WO2019218401A1 - 脆弱拟杆菌或阿克曼粘细菌在制备用于预防或治疗肿瘤的药物中的应用 - Google Patents

脆弱拟杆菌或阿克曼粘细菌在制备用于预防或治疗肿瘤的药物中的应用 Download PDF

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WO2019218401A1
WO2019218401A1 PCT/CN2018/089561 CN2018089561W WO2019218401A1 WO 2019218401 A1 WO2019218401 A1 WO 2019218401A1 CN 2018089561 W CN2018089561 W CN 2018089561W WO 2019218401 A1 WO2019218401 A1 WO 2019218401A1
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bacteroides fragilis
ackermann
tumor
myxobacteria
slime
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PCT/CN2018/089561
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English (en)
French (fr)
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曾谷城
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深圳月曜生命科技有限公司
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Priority to JP2021514459A priority Critical patent/JP2021523947A/ja
Priority to US17/054,836 priority patent/US20210213073A1/en
Priority to EP18918503.6A priority patent/EP3795676A4/en
Priority to AU2018423492A priority patent/AU2018423492A1/en
Publication of WO2019218401A1 publication Critical patent/WO2019218401A1/zh

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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
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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

Definitions

  • the present invention relates to the field of biomedicine, and in particular to the use of Bacteroides fragilis or Ackermann slime bacteria for the preparation of a medicament for the prevention and/or treatment of tumors.
  • Cancer has become the "first killer" of human beings.
  • the World Health Organization's (WHO) Global Cancer Report 2014 predicts that global cancer cases will show rapid growth, from 14 million in 2012 to 19 million in 2025 and 24 million by 2035. .
  • WHO World Health Organization's
  • 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.
  • Breast cancer is currently the most common type of malignant tumor, and it is also the most common disease that captures the health and quality of life of women.
  • the treatment of breast cancer is mainly based on surgical treatment and chemotherapy.
  • tumor immunotherapy techniques showing good clinical effects are roughly divided into two types.
  • the first one is adoptive immune cell therapy.
  • By obtaining immune cells in patients cells with killing ability are induced in vitro according to the nature of tumor antigen targets. It is then reinfused to exert anticancer effects, such as chimeric antigen T cells (CAR-T).
  • CAR-T chimeric antigen T cells
  • the second is antibody-targeted therapy, in which targeted drugs inhibit cancer cells by interacting with specific molecular targets necessary for tumor growth and metastasis.
  • T cell depleting molecular blocking antibody drugs such as CTLA4, PD-1 and PD-L1.
  • CD8+ T cells lymphocytes
  • CD8+ T cells not only have the key function of directly killing tumor cells, but also can significantly enhance the response of tumor patients to immunotherapy techniques such as CAR-T and T cell depletion molecular blocking antibodies, thereby enhancing the efficacy of immunotherapy and how to promote tumors.
  • the infiltration or accumulation of CD8+ T cells in the microenvironment becomes a key scientific and technical problem that needs to be solved in tumor immunotherapy.
  • Probiotics are a class of active microorganisms that are beneficial to the host. After ingesting humans and animals, probiotics can colonize the intestinal mucosa, colonize the gut and prevent harmful microbes from adhering to them.
  • the natural microflora in the intestine promotes the formation of healthy, growable microbial agents in the organism, helping to maintain the health of humans and animals. 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.
  • studies have been carried out on E. coli and Klebsiella pneumoniae, respectively, for use in intestinal and pulmonary cancer, as a "site-specific immunomodulator" and played a more significant role in inhibiting tumor growth.
  • probiotics or intestinal bacteria used to promote the infiltration or accumulation of CD8+ T cells in the tumor microenvironment.
  • 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.
  • Akkermansia muciniphila is a kind of elliptical intestinal bacteria belonging to the genus Microphyllo, anaerobic growth, no flagella, no spores, no movement, Gram-negative bacteria, certain anaerobic ability.
  • Ackermann myxobacteria colonizes mucin layer and can specifically degrade mucin, accounting for 1-3% of total intestinal microbes. It is one of the dominant bacteria in human intestinal tract. In vivo colonization abundance is often negatively correlated with obesity and type 2 diabetes, and plays an important role in body metabolism.
  • intestinal bacteria including Bacteroides fragilis and/or Ackermann myxobacteria to promote CD8+ T cell infiltration and/or accumulation in the tumor microenvironment to achieve tumor prevention and/or treatment.
  • the technical problem to be solved by the present invention is to provide a problem capable of infiltrating and/or accumulating insufficient CD8-positive killer T cells (abbreviation: CD8+ T cells, or CD8+CTL) in the tumor microenvironment in tumor immunotherapy.
