Akkermansia muciniphila and its applications

Abstract

The present invention relates to Akkermansia muciniphila and its applications, belonging to the technical field of microbiology. The Akkermansia muciniphila described in the present invention is Akkermansia muciniphila SYSU-85, which was deposited on August 8, 2024 at the General Microbiological Center of the China General Microbiological Culture Collection Center, and its deposit number is: CGMCC NO. 46070. The Akkermansia muciniphila SYSU-85 of the present invention has a weak dependence on mucin, grows well in BHI medium supplemented with cysteine, and is resistant to oxaliplatin. The Akkermansia muciniphila SYSU-85 of the present invention alleviates the adverse reactions caused by oxaliplatin, and in combination with oxaliplatin or PD-1 inhibitor, significantly slows down the tumor growth curve and significantly reduces the tumor volume, improving the tumor treatment effect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to Akkermansia muciniphila and its applications. Background Art

[0002] Cancer is a serious disease caused by abnormal division and proliferation of normal cells in the human body, threatening the life and health of patients. Most cancer patients are diagnosed at the middle or advanced stage, facing difficulties in treatment, poor prognosis, short survival period, etc. Adopting effective tumor treatment means will greatly improve the prognosis of patients.

[0003] Currently, common tumor treatment means include: radiotherapy / chemotherapy, immunotherapy, small molecule targeted drugs, surgical resection, etc. Among them, immunotherapy is often combined with radiotherapy / chemotherapy to improve the treatment effect. Nowadays, the impact of the gut microbiota on tumors has also attracted increasing attention.

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

[0005] However, the current impact of oxaliplatin on Akkermansia is still blank, and it is also unclear whether Akkermansia muciniphila has an impact on the adverse reactions of oxaliplatin. Moreover, all the currently discovered Akkermansia muciniphila strains are highly dependent on mucin, with high research and production costs; there are also few Akkermansia muciniphila strains available for clinical selection. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide Akkermansia muciniphila for improving the treatment effect of anti-tumor drugs and its applications.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides an Akkermansia muciniphila, which is Akkermansia muciniphila SYSU-85, and was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 8, 2024, with the deposit number: CGMCC NO.46070.

[0009] The Akkermansia muciniphila SYSU-85 of the present invention has weak mucin dependence and can achieve quantitative growth in a culture medium supplemented with oxaliplatin. It can be used in combination with other anti-tumor drugs to enhance the anti-tumor effect and alleviate the toxic and side effects of anti-tumor drugs.

[0010] In a second aspect, the present invention claims the use of the Akkermansia muciniphila in the preparation of anti-tumor drugs.

[0011] In a third aspect, the present invention claims the use of the Akkermansia muciniphila in the preparation of anti-tumor drug synergists.

[0012] Further, the anti-tumor drug synergist is a reagent for alleviating the toxic and side effects of anti-tumor drugs and / or enhancing the anti-tumor effect. The Akkermansia muciniphila SYSU-85 of the present invention can improve the adverse reactions of oxaliplatin and enhance the anti-tumor effect of oxaliplatin.

[0013] In a fourth aspect, the present invention claims the use of the Akkermansia muciniphila in the preparation of immune activators. The Akkermansia muciniphila SYSU-85 of the present invention can significantly promote the expression of the key anti-tumor cytokine CXCL-10 in macrophages RAW264.7 and play an anti-tumor role.

[0014] In a fifth aspect, the present invention claims an anti-tumor drug containing the Akkermansia muciniphila, the fermentation product of the Akkermansia muciniphila, or the inactivated cells of the Akkermansia muciniphila.

[0015] Further, the inactivated cells are heat-inactivated cells.

[0016] Further, the content of the Akkermansia muciniphila in the anti-tumor drug is not less than 10 6 cells.

[0017] Further, the anti-tumor drug further contains platinum-based chemotherapy drugs and / or PD-1 inhibitors.

[0018] Further, the platinum-based chemotherapy drugs include at least one of cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, and lobaplatin.

[0019] In a specific embodiment of the present invention, taking oxaliplatin or a PD-1 inhibitor as an example, the combination of the Akkermansia muciniphila SYSU-85 is used for treatment, which significantly slows down the tumor growth curve and significantly reduces the tumor volume, improving the tumor treatment effect.

