CN113862193B - Acremonium muciniphilum and application thereof in preparation of antitumor drugs - Google Patents
Acremonium muciniphilum and application thereof in preparation of antitumor drugs Download PDFInfo
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- CN113862193B CN113862193B CN202111262055.6A CN202111262055A CN113862193B CN 113862193 B CN113862193 B CN 113862193B CN 202111262055 A CN202111262055 A CN 202111262055A CN 113862193 B CN113862193 B CN 113862193B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a mucin-philin Acremonium (Akkermansia muciniphila) and application thereof in preparing antitumor drugs, and relates to the technical field of biological pharmacy. The mucin-philin Acremonium is preserved in China center for type culture collection (CCTCC.M.2020950), the preservation date is 12 months and 21 days in 2020, and the preservation address is No. 299 of Wuchang district in Wuhan, hubei province. The application is the application of the composition in the aspect of preparing antitumor drugs. The novel strain Akkermansia muciniphila SZ of the mucin-philin Acremonium is obtained through separation, culture and identification, and the fermentation supernatant and the living bacteria of the strain in-vitro fermentation have the effect of obviously inhibiting the proliferation of tumor cells, can be used for preparing products or medicines for diagnosing, preventing and/or treating tumors, and has the advantages of low preparation cost, high preparation efficiency, small side effect and good industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a mucin-philin Acremonium and application thereof in preparation of antitumor drugs.
Background
Colon cancer is one of the common malignant tumors of the digestive system, and the incidence rate is inferior to that of gastric cancer and esophageal cancer, so that the life and health of human beings are seriously affected. The curative effect of the metastatic colon cancer operation is poor, the postoperative recurrence rate is high, and the survival rate of patients who cannot resect tumors for 5 years is almost 0. Drug-targeted therapies, while having great success, still suffer from a number of shortcomings, such as high molecular weight of the antibodies used for targeting, low penetration in tumor tissue, and toxic effects on liver and bone marrow; the immunogenicity is strong; longer preparation process and period, high price, etc. Therefore, the development of new drugs for preventing and treating colon cancer is a hot spot problem of current scientific research work.
It has been found that intestinal bacteria can affect the patient's response to cancer immunotherapy: in patients with a good immune response, there are a large number of "beneficial" bacteria in the gut, such as akkermansia muciniphila (Akkermansia muciniphila), whereas there is no imbalance in the intestinal flora composition of responders, which is associated with impaired immune cell activity. Thus, a healthy intestinal flora is maintained, or the patient may be assisted in combating cancer.
Ackermansia muciniphila (Akkermansia muciniphila) is a beneficial bacterium discovered in recent years, and is a strictly anaerobic gram-negative bacterium isolated and cultured from human feces, and the Ackermania muciniphila is a mucous layer with rich nutrition in the digestive tract, and the Ackermana muciniphila has the effects of losing weight, resisting inflammation and improving metabolism, can regulate the immune response of an organism, and can maintain the metabolic balance in vivo. At present, akkermansia muciniphila bacteria have research reports in various disease fields such as cancer, diabetes, obesity, premature senility, freezing syndrome, epilepsy, IBD, hypertension, autism and the like.
Chinese patent CN110693917a discloses the application of Akkermansia muciniphila bacteria in the preparation of antidepressant or health product.
Chinese patent CN201811541031.2 discloses a murine Akkermansia muciniphila 139 strain for promoting lymphocyte regulatory T cell differentiation and for alleviating enteritis.
At present, no report shows that Akkermansia muciniphila bacteria have direct anticancer effect.
Disclosure of Invention
The invention provides a mucin-philin Acremonium (Akkermansia muciniphila SZ) strain and application thereof in preparing products or medicines for diagnosing, preventing and/or treating tumors, aiming at solving the problems of large side effect, long preparation period, high cost and the like of the existing antitumor medicines.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
on the one hand, the invention discloses a mucin-philic Acremonium, which is preserved in China Center for Type Culture Collection (CCTCC) NO: M2020950, the preservation date is 12 months in 2020, 21 days, and the preservation address is No. 299 of Wuchang district in Wuhan, hubei province.