  • CD8+ T cells or CD8+CTL
  • the present invention provides the use of Bacteroides fragilis or Akkermansia muciniphila for the preparation of a medicament for preventing and/or treating a tumor, wherein the medicament promotes tumor micro Infiltration and/or accumulation of CD8-positive killer T lymphocytes in the environment.
  • Bacteroides fragilis or Ackermann myxobacteria is any one of the following: Bacteroides fragilis or Ackermania viscobacteria; after genetic recombination, transformation or modification, attenuation, chemical treatment, physics Treated or inactivated Bacteroides fragilis or Ackermann myxobacteria; Bacteroides fragilis or Ackermann myxobacteria lysate; and/or Bacteroides fragilis or Ackermann slime culture supernatant.
  • the tumor may be various solid tumors such as, but not limited to, breast cancer, or about liver, lung, skin, oral cavity, esophagus, stomach, intestine, kidney, prostate, brain, nervous system, bladder, lymph.
  • Any one or more of tumors of the pancreas, such as solid tumors such as lung cancer, melanoma tumor, liver cancer, prostate cancer, fibrosarcoma, bladder sarcoma, and glioma.
  • the present invention also provides a method for preventing and/or treating tumors by using Bacteroides fragilis or Akkermansia muciniphila to promote infiltration and/or accumulation of CD8-positive killer T lymphocytes in a tumor microenvironment. .
  • Bacteroides fragilis or Ackermann myxobacteria is any one of the following: Bacteroides fragilis or Ackermania viscobacteria; after genetic recombination, transformation or modification, attenuation, chemical treatment, physics Treated or inactivated Bacteroides fragilis or Ackermann myxobacteria; Bacteroides fragilis or Ackermann myxobacteria lysate; and/or Bacteroides fragilis or Ackermann slime culture supernatant.
  • methods of promoting infiltration and/or accumulation of CD8+ T cells in a tumor microenvironment, thereby preventing and/or treating tumors and other methods of promoting infiltration and/or accumulation of CD8+ T cells in a tumor microenvironment Combinations of methods for preventing and/or treating tumors.
  • the other methods of promoting infiltration and/or accumulation of CD8+ T cells in a tumor microenvironment, thereby preventing and/or treating a tumor include, but are not limited to, chemotherapy, radiation therapy, gene therapy, surgery Or a combination thereof.
  • compositions comprising Bacteroides fragilis or Ackermania.
  • the therapeutic and prophylactic composition comprises Bacteroides fragilis or Ackermania.
  • the therapeutic and prophylactic compositions do not contain other microbial strains.
  • the Bacteroides fragilis or Ackermann myxobacteria can delay tumor growth.
  • the tumor is a solid tumor.
  • the tumor includes, but is not limited to, breast cancer.
  • the present invention provides a pharmaceutical composition for preventing and/or treating a tumor, wherein the pharmaceutical composition comprises a pharmaceutically effective amount of Bacteroides fragilis or Ackerman's slime bacterium and In a pharmaceutically acceptable carrier, and promotes the infiltration and/or accumulation of CD8-positive killer T lymphocytes in the tumor microenvironment.
  • the Bacteroides fragilis or Ackermann slime bacteria is an active ingredient.
  • the Bacteroides fragilis or Ackermann slime bacterium is any one of the following: Bacteroides fragilis or Acrylic myxobacteria; after genetic recombination, modification or modification, Attenuated, chemically treated, physically treated or inactivated Bacteroides fragilis or Ackermann myxobacteria; Bacteroides fragilis or Ackermann myxobacteria lysate; and/or Bacteroides fragilis or Ackermann slime 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 preventing and/or treating a tumor, wherein the food comprises Bacteroides fragilis or Ackermann slime bacteria, and promotes CD8 positivity in a tumor microenvironment. Infiltration and/or accumulation of lethal T lymphocytes.
  • the Bacteroides fragilis or Ackermann slime bacterium is any one of the following: Bacteroides fragilis or Acrylic myxobacteria; after genetic recombination, transformation or modification, attenuation, Chemically treated, physically treated or inactivated Bacteroides fragilis or Ackerman's slime bacteria; Bacteroides fragilis or Ackermann myxobacteria lysate; and/or Bacteroides fragilis or Ackermann slime culture supernatant.
  • the present invention also provides a food additive for preventing and/or treating a tumor, characterized in that the food additive comprises Bacteroides fragilis or Ackermania, and promotes tumor micro Infiltration and/or accumulation of CD8-positive killer T lymphocytes in the environment.
  • the Bacteroides fragilis or Ackermann slime bacterium is any one of the following: Bacteroides fragilis or Ackermann bacteria live cells; genetically recombined, modified or modified, attenuated , chemically treated, physically treated or inactivated Bacteroides fragilis or Ackerman's slime bacteria; Bacteroides fragilis or Ackerman's slime lysate; and/or Bacteroides fragilis or Ackerman's slime culture supernatant.
