CN116103196B - Active ruminococcus and application thereof - Google Patents
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Abstract
The invention relates to the technical field of microorganisms, in particular to active ruminococcus and application thereof. The preservation number of the active ruminococcus (Ruminococcus gnavus) WS29149 provided by the invention is CGMCC No.40452. The active ruminococcus WS29149 can be used for treating colon cancer tumors, and the defect of traditional fecal bacteria transplantation treatment is avoided.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to active ruminococcus and application thereof.
Background
The intestinal tract is one of the organs of the human body which is in contact with the external environment most closely, and environmental microorganisms enter the intestinal tract to form symbiosis with the host through diet and other modes. The research shows that the number of microorganisms in intestinal tracts is 10 times of human cells, and the number of genes is hundreds of times of human cells. Thus the gut microorganism is called the largest "organ" of the human body, while the gut microorganism genome is also called the "second genome of the human body". There are approximately 5000 microorganisms present in the human gut commensal according to the current definition and classification of microorganisms. With regard to such important intestinal microorganisms, very little is known, wherein more than half of the microorganisms cannot be obtained as a single strain by pure culture. It is therefore difficult to study the function of these microorganisms in the host, and to explore the mechanism of interaction with the host, and to use them effectively.
In addition, the intestinal tract is also an important immune organ of the human body. Many immune cells, including myeloid cells such as macrophages, DC cells, neutrophils, and lymphocytes such as innate lymphocytes, T/B cells, and NK cells reside in the intestinal tissue. Since microorganisms and immune cells in the intestinal tract are in intimate contact, there is a close relationship between the two. Microorganisms can influence the development and differentiation of immune cells by self surface antigens, generated metabolites and changed tissue microenvironment, and gene expression, and finally change the immune cell functions, and influence the immune activation and immune monitoring functions of hosts.
Intestinal microorganisms are closely related to various diseases of the human body, wherein the occurrence of colon cancer and intestinal microorganisms are inseparable. According to the investigation, colon cancer is the cancer with the third highest mortality rate, and many people are diagnosed and killed each year. However, no effective treatment is currently found in humans. Currently, colon cancer is treated mainly by radiotherapy, surgical excision, chemotherapy, immunotherapy and other modes. However, radiotherapy and surgical excision have serious adverse effects on the patient's body, resulting in weakness. Long-term chemotherapy can lead to drug resistance of tumor cells, or some patients are insensitive to drugs, so that the effect is very little. Immunotherapy may be an effective treatment for certain types of tumors and certain cancer patients, and can utilize the immune system to increase the killing function of toxic T cells, thereby effectively inhibiting tumors. Currently there are mainly some therapies targeting toxic T lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1) and its ligand (PD-L1). Good effects are obtained in melanoma, renal cell carcinoma and non-small cell lung cancer. However, other cancers (including colon cancer) respond poorly to immune-targeted therapies, and the degree of therapeutic response varies significantly from individual to individual. In order to improve the therapeutic effect, further investigation of its potential influencing factors is required.
Colon cancer is reported to develop well in the distal colon, and is significantly affected by genetic factors, diet and lifestyle. In addition, long-term intestinal inflammation also increases the incidence of cancer. These factors all cause a significant change in intestinal microorganisms. High carbohydrate diets result in increased microorganisms of the phylum firmicutes and actinomycetes, while resistant starch intake is closely related to increased microorganisms of the family chaetoviridae, and changes in these microorganisms can be beneficial in maintaining intestinal homeostasis and host health. While high protein and high fat diets cause an increase in bacteroides microorganisms, resulting in a reduced intestinal microbial diversity. The reduction of intestinal microbial diversity can cause intestinal hypoimmunity and intestinal inflammation, which is unfavorable for host health. Furthermore, it was found in previous studies that the development and progression of colorectal cancer is accompanied by a change in flora, and that the comparison of intestinal microorganisms and healthy persons in colorectal cancer patients is significantly reduced, and that the effects of intestinal flora are significantly evident, so that a series of studies have been conducted around the screening, identification and functional studies of related flora promoting colorectal tumors. It was found that Fusobacterium nucleatum (Fusobacterium nucleatum, fn) was able to promote the occurrence of colorectal cancer. By inhibiting the activation of immune cells, promoting the infiltration of myeloid cells, regulating autophagy and promoting the proliferation of tumor cells, the enrichment of Fusobacterium nucleatum in the intestinal tract accelerates the deterioration and metastasis of tumor cells. Anaerobic streptococcus mutans (Peptostreptococcus anaerobius, pa) has been found in recent studies to selectively adhere to colorectal epithelial cells, promote cell proliferation and through activation of NF-kB and promote inflammation and tumor progression. While streptococcus, lactobacillus and staphylococcus in tumor cells increase metastasis of cancer cells. Intestinal microorganisms not only promote tumor development and metastasis, but also affect the therapeutic effect. Bifidobacterium pseudolongum, lactobacillus johnsonii and Olsenella species can affect immunotherapeutic responses. Bacteria expressing a particular cytidine deaminase (CDD) can increase the resistance of a patient to chemotherapy, and antibiotic treatment can significantly enhance the effect of chemotherapy.