[0020] Furthermore, the anti-tumor drug includes at least one drug for anti-adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, pancreatic cancer, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, glioblastoma multiforme, glioma, head and neck tumor, renal chromophobe cell carcinoma, hybrid renal carcinoma, renal carcinoma, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal adenocarcinoma, sarcoma, cutaneous melanoma, gastric cancer, gastrointestinal stromal tumor, esophageal cancer, testicular cancer, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, and soft tissue sarcoma.

[0021] In a specific embodiment of the present invention, taking colon cancer cells MC38 and CT26 as examples for experiments, the tumor growth curve can be significantly slowed down, the tumor volume can be significantly reduced, and the tumor growth can be inhibited.

[0022] In a specific embodiment of the present invention, taking gastric cancer cell line AGS as an example for experiments, the survival rate of gastric cancer cell line AGS can be significantly reduced.

[0023] Furthermore, the anti-tumor drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) Akkermansia muciniphila SYSU-85 of the present invention is weakly dependent on mucin and grows well in BHI medium supplemented with cysteine.

[0026] (2) Akkermansia muciniphila SYSU-85 of the present invention is resistant to oxaliplatin and can achieve quantitative growth in a medium supplemented with oxaliplatin.

[0027] (3) Akkermansia muciniphila SYSU-85 of the present invention combined with oxaliplatin significantly slows down the tumor growth curve and significantly reduces the tumor volume, improves the tumor treatment effect, and expands the scope of clinical drug use.

[0028] (4) 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.

[0029] (5)The Akkermansia muciniphila SYSU-85 of the present invention or its combination with a PD-1 inhibitor significantly slows down the tumor growth curve, significantly reduces the tumor volume, improves the tumor treatment effect, and expands the scope of clinical drug use. Description of the Drawings

[0030] Figure 1 It is the result of protein spectrum comparison between SYSU-85 and ATCC BAA-835 in Example 2.

[0031] Figure 2 It is the culture result of SYSU-85 in BHI in Example 3. Among them, A is the bacterial liquid cultured for 24 h; B is the colony morphology on the solid medium; C is the bacterial cells after centrifugation, where the left is the blank medium cultured under the same conditions and the right is SYSU-85; D is the microscopic examination diagram of SYSU-85 after Gram staining.

[0032] Figure 3 It is the growth situation of SYSU-85 in the medium supplemented with different concentrations of oxaliplatin in Example 5, compared with Akkermansia muciniphila ATCC BAA-835. Among them, A is the situation where the oxaliplatin concentrations are 15.63 μM, 7.81 μM, 3.91 μM, 1.95 μM, and 0 μM respectively; B is the situation where the oxaliplatin concentrations are 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, and 0 μM respectively.

[0033] Figure 4 It is the effect of SYSU-85 alone or in combination with oxaliplatin on subcutaneous tumor implantation of MC38 in mice in Example 6. Among them, A is the change of tumor volume in each group over time; B is the tumor photo.

[0034] Figure 5 It is the survival curve of mice intragastrically administered or not with SYSU-85 and heat-inactivated SYSU-85 under the condition of intraperitoneal injection of high-dose oxaliplatin in Example 7.

[0035] Figure 6 It is the effect of SYSU-85 alone or in combination with a PD-1 inhibitor on subcutaneous tumor implantation of CT26 in mice in Example 8. Among them, A is the change of tumor volume in each group over time; B is the tumor photo.

[0036] Figure 7 It is the IC of oxaliplatin on gastric cancer cells AGS in Example 9 50And the effects of the combination of oxaliplatin with SYSU-85 or ATCC BAA-835. Among them, A is the effect of different concentrations of oxaliplatin on gastric cancer cell line AGS; B is the effect of the combination of 18 μM oxaliplatin with SYSU-85 or ATCC BAA-835 on gastric cancer cell line AGS.

[0037] Figure 8 It is the result of SYSU-85 promoting macrophage to express CXCL-10 in Example 10.

[0038] In the above figures, " " indicates a significant difference between two groups with p < 0.05; " " indicates a significant difference between two groups with p < 0.01; " " indicates a significant difference between two groups with p < 0.0001. Detailed implementation manners

[0039] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.

[0040] 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.