  • the present invention also provides a food additive for preventing and/or treating a tumor, wherein the health care product comprises Bacteroides fragilis or Ackermann slime bacteria, and promotes tumor microenvironment Infiltration and/or accumulation of CD8-positive killer T lymphocytes.
  • the Bacteroides fragilis or Ackerman's slime bacterium is any one of the following: Bacteroides fragilis or Acrylic myxobacteria; after genetic recombination, modification or modification, attenuation , chemically treated, physically treated or inactivated Bacteroides fragilis or Ackerman's slime bacteria; Bacteroides fragilis or Ackerman's slime lysate; and/or Bacteroides fragilis or Ackerman's slime culture supernatant.
  • the invention establishes a mouse breast cancer model by a transplant tumor research method, and detects and identifies the action of Bacteroides fragilis or Ackermann myxobacteria in a mouse breast cancer model, and the invention proves that the Bacteroides fragilis or Ackermann myxobacteria can significantly inhibit the survival of breast cancer in vitro, and can effectively inhibit the growth of transplanted tumors in mice, suggesting that Bacteroides fragilis or Ackermann slime bacteria have important development and application value in the clinical treatment of tumors.
  • Figure 1 is a schematic diagram showing the experimental procedure for detecting the accumulation of CD8+ T cells in the tumor microenvironment and the therapeutic effect of Bacteroides fragilis and inactivation of Bacteroides fragilis in a mouse breast cancer model.
  • Figure 2 is a typical flow cytometric analysis of mice in each group after administration of Bacteroides fragilis and inactivation of Bacteroides fragilis in breast cancer cell-transplanted mice.
  • the right quadrant is CD8+ T cells, right quadrant The numbers show the percentage of CD8+ T cells in the total cells in the tumor microenvironment.
  • Figure 3 is a graphical representation of the percentage of CD8+ T cells as a percentage of total cells in the tumor microenvironment after administration of Bacteroides fragilis and inactivation of Bacteroides fragilis in mice transplanted with breast cancer cells.
  • Figure 4 is a comparison of tumor size of breast cancer cells after treatment with Bacteroides fragilis and inactivated Bacteroides.
  • Figure 5 is a statistical analysis of the comparison of tumor size of breast cancer cells in mice treated with Bacteroides fragilis and inactivated Bacteroides.
  • Fig. 6 is a schematic diagram showing the experimental procedure for detecting the accumulation of CD8+ T cells in the tumor microenvironment and the therapeutic effect of Acmemann myxobacteria in a mouse breast cancer model.
  • Figure 7 is a typical flow cytometric analysis of one mouse per group after administration of Ackermann myxobacteria in mice transplanted with breast cancer cells.
  • the right quadrant is CD8+ T cells, and the number in the right quadrant is CD8+ T cells account for the percentage of total tumor cells.
  • Figure 8 is a graphical representation of the percentage of total cells in the tumor microenvironment of CD8+ T cells following administration of Ackerman's mucobacteria in breast cancer cell transplanted mice.
  • Figure 9 is a comparison of tumor size of mouse breast cancer after treatment with Ackermann slime bacteria.
  • Figure 10 is a graphical analysis of the comparison of tumor size of mouse breast cancers after treatment with Ackermann myxobacteria.
  • the food additive, after administration to the subject can be applied to the indications described above and exhibit the functions described above, all of the dosage forms within the scope of the invention have been tested, hereinafter, simply It is to be understood that only a few of the embodiments are described in the embodiments, which should not be construed as limiting the invention.
  • Bacteroides fragilis or Ackerman's slime bacteria referred to in the present invention include, but are not limited to, any one of the following: Bacteroides fragilis or Acrylic myxobacteria; after genetic recombination, modification or modification, attenuation, chemistry Bacteroides or Acrylic myxobacteria that are treated, physically treated or inactivated; Bacteroides fragilis or Ackerman's slime lysate; and/or Bacteroides fragilis or Ackerman's slime culture supernatant.
  • the tumor is a solid tumor.
  • the tumor includes, but is not limited to, a breast tumor.
  • the pharmaceutical composition for antitumor provided by the present invention includes a pharmaceutically effective amount of Bacteroides fragilis or Ackermann slime bacteria.
  • the "pharmaceutically effective dose” is referred to as 10 6 - 10 10 CFU, preferably 10 9 CFU.
  • the Bacteroides fragilis or Ackermann slime bacterium includes any one of the following: Bacteroides fragilis or Ackermann bacteria live cells; genetic recombination, modification or modification, attenuation, chemical treatment, physical treatment or inactivation Bacteroides fragilis or Ackermann myxobacteria; Bacteroides fragilis or Ackermann myxobacteria lysate; and/or Bacteroides fragilis or Ackermann slime 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 or Ackermann slime bacterium of the present invention can also be made into foods, health care products or food additives.
