Ackermansiella muciniphila and application thereof

Abstract

The invention relates to Ackermania muciniphila and application thereof, and belongs to the technical field of microorganisms. According to the present invention, the ackermania muciniphilaSYSU-85 is adopted as the ackermania muciniphilaSYSU-85, and has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 8, 2024, and the preservation number of the ackermania muciniphilaSYSU-85 is CGMCC NO.46070; the Ackermania muciniphila SYSU-85 disclosed by the invention is weak in dependence on mucin, grows well in a BHI culture medium added with cysteine, and is resistant to oxaliplatin. The Ackermansiella muciniphila SYSU-85 provided by the invention alleviates adverse reactions caused by oxaliplatin, and is combined with oxaliplatin or a PD-1 inhibitor to significantly slow down a tumor growth curve and significantly reduce the tumor volume, thereby improving the tumor treatment effect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to Akkermansia muciniphila and application thereof. Background Art

[0002] Cancer is a serious disease caused by abnormal division and proliferation of normal cells in the human body, which threatens the life and health of patients. Most cancer patients are already in the middle or late stages when diagnosed, facing difficulties such as difficult treatment, poor prognosis, and short survival. Effective tumor treatment methods can greatly improve the prognosis of patients.

[0003] At present, commonly used tumor treatment methods include: radiotherapy / chemotherapy, immunotherapy, small molecule targeted drugs and surgical resection. Among them, immunotherapy is often combined with radiotherapy / chemotherapy to improve the treatment effect. Nowadays, the impact of intestinal flora on tumors has also attracted more and more attention.

[0004] Akkermansia muciniphila is an intestinal bacterium with a wide range of probiotic effects. Studies have shown that the reduced abundance of Akkermansia muciniphila is associated with obesity, neurological diseases, etc., and is closely related to the occurrence and treatment of colon cancer, lung cancer, and prostate cancer. The combination of Akkermansia muciniphila with PD-1 / PD-L1 checkpoint inhibitors or with oxaliplatin can slow tumor progression in mice, making it a current "star strain."

[0005] However, the effect of oxaliplatin on Akkermansia is still unknown, and it is unclear whether muciniphila Akkermansia has an effect on the adverse reactions of oxaliplatin. In addition, the muciniphila Akkermansia found so far are all highly dependent on mucins, and the research and production costs are high; there are also relatively few muciniphila Akkermansia strains available for clinical selection. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a muciniphilic Akkermansia and its application for improving the therapeutic effect of anti-tumor drugs.

[0007] To achieve the above object, the technical solution adopted by the present invention is: In a first aspect, the present invention provides an Akkermansia muciniphila, wherein the Akkermansia muciniphila is Akkermansia muciniphilaSYSU-85, which has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on August 8, 2024, and its deposit number is: CGMCC NO.46070.

[0008] The Akkermansia muciniphila SYSU-85 of the present invention has weak dependence on mucin and can achieve quantitative growth in a culture medium added with oxaliplatin. It can be used in combination with other anti-tumor drugs to enhance the anti-tumor effect and alleviate the toxic side effects of the anti-tumor drugs.

[0009] In a second aspect, the present invention seeks to protect the use of the Akkermansia muciniphila in the preparation of anti-tumor drugs.

[0010] In a third aspect, the present invention seeks to protect the use of the Akkermansia muciniphila in the preparation of an anti-tumor drug synergist.

[0011] Furthermore, the anti-tumor drug synergist is an agent that alleviates the toxic and side effects of anti-tumor drugs and / or improves the anti-tumor effect. The Akkermansia muciniphila SYSU-85 of the present invention can improve the adverse reactions of oxaliplatin and improve the anti-tumor effect of oxaliplatin.

[0012] In a fourth aspect, the present invention claims the use of the Akkermansia muciniphila in the preparation of an immune activator. The Akkermansia muciniphila SYSU-85 of the present invention can significantly promote the expression of the key anti-tumor cytokine CXCL-10 by macrophages RAW264.7, thereby exerting an anti-tumor effect.