In addition to promoting the development and progression of colorectal cancer, probiotics that inhibit tumorigenesis are also present in intestinal commensal bacteria, but the existing studies are still shallow. Studies have shown that Bifidobacterium pseudolongum (Bifidobacterium pseudolongum) and Lactobacillus johnsonii (Lactobacillus johnsonii) are able to promote tumor immunotherapeutic effects. Acremonium muciniphilum (Akkermansia muciniphila) can inhibit occurrence and development of colorectal cancer, inhibit PD-1 expression by promoting the number of toxic T cells, and improve the activity of the T cells, thereby inhibiting colorectal cancer. Microorganisms of the genus ruminococcus have been found to have potential probiotic value in recent years of research. Some can reduce intestinal inflammation, some can produce ursodeoxycholic acid (UDCA) to protect intestinal barrier. However, whether the ruminococcus bacteria participate in the regulation and control process of intestinal cancer occurrence and development in intestinal tissues, and particularly the regulation and control of the immunity and metabolic process of tumor microenvironment have not been revealed yet.
In addition, metabolites in colorectal cancer tumor microenvironment are key to influencing tumorigenesis and development, and the carcinotropic bacteria can promote tumor development through various metabolites (such as hydrogen sulfide, secondary bile acid, polyamine and the like) produced. The metabolic substances generated by the tumor cells can also inhibit the functions of tumor immunity monitoring cells, thereby promoting the occurrence and development of tumors. How the ruminococcus microorganism modulates the metabolites in the colorectal cancer tumor microenvironment, how it modulates the critical immune cell types of tumor immune monitoring, and potentially the specific strains and functions that modulate the tumor tissue microenvironment, are to be further studied and identified.
At present, in order to increase colorectal cancer treatment effect and slow down disease process, intestinal microorganisms of healthy people are transplanted into intestinal tracts of cancer patients clinically in a fecal fungus transplanting mode. The effect of chemotherapy, radiotherapy and immunotherapy on partial patients after the fecal bacteria transplantation is better.
However, feces of healthy people contain a large amount of unknown microorganisms and human metabolites, and the in vivo transplantation of the fecal microorganisms can increase the probability of pathogenicity or bring other adverse effects to patients. Feces collection, preservation and quality control of healthy people are difficult and there is a risk of contamination. In addition, the components for the implantation of the fecal bacteria are complex, the effective components are difficult to explore, and side effects may occur after the implantation. In order to reduce the risk of disease caused by fecal transplantation, it is desirable to transplant specific probiotic microorganisms into the host, reducing the likelihood of secondary infection of the host. Meanwhile, the metabolic waste brought to the human body by the implantation of the fecal bacteria can be reduced. Based on this, there is also a need for continued research to find new probiotics for the treatment of colorectal cancer.
Disclosure of Invention
The invention aims to provide a novel beneficial bacterium capable of inhibiting the occurrence and development of colorectal cancer tumors, thereby providing a novel method for treating intestinal cancers.
The invention screens and separates an intestinal symbiotic bacterium-active ruminococcus (Ruminococcus gnavus) WS29149 enriched in normal colon tissues by comparing the flora difference of normal colon tissues and tumor tissues, and the symbiotic bacterium is a microorganism of the family of the trichomonadaceae and has the strain name of Ruminococcus gnavus. The strain is anaerobic gram-positive bacteria, and the invention further identifies the anti-tumor function (the function of inhibiting tumor and the regulation and control effect on the immunity and metabolic pathway of tumor microenvironment). The anti-tumor effect of Ruminococcus gnavus WS29149 was identified by a mouse subcutaneous tumor experiment and an AOM/DSS induced mouse colon cancer model. Through subcutaneous intratumoral injection and gastric lavage experiments of mice, the change of metabolites and immune cells in tumor tissues is analyzed, and the tumor can inhibit the growth and development of wild type colon cancer tumors of mice, and activate the anti-tumor immunity monitoring function of immune cells.
