WO2023184631A1 - Akkermansia muciniphila and use thereof in preparing anti-tumor drug - Google Patents
Akkermansia muciniphila and use thereof in preparing anti-tumor drug Download PDFInfo
- Publication number
- WO2023184631A1 WO2023184631A1 PCT/CN2022/089199 CN2022089199W WO2023184631A1 WO 2023184631 A1 WO2023184631 A1 WO 2023184631A1 CN 2022089199 W CN2022089199 W CN 2022089199W WO 2023184631 A1 WO2023184631 A1 WO 2023184631A1
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- WIPO (PCT)
- Prior art keywords
- akkermansia muciniphila
- strain
- bacteria
- tumor
- pancreatic
- Prior art date
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Images
Classifications
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- 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
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- 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
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- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- the invention belongs to the field of biomedicine, and specifically relates to Akkermansia muciniphila and the use of Akkermansia muciniphila in the preparation of drugs for preventing and/or treating pancreatic tumors. .
- Pancreatic cancer is one of the common malignant tumors of the digestive system, with high malignancy and extremely poor prognosis.
- the annual incidence of pancreatic cancer in China is about 120,000, and in developed economies such as the United States, the annual incidence is about 60,000.
- new cases of pancreatic cancer in China increased by about 31.6%, and deaths increased by about 43.5%. New cases and deaths were almost the same, and most patients were diagnosed at an advanced stage.
- Pancreatic ductal adenocarcinoma (PDAC) is the most common and very deadly, with a survival rate of less than 2-5% one year after diagnosis. Effective treatment options are urgently needed.
- pancreas is surrounded by the stomach and arteries, so surgery is risky. Resection is only suitable for early-stage tumors (about 15%). After resection, there is still a high chance of cancer recurrence.
- domestic and foreign guidelines mainly recommend albumin-paclitaxel combined with gemcitabine as first-line treatment for PDAC, but the efficacy is limited and chemotherapy resistance is unavoidable.
- Another possible new direction in the treatment of pancreatic cancer is the synergistic effect of immune checkpoint inhibitors combined with chemotherapy.
- the clinical effect of immune checkpoint therapy is still not significant enough. This also shows that the pathological mechanism of pancreatic cancer is different from other solid malignant tumors. there is a big difference.
- clinical trials of new pancreatic tumor therapies targeting the extracellular matrix (ECM) also ended in failure. Therefore, there is an urgent need to develop new therapies against pancreatic cancer.
- ECM extracellular matrix
- pancreatic cancer To develop new treatments for pancreatic cancer, we must re-understand the pathogenesis of pancreatic cancer and develop new treatments based on new mechanisms. More and more evidence confirms that the intestinal microecology, especially the composition, abundance, structure and function of intestinal bacteria, is a key factor in determining human immune homeostasis and affecting the occurrence and development of tumors. For example, some recent scientific research progress shows that supplementing a certain type of selected intestinal bacteria or certain types of intestinal bacteria can effectively enhance the ability to perturb the tumor microenvironment and enhance the efficacy of anti-tumor immunotherapy. However, there is still no report on which intestinal microorganisms can effectively curb the occurrence and development of pancreatic tumors.
- pancreatic cancer research A series of recent advances in pancreatic cancer research have shown that the serum of pancreatic cancer patients contains antibodies against certain gastrointestinal bacteria, and pancreatic tumor tissue contains rich and disordered bacterial or fungal composition, abundance, structure, and function.
- microorganisms, especially gastrointestinal bacteria may be key pathophysiological factors affecting the occurrence and development of pancreatic tumors, revealing whether and how digestive tract or intestinal bacteria affect the occurrence and development of pancreatic tumors, which can provide insights into the development of anti-pancreatic tumors. Provides new and powerful weapons.
- One object of the present invention is to provide an intestinal bacterium that can effectively prevent and/or treat pancreatic tumors in view of the above technical problems to be solved.
- the present invention provides a strain of Akkermansia muciniphila, whose deposit number is CGMCC No. 23185.
- Akkermansia muciniphila is a strain with tumor microenvironment or systemic CD8+ T cell immune function regulation activity.
- Akkermansia muciniphila can inhibit the number of CD8+ T cells in the tumor microenvironment or system and/or the effector function of expressing inflammatory effector factors such as IFN- ⁇ .
- the present invention provides the Akkermansia muciniphila Use of Kermanella in the preparation of medicaments for the prevention and/or treatment of inflammation and diseases causing, accompanying, related, inducing, feedback.
- the Akkermansia muciniphila provided by the present invention can also effectively enhance the infiltration of other anti-tumor drugs (such as small molecule chemotherapy drugs) into the pancreatic tumor microenvironment and thereby enhance the tumor-killing ability of the anti-tumor drugs.
- other anti-tumor drugs such as small molecule chemotherapy drugs
- the Akkermansia muciniphila of the present invention is any one of the following: a clinical isolate of Akkermansia muciniphila; a live or dead strain of Akkermansia muciniphila. Bacteria, bacterial lysates, metabolites, modified bacteria, mutant bacteria and/or mutagen bacteria.
- the inventor's research has found that the above Akkermansia muciniphila have CD8+ T cell immune function regulation activity and thereby affect the expression and secretion of pro-inflammatory or anti-inflammatory factors of the host, thereby affecting inflammation and/or inflammation-induced diseases. Outcomes related to the development and progression of pancreatic tumors.
- the inventor's research also found that these Akkermansia muciniphila have the function of affecting the infiltration amount and half-life of anti-tumor drugs (such as small molecule chemotherapy drugs) into the tumor microenvironment.
- the present invention also provides the use of Akkermansia muciniphila in the preparation of medicaments for preventing and/or treating pancreatic tumors.
- the present invention also provides a composition comprising the Akkermansia muciniphila of the present invention as an active ingredient.
- the composition may also include other types of intestinal bacteria.
- the composition may also include a pharmaceutically acceptable carrier.
- the composition can be food, health care products, additives or pharmaceutical compositions, but is not limited thereto.
- the composition is a pharmaceutical composition, which includes a pharmaceutically effective dose of Akkermansia muciniphila and a pharmaceutically acceptable carrier.
- the present invention also provides a pharmaceutical composition, which includes a pharmaceutically effective dose of Akkermansia muciniphila or its metabolites and a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier includes but is not limited to milk powder, lactose, glucose, sucrose, maltose, sorbitol, mannose, trehalose, galactose, cyclodextrin, starch, gum arabic, calcium phosphate, Alginate, glycerin, sodium glutamate, vitamin C, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, hydroxybenzoic acid Any one or combination of propyl ester, talc, magnesium stearate or mineral oil.
- the Akkermansia muciniphila described in the present invention can be any one or more of the following: live bacteria, dead bacteria, bacterial lysates, metabolites, modified bacteria, Mutant bacteria, mutagenic bacteria, and/or pharmaceutically acceptable salts.
- the salts include acidic or basic inorganic or organic salts.
- the acidic inorganic salts include hydrochloride, sulfate, phosphate, nitrate, carbonate, borate, sulfamate or hydrobromide.
- the basic salt includes sodium salt, potassium salt, lithium salt, magnesium salt, calcium salt or ammonium salt.
- the organic salts include acetate, propionate, butyrate, tartrate, maleate, hydroxymaleate, fumarate, citrate, lactate, mucinate, gluconic acid Salt, benzoate, succinate, oxalate, phenylacetate, methylsulfonate, p-toluenesulfonate, benzenesulfonate, p-aminosalicylate, aspartate, Glutamate, edetate, stearate, palmitate, oleate, laurate, tannate, ascorbate, valerate or alkylammonium salt.
- Akkermansia muciniphila and/or its metabolites according to the present invention can regulate the number and function of T cells including but not limited to CD8+ T cell subsets.
- Akkermansia muciniphila and/or its metabolites according to the present invention can regulate the number and function of T cells including but not limited to CD8+ T cell subsets to tune the body, especially tumors.
- the microenvironment and immune homeostasis help the body fight against tumors, including but not limited to pancreatic tumors.
- Akkermansia muciniphila and/or its metabolites of the present invention can regulate the infiltration and half-life of drugs including but not limited to gemcitabine and other drugs into the tumor microenvironment, thereby enhancing the tumor killing effect of anti-tumor drugs. Function.
- the preparation form of the pharmaceutical composition of the present invention includes, but is not limited to, tablets, capsules, solutions, emulsions, suspensions, powders or granules.
- the pharmaceutical composition of the present invention is administered including but not limited to oral administration, rectal administration or delivery to the colon.
- compositions of the present invention also include pH-sensitive compositions (compositions exhibiting pH-dependent swelling/dissolution properties), which comprise one or more enteric polymers.
- the present invention also provides the use of Akkermansia muciniphila in preparing pharmaceutical compositions for enhancing the efficacy of anti-tumor drugs.
- the anti-tumor drug is a chemotherapeutic drug, including small molecule chemotherapeutic drugs, such as but not limited to any one of gemcitabine, paclitaxel, or a combination thereof.
- the present invention also provides a culture (for example, a culture supernatant) containing Akkermansia muciniphila according to the present invention or a processed product thereof.
- a culture for example, a culture supernatant
- Akkermansia muciniphila according to the present invention or a processed product thereof.