[0013] In a fifth aspect, the present invention seeks protection for an anti-tumor drug, wherein the anti-tumor drug contains the Akkermansia muciniphila, a fermentation product of the Akkermansia muciniphila, or an inactivated bacterial cell of the Akkermansia muciniphila.

[0014] Furthermore, the inactivated bacteria are heat-inactivated bacteria.

[0015] Furthermore, the content of Akkermansia muciniphila in the anti-tumor drug is not less than 10 6 indivual.

[0016] Furthermore, the anti-tumor drug also contains platinum chemotherapy drugs and / or PD-1 inhibitors.

[0017] Furthermore, the platinum chemotherapy drug includes at least one of cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin and lobaplatin.

[0018] In a specific embodiment of the present invention, taking oxaliplatin or PD-1 inhibitor as an example, combined with the Akkermansia muciniphilaSYSU-85 for treatment, the tumor growth curve is significantly slowed down, the tumor volume is significantly reduced, and the tumor treatment effect is improved.

[0019] Furthermore, the anti-tumor drug includes at least one drug against adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, pancreatic cancer, cervical cancer, bile duct cancer, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, multiforme glioma, glioma, head and neck tumor, renal chromophobe cell carcinoma, mixed renal cancer, renal cancer, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal adenocarcinoma, sarcoma, skin melanoma, gastric cancer, gastrointestinal stromal tumor, esophageal cancer, testicular cancer, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma and soft tissue sarcoma.

[0020] In a specific embodiment of the present invention, experiments were conducted using colon cancer cells MC38 and CT26 as examples, which can significantly slow down the tumor growth curve, significantly reduce the tumor volume, and inhibit tumor growth.

[0021] In a specific embodiment of the present invention, taking gastric cancer cell AGS as an example, an experiment was conducted to significantly reduce the survival rate of gastric cancer cell AGS.

[0022] Furthermore, the anti-tumor drug also contains pharmaceutically acceptable carriers and / or excipients.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The Akkermansia muciniphila SYSU-85 of the present invention has a weak dependence on mucin and grows well in a BHI medium supplemented with cysteine.

[0024] (2) The Akkermansia muciniphila SYSU-85 of the present invention is resistant to oxaliplatin and can achieve quantitative growth in a culture medium supplemented with oxaliplatin.

[0025] (3) The Akkermansia muciniphila SYSU-85 combined with oxaliplatin of the present invention significantly slows down the tumor growth curve and significantly reduces the tumor volume, thereby improving the tumor treatment effect and expanding the scope of clinical medication.

[0026] (4) The Akkermansia muciniphila SYSU-85 of the present invention alleviates the adverse reactions caused by oxaliplatin and improves the survival rate of mice over-injected with oxaliplatin.

[0027] (5) The Akkermansia muciniphila SYSU-85 or its combination with a PD-1 inhibitor of the present invention significantly slows down the tumor growth curve and significantly reduces the tumor volume, thereby improving the tumor treatment effect and expanding the scope of clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The comparison results of protein spectra of SYSU-85 and ATCC BAA-835 in Example 2 are shown in FIG.

[0029] Figure 2 The results of SYSU-85 culture in BHI in Example 3. A is the bacterial solution cultured for 24 hours; B is the colony morphology on the solid culture medium; C is the bacterial body after centrifugation, where the left is the blank culture medium cultured under the same conditions, and the right is SYSU-85; D is the microscopic examination of SYSU-85 after Gram staining.

[0030] Figure 3 The growth of SYSU-85 in Example 5 in a culture medium supplemented with different concentrations of oxaliplatin is compared with that of Akkermansia muciniphila ATCC BAA-835. A is when the oxaliplatin concentrations are 15.63 μM, 7.81 μM, 3.91 μM, 1.95 μM and 0 μM, respectively; B is when the oxaliplatin concentrations are 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM and 0 μM, respectively.

[0031] Figure 4 The effect of SYSU-85 alone or in combination with oxaliplatin on subcutaneous tumor MC38 in mice in Example 6. A is the change of tumor volume in each group over time; B is a tumor photo.