In particular, the research of the invention finds that Ruminococcus gnavus WS29149 can improve CD8 in tumors + T cell activity, in turn, limits tumor development and progression. The method mainly improves CD8 by degrading lysoglycerophospholipids in tumor microenvironment + Activity of T cells. Can promote the immunocyte activity in the tumor microenvironment and inhibit the occurrence and development of colorectal cancer by degrading lysoglycerophospholipids.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:
the active ruminococcus (Ruminococcus gnavus) WS29149 provided by the invention is preserved in China general microbiological culture Collection center (CGMCC) at 11 and 22 days of 2022, and has the classification name Ruminococcus gnavus and the preservation number CGMCC No.40452, wherein the address is 1, 3, the institute of microbiology, the national academy of sciences of China, and the China is the North Chen West Lu.
Furthermore, the invention also provides a microbial inoculum containing the active ruminococcus (Ruminococcus gnavus) WS29149 and/or the fermentation product thereof.
The microbial agent can be liquid microbial agent or solid microbial agent, and can be prepared by adding auxiliary materials allowed in the field of microbial agents by adopting a conventional technical means.
The invention proves that Ruminococcus gnavus WS29149 has important functions of inhibiting the occurrence and development of colorectal cancer tumors. Through different mouse models, flow, transcriptome, metabolome sequencing and other technologies, the method proves that the lysoglycerophospholipids in colon tumor can inhibit CD8 for the first time + T cell activity, promotes the development of colorectal cancer. Ruminococcus gnavus WS29149 is capable of decomposing the metabolite and increasing CD8 in tumor + T cell activity, and inhibit the development and progression of tumors. Proves Ruminococcus gnavus WS29149, CD8 + Relationship between T cells and lysoglycerophospholipids. By colonizing colorectal cancer patients with large amount of probiotics Ruminococcus gnavus WS29149, the content of harmful substances lysoglycerophospholipids in tumor is reduced, and CD8 is increased + T cell activity, and thus inhibit the development of tumors.
The invention further provides application of the active ruminococcus (Ruminococcus gnavus) WS29149 or a microbial inoculum in any one of the following aspects:
(1) Preparing a medicament for reducing the number of colorectal cancer tumors;
(2) Preparing a medicament for reducing the tumor volume of colorectal cancer;
(3) Preparing a medicament capable of degrading glycerophospholipids in tumors;
(4) Preparation of CD8 in activatable tumors + A drug for T cell function;
(5) Preparing a medicine for enhancing the anti-tumor immunity;
(6) Preparing a product for treating colorectal cancer.
In the present invention, the tumor is colorectal cancer.
And a pharmaceutical product comprising the aforementioned active ruminococcus (Ruminococcus gnavus) WS29149 and/or a fermentation product thereof.
The pharmaceutical product of the invention may contain adjuvants known in the pharmaceutical field and be administered in doses that provide therapeutic effects.
The invention has the beneficial effects that:
the invention provides a strain of active ruminococcus Ruminococcus gnavus WS29149 which can be used for treating colon cancer tumors. The strain is singly planted for treatment, so that unknown microorganisms transplanted by fecal bacteria and metabolic wastes of human bodies can be reduced from entering the human bodies during the traditional treatment, and the damage to the organisms and the complications caused by the damage to the organisms are avoided. Through enrichment culture, a large number of strains can be obtained, the problems of difficult collection and quality control of faeces of normal people can be avoided, and better treatment effect than faecal fungus transplantation can be obtained through in vivo field planting. And the human body can be planted in a fixed way through an oral way, so that the pain brought to the patient by the implantation of the fecal bacteria is reduced.
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FIG. 1 is a schematic diagram of a process for the isolation and identification of microorganisms in colon tissue of CRC patients.