  • the food, health care product or food additive comprises Bacteroides fragilis or Acrylic fragile live bacteria, genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation of Bacteroides fragilis or Ackerman Any of the myxobacteria, Bacteroides fragilis or Ackerman's slime lysate and/or Bacteroides fragilis or Ackerman's slime culture supernatant.
  • These foods, health supplements or food additives can be used to treat and/or prevent tumors.
  • Step 1 Take a freeze-dried Bacteroides fragilis strain (purchased from ATCC official website), add 200 ⁇ L TSB medium, reconstitute, absorb 20 ⁇ L, blood plate, and anaerobic tank gas control system pumping After anaerobic incubation for 48 h in a biochemical incubator at 37 ° C;
  • Step 2 Pick up the monoclonal colonies into 10 mL TSB medium, 37 ° C, anaerobic culture for 12 h;
  • Step 3 Take 1 bottle of 500mL TSB medium, connect 1% (v/v) strain, 37 ° C, 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.
  • Step 1 Take a freeze-preserved Akkermansia muciniphila strain (purchased from the ATCC official website), add 200 ⁇ L of TSB medium, reconstitute, pipet 20 ⁇ L, blood plate dash, anaerobic tank gas control system After aspiration, the cells were cultured in a biochemical incubator at 37 ° C for 48 h;
  • Step 2 Pick up the monoclonal colonies into 10 mL TSB medium, 37 ° C, anaerobic culture for 12 h;
  • Step 3 Take 1 bottle of 500mL TSB medium, connect 1% (v/v) strain, 37 ° C, 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.
  • Fig. 1 is a schematic diagram showing the experimental procedure for detecting the accumulation of CD8+ T cells and the therapeutic effect of Bacteroides fragilis and inactivating Bacteroides fragilis in the tumor microenvironment.
  • the culture method of Bacteroides fragilis is the same as in Example 1.
  • Step 1 Take a freeze-dried strain (purchased by commercial means), add 200 ul of freeze-preserved culture medium, reconstitute, absorb 20 ul, draw blood plate, and anaerobic tank gas control system after gas extraction in biochemical culture 37 ° C in the box, anaerobic culture for 48h;
  • Step 2 Pick up the monoclonal colonies into 10 mL TSB medium, anaerobic culture for 12 h at 37 ° C;
  • Step 3 Take 1 bottle of 500mL TSB medium, connect 1% (v/v) strain, 37 ° C, anaerobic culture for 48h;
  • Step 4 The bacteria solution was centrifuged, centrifuged with a centrifuge, centrifuged at 6000 rpm, 10 min, washed twice with physiological saline, and finally reconstituted with physiological saline for use and counted for live bacteria.
  • the inactivated bacterial solution was obtained by heating in a water bath at a temperature of 70 ° C 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 Bacteroides fragilis or Ackerman's slime 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 36 BALB/c mice aged 3 to 4 weeks old, in good mental state, purchased from the Experimental Animal Center of Sun Yat-sen University. The mice were randomly divided into 3 groups, 12 in each group, 3 groups were control group (saline), live bacteria gavage group (Bacteroides fragilis), inactivated bacteria gavage group (inactivated Bacteroides fragilis) Inactivated cells), 10 9 CFU of Bacteroides fragilis and controls were administered to 3 groups of mice, and body weight was measured every day.
  • mouse tumor (breast cancer) cells 4T1 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. Cell count, 10 6 cells were inoculated subcutaneously into each mouse, and the mice were continuously intragastrically treated, then the tumor-bearing mice were sacrificed, and the tumor in situ cells were collected and analyzed by flow cytometry. The content of CD8+ T cells in the tumor microenvironment.
  • Fig. 6 is a schematic diagram showing the experimental procedure for detecting the accumulation of CD8+ T cells in the tumor microenvironment by the Ackermann myxobacteria and inactivated Ackermann myxobacteria.
  • the Ackermann slime culture method was the same as in Example 2.
  • Step 1 Take a freeze-dried strain (purchased by commercial means), add 200 ul of freeze-preserved culture medium, reconstitute, absorb 20 ul, draw blood plate, and anaerobic tank gas control system after gas extraction in biochemical culture 37 ° C in the box, anaerobic culture for 48h;
  • Step 2 Pick up the monoclonal colonies into 10 mL TSB medium, anaerobic culture for 12 h at 37 ° C;
  • Step 3 Take 1 bottle of 500mL TSB medium, connect 1% (v/v) strain, 37 ° C, anaerobic culture for 48h;
  • Step 4 The bacteria solution was centrifuged, centrifuged with a centrifuge, centrifuged at 6000 rpm, 10 min, washed twice with physiological saline, and finally reconstituted with physiological saline for use and counted for live bacteria.