- the Akkermansia muciniphila CGMCC No. 23185 of the present invention has better growth and proliferation capabilities than the ATCC strain and can secrete more palm
- Figure 1 shows a statistical diagram comparing the bacterial density (OD600) of the RV02 strain Akkermansia muciniphila of the present invention compared to the ATCC strain Akkermansia muciniphila under anaerobic culture conditions after 24 hours of cultivation. ;
- Figure 2 shows the results of the RV02 strain Akkermansia muciniphila of the present invention compared to the ATCC strain Akkermansia muciniphila under anaerobic culture conditions after culturing for 24 hours, 48 hours, and 72 hours. Comparative statistical chart of palmitoleic acid content in Qingnai;
- Figure 3 shows typical tumor photos showing the inhibitory effect of the RV02 strain Akkermansia muciniphila of the present invention on pancreatic tumors compared with the ATCC strain Akkermansia muciniphila and physiological saline treatment;
- Figure 4 shows a typical tumor statistical diagram of the inhibitory effect of the RV02 strain Akkermansia muciniphila of the present invention on pancreatic tumors compared with the ATCC strain Akkermansia muciniphila and physiological saline treatment;
- Figure 5 shows a typical tumor statistical diagram of the inhibitory effect of the RV02 Akkermansia muciniphila live bacteria on pancreatic tumors compared to the RV02 Akkermansia muciniphila dead bacteria of the present invention
- Figure 6 shows a typical flow cytometry diagram of the number of CD8+T cells in animals with pancreatic tumors suppressed by the RV02 strain Akkermansia muciniphila of the present invention; the ratio of CD8+T cells relative to the total spleen cells is marked in each Above the flow cytometry chart, the saline group is: 9.99%, the ATCC strain is: 7.45%; the RV02 strain is: 6.05%;
- Figure 7 shows a statistical diagram of the effect of Akkermansia muciniphila of the RV02 strain of the present invention on inhibiting the number of CD8+T cells in animals with pancreatic tumors;
- Figure 8 shows a statistical graph showing that the RV02 strain of Akkermansia muciniphila of the present invention inhibits IFN- ⁇ secretion levels in pancreatic tumor animals;
- Figure 9 shows a statistical diagram of the effect of Akkermansia muciniphila of the RV02 strain of the present invention on increasing gemcitabine (GEM) or paclitaxel (PTX) inhibition of pancreatic tumors;
- GEM gemcitabine
- PTX paclitaxel
- Figure 10 shows the effect of the RV02 strain of Akkermansia muciniphila of the present invention in significantly increasing the amount of gemcitabine in the tumor microenvironment
- Figure 11 shows the effect of the RV02 strain of Akkermansia muciniphila of the present invention on significantly enhancing the half-life of gemcitabine in the tumor microenvironment.
- CGMCC General Microbiology Center of the China Microbial Culture Collection Committee (referred to as CGMCC, address: Beichen West Road, Chaoyang District, Beijing) on August 24, 2021 No. 3, Yard No. 1), the collection number is CGMCC No. 23185.
- Example 1 Screening, isolation and identification of Akkermansia muciniphila RV02
- culturomics technology was used to further analyze, screen, and identify intestinal bacteria, and a type of Akkermansia muciniphila (named strain RV02) was discovered and isolated from healthy humans.
- Akkermansia muciniphila is a bacterium that colonizes the mucus layer in the human intestine and can degrade mucin. It was isolated from human feces by Derrien in 2004 and is present in the human digestive tract, accounting for about 3- 5% is a representative of the Verrucomicrobia phylum, belonging to the genus Akkermansia, and is a Gram-negative strict anaerobe without plasmids. Low levels of Akkermansia muciniphila in the intestine can lead to thinning of the mucosal layer, resulting in a weakened intestinal barrier function, making it easier for intestinal toxins to invade the body.
- Akkermansia muciniphila As a bacterium that prefers to colonize the intestinal mucus layer, Akkermansia muciniphila not only degrades mucin to maintain its own abundance, but also stimulates the thickening of the body's mucus layer to maintain the body's health.
- the present invention is a new strain of Akkermansia muciniphila RV02 selected from a large number of Akkermansia muciniphila strains. Through anaerobic culture, growth curve verification, staining microscopy and animal experiments, it is found that: Compared with the existing published standard strains of Akkermansia muciniphila, RV02 has stronger growth ability, stronger function of inhibiting the occurrence and development of pancreatic and other tumors, and more prominent regulation of the secretion of inflammatory factor INF- ⁇ . and the number and functional capacity of CD8+ T cells.
- Colony characteristics Akkermansia muciniphila RV02 of the present invention is spread on Mucin solid medium and cultured. The colonies are white and slightly convex, with a diameter of about 1-2 mm.
- RV02 The screening and isolation methods for RV02 are as follows:
- Mucin medium The preparation method of Mucin medium is as follows: Weigh 3.85g of brain heart infusion medium (BHI), 0.4g of mucin (Mucin), and 0.05g of L-cysteine, dissolve them in 100 mL of water, and autoclave at 121°C for 20 minutes.
- BHI brain heart infusion medium
- Mucin mucin
- L-cysteine L-cysteine
- (1) Collect 0.5g of a healthy fresh stool sample from the subject, place it in a beaker containing 4.5ml of physiological saline, mix the feces and physiological saline, and then dilute it to obtain a diluent. Take 1 ml of the above dilution and inoculate it into 9 ml of Mucin liquid culture medium, and culture it anaerobically at 37°C for 5 days to obtain the bacterial liquid.
- Reverse primer 5’-GAGTGTTCCGATATCTACGCATTTCA-3’ (SEQ ID NO: 2)
- Amplification system (20 ⁇ L): PCR master mix ( Master Mix (With Dye)) 7 ⁇ L, template DNA 1 ⁇ L, forward primer 1 ⁇ L, reverse primer 1 ⁇ L, sterile deionized water 10 ⁇ L.
- the PCR reaction procedure is as follows:
- the PCR product was electrophoresed on a 2% w/v agarose gel at 100 volts for 20 minutes.
- PCR products were identified by nucleotide sequencing, and the sequence results were compared by BLAST in Genbank. Samples positive for Akkermansia muciniphila were selected for the next step.
- RV02 strain the Akkermansia muciniphila strain provided by the present invention (named: RV02 strain) with the ATCC Akkermansia muciniphila strain (ATCC BAA-835 (Akkermansia muciniphila Derrien et al.), purchased from Beina Bio)'s 16S rDNA sequence, growth kinetic curve and other major microbial indicators, and then use mass spectrometry and non-targeted metabolome detection technology to analyze the metabolic small molecules (such as palmitoleic acid) in their culture supernatants.
- ATCC BAA-835 Akkermansia muciniphila Derrien et al.
- the RV02 strain has a higher bacterial density (OD600) than the ATCC strain Akkermansia muciniphila at the same culture time point in the liquid medium (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001, the following results are the same), which shows that the RV02 strain of Akkermansia muciniphila of the present invention has stronger growth activity and ability than the ATCC strain of Akkermansia muciniphila.
- the palmitoleic acid content in the culture supernatant of the RV02 strain was significantly higher than that in the culture supernatant of the ATCC strain, indicating that the RV02 strain has stronger resistance to Akkermansia muciniphila than the ATCC strain.
- the ability to express or produce immunomodulatory substances such as palmitoleic acid shows that the RV02 strain of Akkermansia muciniphila discovered in the present invention may have advantageous growth and stronger immunomodulatory functions obtained in the body environment.
- mice Twenty-four 1-month-old C57BL/6 mice were purchased from the Guangdong Provincial Experimental Animal Center. The mice were randomly divided into 3 groups, with 8 mice in each group. The mice were given drinking water containing ampicillin (1mg/ml), streptomycin (5mg/ml) and colistin (1mg/ml) for one week. After one week, The mice in each group were subcutaneously injected with the same number of Panc02 pancreatic tumor cells (purchased from Beina Biotechnology, Cat. No. BNCC338034). The first group was gavaged with normal saline as a control, and the second group was gavaged with ATCC strain Akkermansia muciniphila alive.
- the tumor volume of mice administered with the ATCC strain of Akkermansia muciniphila was significantly smaller than that of the saline control group, but the tumor volume of mice injected with live bacteria of the RV02 strain of Akkermansia muciniphila
- the tumors in mice with the ATCC strain Akkermansia muciniphila were smaller than those in mice. This shows that oral administration of the live Akkermansia muciniphila strain RV02 of the present invention can effectively inhibit the growth of pancreatic tumors.
- mice Twenty-four 1-month-old C57BL/6 mice were purchased from the Guangdong Provincial Experimental Animal Center. The mice were randomly divided into 3 groups, with 8 mice in each group. The mice were given drinking water containing ampicillin (1mg/ml), streptomycin (5mg/ml) and colistin (1mg/ml) for one week. After one week, The mice in each group were subcutaneously injected with the same number of Panc02 pancreatic tumor cells (purchased from Beina Biotechnology, Cat. No.
- BNCC338034 and the first group was orally administered an equal amount of live bacteria of Akkermansia muciniphila strain RV02 (1 ⁇ 10 9 CFU /time/mouse), the second group was gavaged with an equal amount of dead bacteria of Akkermansia muciniphila strain RV02 (1 ⁇ 10 9 CFU/time/mouse), once every two days for a total of Oral administration for 4 weeks. Normal saline was administered intragastrically as a control. The volume and weight of pancreatic tumors in each group were then analyzed.
- the preparation method of the RV02 strain Akkermansia muciniphila dead bacteria according to the present invention is: placing the RV02 strain Akkermansia muciniphila live bacteria in a 75°C water bath for 10 minutes for pasteurization, and then obtain Dead bacteria of Akkermansia muciniphila strain RV02.
- the tumor volume of mice given live RV02 Akkermansia muciniphila strain and dead Akkermansia muciniphila strain RV02 strain was significantly smaller, but The tumor volume of mice infused with live bacteria of the RV02 Akkermansia muciniphila strain was slightly smaller than that of mice with dead bacteria of the RV02 Akkermansia muciniphila strain, indicating that the dead bacteria have similar tumor inhibition effects to the live bacteria. Effect. This shows that oral administration of both live and dead Akkermansia muciniphila strain RV02 of the present invention can effectively inhibit the growth of pancreatic tumors.
- the RV02 strain of Akkermansia muciniphila live bacteria provided by the present invention can more effectively inhibit the number of CD8+ T cells and the secretion of the inflammatory factor IFN- ⁇ , thereby inhibiting the pancreas.
- This discovery provides new ideas for the treatment of pancreatic tumors.