FIG. 2 shows the effect of different bacteria on MC38 subcutaneous tumor volume. In the figure, A is a comparison photograph of subcutaneous tumor size after PBS injection, rg, pa and Rg+Pa; panel B is statistics of subcutaneous Tumor volume (Tumor volume) following PBS injection, rg, pa and rg+pa; in the graph C, the size of subcutaneous tumor after PBS injection, rg, fn and Rg+Fn is compared; in panel D is the subcutaneous tumor volume statistic after injection of PBS, rg, fn and Rg+Fn. Data represent at least three independent experiments, at least 3 replicates per group.
FIG. 3 shows the effect of different bacteria on in situ colon cancer tumorigenesis in mice. The A in the figure is the result of AOM/DSS induction experiment, wherein the left graph is the graph showing the colon of the mice after oral cavity gastric lavage PBS, rg, fn, rg+Fn, pa and Rg+Pa; the right panel shows the Tumor number (Tumor number) statistics. In the figure B is Apc min In the mouse model experiment, the statistical result of tumor number after oral cavity gastric lavage PBS, pa and Rg+Pa. Data represent at least three independent experiments, 3 replicates per group.
FIG. 4 shows the results of flow-through detection of the effect of different bacteria on tumor-infiltrating immune cell numbers and function. Panel A shows CD8 in MC38 subcutaneous tumor after PBS, rg, pa, rg+Pa, fn, rg+fn treatment + T cell number change results; in the figure B is CD4 + T cell number change results; c in the graph shows the results of the change in the number of B cells; d in the figure is NK cell number change junctionFruit; in the graph, E is the result of the change in the number of DC cells; f in the graph shows the results of the change in macrophage number. In the figure, G is CD8 infiltrated by MC38 subcutaneous tumor + T cell granzyme B (GZMB) expression; in the figure, H is CD8 infiltrated by MC38 subcutaneous tumor + T cell gamma interferon (IFN- γ) expression; in the figure, I is CD8 infiltrated by MC38 subcutaneous tumor + T cell tumor necrosis factor (TNF alpha) expression. Data represent at least three independent experiments, at least 3 replicates per group.
FIG. 5 shows the results of flow-through detection of the effect of different bacteria on tumor-infiltrating immune cell numbers and function. In the graph A, UPLC-MS detects the metabolite changes in PBS, rg, pa and MC38 subcutaneous tumor interstitial fluid after Rg+Pa treatment, and volcanic graph shows the differential metabolites in Rg vs. PBS and Rg+Pa vs. Pa tumor interstitial fluid; in the graph, B is a line graph showing the variation trend of the reduced metabolites in the Rg vs. PBS group and the Rg+Pa vs. Pa group, and the broken line represents the variation trend of the glycerophospholipid metabolism; panel C shows the effect of different glycerophospholipid metabolites on MC38 subcutaneous tumor size; panel D shows the effect of different glycerophospholipid metabolites on CD8 infiltration of MC38 subcutaneous tumor + Results of T cell numbers; in the figure, E is the CD8 of the influence of different glycerophospholipid metabolites on MC38 subcutaneous tumor infiltration + Results of T cell granzyme B (left) and IFN-gamma expression (right); panel F shows the results of UPLC-MS detection of changes in the content of LysoPA and LysoPC (18:0) in the interstitial fluid of MC38 subcutaneous tumor treated with Rg and PBS; in the graph, G is PBS, lysoPA, lysoPA +Rg, lysoPA+Pa, lysoPC, lysoPC+Rg, and LysoPC+Pa affect the result of MC38 subcutaneous tumor size; in the figure, H is PBS, lysoPA, lysoPA +Rg, lysoPA+Pa, lysoPC, lysoPC+Rg, and LysoPC+Pa affects MC38 subcutaneous tumor CD8 + T cell infiltration results. In the graph, I is PBS, lysoPA, lysoPA +Rg, lysoPA+Pa, lysoPC, lysoPC+Rg, and LysoPC+Pa affects MC38 subcutaneous tumor CD8 + Results of T cell granzyme B (left) and IFN-gamma expression (right); j in the figure is PBS, lysoPA, lysoPA +Rg, lysoPA+Pa, lysoPC, lysoPC+Rg, lysoPC+Pa affects CD8 in MC38 subcutaneous tumor + Results of killing function of T cells. Data represent at least three independent experiments, at least 3 replicates per group.