  • the inactivated bacterial solution was obtained by heating in a water bath at a temperature of 70 ° C for 30 minutes.
  • Ackerman's slime culture medium was treated by sonication using a sonicator, and was broken for 2 seconds, stopped for 5 seconds, and continued for 20 minutes to obtain Ackerman's slime lysate.
  • the Ackerman slime culture medium was centrifuged by a centrifuge, and the centrifugation conditions were 6000 rpm for 10 min to obtain an Ackerman slime culture supernatant.
  • mice 24 BALB/c mice aged 3 to 4 weeks old, in good mental state, purchased from the Experimental Animal Center of Sun Yat-sen University. The mice were randomly divided into two groups, 12 in each group. The two groups were the control group and the live bacteria gavage group (Ackman's viscobacteria live cells), and the two groups of mice were given 10 9 CFU of Ake. Mangic bacteria and controls were measured daily for body weight. Subsequently, mouse tumor (breast cancer) cells 4T1 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.
  • DPBS DPBS
  • Figure 2 is a typical flow cytometric analysis of one mouse per group after administration of Bacteroides fragilis in breast cancer cell-transplanted mice, and the number in the right quadrant shows that CD8+ T cells account for cells in the tumor microenvironment. Percentage chart. From the flow cell analysis quadrant map, it can be seen that the relative amount of CD8+ T cells is increased by about 20-fold compared to the saline control group, Bacteroides fragilis and B. fragilis inactivated.
  • Figure 3 is a graph showing the statistical analysis of the percentage of CD8+ T cells in tumor microenvironment cells after administration of Bacteroides fragilis in breast cancer cell-transplanted mice.
  • Figure 7 is a typical flow cytometric analysis of one mouse per group after administration of Ackermann myxobacteria in breast cancer cell-transplanted mice, and the number in the right quadrant shows CD8+ T cells in the tumor microenvironment A graph of the percentage of cells. From the flow cell analysis quadrant map, it can be seen that Ackerman's slime bacteria increased the relative amount of CD8+ T cells by more than 13 times than the saline control group.
  • Figure 8 is a graphical representation of the percentage of cells in the tumor microenvironment of CD8+ T cells after administration of Ackerman's mucobacteria in breast cancer cell transplanted mice.

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Abstract

提供了脆弱拟杆菌或阿克曼粘细菌在制备用于预防和/或治疗肿瘤的药物中的应用，该药物促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。还提供了用于预防和/或治疗肿瘤的药物组合物、食品、保健品或食品添加剂中的应用，该药物组合物、食品、保健品或食品添加剂包含脆弱拟杆菌或阿克曼粘细菌并促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。

Description

脆弱拟杆菌或阿克曼粘细菌在制备用于预防或治疗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阿克曼粘细菌在制备用于预防和/或治疗肿瘤的药物中的应用。