- mice Thirty-six 1-month-old C57BL/6 mice were purchased from the Guangdong Provincial Experimental Animal Center. The mice were randomly divided into 6 groups, with 6 mice in each group. The mice were given drinking water containing ampicillin (1mg/ml), streptomycin (5mg/ml) and colistin (1mg/ml) for one week. After one week, The mice in each group were subcutaneously inoculated with the same number of Panc02 pancreatic tumor cells. The first group was given normal saline as a control, and the second group was given RV02 strain of Akkermansia muciniphila live bacteria (1 ⁇ 10 9 CFU/time).
- group 3 was orally administered live bacteria of Akkermansia muciniphila strain RV02 (1 ⁇ 10 9 CFU/time/mouse) and intraperitoneally injected with gemcitabine (GEM, 50mg/Kg/time) /week), the 4th group was intraperitoneally injected with gemcitabine (GEM, 50mg/Kg/time/week), and the 5th group was intragastrically injected with live bacteria of Akkermansia muciniphila strain RV02 (1 ⁇ 10 9 CFU/time/ mice) and received intraperitoneal injection of Paclitaxel (PTX). Group 6 received intraperitoneal injection of Paclitaxel, and the intestinal bacteria were gavaged every two days for a total of 4 weeks. Then the volume and weight of pancreatic tumors, as well as mouse tumors, were analyzed. The immune function and status of internal and peripheral CD4+T cells and CD8+T cells.
- mice Sixty-four 1-month-old C57BL/6 mice were purchased from the Guangdong Provincial Experimental Animal Center. The mice were randomly divided into 4 groups, with 16 mice in each group. The mice were given drinking water containing ampicillin (1mg/ml), streptomycin (5mg/ml) and colistin (1mg/ml) for one week. After one week, The mice in each group were subcutaneously inoculated with the same number of Panc02 pancreatic tumor cells. The first group was given normal saline as a control, and the second group was given RV02 strain of Akkermansia muciniphila live bacteria (1 ⁇ 10 9 CFU/time).
- group 3 was orally administered live bacteria of Akkermansia muciniphila strain RV02 (1 ⁇ 10 9 CFU/time/mouse) and intraperitoneally injected with gemcitabine (GEM, 50mg/Kg/time) ), group 4 received intraperitoneal injection of gemcitabine (GEM, 50 mg/Kg/time).
- Live bacteria of Akkermansia muciniphila strain RV02 were administered into the stomach once every two days for a total of 4 weeks. Before the administration of live bacteria of Akkermansia muciniphila strain RV02 and after 1 week, 2 weeks, 3 weeks and 4 weeks of live bacteria of Akkermansia muciniphila strain RV02, 4 animals from each group were taken each time. Mice, take the tumors, peripheral blood, spleen, intestines and other tissues of the mice to analyze and compare the content and half-life of gemcitabine in different parts of the mouse's body (especially within the tumor tissue).
- the RV02 strain of Akkermansia muciniphila of the present invention can not only effectively inhibit the occurrence and development of pancreatic tumors, but also enhance the infiltration of anti-tumor drugs (such as chemotherapy drugs) into the pancreatic tumor microenvironment, thereby solving the problem of pancreatic tumor chemotherapy. It is difficult for drugs to reach and kill tumor cells.
- anti-tumor drugs such as chemotherapy drugs
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Abstract
An Akkermansia muciniphila, with an accession number of CGMCC No. 23185. Use of the Akkermansia muciniphila in preparing a drug for preventing and/or treating a pancreatic tumor. A composition, comprising the Akkermansia muciniphila as an active ingredient. Use of the Akkermansia muciniphila in preparing a pharmaceutical composition for enhancing the efficacy of an anti-tumor drug. A culture or a processing product thereof comprising the Akkermansia muciniphila. The Akkermansia muciniphila has better growth and proliferation ability, can secrete more key immunoregulatory substances such as palmitoleic acid and the like, more effectively inhibit the number of CD8+ T cells to further inhibit the growth of pancreatic tumor cells, and also significantly increase the concentration of a chemotherapeutic drug in a pancreatic tumor microenvironment, so that the efficacy of the anti-tumor drug is enhanced.
Description
本发明属于生物医药领域，具体涉及一种嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)，以及该嗜粘蛋白阿克曼氏菌在制备用于预防和/或治疗胰腺肿瘤的药物中的应用。The invention belongs to the field of biomedicine, and specifically relates to Akkermansia muciniphila and the use of Akkermansia muciniphila in the preparation of drugs for preventing and/or treating pancreatic tumors. .
胰腺癌是常见的消化系统恶性肿瘤之一，恶性程度高、预后极差。中国胰腺癌年发病人数约12万人，发达经济体例如美国的年发病人数约6万人。2015年至2020年，中国胰腺癌新发病例增长31.6％左右，死亡病例增加43.5％左右，患者新发病例与死亡病例几乎持平，患者确诊时多为晚期。胰腺导管腺癌(PDAC)最为常见而且非常致命，确诊一年后存活率低于2-5％，亟需有效的治疗方案。Pancreatic cancer is one of the common malignant tumors of the digestive system, with high malignancy and extremely poor prognosis. The annual incidence of pancreatic cancer in China is about 120,000, and in developed economies such as the United States, the annual incidence is about 60,000. From 2015 to 2020, new cases of pancreatic cancer in China increased by about 31.6%, and deaths increased by about 43.5%. New cases and deaths were almost the same, and most patients were diagnosed at an advanced stage. Pancreatic ductal adenocarcinoma (PDAC) is the most common and very deadly, with a survival rate of less than 2-5% one year after diagnosis. Effective treatment options are urgently needed.
胰腺被胃与动脉包围，手术风险大，切除术只适用于早期肿瘤(大约15％)，切除后，癌症仍有极大机率复发。目前国内外指南主要推荐白蛋白紫杉醇联合吉西他滨作为PDAC的一线治疗，但疗效有限且化疗耐药作用难以避免。另一个可能的胰腺癌治疗新方向是免疫检查点抑制剂联合化疗的协同增效，然而，事实上免疫检查点疗法临床上收效仍然不够显著，这同时说明胰腺癌的病理机制与其他实体恶性肿瘤有很大的不同。此外，靶向针对细胞外基质(ECM)的新型胰腺肿瘤疗法临床试验也以失败告终。因此，亟需开发抗胰腺癌的新型疗法。The pancreas is surrounded by the stomach and arteries, so surgery is risky. Resection is only suitable for early-stage tumors (about 15%). After resection, there is still a high chance of cancer recurrence. At present, domestic and foreign guidelines mainly recommend albumin-paclitaxel combined with gemcitabine as first-line treatment for PDAC, but the efficacy is limited and chemotherapy resistance is unavoidable. Another possible new direction in the treatment of pancreatic cancer is the synergistic effect of immune checkpoint inhibitors combined with chemotherapy. However, in fact, the clinical effect of immune checkpoint therapy is still not significant enough. This also shows that the pathological mechanism of pancreatic cancer is different from other solid malignant tumors. there is a big difference. In addition, clinical trials of new pancreatic tumor therapies targeting the extracellular matrix (ECM) also ended in failure. Therefore, there is an urgent need to develop new therapies against pancreatic cancer.
要开发胰腺癌的新型疗法，必须重新认识胰腺癌的发病机理，并从新的机理入手去开发新的疗法。越来越多的证据证实，肠道微生态，特别是肠道菌的构成、丰度、结构与功能是决定人体免疫稳态并影响肿瘤发生、发展的关键因素。例如，最新一些科研进展显示，补充优选的某种或某几种肠道菌可以有效地增强对肿瘤微环境的扰动能力，并增强抗肿瘤免疫疗法的疗效。但是，究竟何种肠道微生物能够有效遏制胰腺肿瘤的发生发展仍然未见有报道。To develop new treatments for pancreatic cancer, we must re-understand the pathogenesis of pancreatic cancer and develop new treatments based on new mechanisms. More and more evidence confirms that the intestinal microecology, especially the composition, abundance, structure and function of intestinal bacteria, is a key factor in determining human immune homeostasis and affecting the occurrence and development of tumors. For example, some recent scientific research progress shows that supplementing a certain type of selected intestinal bacteria or certain types of intestinal bacteria can effectively enhance the ability to perturb the tumor microenvironment and enhance the efficacy of anti-tumor immunotherapy. However, there is still no report on which intestinal microorganisms can effectively curb the occurrence and development of pancreatic tumors.
最近胰腺癌研究的一系列进展显示，胰腺癌患者的血清中含有针对某些消化道细菌的抗体，胰腺肿瘤组织内含有丰富而又紊乱的细菌或者真菌构成、丰度、结构与功能。这些科学进展提示，微生物，特别是消化道细菌，可能是影响胰腺肿瘤发生发展的关键病理生理因素，揭示消化道或者肠道细菌是否并如何影响胰腺肿瘤的发生发展，从而可以为开发抗胰腺肿瘤提供全新的有力武器。A series of recent advances in pancreatic cancer research have shown that the serum of pancreatic cancer patients contains antibodies against certain gastrointestinal bacteria, and pancreatic tumor tissue contains rich and disordered bacterial or fungal composition, abundance, structure, and function. These scientific advances suggest that microorganisms, especially gastrointestinal bacteria, may be key pathophysiological factors affecting the occurrence and development of pancreatic tumors, revealing whether and how digestive tract or intestinal bacteria affect the occurrence and development of pancreatic tumors, which can provide insights into the development of anti-pancreatic tumors. Provides new and powerful weapons.
然而，目前尚未见到任何利用干预消化道或者肠道微生态进行胰腺肿瘤预防或者治疗的 报道。However, there have not yet been any reports on the use of intervention in the digestive tract or intestinal microecology for the prevention or treatment of pancreatic tumors.
发明内容Contents of the invention
本发明的一个目的是针对以上要解决的技术问题，提供一种能够针对胰腺肿瘤进行有效预防和/或治疗的肠道菌。One object of the present invention is to provide an intestinal bacterium that can effectively prevent and/or treat pancreatic tumors in view of the above technical problems to be solved.
为了实现以上目的，本发明提供了一株嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)，其保藏号为CGMCC No.23185。In order to achieve the above objects, the present invention provides a strain of Akkermansia muciniphila, whose deposit number is CGMCC No. 23185.