[0032] Figure 5 The survival curves of mice in Example 7 were given intraperitoneal injection of high-dose oxaliplatin, and were given or not given SYSU-85 or heat-inactivated SYSU-85.

[0033] Figure 6 The effect of SYSU-85 alone or in combination with PD-1 inhibitor on subcutaneous tumor CT26 in mice in Example 8. A is the change of tumor volume in each group over time; B is a tumor photo.

[0034] Figure 7 is the IC of oxaliplatin against gastric cancer cell AGS in Example 9 50And the effect of oxaliplatin combined with SYSU-85 or ATCC BAA-835. Among them, A is the effect of different concentrations of oxaliplatin on gastric cancer cell AGS; B is the effect of 18μM oxaliplatin combined with SYSU-85 or ATCC BAA-835 on gastric cancer cell AGS.

[0035] Figure 8 The results of SYSU-85 promoting the expression of CXCL-10 in macrophages in Example 10 are shown.

[0036] In the above figure, "Indicates that there is a significant difference between the two groups, p < 0.05;" "Indicates that there is a significant difference between the two groups, p < 0.01;" ” indicates a significant difference between the two groups (p < 0.0001). DETAILED DESCRIPTION

[0037] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0038] In the following examples and comparative examples, SYSU-85 refers to Akkermansia muciniphila SYSU-85; ATCC BAA-835 refers to Akkermansia muciniphila ATCC BAA-835.

[0039] Prepare brain heart infusion (BHI) medium: take 37 g BHI medium powder and add it to 1 L water (1 g cysteine ​​can be added) to fully dissolve, place it in an autoclave at 115°C for 20 min (for solid culture medium, add 15 g / L agar), cool it and set it aside; BHI medium powder was purchased from Oxoid.

[0040] Example 1 Isolation and Identification of SYSU-85 1. Take an appropriate amount of feces from a patient who was treated with toripalimab combined with SOX (oxaliplatin + S-1) and whose gastric cancer area was significantly reduced and met the response criteria of partial response (PR) to chemotherapy. After gradient dilution with phosphate buffered saline (PBS), take 100 μL and evenly spread it on brain heart infusion medium, and culture it at 37°C under anaerobic conditions for 48-72 h.

[0041] 2. Perform Gram staining on the single colony cultured in step 1 and examine under a microscope: pick a single colony and add it to 20 μL PBS, blow and mix to obtain a bacterial suspension; take 15 μL of the bacterial suspension and spread it on a new BHI plate, and culture it at 37°C under anaerobic conditions for 48-72 hours. Take the remaining 5 μL of bacterial suspension and drop it in the center of the slide, bake it evenly with an alcohol lamp to fix it, add crystal violet staining solution to cover it for 1 minute, rinse it with slow water flow, add iodine solution to mordant for 1 minute, rinse it with slow water flow, add decolorizing alcohol for 20-30 seconds, rinse it with slow water flow, and counterstain it with sand yellow staining solution for 1 minute. After the slide is dry, observe the bacterial morphology with an optical microscope. If the bacterial morphology is consistent, scrape the bacterial cells (SYSU-85) obtained by culturing the bacterial suspension and save them.

[0042] 3. Extraction of bacterial DNA and identification of 16S rRNA: Extract bacterial genomic DNA from the bacteria obtained in step 2 according to the instructions of the bacterial DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.), use the universal primers (27F and 1492R) for bacterial molecular identification of 16S rRNA, and use the bacterial genomic DNA extracted from the kit as a template, LA enzyme for PCR amplification, and the PCR amplification program is: 95℃ pre-denaturation for 1 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 90 s, 25 cycles; 72℃ final extension for 7 min. The amplified product was identified by agarose gel electrophoresis to have a band at 1500 bp, and no band appeared in the negative control, and was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0043] The sequencing results were spliced ​​by SeqMan software and sequence searched using the BLAST tool in the NCBI database. It was found that the sequence similarity between the screened SYSU-85 and ATCC BAA-835 was 99.92%, and there was a base mutation at the 1170th base of 16S rRNA, where the nucleotide base mutated from G to A.

