A strain of Lactobacillus mucosae and its application in the preparation of drugs for treating cancer

Abstract

The present invention relates to a strain of Limosilactobacillus mucosae and its application in the preparation of drugs for treating cancer, belonging to the field of biomedical technology. The present invention provides a strain of Limosilactobacillus mucosae XA-1405, which can significantly reduce the growth rate of colon tumors, can significantly increase the response efficiency of PD-1 inhibitors, further reduce the growth rate of colon tumors in the presence of PD-1 inhibitors, can significantly increase the infiltration of CD8+ T cells in colon cancer tumor tissues, can significantly promote CD8+ T cells to secrete TNF-α, and has a significant inhibitory effect on the proliferation of gastric cancer cell line AGS and colon cancer cell line HCT116. It can be seen that Limosilactobacillus mucosae XA-1405 has great application prospects in the preparation of drugs for treating cancer.

Description

Technical Field

[0001] The present invention relates to a Limosilactobacillus mucosae and its application in the preparation of a drug for treating cancer, belonging to the field of biomedical technology. Background Art

[0002] A tumor refers to a disease caused by abnormal growth of its cells in an organism due to various factors, and is divided into malignant tumors and benign tumors according to its characteristics and harm to the organism. Malignant tumors, also known as cancers, have biological characteristics such as abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness, and metastasis.

[0003] The PD-1 / PD-L1 immunotherapy is a type of anti-cancer immunotherapy that treats various types of cancers and improves the overall survival of patients by blocking the PD1 / PD-L1 signaling pathway to cause cancer cell death. However, there are currently many problems in the clinical application of the PD-1 / PD-L1 immunotherapy, and not all patients are suitable for this therapy.

[0004] For example, since the initiation of clinical trials of PD-1 inhibitors, 15-20% of patients have developed drug resistance; moreover, PD-1 inhibitors have relatively strong side effects; in addition, some patients do not respond to the PD-1 / PD-L1 immunotherapy, and this secondary drug resistance phenomenon caused by low response efficiency also limits the clinical application of the PD-1 / PD-L1 immunotherapy (see the literature: Jin-Yu Sun et al. 2005).

[0005] Therefore, there is an urgent need to find a drug for treating cancer that can effectively treat cancer with low drug resistance and side effects, or there is an urgent need to find a method that can reduce the drug resistance and side effects of PD-1 inhibitors and improve the response efficiency of the PD-1 / PD-L1 immunotherapy. Summary of the Invention

[0006] To solve the above problems, the present invention provides a Limosilactobacillus mucosae XA-1405, which is deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 40306 and the deposit date of September 5, 2022.

[0007] The Lactobacillus mucosae XA-1405 is derived from frozen fecal samples of healthy individuals in the Shenzhen area. After sequencing analysis, the 16S rDNA sequence of this strain is shown in SEQ ID NO.1. The sequenced sequence was compared with nucleic acid sequences in NCBI, and the results showed that the strain was Lactobacillus mucosae, which was named Lactobacillus mucosae XA-1405.

[0008] The present invention also provides the use of the above-mentioned Lactobacillus mucosae XA-1405 in the preparation of a drug for treating cancer.

[0009] In one embodiment of the present invention, the cancer is a cancer caused by solid tumors; the cancer caused by solid tumors is gastric cancer, rectal cancer or colon cancer.

[0010] In one embodiment of the present invention, the drug further contains a PD-1 inhibitor.

[0011] In one embodiment of the present invention, the drug further contains a drug carrier and / or pharmaceutical excipients.

[0012] In one embodiment of the present invention, the drug carrier includes microcapsules, microspheres, nanoparticles and / or liposomes.

[0013] In one embodiment of the present invention, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids and / or release retardants.

[0014] In one embodiment of the present invention, the dosage form of the drug is powder, granule, capsule, tablet, pill or oral liquid.

[0015] In one embodiment of the present invention, in the drug, the viable count of Lactobacillus mucosae XA-1405 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0016] The present invention also provides a drug for treating cancer, which contains the above-mentioned Lactobacillus mucosae XA-1405.