本发明所述的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)是具有肿瘤微环境或者系统性CD8+T细胞免疫功能调控活性的菌株。该嗜粘蛋白阿克曼氏菌能够抑制肿瘤微环境或者系统CD8+T细胞的数量和/或表达IFN-γ等炎症效应因子的效应功能，为此，本发明提供了所述嗜粘蛋白阿克曼氏菌在制备用于预防和/或治疗炎症及其导致、伴随、相关、诱导、反馈的疾病的药物中的应用。与此同时，本发明提供的嗜粘蛋白阿克曼氏菌还能够有效增强其它抗肿瘤药物(如小分子化疗药物)在胰腺肿瘤微环境内的浸润进而增强该抗肿瘤药物的杀肿瘤能力。Akkermansia muciniphila according to the present invention is a strain with tumor microenvironment or systemic CD8+ T cell immune function regulation activity. Akkermansia muciniphila can inhibit the number of CD8+ T cells in the tumor microenvironment or system and/or the effector function of expressing inflammatory effector factors such as IFN-γ. To this end, the present invention provides the Akkermansia muciniphila Use of Kermanella in the preparation of medicaments for the prevention and/or treatment of inflammation and diseases causing, accompanying, related, inducing, feedback. At the same time, the Akkermansia muciniphila provided by the present invention can also effectively enhance the infiltration of other anti-tumor drugs (such as small molecule chemotherapy drugs) into the pancreatic tumor microenvironment and thereby enhance the tumor-killing ability of the anti-tumor drugs.
优选地，本发明所述的嗜粘蛋白阿克曼氏菌为以下中的任意一种：嗜粘蛋白阿克曼氏菌的临床分离菌株；嗜粘蛋白阿克曼氏菌的活菌、死菌、细菌裂解物、代谢产物、改造菌、突变菌和/或诱变菌。发明人研究发现，以上这些嗜粘蛋白阿克曼氏菌具有CD8+T细胞免疫功能调控活性并进而影响宿主的促炎或抑炎因子的表达和分泌，进而影响炎症和/或由炎症引起的相关胰腺肿瘤发生发展的结局。发明人的研究还发现了以上这些嗜粘蛋白阿克曼氏菌具有影响抗肿瘤药物(如小分子化疗药物)到肿瘤微环境的浸润量及半衰期的功能。Preferably, the Akkermansia muciniphila of the present invention is any one of the following: a clinical isolate of Akkermansia muciniphila; a live or dead strain of Akkermansia muciniphila. Bacteria, bacterial lysates, metabolites, modified bacteria, mutant bacteria and/or mutagen bacteria. The inventor's research has found that the above Akkermansia muciniphila have CD8+ T cell immune function regulation activity and thereby affect the expression and secretion of pro-inflammatory or anti-inflammatory factors of the host, thereby affecting inflammation and/or inflammation-induced diseases. Outcomes related to the development and progression of pancreatic tumors. The inventor's research also found that these Akkermansia muciniphila have the function of affecting the infiltration amount and half-life of anti-tumor drugs (such as small molecule chemotherapy drugs) into the tumor microenvironment.
另一方面，本发明还提供了所述嗜粘蛋白阿克曼氏菌在制备用于预防和/或治疗胰腺肿瘤的药物中的应用。On the other hand, the present invention also provides the use of Akkermansia muciniphila in the preparation of medicaments for preventing and/or treating pancreatic tumors.
另一方面，本发明还提供了一种组合物，其包括本发明所述的嗜粘蛋白阿克曼氏菌作为活性成分。优选地，该组合物还还可以包括其他种类的肠道菌。优选地，该组合物还可以包括药学上可接受的载体。In another aspect, the present invention also provides a composition comprising the Akkermansia muciniphila of the present invention as an active ingredient. Preferably, the composition may also include other types of intestinal bacteria. Preferably, the composition may also include a pharmaceutically acceptable carrier.
优选地，该组合物可以是食品、保健品、添加剂或药物组合物，但不仅限于于此。Preferably, the composition can be food, health care products, additives or pharmaceutical compositions, but is not limited thereto.
优选地，该组合物为药物组合物，该药物组合物包括药学有效剂量的嗜粘蛋白阿克曼氏菌以及在药学上可接受的载体。Preferably, the composition is a pharmaceutical composition, which includes a pharmaceutically effective dose of Akkermansia muciniphila and a pharmaceutically acceptable carrier.
另一方面，本发明还提供了一种药物组合物，该药物组合物包括药学有效剂量的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)或其代谢物以及在药学上可接受的载体。On the other hand, the present invention also provides a pharmaceutical composition, which includes a pharmaceutically effective dose of Akkermansia muciniphila or its metabolites and a pharmaceutically acceptable carrier.
优选地，所述药学上可接受的载体包括但不限于奶粉、乳糖、葡萄糖、蔗糖、麦芽糖、山梨糖醇、甘露糖、海藻糖、半乳糖、环糊精、淀粉、阿拉伯胶、磷酸钙、藻酸盐、甘油、谷氨酸钠、维生素C、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任意一种或多种的组合。Preferably, the pharmaceutically acceptable carrier includes but is not limited to milk powder, lactose, glucose, sucrose, maltose, sorbitol, mannose, trehalose, galactose, cyclodextrin, starch, gum arabic, calcium phosphate, Alginate, glycerin, sodium glutamate, vitamin C, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, hydroxybenzoic acid Any one or combination of propyl ester, talc, magnesium stearate or mineral oil.
本发明所述是嗜粘蛋白阿克曼氏菌可以是以下中的任意一种或多种：嗜粘蛋白阿克曼氏菌的活菌、死菌、细菌裂解物、代谢产物、改造菌、突变菌、诱变菌、和/或药学上可接受的盐。The Akkermansia muciniphila described in the present invention can be any one or more of the following: live bacteria, dead bacteria, bacterial lysates, metabolites, modified bacteria, Mutant bacteria, mutagenic bacteria, and/or pharmaceutically acceptable salts.
所述盐包括酸式或碱式无机盐或有机盐。The salts include acidic or basic inorganic or organic salts.
所述酸式无机盐包括盐酸盐、硫酸盐、磷酸盐、硝酸盐、碳酸盐、硼酸盐、氨基磺酸盐或氢溴酸盐。所述碱式盐包括钠盐、钾盐、锂盐、镁盐、钙盐或铵盐。The acidic inorganic salts include hydrochloride, sulfate, phosphate, nitrate, carbonate, borate, sulfamate or hydrobromide. The basic salt includes sodium salt, potassium salt, lithium salt, magnesium salt, calcium salt or ammonium salt.
所述有机盐包括乙酸盐、丙酸盐、丁酸盐、酒石酸盐、马来酸盐、羟基马来酸盐、富马酸盐、柠檬酸盐、乳酸盐、粘液酸盐、葡萄糖酸盐、苯甲酸盐、琥珀酸盐、草酸盐、苯乙酸盐、甲基磺酸盐、对甲苯磺酸盐、苯磺酸盐、对氨基水杨酸盐、天门冬氨酸盐，谷氨酸盐、依地酸盐、硬脂酸盐、棕榈酸盐、油酸盐、月桂酸盐、鞣酸盐、抗坏血酸盐、戊酸盐或烷铵盐。The organic salts include acetate, propionate, butyrate, tartrate, maleate, hydroxymaleate, fumarate, citrate, lactate, mucinate, gluconic acid Salt, benzoate, succinate, oxalate, phenylacetate, methylsulfonate, p-toluenesulfonate, benzenesulfonate, p-aminosalicylate, aspartate, Glutamate, edetate, stearate, palmitate, oleate, laurate, tannate, ascorbate, valerate or alkylammonium salt.
此外，本发明所述的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)和/或其代谢物可以调节包括但不限于CD8+T细胞亚群等T细胞的数量及功能。In addition, Akkermansia muciniphila and/or its metabolites according to the present invention can regulate the number and function of T cells including but not limited to CD8+ T cell subsets.
此外，本发明所述的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)和/或其代谢物可以调控包括但不限于CD8+T细胞亚群等T细胞的数量及功能以调谐机体特别是肿瘤微环境及的免疫稳态，有助于机体对抗包括但不限于胰腺肿瘤在内的肿瘤。In addition, Akkermansia muciniphila and/or its metabolites according to the present invention can regulate the number and function of T cells including but not limited to CD8+ T cell subsets to tune the body, especially tumors. The microenvironment and immune homeostasis help the body fight against tumors, including but not limited to pancreatic tumors.
此外，本发明所述的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)和/或其代谢物可以调控包括但不限于吉西他滨等药物到肿瘤微环境的浸润、半衰期进而增强抗肿瘤药物的杀肿瘤功能。In addition, Akkermansia muciniphila and/or its metabolites of the present invention can regulate the infiltration and half-life of drugs including but not limited to gemcitabine and other drugs into the tumor microenvironment, thereby enhancing the tumor killing effect of anti-tumor drugs. Function.
优选地，本发明所述的药物组合物的制剂形式包括但不限于片剂、胶囊剂、溶液、乳液、混悬液、粉剂或颗粒剂。Preferably, the preparation form of the pharmaceutical composition of the present invention includes, but is not limited to, tablets, capsules, solutions, emulsions, suspensions, powders or granules.
优选地，本发明所述的药物组合物的给药方式包括但不限于口服给药、直肠给药或递送至结肠。Preferably, the pharmaceutical composition of the present invention is administered including but not limited to oral administration, rectal administration or delivery to the colon.
优选地，本发明所述的药物组合物还包括pH敏感组合物(表现出pH依赖的溶胀/溶解特性的组合物)，其包含一种或多种肠溶聚合物。Preferably, the pharmaceutical compositions of the present invention also include pH-sensitive compositions (compositions exhibiting pH-dependent swelling/dissolution properties), which comprise one or more enteric polymers.
