS重悬细胞。细胞计数，将107个细胞右腋皮下接种到每只小鼠。并持续对小鼠分别进行灌胃治疗，1周后将荷瘤小鼠处死，剖取皮下瘤块，测量肿瘤大小。结果显示，脆弱拟杆菌及其灭活菌均能对小鼠肿瘤的形成以及生长有明显的抑制作用，(图2及图3)。此外，图4实验结果表明，对照组中小鼠肿瘤(黑色素瘤)随着时间肿块逐渐变大，灌胃脆弱拟杆菌各组的肿瘤生长速度明显减缓、不形成肿瘤或者肿瘤体积显著小于对照盐水组(且差异具有统计学意义，p＜0.01)，表明脆弱拟杆菌在体内诱导机体产生了抗肿瘤效应，抑制肿瘤生长，对于防治黑色素瘤等肿瘤 具有很好的效果。
以上内容是结合本发明创造的优选实施方式对所提供技术方案所作的进一步详细说明，不能认定本发明创造具体实施只局限于上述这些说明，对于本发明创造所属技术领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明创造的保护范围。

Claims (11)

  1. 脆弱拟杆菌(Bacteroides fragilis)在治疗和/或预防肿瘤中的应用。
  2. 根据权利要求1的应用，其特征在于，所述脆弱拟杆菌为以下中的任意一种：脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
  3. 根据权利要求1，其特征在于，所述肿瘤为各种实体瘤。
  4. 脆弱拟杆菌在制备用于治疗和/或预防肿瘤的药物中的用途，其特征在于，所述肿瘤是黑色素瘤、或关于乳房乳腺、肝、肺、皮肤、口腔、食道、胃、肠道、肾、前列腺、脑、神经系统、膀胱、淋巴、胰腺等的肿瘤。
  5. 一种用于治疗和/或预防肿瘤的药物组合物，其特征在于，所述药物组合物包括药学有效剂量的脆弱拟杆菌及其在药学上可接受的载体。
  6. 根据权利要求3的药物组合物，其特征在于，所述脆弱拟杆菌为以下中的任意一种：脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
  7. 根据权利要求3所述的药物组合物，其特征在于，所述药物组合物为片剂、胶囊剂、口服液或冻干粉剂。
  8. 根据权利要求3所述的药物组合物，其特征在于，所述药学上可接受的载体为脱脂奶、乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖、海藻糖、淀粉、阿拉伯胶、磷酸钙、藻酸盐、明胶、硅酸钙、细结晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁或矿物油中的一种或多种的混合物。
  9. 一种用于治疗和/或预防肿瘤的食品，其特征在于，所述食品包括以下中的任意一种：脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
  10. 一种用于治疗和/或预防肿瘤的保健品，其特征在于，所述保健品包括以下中的任意一种：脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
  11. 一种用于治疗和/或预防肿瘤的食品添加剂，其特征在于，所述食品添加剂包括 以下中的任意一种：脆弱拟杆菌活菌体；经过基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌；脆弱拟杆菌裂解物；和/或脆弱拟杆菌培养上清液。
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