The above-mentioned mucin-philin Acremonium (Akkermansia muciniphila SZ) was obtained as follows: the applicant's previous research found that the abundance of Akkermansia muciniphila bacteria in the fecal sample of chinese intestinal cancer patients was significantly lower than in healthy people. In particular, in the study of the relationship between intestinal flora and BMI (body weight factor), the applicant found a volunteer who had not been fat in eating, had bacteria in the intestinal tract which had not been found by others, isolated and cultured the bacteria, and then sequenced the full length of the 16S rRNA gene, and the sequence results were compared and analyzed by NCBI database to identify the strain as a new human Akkermansia muciniphila strain, which was designated Akkermansia muciniphila SZ.
Some embodiments, which are preferred but not limiting to the scope of the invention, may be:
the culture medium can be 38.5g/L of BHI, 2g/L, L-cysteine of mucin and 0.3g/L of agar powder and 17g/L;
the culture conditions may be anaerobic culture at 37℃for 96 hours;
the basis for judging whether the strain is separated and purified can be as follows: gram negative bacteria, colony morphology; the colony is small, round, convex, opaque, white, smooth in surface and non-hemolytic.
In another aspect, the invention discloses a pharmaceutical formulation comprising the above living, dead, cell disruption, fermentation supernatant, engineered, mutant and/or mutagenic bacteria of Acremodelling protein Acremodelling bacteria CCTCC NO: M2020950.
Specifically, the culture method of the living bacteria of the mucin-philic Acremonium CCTCC NO: M2020950 comprises the following steps:
s1, inoculating mucin-philic Acremonium CCTCC NO: M2020950;
s2, anaerobic culture is carried out to obtain the living bacteria of the Acremonium muciniphilum CCTCC NO: M2020950.
Some embodiments, which are preferred but not limiting to the scope of the invention, may be:
the medium components may be BHI 38.5g/L and mucin 2g/L.
Specifically, the preparation method of the colistin-philic Acremonium CCTCC NO: M2020950 dead bacteria comprises the step of pasteurizing the colistin-philic Acremonium CCTCC NO: M2020950 live bacteria in a water bath at 75 ℃ for 10min to obtain the colistin-philic Acremonium CCTCC NO: M2020950 dead bacteria.
Specifically, the cell disruption product is obtained by destroying cell membranes and cell walls of the Alkermansia muciniphila CCTCC NO: M2020950 by external force, and the discharged cell content and the destroyed cell membranes and cell walls are the cell disruption product.
Specifically, the preparation method of the fermentation supernatant comprises the following steps:
s1, inoculating mucin-philic Acremonium CCTCC NO: M2020950;
s2, anaerobic culture;
s3, centrifuging the culture obtained in the step S2, collecting fermentation supernatant, filtering and sterilizing to obtain the mucin-philin Acremonium CCTCC NO: M2020950 fermentation supernatant.
Specifically, the modified bacteria, mutant bacteria and/or mutant bacteria are new strains obtained by modifying the genotype of the original strain through natural mutation, mutagenesis and artificial design on the basis of the mucin-philin Acremodelling bacteria CCTCC NO: M2020950.
In still another aspect, the invention also discloses a gene coding product which is coded by the gene on the genome of the above-mentioned mucin-philin Acremonium CCTCC NO: M2020950, and can diagnose, prevent and/or treat tumors.
Specifically, the above gene-encoded products include RNA and protein.
The invention also discloses application of the akkermansia muciniphila CCTCC NO: M2020950 in preparing products or medicines for diagnosing, preventing and/or treating tumors.
In still another aspect, the invention also discloses application of the pharmaceutical preparation in preparing products or medicines for diagnosing, preventing and/or treating tumors.
Some embodiments, which are preferred but not limiting to the scope of the invention, may be:
based on the in vitro inhibition effect on colon cancer cells HT29 and HCT116, the pharmaceutical preparation of the product or the medicament for diagnosing, preventing and/or treating tumors can comprise fermentation supernatant of the mucin-philin Acremodelling bacteria CCTCC NO: M2020950, viable bacteria of the mucin-philin Acremodelling bacteria CCTCC NO: M2020950 and dead bacteria of the mucin-philin Acremodelling bacteria CCTCC NO: M2020950; preferably, the fermentation supernatant of Alkermansia muciniphila CCTCC NO: M2020950.