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》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。全球每年新增癌症患者约700万人，死亡的肿瘤患者人数约500万，平均每6秒钟就有一人死于肿瘤。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。乳腺癌目前是恶性肿瘤里最常见的一种，也是夺取广大女性生命健康和生活质量的最常见的疾病。乳腺癌治疗方法主要以手术治疗、化疗为主，随着基因诊断个性化治疗的开展，乳腺癌患者预后效果尚可，但由于种种原因，大部分患者疗效不尽人意，因此，抗乳腺癌药物的研究具有重大意义。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”。如今，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。
目前显示较好临床效果的肿瘤免疫治疗技术大致分为两种，第一种是过继免疫细胞疗法，通过获得患者体内的免疫细胞，根据肿瘤抗原靶标的性质在体外诱导出具有杀伤能力的细胞，再回输体内发挥抗癌作用，例如嵌合抗原T细胞(CAR-T)等。第二种是抗体靶向治疗法，靶向药物通过与肿瘤生长转移过程中必需的特定分子靶点相互作用来抑制癌细胞。目前已有CTLA4、PD-1、PD-L1等T细胞耗竭分子阻断性抗体药物。虽然这些阻断性抗体肿瘤治疗在部分患者及部分瘤种上显示了一定的临床效果，但是阻断性抗体药物对相当比例的患者仍然无效或者低效，而且肿瘤微环境中CD8阳性杀伤性T淋巴细胞(简称：CD8+T细胞)的浸润或者积累缺乏是免疫治疗效果欠佳的关键原因之一。鉴于CD8+T细胞不仅具有关键的直接杀伤肿瘤细胞的功能而且可以显著增强肿瘤患者对CAR-T及T细胞耗竭分子阻断性抗体等免疫治疗技术的响应从而增强免疫治疗的疗效，如何促进肿瘤微环境中CD8+T细胞的浸润或者 积累成为肿瘤免疫治疗中亟需解决的关键科学与技术问题。
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，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、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。但是，目前没有益生菌或肠道细菌用于促进肿瘤微环境中CD8+T细胞的浸润或者积累的相关报道。
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。
阿克曼粘细菌(Akkermansia muciniphila)是一类椭圆形的肠道细菌，属于疣微菌门，厌氧生长，无鞭毛，不产芽孢，不运动，革兰氏阴性菌，具有一定的厌氧能力。阿克曼粘细菌定植在粘液层中可特异性降解粘蛋白，占肠道微生物总量的1-3％，是人体肠道中的优势菌群之一，目前研究发现阿克曼粘细菌在人体内定植丰度与肥胖和Ⅱ型糖尿病常呈负相关，对机体代谢有着重要作用。
但是，目前没有关于利用包括脆弱拟杆菌和/或阿克曼粘细菌等肠道细菌促进肿瘤微环境中CD8+T细胞浸润和/或积累进而实现肿瘤预防和/或治疗的报道。
发明内容
本发明所要解决的技术问题是针对目前肿瘤免疫治疗中肿瘤微环境中CD8阳性杀伤性T细胞(简称：CD8+T细胞，或CD8+CTL)浸润和/或积累不足的难题，提供一种能够促进肿瘤微环境中CD8+T细胞的浸润或积累进而预防和/或治疗肿瘤的药物。
为了实现上述目的，本发明提供了脆弱拟杆菌(Bacteroides fragilis)或阿克曼粘细菌(Akkermansia muciniphila)在制备用于预防和/或治疗肿瘤的药物中的应用，其中，所述药物促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。
优选地，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
优选地，明所述肿瘤可以为各种实体瘤，例如但不限于乳腺癌、或关于肝、肺、皮肤、口腔、食道、胃、肠道、肾、前列腺、脑、神经系统、膀胱、淋巴、胰腺的肿瘤中的任意一种或多种，例如肺癌、黑色素肿瘤、肝癌、前列腺癌、纤维肉瘤、膀胱肉瘤及神经胶质瘤等实体瘤。
本发明还提供了利用脆弱拟杆菌(Bacteroides fragilis)或阿克曼粘细菌(Akkermansia muciniphila)促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累进而预防和/或治疗肿瘤的方法。
优选地，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
在某些实施方案中，将促进肿瘤微环境中CD8+T细胞的浸润和/或积累进而预防和/或治疗肿瘤的方法与其他促进肿瘤微环境中CD8+T细胞的浸润和/或积累进而预防和/或治疗肿瘤的方法进行组合。在某些实施方案中，所述其它促进肿瘤微环境中CD8+T细胞的浸润和/或积累进而预防和/或治疗肿瘤的方法包括但不限于是化学疗法、放射疗法、基因疗法、外科手术或其组合。
本文还描述了包含脆弱拟杆菌或阿克曼粘细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或阿克曼粘细菌。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，所述脆弱拟杆菌或阿克曼粘细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：乳腺癌。
根据本发明的一个方面，本发明还提供了一种用于预防和/或治疗肿瘤的药物组合物，其中，所述药物组合物包括药学有效剂量的脆弱拟杆菌或阿克曼粘细菌及其在药学上可接受的载体，并促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。其中，所述脆弱拟杆菌或阿克曼粘细菌为活性成分。
优选地，上述的药物组合物中，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
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预防和/或治疗肿瘤的食品，其中，所述食品包括脆弱拟杆菌或阿克曼粘细菌，并促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。
优选地，上述食物中，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
为了更好地实现上述目的，本发明还提供了一种用于预防和/或治疗肿瘤的食品添加剂，其特征在于，所述食品添加剂包括脆弱拟杆菌或阿克曼粘细菌，并促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。