In the present figures, data are shown as mean ± SEM, and statistical analysis was performed using unpaired parametric t-test. Wherein P < 0.05; * P < 0.01; * P < 0.001; ns, the unpaired parameter t-test is not significant.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 acquisition of active ruminococcus Ruminococcus gnavus WS29149
1. Screening of symbiotic bacteria in intestinal tissues
1) Tumor tissue samples were from CRC patients in the university of si, chinese adult. Normal, paracancerous and cancerous colon tissue was excised from each of the 32 CRC patients. The obtained tissue samples were rinsed with sterile PBS at an ultra clean bench, at least 3 times per sample.
2) After washing with PBS, the tissue samples were minced with scissors and homogenized with a homogenizer, and sample DNA was extracted according to the instructions of QIAamp DNA Mini Kit (QIAGEN). PBS to flush the scissors and homogenizer served as a negative control. The ribosomal 16S rDNA V4 region was amplified using the universal primers 515F and 806R. A sequencing library was created using Ion Plus Fragment Library Kit, 48, rxns (Thermo Scientific) and sequenced using the IonS5TM XL platform (Thermo Fisher Scientific).
Quality control of original reads was performed using Cutadapt (Version 1.9.1), and chimeric sequences were removed using UCHIME. Clear reads were obtained and analyzed (using Uparse software version 7.0.1001) and classified as identical OTUs with a similarity of > 97%. Alpha-diversity index (including ACE, chao1, shannon, et al) was calculated using the MOTHUR program. QIIME 2 calculated the weighted-unirad distance of PCoA and Shannon index. The colony function was predicted using PICRUSt2 and the co-occurrence network was visualized using Cytoscape.
2. Isolation, identification and cultivation of symbiotic bacteria
1) Modified GAM medium (medium composition: modified GAM broth (purchased from Haibo organism) 60g, D-galactose 0.5g, D-fructose 0.5g, D-mannose 0.5g, D-cellobiose 0.5g, D-trehalose 0.5g, L-rhamnose 0.5g, isomaltulose 0.5g, inulin 0.5g, cysteine 0.5g, arginine 0.5g, tryptophan 0.3g, na 2 HCO 3 2g, 2.46g of sodium acetate, 5mL of hemin (0.1 g/mL), 1mg of resazurin, 100mL of clarified rumen fluid and 900mL of water) are added into an anaerobic culture device, and the device added with the culture medium is subjected to oxygen substitution and filled with nitrogen to form an anaerobic culture medium. Sterilizing the culture medium with oxygen at high temperature, cooling, and standing at room temperature for use.
2) In an anaerobic platform, normal, paracancerous and cancerous colon tissue is minced with scissors and each type of tissue sample is homogenized separately by stirring with a homogenizer. Homogenized samples were resuspended in 1mL of PBSC (1 XPBS+1% cysteine), 100. Mu.L was plated, and each sample was plated on 3 GAM plates.
3) The coated plates were placed in an anaerobic incubator and incubated at 37℃for 3 days. After 3 days, the single clone was selected for 16s strain identification, amplified with universal primers 27F and 1492R and then sanger sequenced. Blast comparison is carried out on the obtained gene sequence. Separating to obtain a strain of active ruminococcus Ruminococcus gnavus WS29149.
A schematic diagram of the colon tissue microorganism separation and identification flow of the CRC patient is shown in figure 1.
Example 2 functional identification and use of symbiotic bacteria Ruminococcus gnavus WS29149 to inhibit colorectal cancer
1. Extracting Ruminococcus gnavus WS29149 bacterial liquid stored in anaerobic culture tube with sterile syringe, injecting into sterilized and cooled GAM culture medium, and culturing in anaerobic incubator at 37deg.C for 48-72 hr.
2. The bacteria after the expansion culture were centrifuged at 5000rpm, the supernatant was removed and resuspended in anaerobic 1% PBS.