[0017] In one embodiment of the present invention, the cancer is a cancer caused by solid tumors; the cancer caused by solid tumors is gastric cancer, rectal cancer or colon cancer.

[0018] In one embodiment of the present invention, the drug further contains a PD-1 inhibitor.

[0019] In one embodiment of the present invention, the drug further contains a drug carrier and / or pharmaceutical excipients.

[0020] In one embodiment of the present invention, the drug carrier includes microcapsules, microspheres, nanoparticles, and / or liposomes.

[0021] In one embodiment of the present invention, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and / or release retardants.

[0022] In one embodiment of the present invention, the dosage form of the drug is powder, granule, capsule, tablet, pill, or oral liquid.

[0023] In one embodiment of the present invention, in the drug, the viable count of Limosilactobacillus mucosae XA-1405 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0024] The technical solution of the present invention has the following advantages:

[0025] The present invention provides a strain of Limosilactobacillus mucosae XA-1405, and this Limosilactobacillus mucosae XA-1405 can treat cancer, specifically manifested in:

[0026] (1) It can significantly reduce the growth rate of colon tumors;

[0027] (2) It can significantly increase the response efficiency of the PD-1 inhibitor and further reduce the growth rate of colon tumors in the presence of the PD-1 inhibitor;

[0028] (3) It can significantly increase the infiltration of CD8+ T cells in colon cancer tumor tissues;

[0029] (4) It can significantly promote CD8+ T cells to secrete TNF-α;

[0030] (5) It has a significant inhibitory effect on the proliferation of gastric cancer cell line AGS and colon cancer cell line HCT116.

[0031] It can be seen that Limosilactobacillus mucosae XA-1405 has great application prospects in the preparation of drugs for treating cancer.

[0032] In addition, Limosilactobacillus mucosae XA-1405 belongs to intestinal probiotics, can improve the intestinal flora, and has small advantages in terms of drug resistance and side effects.

[0033] Biological Material Deposit

[0034] A strain of Limosilactobacillus mucosae XA-1405, taxonomically named Limosilactobacillus mucosae, was deposited in the China General Microbiological Culture Collection Center on September 5, 2022, with the deposit number CGMCC No. 40306 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the Drawings

[0035] Figure 1 : Line graph of the volume change of tumors in mice of different groups. Figure 1 In, the inter-group differences in tumor volume were analyzed by Two-way ANOVA analysis, * indicates p < 0.05, and *** indicates p < 0.001.

[0036] Figure 2 : Photos of tumors in mice of different groups.

[0037] Figure 3 : Statistical results of the weights of tumors in mice of different groups. Figure 3 In, nc indicates no statistical difference, * indicates p < 0.05, and *** indicates p < 0.001.

[0038] Figure 4 : Flow cytometry statistical analysis results of Limosilactobacillus mucosae XA-1405. Figure 4 In, the Mann-Whitney test was used, and * indicates p < 0.05.

[0039] Figure 5 : Effect of Limosilactobacillus mucosae XA-1405 on the secretion of Granzyme B by CD8+ T cells. Figure 5 In, ** indicates p < 0.01, and ns indicates no significant difference.

[0040] Figure 6 : Effect of Limosilactobacillus mucosae XA-1405 on the secretion of IFN-γ by CD8+ T cells. Figure 6 In, ** indicates p < 0.01, and ns indicates no significant difference.

[0041] Figure 7:Effect of Lactobacillus mucosae XA-1405 on TNF-α secretion by CD8+ T cells. Figure 7 In the figure, * indicates p < 0.05, ** indicates p < 0.01, and ns indicates no significant difference.

[0042] Figure 8 :Effect of Lactobacillus mucosae XA-1405 on the proliferation of gastric cancer cell line AGS. Figure 8 In the figure, *** indicates p < 0.001.

[0043] Figure 9 :Effect of Lactobacillus mucosae XA-1405 on the proliferation of gastric cancer cell line HCT116. Figure 9 In the figure, *** indicates p < 0.001. Detailed implementation manner

[0044] The following embodiments are provided to better further understand the present invention. It is not limited to the best implementation manner, and does not limit the content and protection scope of the present invention. Any product that is the same or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.