[0041] Prepare brain heart infusion medium (BHI): Take 37 g of BHI medium powder and add it to 1 L of water (1 g of cysteine can be added), fully dissolve it, place it in a high-pressure sterilizer at 115 °C for 20 min (for solid medium, add 15 g / L of agar), and cool for later use; the BHI medium powder is purchased from Oxoid.

[0042] Example 1 Isolation and identification of SYSU-85

[0043] 1. Take an appropriate amount of feces from a patient who has received treatment with the combination of toripalimab and SOX (oxaliplatin + tegafur) and whose gastric cancer range has significantly shrunk and meets the response standard of partial response (PR) to chemotherapy. After gradient dilution with phosphate buffer solution (PBS), take 100 μL and evenly coat it on the brain heart infusion medium, and culture it at 37 °C under anaerobic conditions for 48 - 72 h.

[0044] 2. Gram stain and microscopic examination of the single colonies after culturing in Step 1: Pick a single colony into 20 μL of PBS, pipette and mix well 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 h. Take the remaining 5 μL of the bacterial suspension and drop it in the center of a glass slide, bake it evenly with an alcohol lamp for fixation, drop crystal violet staining solution to cover for 1 min, rinse with slow running water, drop iodine solution for mordanting for 1 min, rinse with slow running water, drop decolorizing alcohol for 20 - 30 s, rinse with slow running water, and counterstain with safranin staining solution for 1 min. After the glass slide is dry, observe the bacterial morphology with an optical microscope. If the bacterial morphologies are consistent, scrape off the bacteria (SYSU - 85) obtained from the culture of the bacterial suspension and store them.

[0045] 3. Extraction of bacterial DNA and 16S rRNA identification: Extract the 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 for bacterial molecular identification of 16S rRNA (27F and 1492R), and use the bacterial genomic DNA extracted by the kit as a template for PCR amplification with LA enzyme. The PCR amplification program is as follows: pre - denaturation at 95 °C for 1 min; denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 90 s, cycle 25 times; final extension at 72 °C for 7 min. The amplified product is identified by agarose gel electrophoresis, and a band appears at 1500 bp, and no band appears in the negative control. Then send it to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0046] After the sequencing results are spliced by SeqMan software, use the BLAST tool in the NCBI database for sequence retrieval. It is found that the screened SYSU - 85 has a sequence similarity of 99.92% with the ATCC BAA - 835 sequence, and there is a base mutation at the 1170th base of 16S rRNA, where the nucleotide base at this site changes from G to A.

[0047] The nucleotide sequence of bacterial 16S rRNA is shown as SEQ ID NO: 1, the nucleotide sequence of 23S rRNA is shown as SEQ ID NO: 2, and the nucleotide sequence of 5S rRNA is shown as SEQ ID NO: 3.

[0048] Example 2 ANI analysis and proteomic analysis of SUSU85

[0049] I. Experimental method

[0050] After ultrasonic lysis, acetone precipitation, enzymatic hydrolysis and desalting of the strain SYSU - 85 and ATCC BAA - 835 screened in Example 1 respectively, perform proteomic analysis by high - performance nanoflow liquid chromatography - ultra - high resolution mass spectrometry (School of Pharmacy, Sun Yat - sen University).

[0051] ANI analysis (Average Nucleotide Identity) was performed on SYSU-85 and ATCC BAA-835.

[0052] A proteomic study was conducted on ATCC BAA-835 and SYSU-85 strains. After excluding data with medium and low confidence, proteins with an abundance ratio of SYSU-85:ATCC BAA-835 greater than 2 were selected to draw a KEGG enrichment analysis map.

[0053] II. Experimental Results

[0054] 1. The results showed that the ANI value between SYSU-85 and Akkermansia muciniphila ATCC BAA-835 of the genus Akkermansia was 98.72%, showing certain differences.