[0044] The nucleotide sequence of bacterial 16S rRNA is shown in SEQ ID NO: 1, the nucleotide sequence of 23S rRNA is shown in SEQ ID NO: 2, and the nucleotide sequence of 5S rRNA is shown in SEQ ID NO: 3.

[0045] Example 2 ANI analysis and proteomic analysis of SUSU85 1. Experimental Methods The strains SYSU-85 and ATCC BAA-835 obtained in Example 1 were subjected to ultrasonic lysis, acetone precipitation, enzymatic hydrolysis and desalting, respectively, and then protein spectrum analysis was performed using high performance nanoflow liquid chromatography-ultra-high resolution mass spectrometry (School of Pharmacy, Sun Yat-sen University).

[0046] ANI analysis (Average Nucleotide Identity) was performed on SYSU-85 and ATCC BAA-835.

[0047] The proteomic analysis of ATCC BAA-835 and SYSU-85 strains was performed. After excluding the data with medium and low confidence levels, SYSU-85: ATCC BAA-835 proteins with abundance greater than 2 were selected to draw KEGG enrichment analysis diagrams.

[0048] 2. Experimental Results 1. The results showed that the ANI value of SYSU-85 was 98.72% compared with that of Akkermansia muciniphila ATCC BAA-835 of the genus Akkermansia, which had a certain difference.

[0049] SYSU-85 was named Akkermansia muciniphilaSYSU-85 and was deposited in the General Microbiology Center of China Culture Collection Administration on August 8, 2024. Its deposit number is CGMCC NO.46070, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0050] 2. A total of 958 proteins were analyzed for SYSU-85 and 1234 proteins were analyzed for ATCC BAA-835. Subsequently, SYSU-85-specific proteins and proteins with a content greater than 2 times that of ATCC BAA-835 were screened, and 374 related proteins were obtained. KEGG enrichment analysis was performed online at bioinfo.org / kobas ( Figure 1 ), indicating that there are proteome differences between the two strains. The comparison found that the "Mucin-associated surface protein (MASP)" (Uniprot: A0A4Q5IJY4) related to bacterial mucin was significantly enriched in the proteome of ATCC BAA-835 but lacked in the proteome of SYSU-85. In addition, SYSU-85 does not contain the Amuc_1434 protein from ATCC BAA-835 described in patent CN118063568A, indicating that the anti-tumor effect of SYSU-85 has different mechanisms.

[0051] Example 3 SYSU-85 is weakly dependent on mucin 1. Experimental Methods 1. Inoculate strain SYSU-85 into BHI liquid medium for activation and culture at 37°C under anaerobic conditions for 24 h to obtain seed solution.

[0052] 2. Inoculate the seed solution obtained in step 1 into BHI liquid culture medium at an inoculum volume of 2% (v / v) and culture anaerobically at 37°C for 24 h. Set up another blank BHI liquid culture medium without inoculation of any strain and culture it under the same conditions.

[0053] 3. After 24 h of incubation in step 2, take 1 mL of bacterial solution and add it to two centrifuge tubes. Centrifuge at 1,2000 rpm for 1 min. Record the bacterial solution concentration and bacterial cell count. Figure 2 As shown in A, it is the bacterial solution cultured for 24 hours; Figure 2 As shown in B, it is the colony morphology on solid culture medium; Figure 2 As shown in C, the bacteria after centrifugation, where the left is the blank culture medium cultured under the same conditions, and the right is SYSU-85.

[0054] 4. Perform Gram staining on the bacterial solution of SYSU-85 according to step 2 in Example 1.

[0055] 2. Experimental Results Gram staining results Figure 2 As shown in D, SYSU-85 is a Gram-positive bacterium.

[0056] Consistent with the results of the proteome analysis in Example 2, SYSU-85 has a low dependence on mucin. The culture conditions of ATCC BAA-835 are usually BHI medium with added mucin, and the growth rate is slow. SYSU-85 can be cultured in a good state using only BHI medium, and the bacterial count is large, which is convenient for subsequent experimental research and industrial cultivation.