[0045] MRS solid medium: Peptone 10.0 g / L, beef extract powder 5.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, yeast extract powder 4.0 g / L, ammonium citrate tribasic 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.2 g / L, Tween 80 0.01 mL / L, agar 15.0 g / L, manganese sulfate 0.05 g / L, pH 6.2.

[0046] MRS liquid medium: Peptone 10.0 g / L, beef extract powder 5.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, yeast extract powder 4.0 g / L, ammonium citrate tribasic 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.2 g / L, Tween 80 0.01 mL / L, manganese sulfate 0.05 g / L, pH 6.2.

[0047] YCFA solid medium: Tryptone 10.0 g / L, Yeast Extract 2.5 g / L, Sodium Bicarbonate 4.0 g / L, Glucose 2.0 g / L, Maltose 2.0 g / L, Cellobiose 2.0 g / L, Cysteine Hydrochloride 1.0 g / L, Dipotassium Hydrogen Phosphate 0.25 g / L, Potassium Dihydrogen Phosphate 0.45 g / L, Ammonium Sulfate 0.9 g / L, Sodium Chloride 0.9 g / L, Magnesium Sulfate Heptahydrate 0.09 g / L, Calcium Chloride Dihydrate 0.09 g / L, Resazurin 1.0 mL / L, Hemin 0.01 g / L, VFAmix 6.2 mL / L (VFAmix: Acetic Acid 17.0 mL, Propionic Acid 6.0 mL, n-Valeric Acid 1.0 mL, Iso-Valeric Acid 1.0 mL, Iso-Butyric Acid 1.0 mL), vitamin solution I 1.0 mL / L (vitamin solution I: Biotin 5.0 mg, Vitamin B12 5.0 mg, p-Aminobenzoic Acid 15.0 mg, Folic Acid VB2 5.0 mg, Vitamin B6 Hydrochloride 75 mg), Agar 15.0 g / L, made up to 1 L with Distilled water, pH 7.0.

[0048] BHI liquid medium: Tryptone 10.0 g / L, Sodium Chloride 5.0 g / L, Disodium Hydrogen Phosphate 2.5 g / L, Dextrose 2.0 g / L, Heart Extract Powder 9.8 g / L, Brains Extract Powder 7.7 g / L, pH 7.4.

[0049] The digestive fluids involved in the following examples are as follows:

[0050] 0.25% Trypsin Digestive Fluid: First, weigh 2.5 g of porcine trypsin (purchased from Gibco) and 0.2 g of EDTA and dissolve them in PBS buffer (purchased from Solarbio, product number P1020), then adjust the pH to 7.4 with HCl, and finally make up to 1 L with PBS buffer to obtain 0.25% Trypsin Digestive Fluid.

[0051] The preparation methods of the bacterial suspensions, culture supernatants and dead bacteria involved in the following examples are as follows:

[0052] Take the mucous membrane Lactobacillus mucosae bacterial liquid and inoculate it into MRS liquid medium at an inoculation amount of 4% (v / v). Incubate it statically in a constant temperature incubator at 37 °C for 3 days to obtain a culture solution; centrifuge the culture solution at 8000 g for 10 min to obtain the supernatant of Lactobacillus mucosae culture and Lactobacillus mucosae cells; wash the Lactobacillus mucosae cells with physiological saline and then resuspend them in PBS buffer (purchased from Solarbio, product number P1020) until the bacterial concentration reaches 1×10 8 CFU / mL to obtain a Lactobacillus mucosae bacterial suspension, and store the Lactobacillus mucosae bacterial suspension at -80 °C for later use; boil the Lactobacillus mucosae cells at 100 °C for 15 min to obtain heat-killed Lactobacillus mucosae; resuspend the heat-killed Lactobacillus mucosae in MRS liquid medium until the bacterial concentration reaches 1×10 8 CFU / mL to obtain a heat-killed Lactobacillus mucosae suspension.