[0055] SYSU-85 was named Akkermansia muciniphila SYSU-85 and was deposited in the General Microbiology Center of the China Microbial Culture Collection Center on August 8, 2024. Its deposit number is CGMCC NO.46070, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0056] 2. A total of 958 proteins were analyzed in SYSU-85, and a total of 1234 proteins were analyzed in ATCC BAA-835. Subsequently, proteins unique to SYSU-85 and those with a content more than twice that of ATCC BAA-835 were screened to obtain 374 related proteins, and online KEGG enrichment analysis was performed at bioinfo.org / kobas ( Figure 1 ), indicating proteomic differences between the two strains. It was found by comparison that "Mucin-associated surface protein (MASP)" (Uniprot: A0A4Q5IJY4) related to bacterial mucin was significantly enriched in the proteome of ATCC BAA-835 but lacking in the proteome of SYSU-85. And SYSU-85 does not contain the Amuc_1434 protein from ATCC BAA-835 described in patent CN118063568A, indicating that SYSU-85 has a different mechanism of anti-tumor action.

[0057] Example 3 SYSU-85 has weak dependence on mucin

[0058] I. Experimental Methods

[0059] 1. Inoculate strain SYSU-85 into BHI liquid medium for activation, and culture it at 37°C under anaerobic conditions for 24 h to obtain a seed solution.

[0060] 2. Inoculate the seed solution obtained in step 1 into BHI liquid medium at an inoculation amount of 2% (v / v), and culture it at 37°C under anaerobic conditions for 24 h; additionally, set up a blank BHI liquid medium without inoculating any strain and culture it under the same conditions.

[0061] 3. After culturing for 24 h in step 2, respectively pipette 1 mL of the bacterial solution into two centrifuge tubes and centrifuge at 2000 rpm for 1 min. Record the bacterial solution concentration and the amount of bacteria. As shown in A in Figure 2 , it is the bacterial solution cultured for 24 h; as shown in B in Figure 2 , it is the colony morphology on the solid medium; as shown in C in Figure 2 , it is the bacteria after centrifugation. Among them, the left is the blank medium cultured under the same conditions, and the right is SYSU-85.

[0062] 4. Perform Gram staining on the bacterial solution of SYSU-85 according to step 2 in Example 1.

[0063] II. Experimental Results

[0064] The result of Gram staining is as shown in D in Figure 2 , indicating that SYSU-85 is a Gram-positive bacterium.

[0065] Consistent with the proteomic analysis result of Example 2, SYSU-85 has a low dependence on mucin. The culture conditions of ATCC BAA-835 are usually BHI medium supplemented with mucin, and the growth rate is slow. SYSU-85 can be cultured in a good state and with a large amount of bacteria only using BHI medium, which is convenient for subsequent experimental research and industrial cultivation.

[0066] Example 4 Drug Sensitivity Test of SYSU-85

[0067] I. Experimental Method

[0068] Take SYSU-85 and ATCC BAA-835 in the logarithmic growth phase and inoculate them into sterile PBS respectively to obtain the bacterial solutions to be tested. Dip a sterile cotton swab into the bacterial solutions to be tested and evenly coat them on BHI and BHI plates supplemented with mucin (0.5 - 2%, m / v);

[0069] After the bacterial solutions to be tested on the plates are absorbed, use forceps to evenly place the corresponding drug sensitivity test paper on the plates, and culture them upright in a 37°C bacterial incubator for 48 h, and measure the diameter of the inhibition zone.

[0070] II. Experimental Results

[0071] As shown in Table 1, SYSU-85 is insensitive to levofloxacin and penicillin, while ATCC BAA-835 is sensitive to levofloxacin and penicillin.

[0072] Table 1 Inhibition zone diameters of SYSU-85 and ATCC BAA-835 in drug susceptibility tests

[0073]

[0074] Example 5 Effect of oxaliplatin on SYSU-85

[0075] I. Experimental method

[0076] 1. Pipette 100 μL of 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 using the serial dilution method. Set three parallels for each concentration. Additionally, set a column with 0 μM oxaliplatin and a blank group without bacteria.

[0077] 2. Add SYSU-85 and ATCC BAA-835 into the culture medium respectively. After anaerobic incubation at 37 °C for 24 h, use a microplate reader to measure the absorbance value of each well at 600 nm.

[0078] II. Experimental results

[0079] As Figure 3 shown in A of Figure 3 and B of

[0080] the above, low concentrations of oxaliplatin can promote the growth of SYSU-85, and the inhibitory effect of high concentrations of oxaliplatin on SYSU-85 is not obvious. The growth status of SYSU-85 is always better than that of ATCC BAA-835.