[0057] Example 4 Drug sensitivity test of SYSU-85 1. Experimental Methods Take SYSU-85 and ATCC BAA-835 in the logarithmic growth phase and inoculate them in sterile PBS to obtain the test bacterial solution. Use a sterile cotton swab to dip the test bacterial solution and evenly spread it on BHI and BHI plates with added mucin (0.5-2%, m / v). After the test bacterial solution on the plate is absorbed, use tweezers to take the corresponding drug-sensitive paper and place it evenly on the plate. Incubate it upright in a 37℃ bacterial incubator for 48 h and measure the diameter of the inhibition zone.

[0058] 2. Experimental Results As shown in Table 1, SYSU-85 was not sensitive to levofloxacin and penicillin, but ATCC BAA-835 was sensitive to levofloxacin and penicillin.

[0059] Table 1 Diameters of inhibition zones in drug sensitivity tests of SYSU-85 and ATCC BAA-835 Example 5 Effect of Oxaliplatin on SYSU-85 1. Experimental Methods 1. Pipette 100 μL BHI into a 96-well plate, and dilute oxaliplatin to final concentrations of 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, 15.63 μM, 7.81 μM, 3.91 μM and 1.95 μM by half-dilution method, with three parallels for each concentration. In addition, set up a column with 0 μM oxaliplatin and no bacteria as a blank group.

[0060] 2. Add SYSU-85 and ATCC BAA-835 to the culture medium respectively. After culturing at 37°C under anaerobism for 24 h, use a microplate reader to measure the absorbance of each well at 600 nm.

[0061] 2. Experimental Results like Figure 3 A and Figure 3 As shown in Figure B, low concentrations of oxaliplatin can promote the growth of SYSU-85, and high concentrations of oxaliplatin have little inhibitory effect on SYSU-85. The growth of SYSU-85 is always better than that of ATCC BAA-835.

[0062] The above results indicate that the Akkermansia muciniphila SYSU-85 provided by the present invention is an oxaliplatin-promoted growth Akkermansia muciniphila, which can achieve quantitative growth when used in combination with oxaliplatin.

[0063] Example 6 Antitumor effect of SYSU-85 combined with oxaliplatin on subcutaneous MC38 in mice 1. Experimental Methods 1. 5-8 weeks old C57BL / 6 male mice (Experimental Animal Center of Sun Yat-sen University) were selected and randomly divided into four groups after one week of adaptive feeding, namely: control group with intraperitoneal injection of PBS + intragastric administration of PBS, group with intraperitoneal injection of oxaliplatin, group with intragastric administration of SYSU-85, and experimental group with intraperitoneal injection of oxaliplatin + intragastric administration of SYSU-85. Each mouse was subcutaneously inoculated with 10 6 After tumor formation, for the group that needed intraperitoneal injection of oxaliplatin, each mouse was intraperitoneally injected with 100 μL of 6 mg / mL oxaliplatin every two days, and for the experimental group that received intragastric administration of SYSU-85, each mouse was intraperitoneally administered with 100 μL of 1×10 7 CFU of SYSU-85 (take SYSU-85 bacterial solution and centrifuge it, resuspend and wash the centrifugal pellet with PBS, and dilute it with PBS to 1×10 8CFU / mL), and each mouse in the control group was synchronously gavaged or intraperitoneally injected with an equal volume of PBS.

[0064] 2. The size of the tumor was measured on days 9, 12, 15, 18 and 21 after implantation. The mice were killed on day 21 and the tumors were removed and photographed.

[0065] 2. Experimental Results like Figure 4 A and Figure 4 As shown in Figure B, SYSU-85 combined with oxaliplatin significantly inhibited the growth of MC38 tumors. Specifically, SYSU-85 combined with oxaliplatin significantly slowed down the tumor growth curve and significantly reduced the tumor volume. The above results show that SYSU-85 combined with oxaliplatin has a good anti-tumor effect and has clinical application value.