[0053] Experimental Example 1: Obtaining of Lactobacillus mucosae XA-1405

[0054] This experimental example provides the process for obtaining Lactobacillus mucosae XA-1405, and the specific process is as follows:

[0055] In an anaerobic workbench, take 29 frozen fecal samples from healthy people in the Shenzhen area, mix them evenly by equal volume to obtain a mixed sample; suck 0.5 mL of the mixed sample into an anaerobic blood culture bottle, and after statically incubating it in a constant temperature incubator at 37 °C for 3 days, first suck 0.1 mL of the mixed sample cultured for 3 days into 0.9 mL of PBS buffer (purchased from Solarbio, product number P1020) containing 1 g / L L-cysteine hydrochloride to obtain 10 -1 dilution, and then suck 0.1 mL of the 10 -1 dilution into 0.9 mL of PBS buffer containing 1 g / L L-cysteine hydrochloride to obtain 10 -2 dilution. According to this operation, successively obtain 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 dilutions; suck 10 -5 dilution, 10 -6 dilution and 10 -7The diluent was coated on the deoxygenated YCFA solid medium at a coating amount of 100 μL / plate (each dilution of the diluent corresponded to 3 deoxygenated YCFA solid media). After static culture in a constant temperature incubator at 37 °C for 3 days, single colonies were picked and inoculated into the deoxygenated BHI liquid medium, and then statically cultured in a constant temperature incubator at 37 °C for 3 days to obtain bacterial solutions. After numbering each strain corresponding to each bacterial solution, Gram staining, strain identification, physiological and biochemical experiments, and genomic identification and analysis were carried out according to the steps described in the textbook "Microbiology" (edited by Shen Ping and Chen Xiangdong). Strains with typical characteristics of Limosilactobacillus mucosae were selected to obtain strain XA-1405.

[0056] The process of strain identification is as follows:

[0057] Take the cells of XA-1405, extract the genome of XA-1405 using a bacterial genomic DNA extraction kit, and use the 27F / 1492R primer pair with sequences shown in SEQ ID NO.2 and SEQ ID NO.3 respectively. Using the extracted genome of XA-1405 as a template for amplification, the 16S rRNA of XA-1405 was obtained (the 16S rDNA sequence of XA-1405 is shown in SEQ ID NO.1). The 16S rDNA of XA-1405 was subjected to nucleic acid sequence alignment using the Blastn program of NCBI. The results showed that this strain was Limosilactobacillus mucosae, and it was named Limosilactobacillus mucosae XA-1405.

[0058] Experimental Example 2: Effect of Limosilactobacillus mucosae XA-1405 on tumor growth in a colon cancer model mouse

[0059] This experimental example provided an experiment on the effect of Limosilactobacillus mucosae XA-1405 on tumor growth in a colon cancer model mouse. The experimental process was as follows:

[0060] Twenty C57BL / 6J mice (female, 5 weeks old, weighing 16 - 20 g, purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into 4 groups, with 5 mice in each group. The 4 groups were: blank control group (NC), PD-1 treatment group intragastrically administered with PD-1 inhibitor, Limosilactobacillus mucosae single bacteria treatment group (Mu) intragastrically administered with Limosilactobacillus mucosae XA-1405 bacterial suspension, and Limosilactobacillus mucosae + PD-1 combination treatment group (Mu + PD-1) intragastrically administered with PD-1 inhibitor and Limosilactobacillus mucosae XA-1405 bacterial suspension.