[0081] Example 6 Antitumor effect of SYSU-85 combined with oxaliplatin on subcutaneous MC38 in mice

[0082] I. Experimental method

[0083] 1. Five - to - eight - week - old male C57BL / 6 mice (from the Experimental Animal Center of Sun Yat - sen University) were selected. After one - week of adaptive feeding, they were randomly divided into four groups: a control group with intraperitoneal injection of PBS + gavage of PBS, an oxaliplatin - intraperitoneal - injection group, a SYSU - 85 - gavage group, and an experimental group with intraperitoneal injection of oxaliplatin + gavage of SYSU - 85. Each mouse was subcutaneously inoculated with 10 6 MC38 colon cancer cells per mouse. After tumor formation, for the groups that needed intraperitoneal injection of oxaliplatin, each mouse was intraperitoneally injected with 100 μL of oxaliplatin at a concentration of 6 mg / mL every two days. For the experimental group with gavage of SYSU - 85, each mouse was gavaged with 100 μL of SYSU - 85 with a bacterial content of 1×10 7 CFU (the SYSU - 85 bacterial solution was centrifuged, resuspended and washed with PBS, and centrifuged again to precipitate, and then diluted with PBS to 1×10 8 CFU / mL). The control group was gavaged and intraperitoneally injected with the same volume of PBS synchronously for each mouse.

[0084] 2. On the 9th, 12th, 15th, 18th, and 21st days after tumor implantation, the size of the tumor - bearing mice was measured. On the 21st day, the mice were sacrificed, and the tumors were removed for photographing.

[0085] II. Experimental Results

[0086] As shown in A of Figure 4 and B of Figure 4 , SYSU - 85 combined with oxaliplatin significantly inhibited the growth of MC38 tumors. Specifically, SYSU - 85 combined with oxaliplatin made the tumor growth curve significantly slower and the tumor volume significantly smaller. The above results indicate that SYSU - 85 combined with oxaliplatin has good anti - tumor effects and has clinical application value.

[0087] Example 7 SYSU - 85 Alleviates the Toxic Side Effects of Oxaliplatin

[0088] I. Experimental Methods

[0089] Five - to - eight - week - old male C57BL / 6 mice (from the Experimental Animal Center of Sun Yat - sen University) were selected. After one - week of adaptive feeding, they were randomly divided into four groups: a control group with intraperitoneal injection of PBS + gavage of PBS, an oxaliplatin - intraperitoneal - injection group, an experimental group with intraperitoneal injection of oxaliplatin + gavage of SYSU - 85, and an experimental group with intraperitoneal injection of oxaliplatin + gavage of heat - inactivated SYSU - 85. For the groups that needed intraperitoneal injection of oxaliplatin, each mouse was intraperitoneally injected with 100 μL of oxaliplatin at a concentration of 60 mg / mL. For the experimental group with gavage of SYSU - 85, each mouse was gavaged with 100 μL of SYSU - 85 with a bacterial content of 1×10 7 CFU (the SYSU - 85 bacterial solution was centrifuged, resuspended and washed with PBS, and centrifuged again to precipitate, and then diluted with PBS to 1×10 8CFU / mL). Mice in the heat-inactivated group were gavaged with the same number of SYSU-85 inactivated at 70 °C for 30 min, and mice in the control group were simultaneously gavaged and intraperitoneally injected with 100 μL of PBS. The death of mice was recorded daily.

[0090] II. Experimental Results

[0091] As Figure 5 shown, high-dose oxaliplatin can cause the death of mice, while SYSU-85, especially heat-inactivated SYSU-85, can alleviate the toxic and side effects of oxaliplatin.

[0092] The above results indicate that when SYSU-85 is used in combination with oxaliplatin, it can not only enhance the anti-tumor effect of oxaliplatin but also alleviate the toxic and side effects of oxaliplatin.