优选地，上述食品添加剂中，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
为了更好地实现上述目的，本发明还提供了一种用于预防和/或治疗肿瘤的食品添加剂，其中，所述保健品包括脆弱拟杆菌或阿克曼粘细菌，并促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。
优选地，上述保健品中，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
本发明以移植性肿瘤研究法建立小鼠乳腺癌模型，通过在小鼠乳腺癌模型中对脆弱拟杆菌或阿克曼粘细菌的作用进行检测和鉴定，本发明通过实验证明，脆弱拟杆菌或阿克曼粘细 菌能够显著抑制体外乳腺癌的存活，并能有效抑制小鼠体内移植肿瘤的生长，提示脆弱拟杆菌或阿克曼粘细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为在小鼠乳腺癌模型中检测脆弱拟杆菌及灭活脆弱拟杆菌促进肿瘤微环境中CD8+T细胞的积累及其治疗作用实验流程示意图。
图2为乳腺癌细胞移植的小鼠中施用脆弱拟杆菌及灭活脆弱拟杆菌后每组一只小鼠的典型的流式细胞分析图,右侧象限为CD8+T细胞，右侧象限的数字显示的为CD8+T细胞占肿瘤微环境内总体细胞的百分比。
图3为乳腺癌细胞移植的小鼠中施用脆弱拟杆菌及灭活脆弱拟杆菌后CD8+T细胞占肿瘤微环境内总体细胞的百分比的统计分析图。
图4为脆弱拟杆菌及灭活脆弱拟杆菌治疗后小鼠乳腺癌肿瘤大小对比图。
图5为脆弱拟杆菌及灭活脆弱拟杆菌治疗后小鼠乳腺癌肿瘤大小对比图的统计分析图。
图6为在小鼠乳腺癌模型中检测阿克曼粘细菌促进肿瘤微环境中CD8+T细胞的积累及其治疗作用实验流程示意图。
图7为乳腺癌细胞移植的小鼠中施用阿克曼粘细菌后每组一只小鼠的典型的流式细胞分析图,右侧象限为CD8+T细胞，右侧象限的数字显示的为CD8+T细胞占肿瘤总体细胞的百分比。
图8为乳腺癌细胞移植的小鼠中施用阿克曼粘细菌后CD8+T细胞占肿瘤微环境内总体细胞的百分比的统计分析图。
图9为阿克曼粘细菌治疗后小鼠乳腺癌肿瘤大小对比图。
图10为阿克曼粘细菌治疗后小鼠乳腺癌肿瘤大小对比图的统计分析图。
具体实施方式
下面将结合附图和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阿克曼粘细菌或含有本发明的脆弱拟杆菌或阿克曼粘细菌的药物组合物、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或阿克曼粘细菌包括但不限于以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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 脆弱拟杆菌或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
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：乳腺肿瘤。
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或阿克曼粘细菌。其中，所称的“药学有效剂量”为10 6-10 10CFU，优选为10 9CFU。所述脆弱拟杆菌或阿克曼粘细菌包括以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。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或阿克曼粘细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或阿克曼粘细菌活菌体、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或阿克曼粘细菌、脆弱拟杆菌或阿克曼粘细菌裂解物和/或脆弱拟杆菌或阿克曼粘细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(购自ATCC官网)，加入200μL TSB培养基，复溶，吸取20μL，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℃、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 阿克曼粘细菌培养
培养方法
步骤1：取一支冻干保存阿克曼粘细菌(Akkermansia muciniphila)菌种(购自ATCC官 网)，加入200μL TSB培养基，复溶，吸取20μL，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℃、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3 脆弱拟杆菌促进肿瘤微环境中CD8+T细胞浸润和/或积累及治疗肿瘤作用实验
图1为检测脆弱拟杆菌及灭活脆弱拟杆菌促进肿瘤微环境中CD8+T细胞的积累及其治疗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(通过商业途径购买)，加入200ul冻干保存菌培养基，复溶，吸取20ul，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℃、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，用离心机进行离心，离心条件为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℃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，获得脆弱拟杆菌或阿克曼粘细菌裂解液。
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：3～4周龄BALB/c小鼠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(生理盐水)、活菌灌胃组(脆弱拟杆 菌活菌体)、灭活菌灌胃组(灭活脆弱拟杆菌灭活菌体)，对3组小鼠分别灌胃10 9CFU的脆弱拟杆菌及对照，每天测定体重。随后待小鼠肿瘤(乳腺癌)细胞4T1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10 6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，然后处死荷瘤小鼠，分别收集肿瘤原位细胞，利用流式细胞术检测分析肿瘤微环境中CD8+T细胞的含量。