3. And (3) functional verification:
subcutaneous tumor-bearing mice, AOM/DSS induced in situ colorectal tumor mice and Apc min Spontaneous colorectal cancer mice, after colonization with this strain, had significantly smaller tumors and significantly reduced numbers. Tumor-infiltrating CD8 + T cells are significantly increased. And the strain can degrade glycerophospholipids in tumor to promote CD8 + T cell function, the specific validation process is as follows:
(1) Intestinal symbiotic Ruminococcus gnavus WS29149 obviously inhibits development of subcutaneous tumor of mice
A greater amount of Ruminococcus gnavus WS29149 was obtained after identification and enrichment culture by the procedure described above. BALB/c mice were subcutaneously tumor-bearing with MC38 mice colon cancer cells and grown to a subcutaneous tumor size of approximately 5mm by 5mm after 5-7 days. On day 7, mice were counted for subcutaneous tumor size and PBS (negative control), ruminococcus gnavus WS29149 (Rg), peptostreptococcus anaerobius GIM 1.536.536 (Pa), fusobacterium nucleatum CGMCC 1.2526 (Fn), pa+rg (1:1 ratio of viable count to 1:1) and fn+rg (1:1 ratio of viable count to), respectively, were started. The injections were continued for 3 weeks, twice weekly (monday and monday), 100 μl of PBS was injected for each negative control group, and the remaining groups were injected at a concentration of 1×10 at 100 μl 9 cell/mL bacterial liquid. Tumor sizes were counted prior to each injection and, based on the results, mice injected with Ruminococcus gnavus WS29149 were found to have significantly smaller tumor sizes than PBS groups. Mice injected with pa+rg have significantly smaller tumor sizes than the Pa group. Mice injected with rg+fn had significantly smaller tumor sizes than the Fn group (fig. 2).
(2) Intestinal symbiotic bacteria Ruminococcus gnavus WS29149 obviously inhibit the induction and spontaneous colon cancer tumor development of mice
The mouse MC38 subcutaneous tumor model proves that Rg can inhibit the development of colorectal tumors. To further demonstrate this phenomenon, induction and Apc by AOM/DSS min The mouse model (purchased from Jiangsu Jiugao Biotech Co., ltd.) was experimentally verified.
The experimental verification steps of AOM/DSS induced colorectal cancer mice are as follows: BALB/c mice aged 6-8 weeks were drinkable with antibiotics (ampicillin, ten thousand)Vancomycin, metronidazole, gentamicin, and neomycin sulfate 0.1 g/L) for 2 weeks. b. Mice were intraperitoneally injected with azoxymethane (AOM, 10mg/kg body weight). c. The following day, mice were dosed with water containing 2.5% DSS for one week. d. After one week, the mice were given plain purified water for two weeks. Mice were perfused every other day with 100 μl of sterile PBS or 1×10 strength in two weeks with purified water 8 Bacterial dilutions of cells/mL (Rg, fn, rg+Fn (1:1), pa or Rg+Pa (1:1)). Steps c and d above were repeated three times and sacrificed one week after the last treatment for subsequent experimental analysis. The results are shown in FIG. 3 at A.
Apc min The experimental verification treatment of the mouse model is as follows: apc to 6-8 weeks of age Min/+ Mice were supplied with water containing antibiotics for 2 weeks and then changed back to plain purified water. The following day of normal drinking water administration to mice, 100 μl of sterile PBS or 1x10 concentration was gavaged every other day 8 Bacterial dilutions (Pa or Rg+Pa (1:1)) of cells/mL for 5 weeks. One week after stopping the gastric lavage, the cells were sacrificed and analyzed in subsequent experiments. The results are shown in FIG. 3B.
By lavage of Rg, the size and number of AOM/DSS induced colon tumors can be significantly reduced. Fn and Pa in AOM/DSS induction and Apc min Both in the mouse model, an increase in tumor number was caused, and Rg was able to inhibit promotion of tumors by Fn and Pa (FIG. 3).