另一方面，本发明还提供了所述的嗜粘蛋白阿克曼氏菌在制备用于增强抗肿瘤药物功效的医药组合物方面的应用。On the other hand, the present invention also provides the use of Akkermansia muciniphila in preparing pharmaceutical compositions for enhancing the efficacy of anti-tumor drugs.
优选地，所述抗肿瘤药物是化疗药物，包括小分子化疗药物，例如但不限于吉西他滨、紫杉醇中的任意一种或其组合。Preferably, the anti-tumor drug is a chemotherapeutic drug, including small molecule chemotherapeutic drugs, such as but not limited to any one of gemcitabine, paclitaxel, or a combination thereof.
另一方面，本发明还提供了一种包含本发明所述的嗜粘蛋白阿克曼氏菌的培养物(例如培养上清)或其加工物。On the other hand, the present invention also provides a culture (for example, a culture supernatant) containing Akkermansia muciniphila according to the present invention or a processed product thereof.
与现有的普通阿克曼氏菌相比，本发明的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)CGMCC No.23185具有比ATCC株更好的生长和增殖能力，能够分泌更多的棕榈油酸等关键免疫调节物质的能力，并且能够更有效地抑制CD8+T细胞的数量进而遏制胰腺肿瘤细胞的生长，还能够使胰腺肿瘤微环境内的化疗药物浓度得到显著增加，从而增强抗肿瘤药物的功效。Compared with the existing Akkermansia vulgaris, the Akkermansia muciniphila CGMCC No. 23185 of the present invention has better growth and proliferation capabilities than the ATCC strain and can secrete more palm The ability of key immunomodulatory substances such as oleic acid to more effectively inhibit the number of CD8+ T cells and thus curb the growth of pancreatic tumor cells. It can also significantly increase the concentration of chemotherapy drugs in the pancreatic tumor microenvironment, thereby enhancing anti-tumor Efficacy of the drug.
图1示出了本发明的RV02株嗜粘蛋白阿克曼氏菌相比ATCC株嗜粘蛋白阿克曼氏菌在厌氧培养条件下，培养24小时后，细菌密度(OD600)比较统计图；Figure 1 shows a statistical diagram comparing the bacterial density (OD600) of the RV02 strain Akkermansia muciniphila of the present invention compared to the ATCC strain Akkermansia muciniphila under anaerobic culture conditions after 24 hours of cultivation. ;
图2示出了本发明的RV02株嗜粘蛋白阿克曼氏菌相比ATCC株嗜粘蛋白阿克曼氏菌在厌氧培养条件下，培养24小时、48小时、72小时后，培养上清内棕榈油酸的含量比较统计图；Figure 2 shows the results of the RV02 strain Akkermansia muciniphila of the present invention compared to the ATCC strain Akkermansia muciniphila under anaerobic culture conditions after culturing for 24 hours, 48 hours, and 72 hours. Comparative statistical chart of palmitoleic acid content in Qingnai;
图3示出了本发明的RV02株嗜粘蛋白阿克曼氏菌相比ATCC株嗜粘蛋白阿克曼氏菌及生理盐水处理对胰腺肿瘤的抑制效果的典型肿瘤照片图；Figure 3 shows typical tumor photos showing the inhibitory effect of the RV02 strain Akkermansia muciniphila of the present invention on pancreatic tumors compared with the ATCC strain Akkermansia muciniphila and physiological saline treatment;
图4示出了本发明的RV02株嗜粘蛋白阿克曼氏菌相比ATCC株嗜粘蛋白阿克曼氏菌及生理盐水处理对胰腺肿瘤的抑制效果的典型肿瘤统计图；Figure 4 shows a typical tumor statistical diagram of the inhibitory effect of the RV02 strain Akkermansia muciniphila of the present invention on pancreatic tumors compared with the ATCC strain Akkermansia muciniphila and physiological saline treatment;
图5示出了本发明的RV02株嗜粘蛋白阿克曼氏菌活菌相比RV02株嗜粘蛋白阿克曼氏菌死菌对胰腺肿瘤的抑制效果的典型肿瘤统计图；Figure 5 shows a typical tumor statistical diagram of the inhibitory effect of the RV02 Akkermansia muciniphila live bacteria on pancreatic tumors compared to the RV02 Akkermansia muciniphila dead bacteria of the present invention;
图6示出了本发明的RV02株嗜粘蛋白阿克曼氏菌抑制胰腺肿瘤动物中CD8+T细胞的数量典型流式细胞图；CD8+T细胞相对于总体脾脏细胞的比例标注在每一个流式细胞图的上方，盐水组为：9.99％，ATCC株为：7.45％；RV02株为：6.05％；Figure 6 shows a typical flow cytometry diagram of the number of CD8+T cells in animals with pancreatic tumors suppressed by the RV02 strain Akkermansia muciniphila of the present invention; the ratio of CD8+T cells relative to the total spleen cells is marked in each Above the flow cytometry chart, the saline group is: 9.99%, the ATCC strain is: 7.45%; the RV02 strain is: 6.05%;
图7示出了本发明的RV02株嗜粘蛋白阿克曼氏菌抑制胰腺肿瘤动物中CD8+T细胞的数量的效果的统计图；Figure 7 shows a statistical diagram of the effect of Akkermansia muciniphila of the RV02 strain of the present invention on inhibiting the number of CD8+T cells in animals with pancreatic tumors;
图8示出了本发明的RV02株嗜粘蛋白阿克曼氏菌抑制胰腺肿瘤动物中IFN-γ分泌水平的统计图；Figure 8 shows a statistical graph showing that the RV02 strain of Akkermansia muciniphila of the present invention inhibits IFN-γ secretion levels in pancreatic tumor animals;
图9示出了本发明的RV02株嗜粘蛋白阿克曼氏菌增加吉西他滨(GEM)或紫杉醇(PTX)对胰腺肿瘤抑制的效果统计图；Figure 9 shows a statistical diagram of the effect of Akkermansia muciniphila of the RV02 strain of the present invention on increasing gemcitabine (GEM) or paclitaxel (PTX) inhibition of pancreatic tumors;
图10示出了本发明的RV02株嗜粘蛋白阿克曼氏菌显著增加肿瘤微环境内吉西他滨量的效果；Figure 10 shows the effect of the RV02 strain of Akkermansia muciniphila of the present invention in significantly increasing the amount of gemcitabine in the tumor microenvironment;
图11示出了本发明的RV02株嗜粘蛋白阿克曼氏菌显著增强肿瘤微环境内吉西他滨半衰期的效果。Figure 11 shows the effect of the RV02 strain of Akkermansia muciniphila of the present invention on significantly enhancing the half-life of gemcitabine in the tumor microenvironment.
本发明的嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)RV02已于2021年8月24日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称为CGMCC，地址：北京市朝阳区北辰西路1号院3号)，保藏号为CGMCC No.23185。Akkermansia muciniphila RV02 of the present invention has been deposited in the General Microbiology Center of the China Microbial Culture Collection Committee (referred to as CGMCC, address: Beichen West Road, Chaoyang District, Beijing) on August 24, 2021 No. 3, Yard No. 1), the collection number is CGMCC No. 23185.
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明，而非用于限制本发明的范围。为简明起见，以下实施例中对本领域技术人员熟知的常规技术操作(如流式细胞仪检测、质谱分析检测、非靶向代谢组检测技术等)的具体步骤并未进行详述，但应理解，这些操作均为本领域技术人员所知并且是可实现的。The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the sake of simplicity, the specific steps of conventional technical operations (such as flow cytometry detection, mass spectrometry detection, non-targeted metabolome detection technology, etc.) well known to those skilled in the art are not described in detail in the following examples, but it should be understood that , these operations are known to those skilled in the art and can be implemented.
实施例1：嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)RV02的筛选、分离和鉴定Example 1: Screening, isolation and identification of Akkermansia muciniphila RV02
招募胰腺导管癌患者进行实验，并将其与健康人作比较，利用16S rRNA测序等方法发现了健康人群粪标本中具有更高丰度的嗜粘蛋白阿克曼氏菌。Patients with pancreatic ductal cancer were recruited for experiments and compared with healthy people. Using methods such as 16S rRNA sequencing, it was found that Akkermansia muciniphila was more abundant in fecal samples of healthy people.
进一步利用培养组学技术对其肠道菌进行进一步的分析、筛查、鉴定，从健康人体内发现并分离了一种嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila，命名为RV02株)。Furthermore, culturomics technology was used to further analyze, screen, and identify intestinal bacteria, and a type of Akkermansia muciniphila (named strain RV02) was discovered and isolated from healthy humans.
嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)是人体肠道中一种定植在粘液层可降解粘蛋白的细菌，2004年由Derrien从人类粪便中分离，存在于人体消化道中，约占比3-5％，是疣微菌门的代表，从属于Akkermansia属，为革兰氏阴性严格厌氧菌无质粒。肠道中低水平的嗜粘蛋白阿克曼氏菌可导致粘膜层的变薄，从而导致肠道屏障功能减弱，使肠道内的毒素更容易侵入人体。作为偏好定植在肠道粘液层中的细菌，嗜粘蛋白阿克曼氏菌既降解粘蛋白维持自身丰度，又刺激机体粘液层增厚，维持机体健康状况。Akkermansia muciniphila is a bacterium that colonizes the mucus layer in the human intestine and can degrade mucin. It was isolated from human feces by Derrien in 2004 and is present in the human digestive tract, accounting for about 3- 5% is a representative of the Verrucomicrobia phylum, belonging to the genus Akkermansia, and is a Gram-negative strict anaerobe without plasmids. Low levels of Akkermansia muciniphila in the intestine can lead to thinning of the mucosal layer, resulting in a weakened intestinal barrier function, making it easier for intestinal toxins to invade the body. As a bacterium that prefers to colonize the intestinal mucus layer, Akkermansia muciniphila not only degrades mucin to maintain its own abundance, but also stimulates the thickening of the body's mucus layer to maintain the body's health.