[0093] Example 8 Anti-tumor Effect of SYSU-85 Combined with PD-1 (Programmed Death Receptor 1) Inhibitor on Subcutaneous CT26 in Mice

[0094] I. Experimental Methods

[0095] 1. Five- to eight-week-old male C57BL / 6 mice (Experimental Animal Center of Sun Yat-sen University) were randomly divided into four groups after one week of adaptive feeding: a control group intraperitoneally injected with PBS + gavaged with PBS, a group intraperitoneally injected with PD-1 inhibitor, a group gavaged with SYSU-85, and an experimental group intraperitoneally injected with PD-1 inhibitor + gavaged with SYSU-85. Each mouse was subcutaneously inoculated with 10 6 CT26 colon cancer cells per mouse. After tumor formation, for the groups that needed to be intraperitoneally injected with PD-1 inhibitor, each mouse was intraperitoneally injected with 60 μg of PD-1 inhibitor every two days, and for the experimental group gavaged with SYSU-85, each mouse was gavaged with 100 μL of SYSU-85 with a bacterial content of 1 × 10 7 CFU (the SYSU-85 bacterial solution was centrifuged, resuspended and washed with PBS, and diluted to 1 × 10 8 CFU / mL) with PBS. Mice in the control group were simultaneously gavaged and intraperitoneally injected with the same volume of PBS.

[0096] 2. The size of the tumor-bearing mice was measured on the 7th, 10th, 13th, 16th, and 19th days after tumor implantation. The mice were sacrificed on the 19th day, and the tumors were removed and photographed.

[0097] II. Experimental Results

[0098] As Figure 6 in A and Figure 6As shown in B of , SYSU-85 combined with a PD-1 inhibitor has a significant effect on inhibiting the growth of CT26 tumors. Specifically, SYSU-85 combined with PD-1 significantly slows down the tumor growth curve and significantly reduces the tumor volume. The above results indicate that SYSU-85 combined with a PD-1 inhibitor has a good anti-tumor effect and has clinical application value.

[0099] Example 9 SYSU-85 combined with oxaliplatin inhibits the proliferation of gastric cancer cells AGS

[0100] I. Experimental method

[0101] 1. Uniformly seed 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 wall.

[0102] 2. Replace the medium with DMEM medium containing 500 μM to 0 μM (serially diluted concentration, specific concentrations are shown in Example 5) oxaliplatin and culture for 48 h.

[0103] 3. Remove the supernatant, and detect and calculate the cell survival rate using the CCK8 method.

[0104] 4. After repeating steps 1 and 2, add 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) respectively. The multiplicity of infection MOI is 10:1. After co-culturing with the cells for 48 h, remove the supernatant, and detect and calculate the cell survival rate using the CCK8 method.

[0105] II. Experimental results

[0106] As Figure 7 shown in A of , the IC 50 is 18.89 μM; as Figure 7 shown in B of , the killing effect of oxaliplatin combined with SYSU-85 on gastric cancer cells AGS is the highest.

[0107] Example 10 SYSU-85 promotes the expression of CXCL-10 (CXC chemokine 10) in macrophages RAW264.7

[0108] I. Experimental method

[0109] 1. Uniformly seed macrophages RAW264.7 in a six-well plate and culture them in DMEM medium containing 10% (v / v) fetal bovine serum for 24 h.

[0110] 2. Add SYSU-85 to the RAW264.7 cultured in Step 1 at a MOI of 10:1 and co-culture for 24 h.

[0111] 3. Aspirate the supernatant of the cell culture medium in the six-well plate from Step 2, centrifuge at 3,000×g for 10 min, and transfer the supernatant to a new centrifuge tube.

[0112] 4. Use a CXCL-10 ELISA kit (Linker Biotechnology) to measure the content of CXCL-10 in the supernatant of the cell culture medium.

[0113] II. Experimental Results

[0114] As Figure 8 shown, SYSU-85 significantly promotes the expression of CXCL-10 in RAW264.7. This indicates that SYSU-85 activates macrophages to produce key anti-tumor cytokines and plays an anti-tumor role.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An Akkermansia muciniphila, characterized in that, The Akkermansia muciniphila is Akkermansia muciniphila SYSU-85, which was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on August 8, 2024, and its deposit number is: CGMCC NO. 46070.

2. Use of the Akkermansia muciniphila described in claim 1 in combination with oxaliplatin in the preparation of a drug for treating colorectal cancer and / or gastric cancer.

3. A drug for treating colorectal cancer and / or gastric cancer, characterized in that, The drug contains the Akkermansia muciniphila described in claim 1 and oxaliplatin.

4. The drug according to claim 3, characterized in that, The drug further comprises a pharmaceutically acceptable carrier and / or excipient.

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