实施例4 阿克曼粘细菌促进肿瘤微环境中CD8+T细胞浸润和/或积累及治疗肿瘤作用实验
图6为检测阿克曼粘细菌及灭活阿克曼粘细菌促进肿瘤微环境中CD8+T细胞的积累及其治疗作用实验流程示意图。
1、培养方法
阿克曼粘细菌培养方法同实施例2。
2、样品准备
1)阿克曼粘细菌活菌体的制备
步骤1：取一支冻干保存菌种(通过商业途径购买)，加入200ul冻干保存菌培养基，复溶，吸取20ul，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℃、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，用离心机进行离心，离心条件为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)阿克曼粘细菌灭活菌体
在温度70℃水浴锅中加热30min获得灭活菌液。
3)阿克曼粘细菌裂解液
阿克曼粘细菌培养菌液，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，获得阿克曼粘细菌裂解液。
4)阿克曼粘细菌培养上清液
阿克曼粘细菌培养菌液，用离心机进行离心，离心条件为6000rpm、10min，获得阿克曼粘细菌培养上清液。
3、阿克曼粘细菌对肿瘤防治作用的小鼠实验
实验动物：3～4周龄BALB/c小鼠24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2组，每组12只，2组分别为对照组、活菌灌胃组(阿克曼粘细菌活菌体)， 对2组小鼠分别灌胃10 9CFU的阿克曼粘细菌及对照，每天测定体重。随后待小鼠肿瘤(乳腺癌)细胞4T1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10 6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，处死荷瘤小鼠，分别收集肿瘤原位细胞，利用流式细胞术检测分析肿瘤微环境中CD8+T细胞的含量。
结果分析：
单个小鼠的典型的流式检测结果及每组小鼠多只的统计结果见图2、图3、图7、图8。
图2是乳腺癌细胞移植的小鼠中施用脆弱拟杆菌后每组一只小鼠的典型的流式细胞分析图,右侧象限的数字显示的为CD8+T细胞占肿瘤微环境中细胞的百分比情况图。从流式细胞分析象限图可以看出，相对比生理盐水对照组，脆弱拟杆菌和灭活脆弱拟杆菌增加了CD8+T细胞相对量约20倍。图3是乳腺癌细胞移植的小鼠中施用脆弱拟杆菌后CD8+T细胞占肿瘤微环境细胞的百分比的统计分析图。从统计图可以看出，相对比生理盐水对照组，脆弱拟杆菌和灭活脆弱拟杆菌显著增加了肿瘤微环境中CD8+T细胞的数量。统计分析图中，*表示student t-test p<0.05，**表示student t-test p<0.01。p<0.05具有统计学差异意义。每种处理组有12只小鼠。
图7是乳腺癌细胞移植的小鼠中施用阿克曼粘细菌后每组一只小鼠的典型的流式细胞分析图,右侧象限的数字显示的为CD8+T细胞占肿瘤微环境中细胞的百分比情况图。从流式细胞分析象限图可以看出，相对比生理盐水对照组，阿克曼粘细菌增加了CD8+T细胞相对量约13倍以上。图8是乳腺癌细胞移植的小鼠中施用阿克曼粘细菌后CD8+T细胞占肿瘤微环境中细胞的百分比的统计分析图。从统计图可以看出，相对比生理盐水对照组，阿克曼粘细菌显著增加了肿瘤微环境中CD8+T细胞的数量。统计分析图中，***表示student t-test p<0.001。p<0.001具有统计学差异意义。每种处理组有12只小鼠。
结果显示，脆弱拟杆菌及灭活脆弱拟杆菌或阿克曼粘细菌均能对小鼠肿瘤的形成以及生长有明显的抑制作用(图4、图5、图9、图10)。此外，图4、图5、图9、图10的实验结果表明，灌胃脆弱拟杆菌或阿克曼粘细菌小鼠的肿瘤体积显著小于对照盐水组，表明脆弱拟杆菌及灭活脆弱拟杆菌或阿克曼粘细菌在体内促进肿瘤微环境中CD8+T细胞的浸润和/或积累从而增强了抗肿瘤功能，抑制肿瘤生长，对于防治乳腺癌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 (10)

  1. 脆弱拟杆菌(Bacteroides fragilis)或阿克曼粘细菌(Akkermansia muciniphila)在制备用于预防和/或治疗肿瘤的药物中的应用，其特征在于，所述药物促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。
  2. 根据权利要求1所述的应用，其特征在于，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
  3. 根据权利要求1或2所述的应用，其特征在于，所述肿瘤为实体瘤。
  4. 根据权利要求1或2所述的应用，其特征在于，所述肿瘤是乳腺癌、或关于肝、肺、皮肤、口腔、食道、胃、肠道、肾、前列腺、脑、神经系统、膀胱、淋巴、胰腺的肿瘤中的任意一种或多种。
  5. 一种用于预防和/或治疗肿瘤的药物组合物，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或阿克曼粘细菌及其在药学上可接受的载体，并促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。
  6. 根据权利要求5所述的药物组合物，其特征在于，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
  7. 一种用于预防和/或治疗肿瘤的食品，其特征在于，所述食品包括脆弱拟杆菌或阿克曼粘细菌，并促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。
  8. 根据权利要求7所述的食品，其特征在于，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
  9. 一种用于预防和/或治疗肿瘤的食品添加剂，其特征在于，所述食品添加剂包括脆弱拟杆菌或阿克曼粘细菌，并促进肿瘤微环境中CD8阳性杀伤性T淋巴细胞的浸润和/或积累。
  10. 根据权利要求9所述的食品添加剂，其特征在于，所述脆弱拟杆菌或阿克曼粘细菌为以下中的任意一种：脆弱拟杆菌或阿克曼粘细菌活菌体；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或阿克曼粘细菌；脆弱拟杆菌或阿克曼粘细菌裂解物；和/或脆弱拟杆菌或阿克曼粘细菌培养上清液。
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