本发明是从大量嗜粘蛋白阿克曼氏菌菌株中筛选出的一株新的嗜粘蛋白阿克曼氏菌RV02，经过厌氧培养、生长曲线验证、染色镜检及动物实验发现：与现有的已发表的嗜粘蛋白阿克曼氏菌标准菌株相比，RV02具有更强的生长能力，更强的抑制胰腺等肿瘤发生发展的功能，和更突出的调控炎症因子INF-γ分泌和CD8+T细胞的数量与功能的能力。The present invention is a new strain of Akkermansia muciniphila RV02 selected from a large number of Akkermansia muciniphila strains. Through anaerobic culture, growth curve verification, staining microscopy and animal experiments, it is found that: Compared with the existing published standard strains of Akkermansia muciniphila, RV02 has stronger growth ability, stronger function of inhibiting the occurrence and development of pancreatic and other tumors, and more prominent regulation of the secretion of inflammatory factor INF-γ. and the number and functional capacity of CD8+ T cells.
菌落特征：将本发明的嗜粘蛋白阿克曼氏菌RV02涂布于Mucin固体培养基培养，菌落呈白色微凸状，直径约1-2mm。Colony characteristics: Akkermansia muciniphila RV02 of the present invention is spread on Mucin solid medium and cultured. The colonies are white and slightly convex, with a diameter of about 1-2 mm.
显微镜下形态：嗜粘蛋白阿克曼氏菌RV02进行革兰染色后镜检，镜下呈现椭圆形无鞭毛状，为革兰阴性菌。Morphology under the microscope: Akkermansia muciniphila RV02 was examined under the microscope after Gram staining. Under the microscope, it showed an oval shape without flagella, indicating that it was a Gram-negative bacterium.
RV02的筛选和分离方法如下：The screening and isolation methods for RV02 are as follows:
1、培养基配置1. Medium configuration
Mucin培养基制备方法为：称取3.85g脑心浸液培养基(BHI)、0.4g粘蛋白(Mucin)、0.05g L-半胱氨酸溶于100mL水中，121℃高压灭菌20分钟。The preparation method of Mucin medium is as follows: Weigh 3.85g of brain heart infusion medium (BHI), 0.4g of mucin (Mucin), and 0.05g of L-cysteine, dissolve them in 100 mL of water, and autoclave at 121°C for 20 minutes.
2、分离筛选2. Separation and screening
(1)采集受试人员的健康新鲜粪便样本0.5g，置于盛有4.5ml生理盐水的烧杯中，粪便和生理盐水混匀后稀释得到稀释液。取上述稀释液1ml接种于9ml Mucin液体培养基中，37℃厌氧培养5天后得到菌液。(1) Collect 0.5g of a healthy fresh stool sample from the subject, place it in a beaker containing 4.5ml of physiological saline, mix the feces and physiological saline, and then dilute it to obtain a diluent. Take 1 ml of the above dilution and inoculate it into 9 ml of Mucin liquid culture medium, and culture it anaerobically at 37°C for 5 days to obtain the bacterial liquid.
(2)PCR鉴定(2)PCR identification
嗜粘蛋白阿克曼氏菌特异性PCR引物序列如下Akkermansia muciniphila -specific PCR primer sequences are as follows
正向引物：5’-GCGTAGGCTGTTTCGTAAGTCGTGTGTGAAAG-3’(SEQ ID NO：1)Forward primer: 5’-GCGTAGGCTGTTTCGTAAGTCGTGTGTGAAAG-3’ (SEQ ID NO: 1)
反向引物：5’-GAGTGTTCCCGATATCTACGCATTTCA-3’(SEQ ID NO：2)Reverse primer: 5’-GAGTGTTCCGATATCTACGCATTTCA-3’ (SEQ ID NO: 2)
PCR扩增步骤：PCR amplification steps:
取菌液于PBS中进行稀释，提取细菌DNA，作为PCR的模板DNA，使用上述特异性PCR引物进行扩增。Dilute the bacterial solution in PBS, extract bacterial DNA, use it as template DNA for PCR, and use the above-mentioned specific PCR primers for amplification.
扩增体系(20μL)：PCR预混液(
Master Mix(With Dye))7μL，模板DNA 1μL，正向引物1μL，反向引物1μL，无菌去离子水10μL。
Amplification system (20μL): PCR master mix ( Master Mix (With Dye)) 7 μL, template DNA 1 μL, forward primer 1 μL, reverse primer 1 μL, sterile deionized water 10 μL.
PCR反应程序如下：The PCR reaction procedure is as follows:
PCR产物于2％w/v的琼脂糖凝胶跑电泳，电泳条件为100伏，20分钟。The PCR product was electrophoresed on a 2% w/v agarose gel at 100 volts for 20 minutes.
将PCR产物进行核苷酸测序鉴定，序列结果在Genbank中进行BLAST比对，选取嗜粘 蛋白阿克曼氏菌阳性的样本进行下一步操作。The PCR products were identified by nucleotide sequencing, and the sequence results were compared by BLAST in Genbank. Samples positive for Akkermansia muciniphila were selected for the next step.
挑取PCR反应阳性的菌液样本，划线接种于Mucin固体培养基平皿，刮取生长出的单菌落全部进行16S rDNA基因全长的PCR鉴定，扩增产物的测序结果进行NCBI BLAST，最终筛选获得一株嗜粘蛋白阿克曼氏菌，将其命名为RV02。Pick the bacterial liquid samples with positive PCR reaction, streak and inoculate them on Mucin solid medium plates, scrape out the single colonies that have grown, and conduct PCR identification of the full length of the 16S rDNA gene. The sequencing results of the amplified products are subjected to NCBI BLAST for final screening. A strain of Akkermansia muciniphila was obtained and named RV02.
比较本发明所提供的该株嗜粘蛋白阿克曼氏菌(命名为：RV02株)与ATCC株嗜粘蛋白阿克曼氏菌株(ATCC BAA-835(Akkermansia muciniphila Derrien et al.)，购自北纳生物)的16S rDNA序列、生长动力学曲线等主要微生物指标，然后利用质谱及非靶向代谢组检测技术分析它们培养上清里面的代谢小分子(如棕榈油酸)。Compare the Akkermansia muciniphila strain provided by the present invention (named: RV02 strain) with the ATCC Akkermansia muciniphila strain (ATCC BAA-835 (Akkermansia muciniphila Derrien et al.), purchased from Beina Bio)'s 16S rDNA sequence, growth kinetic curve and other major microbial indicators, and then use mass spectrometry and non-targeted metabolome detection technology to analyze the metabolic small molecules (such as palmitoleic acid) in their culture supernatants.
如图1所示，RV02株比ATCC株嗜粘蛋白阿克曼氏菌在同样的液体培养基内培养时间点具有更高的细菌密度(OD600)(*p<0.05；**p<0.01；***p<0.001，以下结果同)，这说明本发明的RV02株嗜粘蛋白阿克曼氏菌与ATCC株嗜粘蛋白阿克曼氏菌相比具有更强的生长活性与能力。As shown in Figure 1, the RV02 strain has a higher bacterial density (OD600) than the ATCC strain Akkermansia muciniphila at the same culture time point in the liquid medium (*p<0.05; **p<0.01; ***p<0.001, the following results are the same), which shows that the RV02 strain of Akkermansia muciniphila of the present invention has stronger growth activity and ability than the ATCC strain of Akkermansia muciniphila.
如图2所示，RV02株培养上清中的棕榈油酸含量明显高于ATCC株培养上清中的棕榈油酸含量，表明RV02株比ATCC株嗜粘蛋白阿克曼氏菌具有更强的表达或产生棕榈油酸等免疫调节物质的能力。这说明本发明发现的RV02株嗜粘蛋白阿克曼氏菌可能具有优势生长及机体环境下所获得的更强的免疫调节功能。As shown in Figure 2, the palmitoleic acid content in the culture supernatant of the RV02 strain was significantly higher than that in the culture supernatant of the ATCC strain, indicating that the RV02 strain has stronger resistance to Akkermansia muciniphila than the ATCC strain. The ability to express or produce immunomodulatory substances such as palmitoleic acid. This shows that the RV02 strain of Akkermansia muciniphila discovered in the present invention may have advantageous growth and stronger immunomodulatory functions obtained in the body environment.
实施例2：胰腺肿瘤生长实验Example 2: Pancreatic tumor growth experiment
1、RV02株嗜粘蛋白阿克曼氏菌活菌的肿瘤抑制实验1. Tumor inhibition experiment of live bacteria of Akkermansia muciniphila strain RV02
从广东省实验动物中心购买24只1月龄C57BL/6小鼠。将小鼠随机分成3组，每组8只，给予小鼠含氨苄西林(1mg/ml)、链霉素(5mg/ml)和粘菌素(1mg/ml)的饮用水一周，一周后，每组小鼠皮下注射相同数量的Panc02胰腺肿瘤细胞(购自北纳生物，货号BNCC338034)，第1组灌胃生理盐水为对照，第2组灌胃ATCC株嗜粘蛋白阿克曼氏菌活菌(1×10
9CFU/次/只小鼠)，第3组灌胃等量的RV02株嗜粘蛋白阿克曼氏菌活菌(1×10
9CFU/次/只小鼠)，每两天灌胃一次，共灌胃4周。然后分析各组胰腺肿瘤的体积和重量、以及小鼠肿瘤内及外周的IFN-γ等炎症因子水平和CD4+T细胞、CD8+T细胞免疫等T细胞亚群功能及状态。
Twenty-four 1-month-old C57BL/6 mice were purchased from the Guangdong Provincial Experimental Animal Center. The mice were randomly divided into 3 groups, with 8 mice in each group. The mice were given drinking water containing ampicillin (1mg/ml), streptomycin (5mg/ml) and colistin (1mg/ml) for one week. After one week, The mice in each group were subcutaneously injected with the same number of Panc02 pancreatic tumor cells (purchased from Beina Biotechnology, Cat. No. BNCC338034). The first group was gavaged with normal saline as a control, and the second group was gavaged with ATCC strain Akkermansia muciniphila alive. bacteria (1×10 9 CFU/time/mouse), and the third group was orally administered an equal amount of RV02 strain Akkermansia muciniphila live bacteria (1×10 9 CFU/time/mouse), each time The patients were administered intragastrically once every two days for a total of 4 weeks. Then, the volume and weight of pancreatic tumors in each group, as well as the levels of IFN-γ and other inflammatory factors in and around the mouse tumors, and the functions and status of T cell subsets such as CD4+ T cells and CD8+ T cell immunity were analyzed.