[0061] Each group of mice was adaptively fed in an environment with constant temperature and humidity, with free access to water. The experiment started after 7 days of adaptive feeding. After the experiment started, each group of mice was treated with antibiotics for two weeks. The administration method of antibiotic treatment was as follows: drinking water containing an antibiotic aqueous solution with an ampicillin concentration of 1 mg / mL, and gavage with a mixed antibiotic aqueous solution with a neomycin concentration of 10 mg / mL, a metronidazole concentration of 10 mg / mL, and a vancomycin concentration of 5 mg / mL. The gavage dose was 200 μL / mouse / time, and gavage was performed once a day. After the antibiotic treatment ended, the mice in the single-bacteria treatment group and the combined treatment group were gavaged with the bacterial suspension at a dose of 1×10 8 CFU / mouse / time, and the mice in the other groups were gavaged with an equal volume of PBS buffer, once a day. After 7 days of gavage, 5×10 5 MC38 cells (purchased from the ATCC cell bank) were subcutaneously inoculated into each group of mice, and the day of subcutaneous tumor inoculation was recorded as D0 (baseline). After subcutaneous tumor inoculation, the mice in the single-bacteria treatment group and the combined treatment group continued to be gavaged with the bacterial suspension until the end of the experiment, and the mice in the blank control group and the PD-1 treatment group continued to be gavaged with PBS buffer until the end of the experiment. When the average tumor volume reached 50 mm 3 , the mice in the PD-1 treatment group and the combined treatment group began to be injected with the PD-1 inhibitor (purchased from Biocell Company) at a dose of 100 μg / mouse / time, once every three days, for a total of four injections. During the experiment, starting from D0, the size and weight of the tumors of each group of mice were observed and measured every two days, and the tumor volume of each group of mice was calculated according to the formula tumor volume = 1 / 2 × tumor long diameter 2 × tumor short diameter. The measurement and calculation results are shown in Figures 1 - 3 .

[0062] As can be seen from Figures 1 - 3 , the tumors in the blank control group grew rapidly and reached 900 mm 3 at D21; the tumor growth rates in the PD-1 treatment group and the L. mucosae single-bacteria treatment group were significantly lower than those in the blank control group. The tumor volumes of these two groups at D21 were 416 mm 3 , 479 mm 3 respectively; the tumor growth rate in the L. mucosae + PD-1 combined treatment group was significantly lower than those in the blank control group, the PD-1 treatment group, and the L. mucosae single-bacteria treatment group, and the tumor volume at D21 was 109 mm 3 . This result shows that Lactobacillus mucosae XA-1405 can significantly reduce the growth rate of colon tumors, and can significantly increase the response efficiency of the PD-1 inhibitor, and further reduce the growth rate of colon tumors in the presence of the PD-1 inhibitor.

[0063] Experimental Example 3: Effect of Lactobacillus mucosae XA-1405 on the expression of related markers in tumors of colon cancer model mice

[0064] This experimental example provides an experiment on the effect of Lactobacillus mucosae XA-1405 on the expression of related markers in tumors of colon cancer model mice. The experimental procedure is as follows:

[0065] Add 3 mL of RPMI 1640 medium (Corning Cat#10-040-CVR) to each vial of Enzyme D freeze-dried powder in the Tumor Dissociation Kit (Miltenyi, CAT#130-096-730) to obtain Enzyme D solution; add 2.7 mL of RPMI 1640 medium to each vial of Enzyme R freeze-dried powder in the Tumor Dissociation Kit to obtain Enzyme R solution; add 1 mL of Buffer 1 reagent in the Tumor Dissociation Kit (Buffer 1 reagent: 1 part of Fixation / permeabilization Concentrate (4x) diluted with 3 parts of Fixation / Perm Diluent) to each vial of Enzyme A freeze-dried powder in the Tumor Dissociation Kit to obtain Enzyme A solution; add 2.35 mL of RPMI 1640 medium, 100 μL of Enzyme D solution, 50 μL of Enzyme R solution, and 12.5 μL of Enzyme A solution to a gentleMACS C tube to obtain a mixed enzyme solution; take the tumors of each group of mice at D21 obtained in Experimental Example 2; take the tumors, remove non-tumor tissues, cut them into pieces of 2 mm 3 After that, add them to the mixed enzyme solution to obtain a mixture; use a gentleMACS Octo tissue processor to filter, resuspend, and adjust the volume of the mixture to a cell concentration of 1×10 8 cell / μL to obtain a tumor cell suspension.