(3) Intestinal symbiotic Ruminococcus gnavus WS29149 promotes intratumoral CD8 + Infiltration of T cells and activation of immune function
Flow-through detection of immune cells in the subcutaneous tumor obtained in the above detection (1) (see results A-F in FIG. 4). The detection steps are as follows: tumor tissues were cut into small pieces and digested with collagenase II and III (1 mg/ml), DNase I (200 mg/ml; roche) on a shaker for 60 minutes at 37 ℃. The supernatant was passed through a 100- μm cell filter to remove undigested tissue pieces. The filtered liquid was collected in a 50ml tube and centrifuged at 1000g for 5min to remove the supernatant. The obtained cells were resuspended in 1 XPBS and centrifuged at 3000g for 1min. Resuspended with 1ml FACS buffer (1×pbs with 0.5% fbs) for subsequent antibody staining. Different immunocyte staining protocols: CD8 + T cell: CD19-CD4-CD45 + CD3 + CD8 + ；CD4 + T cell: CD19-CD8-CD45 + CD3 + CD4 + The method comprises the steps of carrying out a first treatment on the surface of the B cell: CD3-CD8-CD4-CD45 + CD19 + The method comprises the steps of carrying out a first treatment on the surface of the DC cells: CD19-Gr1 - CD11B - F4/80 - CD45 + CD11c ++ The method comprises the steps of carrying out a first treatment on the surface of the Macrophages: CD19 - CD45 + CD11B + F4/80 + The method comprises the steps of carrying out a first treatment on the surface of the NK cells: CD3 - CD19 - CD127 - CD45 + DX5 + NKp46 + The method comprises the steps of carrying out a first treatment on the surface of the Cells and corresponding antibodies were stained on ice for 60 min, cell sample analysis was performed on a flow cytometer (FACS Aria III, BD), and flow cytometry data was analyzed using Flowjo (V10).
The results show that Rg treatment increases CD8 in tumors + Infiltration of T cells, but for macrophages (Macrophage), DC cells (Dendritic cells), NK cells, CD4 + T cells and B cells have no effect. While Fn and Pa have no effect on immune cell infiltration in tumors.
CD8 further infiltrating tumors + T cells were functionally analyzed for granzyme B, IFN-gamma and TNF alpha (see G-I for results in FIG. 4). For detection of cytokine expression, the digested tumor cells described above were incubated with ionomycin (500 ng/mL), PMA (50 ng/mL) and Brefeldin A (1. Mu.l/mL) in RPMI1640 medium for 4 hours at 37 ℃. Next, the cells were collected for surface antigen staining, CD8 + T cell: CD19 - CD4 - CD45 + CD3 + CD8 + . After staining for surface antigens, cell perforation and fixation was performed by cell fixation and permeation buffer (eBioscience), followed by staining for intracellular antigens (i.e. granzyme B, IFN- γ and tnfα). Cell sample analysis was performed on a flow cytometer (FACS Aria III, BD) and flow cytometry data was analyzed using Flowjo (V10).
Results CD8 in tumor after intratumoral injection of Rg through subcutaneous tumor + The expression of the T cell function related proteins (GZMB, IFN-gamma and TNF alpha) is obviously increased, and the T cell function is activated. CD8 + T cells are very important immune cells in anti-tumor immunity, CD8 + Activation of T cell function facilitates the hostThe tumor cells are cleared, and the anti-tumor immunity is enhanced.
(4) Enteric symbiotic Ruminococcus gnavus WS29149 can degrade glycerophospholipids in tumors
UPLC-MS detection of metabolites in the intratumoral environment (interstitial fluid of tumors) was performed to analyze the effect of the metabolites on tumorigenesis and development, and the results are shown as A-B in FIG. 5. The tumor interstitial fluid is obtained by the following steps: cutting tumor into pieces (3-5 mm) 3 ) Wrapped in a 70- μm nylon mesh filter and then placed on a 50mL Eppendorf tube and centrifuged at 400g for 15 minutes. The tumor interstitial fluid at the bottom of the tube was collected and filtered through a 0.22 μm sterile filter (Millipore). The obtained interstitial fluid was flash frozen in liquid nitrogen and stored at-80 ℃.100uL of tumor interstitial fluid was used for subsequent UPLC-MS assays, which were performed by Shanghai Meiji Biometrics, inc. according to routine methods in the art. Glycerophospholipid metabolites were found to be significantly reduced in Rg treated subcutaneous tumors.
Further, 100uL PBS (control Ctrl) or 100. Mu.g/ml of different glycerophospholipids (LysoPC (17:0), lysoPC (18:0), PC (16:0/18:1), PE (18:0/18:2), lysoPA (0:0/18:1)) were injected subcutaneously (subcutaneous tumor obtained as described above). The volume of the injection in subcutaneous tumor reaches 50mm 3 Starting from left to right, two injections were made at 1 week, three weeks of injection, and tumor volume size was recorded before the last injection. It was found that LysoPA and LysoPC treatment resulted in enlargement of the tumor, whereas tumor infiltrating CD8 + T cells were significantly depleted in both number and function (detection methods described above). The results are shown in FIG. 5 at C-E.