[0066] Example 7 SYSU-85 alleviates the toxic side effects of oxaliplatin 1. Experimental Methods C57BL / 6 male mice aged 5 to 8 weeks (Experimental Animal Center, Sun Yat-sen University) were selected and randomly divided into four groups after one week of adaptive feeding, namely: control group with intraperitoneal injection of PBS + intragastric administration of PBS, group with intraperitoneal injection of oxaliplatin, group with intraperitoneal injection of oxaliplatin + intragastric administration of SYSU-85, and experimental group with intraperitoneal injection of oxaliplatin + intragastric administration of heat-inactivated SYSU-85. For the group requiring intraperitoneal injection of oxaliplatin, each mouse was intraperitoneally injected with 100 μL of 60 mg / mL oxaliplatin, and for the experimental group receiving intragastric administration of SYSU-85, each mouse was intragastricly administered with 100 μL of 1×10 7 CFU of SYSU-85 (take SYSU-85 bacterial solution and centrifuge it, resuspend and wash the centrifugal pellet with PBS, and dilute it with PBS to 1×10 8 CFU / mL), the heat-inactivated group was gavaged with the same amount of SYSU-85 inactivated at 70℃ for 30 min, and each mouse in the control group was synchronously gavaged and intraperitoneally injected with 100μL PBS, and the mortality of mice was recorded every day.

[0067] 2. Experimental Results like Figure 5 As shown, high doses of oxaliplatin can cause death in mice, while SYSU-85, especially heat-inactivated SYSU-85, can alleviate the toxic side effects of oxaliplatin.

[0068] The above results indicate that SYSU-85, when used in combination with oxaliplatin, can not only enhance the anti-tumor effect of oxaliplatin, but also alleviate the toxic side effects of oxaliplatin.

[0069] Example 8 Anti-tumor effect of SYSU-85 combined with PD-1 (programmed death receptor 1) inhibitor on subcutaneous CT26 in mice 1. Experimental Methods 1. 5-8 weeks old C57BL / 6 male mice (Experimental Animal Center of Sun Yat-sen University) were selected and randomly divided into four groups after one week of adaptive feeding, namely: control group with intraperitoneal injection of PBS + intragastric administration of PBS, group with intraperitoneal injection of PD-1 inhibitor, group with intragastric administration of SYSU-85, and experimental group with intraperitoneal injection of PD-1 inhibitor + intragastric administration of SYSU-85. Each mouse was subcutaneously inoculated with 10 6 After tumor formation, for the group that needed intraperitoneal injection of PD-1 inhibitor, each mouse was intraperitoneally injected with 60 μg of PD-1 inhibitor every two days, and for the experimental group that received intragastric administration of SYSU-85, each mouse was intraperitoneally administered with 100 μL of 1×10 7 CFU of SYSU-85 (take SYSU-85 bacterial solution and centrifuge it, resuspend and wash the centrifugal pellet with PBS, and dilute it with PBS to 1×10 8 CFU / mL), and each mouse in the control group was synchronously gavaged or intraperitoneally injected with an equal volume of PBS.

[0070] 2. Measure the size of the tumor on days 7, 10, 13, 16, and 19 after implantation. On day 19, the mice were killed and the tumors were removed and photographed.

[0071] 2. Experimental Results like Figure 6 A and Figure 6 As shown in Figure B, SYSU-85 combined with PD-1 inhibitors has a significant inhibitory effect on CT26 tumor growth. Specifically, SYSU-85 combined with PD-1 significantly slowed down the tumor growth curve and significantly reduced the tumor volume. The above results show that SYSU-85 combined with PD-1 inhibitors has a good anti-tumor effect and has clinical application value.

[0072] Example 9 SYSU-85 combined with oxaliplatin inhibits the proliferation of gastric cancer cells AGS 1. Experimental Methods 1. Evenly plate gastric cancer cells AGS in a 96-well plate at a density of 3000 cells per well and culture them in DMEM medium (Dulbecco's modified eagle medium) containing 10% (v / v) fetal bovine serum for 12 h until they adhere to the plate.