[0066] Take 10 μL of the tumor cell suspension and add it to a flow tube. Then, add 0.25 μg of CD8a antibody (APC-FIRE750 fluorescence) and 0.5 μg of CD45 antibody (BV785 fluorescence) (specific information on dyes and antibodies is shown in Table 1) to the flow tube to prepare a mixed solution. Centrifuge the mixed solution in the flow tube, discard the supernatant, resuspend it to the original volume with 100 μL of Buffer 2 reagent (Buffer 2 reagent: 1 part of Permeabilization Buffer (10x) diluted with 9 parts of pure water), and then load it onto the machine. Use a flow cytometer to collect data, and use Kaluza 2.1 software to analyze the percentages of CD45+ (immune cells) and CD4- / CD8a+ (cytotoxic T cells) in different immune cell subsets. Use Bartlett's test to test for homogeneity of variance and normality. The analysis results are shown in Figure 4 .

[0067] It can be seen from Figure 4 that compared with the PD-1 treatment group, the infiltration of CD8+ T cells (p < 0.05) in the tumor tissues of mice in the L. mucosae single-strain treatment group increased (by 5%). This result indicates that Lactobacillus mucosae XA-1405 mainly kills tumors by increasing the infiltration of CD8+ T cells and mainly kills tumors by increasing the infiltration of CD8+ T cells.

[0068] Table 1 Information on dyes and antibodies

[0069] Fluorescent dye Antibody Catalog number Supplier Clone ISO APC-FIRE750 CD8a 100766 Biolegend 53-6.7 RatIgG2a,κ BV785 CD45 103149 Biolegend 30-F11 RatIgG2b,κ

[0070] Experimental Example 4: Effects of Lactobacillus mucosae XA-1405 on the secretion of Granzyme B, IFN-γ, and TNF-α by CD8+ T cells

[0071] This experimental example provides an experiment on the effects of Lactobacillus mucosae XA-1405 on the secretion of Granzyme B, IFN-γ, and TNF-α by CD8+ T cells. The experimental procedure is as follows:

[0072] Isolate lymphocytes from Cd4 cre Id2 fl / fl mice (Taconic Stock#4196), and purify CD8+ T cells using MACS negative selection (Thermo Fisher Scientific) (purity > 95%). Purified CD8+ T cells were seeded at 1 × 10 6The addition amount of CFU / mL was added to RPMI 1640 medium (Corning Cat#10 - 040 - CVR) containing 10 ng / mL recombinant mouse IL - 2 pre - coated with anti - CD3 (concentration 1 μg / mL) and anti - CD28 (concentration 2 mg / mL), and incubated statically in an incubator at 37°C and 5% (v / v) CO 2 for 48 h; after 48 h of culture, the CD8+ T cells were washed with PBS buffer; after washing, using PBS buffer as the blank control and MRS liquid medium as the negative control, the CD8+ T cells were added to the heat - inactivated dead bacteria suspension of Lactobacillus mucosae and the culture supernatant of Lactobacillus mucosae until the cell concentration reached 1×10 6 CFU / mL, and incubated statically in an incubator at 37°C and 5% (v / v) CO 2 for 24 h; after 24 h of incubation, the contents of Granzyme B, IFN - γ and TNF - α in the incubation supernatant were detected by flow cytometry, and the experimental results are shown in Figures 5 - 7 .

[0073] It can be seen from Figures 5 - 7 that the heat - inactivated dead bacteria of Lactobacillus mucosae XA - 1405 can significantly promote the expression of IFN - γ (p = 0.005) and Granzyme B (p = 0.002) in CD8+ T cells, and the culture supernatant of Lactobacillus mucosae XA - 1405 can significantly promote the expression of TNF - α in CD8+ T cells (p = 0.020) (a 5% increase compared with the negative control group). This result indicates that the metabolites of Lactobacillus mucosae XA - 1405 can effectively promote anti - tumor immunity.

[0074] Experimental Example 5: Effects of Lactobacillus mucosae XA - 1405 on the proliferation of gastric cancer cell line AGS and colon cancer cell line HCT116

[0075] This experimental example provides an experiment on the effects of Lactobacillus mucosae XA - 1405 on the proliferation of gastric cancer cell line AGS and colon cancer cell line HCT116. The experimental procedure is as follows:

[0076] Co - culture experiment of gastric cancer cell line AGS: One vial of gastric cancer cell line AGS cells (purchased from ATCC) was added to a T25 cell flask containing RPMI 1640 medium (Corning Cat#10 - 040 - CVR) with 10% (v / v) fetal bovine serum, 1% (w / v, g / 100 mL) penicillin and 1% (w / v, g / 100 mL) streptomycin, and incubated at 37°C and 5% (v / v) CO 2It was statically cultured in a constant temperature incubator at 37°C for 72 h to obtain adherent AGS cells. After discarding the culture supernatant in the T25 cell flask, the adherent AGS cells in the T25 cell flask were washed with PBS buffer. After the washing was completed, 0.25% trypsin digestion solution was added to the T25 cell flask at an addition amount of 2 mL / flask to soak the adherent AGS cells for 30 s. After the soaking was completed, the 0.25% trypsin digestion solution was discarded, and the adherent AGS cells in the T25 cell flask were digested at 37°C for 2 min. After the digestion was completed, the AGS cells in the T25 cell flask were resuspended with RPMI 1640 medium containing 10% (v / v) fetal bovine serum and pipetted evenly to obtain an AGS suspension with a cell concentration of 2×10 4 CFU / mL; Using PBS buffer as a blank control and Escherichia coli BL21(DE3) (purchased from ATCC) as a negative control, after inoculating the AGS suspension into a 96-well plate at an inoculation amount of 100 μL / well, the cells of Lactobacillus mucosae were inoculated into the 96-well plate at an inoculation amount of 2×10 6 CFU (MOI = 100:1), and it was statically cultured in a constant temperature incubator at 37°C and 5% (v / v) CO 2 . During the culture period, the cell density in the 96-well plate was detected every 24 h for 4 consecutive days. The detection results are shown in Figure 8 ; Among them, the detection method of cell density was as follows: After discarding the culture supernatant in the 96-well plate, 100 μL of RPMI 1640 medium containing 10 μL of CCK-8 reagent (purchased from Dojindo) was added to each well, and it was incubated in a constant temperature incubator at 37°C and 5% (v / v) CO 2 for 2 h. After the incubation was completed, the absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0077] Co-culture experiment of colon cancer cell line HCT116: On the basis of the co-culture experiment of gastric cancer cell line AGS, the AGS cells of gastric cancer cell line were replaced with HCT116 cells of colon cancer cell line (purchased from ATCC). The detection results are shown in Figure 9 ; Among them, the detection method of cell density was as follows: After discarding the culture supernatant in the 96-well plate, 100 μL of RPMI 1640 medium containing 10 μL of CCK-8 reagent (purchased from Dojindo) was added to each well, and it was incubated in a constant temperature incubator at 37°C and 5% (v / v) CO 2 for 2 h. After the incubation was completed, the absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0078] As can be seen from Figures 8 - 9 , the viable bacteria of Lactobacillus mucosae XA-1405 have a significant inhibitory effect on the proliferation of gastric cancer cell line AGS and colon cancer cell line HCT116 (compared with the blank control group, it inhibited by 38%).

[0079] Obviously, the above embodiments are merely examples given for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of this invention.

Claims

1. Use of Limosilactobacillus mucosae in the preparation of a drug for treating colon cancer, characterized in that, the Limosilactobacillus mucosae is deposited in the General Microbiology Center of the China Microbial Culture Collection Center, with the deposit number CGMCC No. 40306.

2. A strain of Limosilactobacillus mucosae, characterized in that, the Limosilactobacillus mucosae is deposited in the General Microbiology Center of the China Microbial Culture Collection Center, with the deposit number CGMCC No. 40306.

3. A drug for treating colon cancer, characterized in that, the drug contains the Limosilactobacillus mucosae described in claim 2.

4. The drug according to claim 3, characterized in that, the drug further contains a PD-1 inhibitor.

5. The drug according to claim 3 or 4, characterized in that, the drug further contains a drug carrier and / or pharmaceutical excipients.

6. The drug according to claim 5, characterized in that, the drug carrier includes microcapsules, microspheres, nanoparticles and / or liposomes.

7. The drug according to claim 5, characterized in that, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, antiadhesives, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids and / or release retardants.

8. The drug according to claim 3, characterized in that, the dosage form of the drug is powder, granule, capsule, tablet, pill or oral liquid.

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