mu.L of a mixture of PBS and 100. Mu.L of lysoPC (18:0), 100. Mu.L of a mixture of PBS and 100. Mu.L of lysoPA (0:0/18:1), 100. Mu.L of Rg (1X 10) 9 cells/mL) and 100 μl LysoPC (18: 0) 100. Mu.L Rg (1X 10) 9 cells/mL) and 100 μl LysoPA (0: 0/18: 1) Is respectively injected into subcutaneous tumor of mice, and the tumor volume reaches 50mm 3 Injection was performed left and right. The injections were continued for 3 weeks, twice weekly. After the last injection, the mice were euthanized the next day and tumor interstitial fluid was obtained. The obtained tumor interstitial fluid is targeted and detected by UPLC-MSThe results of measuring LysoPA and LysoPC are shown in F in fig. 5, which demonstrates that Rg reduces the level of this type of metabolite in the interstitial fluid of tumors.
mu.L of PBS, lysoPC (18:0), lysoPA (0:0/18:1), rg (1X 10) 9 cell/mL) +lysopc (18: 0) Mixed solution, rg (1×10) 9 cell/mL) +lysopa (0: 0/18: 1) Mixed solution, pa (1×10) 9 cell/mL) +lysopc (18: 0) Mixed solution and Pa (1×10) 9 cell/mL) +lysopa (0: 0/18: 1) The mixed solution is respectively injected into subcutaneous tumor of mice, and the tumor volume reaches 50mm 3 Injection was performed left and right. The injections were continued for 3 weeks, twice weekly, and tumor volumes were counted prior to the last injection. The following day after the last injection, mice were euthanized for CD8 in subcutaneous tumors + T cells were subjected to flow detection and functional analysis. The results are shown in FIG. 5 as G-I. Under the action of Rg, lysoPA and LysoPC have no obvious promotion effect on tumors, and CD8 for tumor infiltration + T cell numbers and function return to normal. To further demonstrate CD8 + T cell function is activated and CD8 infiltrated in subcutaneous tumor is selected by flow separation + T cells. Using CytoToxNon-Radioactive Cytotoxicity Assay kit (Promega) for detecting CD8 + Killing function of T cells. CD8 + T cells were co-cultured with MC38 cells in a 1:8 ratio in 96-well plates. After 4 hours of incubation, the supernatant was analyzed for absorbance at 490 nm. The results are shown in FIG. 5J. LysoPA and LysoPC significantly reduce CD8 + T cell killing function, but under Rg action, CD8 + And the recovery and improvement of the T cell killing function are realized.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (9)
1. The active ruminococcus (Ruminococcus gnavus) WS29149 is characterized in that the preservation number is CGMCC No.40452.
2. A microbial inoculum comprising the active ruminococcus (Ruminococcus gnavus) WS29149 of claim 1.
3. Use of the ruminococcus livens (Ruminococcus gnavus) WS29149 of claim 1 or the bacterial agent of claim 2 in the preparation of a product for the treatment of colorectal cancer.
4. Use of the ruminococcus livens (Ruminococcus gnavus) WS29149 of claim 1 or the bacterial agent of claim 2 for the preparation of a medicament for reducing the number of colorectal cancer tumors.
5. Use of the ruminococcus livens (Ruminococcus gnavus) WS29149 of claim 1 or the bacterial agent of claim 2 for the manufacture of a medicament for reducing colorectal cancer tumor volume.
6. Use of the ruminococcus livens (Ruminococcus gnavus) WS29149 of claim 1 or the microbial agent of claim 2 for the preparation of a medicament for degrading glycerophospholipids in a tumor, said tumor being colorectal cancer.
7. The use of the ruminococcus livens (Ruminococcus gnavus) WS29149 of claim 1 or the microbial inoculum of claim 2 for the preparation of CD8 in activatable tumors + Use of a T cell functional drug, said tumor being colorectal cancer.
8. Use of the ruminococcus livens (Ruminococcus gnavus) WS29149 of claim 1 or the microbial agent of claim 2 for the manufacture of a medicament for enhancing anti-tumor immunity, said tumor being colorectal cancer.
9. A medicament for the treatment of colorectal cancer, characterized by comprising active ruminococcus (Ruminococcus gnavus) WS29149 according to claim 1.
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