[0073] 2. Replace the culture medium with DMEM containing 500 μM to 0 μM (half-fold dilution concentration, see Example 5 for specific concentrations) of oxaliplatin and culture for 48 h.

[0074] 3. Remove the supernatant and use CCK8 method to detect and calculate the cell survival rate.

[0075] 4. After repeating steps 1 and 2, PBS (control), oxaliplatin (OXA), ATCC BAA-835, SYSU-85, oxaliplatin combined with ATCC BAA-835 (OXA+ATCC BAA-835), and oxaliplatin combined with SYSU-85 (OXA+SYSU-85) were added respectively, with the multiplicity of infection MOI of 10:1. After co-culture with cells for 48 h, the supernatant was removed and the cell survival rate was detected and calculated using the CCK8 method.

[0076] 2. Experimental Results like Figure 7 As shown in A, IC 50 is 18.89μM; Figure 7 As shown in B, oxaliplatin combined with SYSU-85 has the highest killing effect on gastric cancer cell AGS.

[0077] Example 10 SYSU-85 promotes the expression of CXCL-10 (CXC chemokine 10) in macrophages RAW264.7 1. Experimental Methods 1. Macrophages RAW264.7 were evenly plated in a six-well plate and cultured in DMEM medium containing 10% (v / v) fetal bovine serum for 24 h.

[0078] 2. Add SYSU-85 to the RAW264.7 cultured in step 1 at an MOI of 10:1 and culture for 24 h.

[0079] 3. Pipette the supernatant of the cell culture medium in the six-well plate in step 2, centrifuge at 3,000×g for 10 min, and transfer the supernatant to a new centrifuge tube.

[0080] 4. The CXCL-10 content in the supernatant of cell culture medium was determined using a CXCL-10 ELISA kit (Lianke Biotechnology).

[0081] 2. Experimental Results like Figure 8 As shown, SYSU-85 significantly promoted the expression of CXCL-10 in RAW264.7, indicating that SYSU-85 activated macrophages to produce key anti-tumor cytokines and exerted anti-tumor effects.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A muciniphilic Akkermansia, characterized in that The Akkermansia muciniphila is Akkermansia muciniphilaSYSU-85, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on August 8, 2024, and its deposit number is: CGMCC NO.46070.

2. Use of Akkermansia muciniphila according to claim 1 in the preparation of anti-tumor drugs.

3. Use of Akkermansia muciniphila according to claim 1 in the preparation of an anti-tumor drug synergist.

4. The use according to claim 3, characterized in that: The anti-tumor drug synergist is a reagent that alleviates the toxic and side effects of anti-tumor drugs and / or improves the anti-tumor effect.

5. Use of the Akkermansia muciniphila according to claim 1 in the preparation of an immune activator.

6. An anti-tumor drug, characterized in that: The anti-tumor drug contains the Akkermansia muciniphila according to claim 1, a fermentation product of the Akkermansia muciniphila or an inactivated bacterial cell of the Akkermansia muciniphila.

7. The anti-tumor drug according to claim 6, characterized in that: The anti-tumor drug also contains platinum chemotherapy drugs and / or PD-1 inhibitors.

8. The anti-tumor drug according to claim 7, characterized in that: The platinum chemotherapy drug includes at least one of cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin and lobaplatin.

9. The anti-tumor drug according to claim 6, characterized in that: The anti-tumor drug includes at least one of the drugs for adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, pancreatic cancer, cervical cancer, bile duct cancer, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, multiforme glioma, glioma, head and neck tumor, renal chromophobe cell carcinoma, mixed renal cancer, kidney cancer, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal adenocarcinoma, sarcoma, skin melanoma, gastric cancer, gastrointestinal stromal tumor, esophageal cancer, testicular cancer, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma and soft tissue sarcoma.

10. The anti-tumor drug according to any one of claims 6 to 9, characterized in that: The anti-tumor drug further comprises a pharmaceutically acceptable carrier and / or auxiliary material.

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