如图3、图4所示，施用ATCC株嗜粘蛋白阿克曼氏菌的小鼠肿瘤体积明显小于生理盐水对照组，但是灌RV02株嗜粘蛋白阿克曼氏菌活菌的小鼠肿瘤体积比ATCC株嗜粘蛋白阿克曼氏菌的小鼠肿瘤体积更小。这说明，通过口服本发明的RV02株嗜粘蛋白阿克曼氏菌活菌能够有效抑制胰腺肿瘤的生长。As shown in Figures 3 and 4, the tumor volume of mice administered with the ATCC strain of Akkermansia muciniphila was significantly smaller than that of the saline control group, but the tumor volume of mice injected with live bacteria of the RV02 strain of Akkermansia muciniphila The tumors in mice with the ATCC strain Akkermansia muciniphila were smaller than those in mice. This shows that oral administration of the live Akkermansia muciniphila strain RV02 of the present invention can effectively inhibit the growth of pancreatic tumors.
2、RV02株嗜粘蛋白阿克曼氏菌活菌与死菌对比的肿瘤抑制实验2. Tumor inhibition experiment comparing live and dead bacteria of Akkermansia muciniphila strain RV02
从广东省实验动物中心购买24只1月龄C57BL/6小鼠。将小鼠随机分成3组，每组8只，给予小鼠含氨苄西林(1mg/ml)、链霉素(5mg/ml)和粘菌素(1mg/ml)的饮用水一周，一周后，每组小鼠皮下注射相同数量的Panc02胰腺肿瘤细胞(购自北纳生物，货号BNCC338034)，第1组灌胃等量的RV02株嗜粘蛋白阿克曼氏菌活菌(1×10
9CFU/次/只小鼠)，第2组灌胃等量的RV02株嗜粘蛋白阿克曼氏菌死菌(1×10
9CFU/次/只小鼠)，每两天灌胃一次，共灌胃4周。生理盐水灌胃作为对照。然后分析各组胰腺肿瘤的体积和重量。
Twenty-four 1-month-old C57BL/6 mice were purchased from the Guangdong Provincial Experimental Animal Center. The mice were randomly divided into 3 groups, with 8 mice in each group. The mice were given drinking water containing ampicillin (1mg/ml), streptomycin (5mg/ml) and colistin (1mg/ml) for one week. After one week, The mice in each group were subcutaneously injected with the same number of Panc02 pancreatic tumor cells (purchased from Beina Biotechnology, Cat. No. BNCC338034), and the first group was orally administered an equal amount of live bacteria of Akkermansia muciniphila strain RV02 (1×10 9 CFU /time/mouse), the second group was gavaged with an equal amount of dead bacteria of Akkermansia muciniphila strain RV02 (1×10 9 CFU/time/mouse), once every two days for a total of Oral administration for 4 weeks. Normal saline was administered intragastrically as a control. The volume and weight of pancreatic tumors in each group were then analyzed.
本发明所述的RV02株嗜粘蛋白阿克曼氏菌死菌的制备方法为：将RV02株嗜粘蛋白阿克曼氏菌活菌在75℃水浴中放置10min进行巴氏灭菌，进而得到RV02株嗜粘蛋白阿克曼氏菌死菌。The preparation method of the RV02 strain Akkermansia muciniphila dead bacteria according to the present invention is: placing the RV02 strain Akkermansia muciniphila live bacteria in a 75°C water bath for 10 minutes for pasteurization, and then obtain Dead bacteria of Akkermansia muciniphila strain RV02.
如图5所示，相对于生理盐水对照组，灌胃RV02株嗜粘蛋白阿克曼氏菌活菌和RV02株嗜粘蛋白阿克曼氏菌死菌的小鼠肿瘤体积明显变小，但是灌RV02株嗜粘蛋白阿克曼氏菌活菌的小鼠肿瘤体积比RV02株嗜粘蛋白阿克曼氏菌死菌的小鼠肿瘤体积稍小，表明死菌具有与活菌相近的肿瘤抑制效果。这说明，通过口服本发明的RV02株嗜粘蛋白阿克曼氏菌活菌和死菌均能够有效抑制胰腺肿瘤的生长。As shown in Figure 5, compared with the normal saline control group, the tumor volume of mice given live RV02 Akkermansia muciniphila strain and dead Akkermansia muciniphila strain RV02 strain was significantly smaller, but The tumor volume of mice infused with live bacteria of the RV02 Akkermansia muciniphila strain was slightly smaller than that of mice with dead bacteria of the RV02 Akkermansia muciniphila strain, indicating that the dead bacteria have similar tumor inhibition effects to the live bacteria. Effect. This shows that oral administration of both live and dead Akkermansia muciniphila strain RV02 of the present invention can effectively inhibit the growth of pancreatic tumors.
如图6、图7、图8所示，本发明提供的RV02株嗜粘蛋白阿克曼氏菌活菌可以更有效地抑制CD8+T细胞的数量和炎症因子IFN-γ的分泌进而遏制胰腺肿瘤细胞的生长，此发现为胰腺肿瘤的治疗提供了新思路。As shown in Figures 6, 7, and 8, the RV02 strain of Akkermansia muciniphila live bacteria provided by the present invention can more effectively inhibit the number of CD8+ T cells and the secretion of the inflammatory factor IFN-γ, thereby inhibiting the pancreas. This discovery provides new ideas for the treatment of pancreatic tumors.
实施例3：药物联用实验Example 3: Drug combination experiment
从广东省实验动物中心购买36只1月龄C57BL/6小鼠。将小鼠随机分成6组，每组6只，给予小鼠含氨苄西林(1mg/ml)、链霉素(5mg/ml)和粘菌素(1mg/ml)的饮用水一周，一周后，每组小鼠皮下注射接种相同数量的Panc02胰腺肿瘤细胞，第1组灌胃生理盐水为对照，第2组灌胃RV02株嗜粘蛋白阿克曼氏菌活菌(1×10
9CFU/次/只小鼠)，第3组灌胃RV02株嗜粘蛋白阿克曼氏菌活菌(1×10
9CFU/次/只小鼠)并腹腔注射吉西他滨(Gemcitabine，GEM，50mg/Kg/次/周)，第4组腹腔注射吉西他滨(Gemcitabine,GEM，50mg/Kg/次/周)，第5组灌胃RV02株嗜粘蛋白阿克曼氏菌活菌(1×10
9CFU/次/只小鼠)并腹腔注射紫杉醇(Paclitaxel，PTX)，第6组腹腔注射紫杉醇，肠道菌每两天灌胃一次，共灌胃4周，然后分析胰腺肿瘤的体积和重量、以及小鼠肿瘤内及外周的CD4+T细胞、CD8+T细胞免疫功能及状态。
Thirty-six 1-month-old C57BL/6 mice were purchased from the Guangdong Provincial Experimental Animal Center. The mice were randomly divided into 6 groups, with 6 mice in each group. The mice were given drinking water containing ampicillin (1mg/ml), streptomycin (5mg/ml) and colistin (1mg/ml) for one week. After one week, The mice in each group were subcutaneously inoculated with the same number of Panc02 pancreatic tumor cells. The first group was given normal saline as a control, and the second group was given RV02 strain of Akkermansia muciniphila live bacteria (1×10 9 CFU/time). /mouse), group 3 was orally administered live bacteria of Akkermansia muciniphila strain RV02 (1×10 9 CFU/time/mouse) and intraperitoneally injected with gemcitabine (GEM, 50mg/Kg/time) /week), the 4th group was intraperitoneally injected with gemcitabine (GEM, 50mg/Kg/time/week), and the 5th group was intragastrically injected with live bacteria of Akkermansia muciniphila strain RV02 (1×10 9 CFU/time/ mice) and received intraperitoneal injection of Paclitaxel (PTX). Group 6 received intraperitoneal injection of Paclitaxel, and the intestinal bacteria were gavaged every two days for a total of 4 weeks. Then the volume and weight of pancreatic tumors, as well as mouse tumors, were analyzed. The immune function and status of internal and peripheral CD4+T cells and CD8+T cells.
与此同时，如图9所示，当本发明提供的RV02株嗜粘蛋白阿克曼氏菌活菌与吉西他滨或紫杉醇组合施用的时候，相比单独施用RV02株嗜粘蛋白阿克曼氏菌活菌、吉西他滨或者紫杉醇，肿瘤体积得到显著抑制，这说明本发明提供的RV02株嗜粘蛋白阿克曼氏菌可以有效增强、促进抗肿瘤药物(如抗胰腺肿瘤药物)的疗效。At the same time, as shown in Figure 9, when the live bacteria of Akkermansia muciniphila strain RV02 provided by the present invention are administered in combination with gemcitabine or paclitaxel, compared with the administration of Akkermansia muciniphila strain RV02 alone, Live bacteria, gemcitabine or paclitaxel significantly inhibited tumor volume, which shows that the RV02 strain of Akkermansia muciniphila provided by the present invention can effectively enhance and promote the efficacy of anti-tumor drugs (such as anti-pancreatic tumor drugs).
实施例4：嗜粘蛋白阿克曼氏菌对肿瘤微环境的影响Example 4: Effect of Akkermansia muciniphila on tumor microenvironment
通常，化疗药难以靶向到达胰腺肿瘤微环境进而杀伤肿瘤细胞，这是目前胰腺肿瘤治疗的一大难题。该实施例中分析了灌胃处理嗜粘蛋白阿克曼氏菌是否有助于增加肿瘤微环境内吉西他滨的药物的浓度及吉西他滨药物的半衰期。Usually, it is difficult for chemotherapy drugs to reach the pancreatic tumor microenvironment and kill tumor cells, which is a major problem in the current treatment of pancreatic tumors. In this example, it was analyzed whether intragastric treatment of Akkermansia muciniphila helps increase the concentration of gemcitabine drug in the tumor microenvironment and the half-life of gemcitabine drug.
从广东省实验动物中心购买64只1月龄C57BL/6小鼠。将小鼠随机分成4组，每组16只，给予小鼠含氨苄西林(1mg/ml)、链霉素(5mg/ml)和粘菌素(1mg/ml)的饮用水一周，一周后，每组小鼠皮下注射接种相同数量的Panc02胰腺肿瘤细胞，第1组灌胃生理盐水为对照，第2组灌胃RV02株嗜粘蛋白阿克曼氏菌活菌(1×10
9CFU/次/只小鼠)，第3组灌胃RV02株嗜粘蛋白阿克曼氏菌活菌(1×10
9CFU/次/只小鼠)并腹腔注射吉西他滨(Gemcitabine，GEM，50mg/Kg/次)，第4组腹腔注射吉西他滨(Gemcitabine，GEM，50mg/Kg/次)。RV02株嗜粘蛋白阿克曼氏菌活菌每两天灌胃一次，共灌胃4周。灌胃RV02株嗜粘蛋白阿克曼氏菌活菌之前、灌胃RV02株嗜粘蛋白阿克曼氏菌活菌1周、2周、3周、4周之后，每次取每组4只小鼠，取小鼠的肿瘤、外周血、脾脏、肠道等组织，分析并比较小鼠体内不同部位(特别是肿瘤组织内)吉西他滨药物的含量及其半衰期。
Sixty-four 1-month-old C57BL/6 mice were purchased from the Guangdong Provincial Experimental Animal Center. The mice were randomly divided into 4 groups, with 16 mice in each group. The mice were given drinking water containing ampicillin (1mg/ml), streptomycin (5mg/ml) and colistin (1mg/ml) for one week. After one week, The mice in each group were subcutaneously inoculated with the same number of Panc02 pancreatic tumor cells. The first group was given normal saline as a control, and the second group was given RV02 strain of Akkermansia muciniphila live bacteria (1×10 9 CFU/time). /mouse), group 3 was orally administered live bacteria of Akkermansia muciniphila strain RV02 (1×10 9 CFU/time/mouse) and intraperitoneally injected with gemcitabine (GEM, 50mg/Kg/time) ), group 4 received intraperitoneal injection of gemcitabine (GEM, 50 mg/Kg/time). Live bacteria of Akkermansia muciniphila strain RV02 were administered into the stomach once every two days for a total of 4 weeks. Before the administration of live bacteria of Akkermansia muciniphila strain RV02 and after 1 week, 2 weeks, 3 weeks and 4 weeks of live bacteria of Akkermansia muciniphila strain RV02, 4 animals from each group were taken each time. Mice, take the tumors, peripheral blood, spleen, intestines and other tissues of the mice to analyze and compare the content and half-life of gemcitabine in different parts of the mouse's body (especially within the tumor tissue).
如图10所示，施用本发明的RV02株嗜粘蛋白阿克曼氏菌4周后，胰腺肿瘤微环境内的化疗药物吉西他滨的药物浓度得到显著增加。As shown in Figure 10, 4 weeks after administration of the RV02 strain of Akkermansia muciniphila of the present invention, the drug concentration of the chemotherapy drug gemcitabine in the pancreatic tumor microenvironment was significantly increased.
此外，如图11所示，肿瘤微环境内的化疗药物半衰期得以显著延长。In addition, as shown in Figure 11, the half-life of chemotherapy drugs in the tumor microenvironment is significantly extended.
可见，本发明的RV02株嗜粘蛋白阿克曼氏菌不仅能够有效抑制胰腺肿瘤的发生发展，而且能够增强抗肿瘤药物(如化疗药物)到胰腺肿瘤微环境中的浸润，从而解决胰腺肿瘤化疗药物难以到达进而杀伤肿瘤细胞难题。It can be seen that the RV02 strain of Akkermansia muciniphila of the present invention can not only effectively inhibit the occurrence and development of pancreatic tumors, but also enhance the infiltration of anti-tumor drugs (such as chemotherapy drugs) into the pancreatic tumor microenvironment, thereby solving the problem of pancreatic tumor chemotherapy. It is difficult for drugs to reach and kill tumor cells.
Claims (10)
- 一株嗜粘蛋白阿克曼氏菌(Akkermansia muciniphila)，其特征在于，其保藏号为CGMCC No.23185。A strain of Akkermansia muciniphila is characterized in that its deposit number is CGMCC No. 23185.
- 权利要求1所述的嗜粘蛋白阿克曼氏菌在制备用于预防和/或治疗胰腺肿瘤的药物中的应用。Use of Akkermansia muciniphila according to claim 1 in the preparation of medicaments for preventing and/or treating pancreatic tumors.
- 权利要求1所述的嗜粘蛋白阿克曼氏菌在制备用于预防和/或治疗炎症及其导致、伴随、相关、诱导、反馈的疾病的药物中的应用。Application of the Akkermansia muciniphila described in claim 1 in the preparation of medicaments for preventing and/or treating inflammation and diseases caused, accompanied, related, induced, and fed back.
- 一种组合物，其包括权利要求1所述的嗜粘蛋白阿克曼氏菌的活菌、死菌、细胞裂解物、代谢产物、改造菌、突变菌和/或诱变菌作为活性成分。A composition comprising live bacteria, dead bacteria, cell lysates, metabolites, modified bacteria, mutant bacteria and/or mutagenized bacteria of Akkermansia muciniphila according to claim 1 as active ingredients.
- 根据权利要求4所述的组合物，还包括其他种类的肠道菌。The composition according to claim 4, further comprising other types of intestinal bacteria.
- 根据权利要求4或5所述的组合物，还包括药学上可接受的载体。The composition according to claim 4 or 5, further comprising a pharmaceutically acceptable carrier.
- 根据权利要求4或5所述的组合物，所述药物组合物为胶囊、溶液、悬乳液、袋装粉剂或颗粒剂。The composition according to claim 4 or 5, wherein the pharmaceutical composition is a capsule, solution, suspoemulsion, bagged powder or granule.
- 根据权利要求1所述的嗜粘蛋白阿克曼氏菌在制备用于增强抗肿瘤药物功效的医药组合物方面的应用。Use of Akkermansia muciniphila according to claim 1 in the preparation of pharmaceutical compositions for enhancing the efficacy of anti-tumor drugs.
- 根据权利要求8所述的应用，其特征在于，所述抗肿瘤药物为化疗药物，特别是吉西他滨、紫杉醇中的任意一种或其组合。The application according to claim 8, characterized in that the anti-tumor drug is a chemotherapy drug, especially any one of gemcitabine and paclitaxel or a combination thereof.
- 一种包含权利要求1所述的嗜粘蛋白阿克曼氏菌的培养物或其加工物。A culture containing Akkermansia muciniphila according to claim 1 or a processed product thereof.
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| PCT/CN2022/089199 WO2023184631A1 (en) | 2022-03-28 | 2022-04-26 | Akkermansia muciniphila and use thereof in preparing anti-tumor drug |
Country Status (2)
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| CN (1) | CN116855397A (en) |
| WO (1) | WO2023184631A1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110496140A (en) * | 2018-05-18 | 2019-11-26 | 深圳月曜生命科技有限公司 | Bacteroides fragilis or Ackermam slime bacteria are preparing the application in the drug for preventing or treating tumour |
| WO2021060945A1 (en) * | 2019-09-25 | 2021-04-01 | ㈜헬스바이옴 | Obligate anaerobic human intestinal microbe for cancer treatment, and use thereof |
| CN112933115A (en) * | 2020-12-31 | 2021-06-11 | 江南大学 | Application of Akkermansia muciniphila in treating chronic pancreatitis |
| CN113862193A (en) * | 2021-10-28 | 2021-12-31 | 江西普瑞森基因科技有限公司 | Ackermanella muciniphila and application thereof in preparation of antitumor drugs |
-
2022
- 2022-04-20 CN CN202210416273.9A patent/CN116855397A/en active Pending
- 2022-04-26 WO PCT/CN2022/089199 patent/WO2023184631A1/en unknown
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| CN110496140A (en) * | 2018-05-18 | 2019-11-26 | 深圳月曜生命科技有限公司 | Bacteroides fragilis or Ackermam slime bacteria are preparing the application in the drug for preventing or treating tumour |
| WO2021060945A1 (en) * | 2019-09-25 | 2021-04-01 | ㈜헬스바이옴 | Obligate anaerobic human intestinal microbe for cancer treatment, and use thereof |
| CN112933115A (en) * | 2020-12-31 | 2021-06-11 | 江南大学 | Application of Akkermansia muciniphila in treating chronic pancreatitis |
| CN113862193A (en) * | 2021-10-28 | 2021-12-31 | 江西普瑞森基因科技有限公司 | Ackermanella muciniphila and application thereof in preparation of antitumor drugs |
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| BERTRAND ROUTY; EMMANUELLE LE CHATELIER; LISA DEROSA; CONNIE P M DUONG; MARYAM TIDJANI ALOU; ROMAIN DAILLÈRE; AURÉLIE FLUCKIGER; M: "Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 359, no. 6371, 5 January 2018 (2018-01-05), US , pages 91 - 97, XP055554928, ISSN: 0036-8075, DOI: 10.1126/science.aan3706 * |
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| JEONG JIN-YONG, KIM TAE-BUM, KIM JINJU, CHOI HWI WAN, KIM EO JIN, YOO HYUN JU, LEE SONG, JUN HYE RYEONG, YOO WONBEAK, KIM SEOKHO, : "Diversity in the Extracellular Vesicle-Derived Microbiome of Tissues According to Tumor Progression in Pancreatic Cancer", CANCERS, vol. 12, no. 9, pages 2346, XP093095409, DOI: 10.3390/cancers12092346 * |
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