Based on the in vivo inhibition effect on the colon cancer cell line MC38 in the C57BL/6 mice, the pharmaceutical preparation applied to the preparation of products or medicaments for diagnosing, preventing and/or treating tumors can comprise living bacteria of the mucin-philin Acremonium CCTCC NO: M2020950.
Specifically, the tumor is a digestive system tumor.
In particular, the digestive system tumor is intestinal tumor, stomach tumor, esophageal tumor, pancreatic tumor, gallbladder tumor, liver tumor and/or appendiceal tumor.
In particular, the intestinal tumor is colon tumor, rectal tumor, duodenal tumor and/or small intestine tumor.
Some embodiments, which are preferred but not limiting to the scope of the invention, may be:
based on the in vitro inhibition effect of colon cancer cells HT29 and HCT116 and the in vivo inhibition effect of colon cancer cell line MC38 in C57BL/6 mice, the tumor is preferably colon tumor.
Compared with the prior art, the invention has the following beneficial effects:
1. the mucin-philin Acremonium CCTCC NO: M2020950 provided by the invention is derived from the feces of a volunteer who is not fat to eat, and has the bacteria which are not available to other people in the intestinal tract, and the mucin-philin Acremonium CCTCC NO: M2020950 for treating colon cancer is separated, cultured and identified by combining the high-throughput screening of metagenome of intestinal microbiota with the traditional microbiology and molecular biology and genetics methods, so that the fermentation supernatant and living thalli of the mucin-philin Acremonium CCTCC NO: M2020950 can obviously inhibit the proliferation of tumor cells, and can be used for preparing products or medicines for diagnosing, preventing and/or treating tumors, and the preparation cost is low and the efficiency is high.
2. Since the mucin-philin Acremonium CCTCC NO: M2020950 is derived from the feces of a healthy volunteer, and related drug experiments show that the drug toxicity is not obvious under the administration dosage. Therefore, the antitumor drug prepared from the extract has high biological safety and low side effect.
Preservation description
Preservation address: no. 299 of eight paths in Wuhan City of Hubei province in Wuchang district
Preservation date: 2020, 12 and 21 days
Strain name: acremonium muciniphilum
Latin name: akkermansia muciniphila
Strain number: SZ (SZ)
Preservation mechanism: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (cctccc)
Accession numbers of the preservation center: CCTCC NO: M2020950
Drawings
FIG. 1 is a colony morphology diagram of the Acremonium muciniphilum CCTCC NO: M2020950 after anaerobic culture;
FIG. 2 is a gram-stain microscopic image of the Acremonium muciniphilum CCTCC NO: M2020950 of the present invention;
FIG. 3 is a graph showing the growth of the inventive Acremonium muciniphilum CCTCC NO: M2020950;
FIG. 4 is a graph showing the results of in vitro pharmacological experiments on colon cancer cells HCT116 obtained from fermentation supernatant of Acremodelling protein CCTCC NO: M2020950 in example 2 of the present invention;
FIG. 5 is a graph showing the results of in vitro experiments on the results of the in vitro experiments on colon cancer cells HCT116 of the living and dead bacteria of the Acremonium muciniphilum CCTCC NO: M2020950 in example 2 of the present invention;
FIG. 6 is a graph showing the results of in vitro pharmacological experiments on colon cancer cells HT29 from fermentation supernatant of Acremonium muciniphilum CCTCC NO: M2020950 in example 2 of the present invention;
FIG. 7 is a graph showing the results of in vitro pharmacological experiments on colon cancer cells HT29 of the living and dead bacteria of the Acremonium muciniphilum CCTCC NO: M2020950 in example 2 of the present invention;
FIG. 8 is a graph showing the trend of the body weight change of mice in the in vivo efficacy experiment of the living organism of Acremodelling protein CCTCC NO: M2020950 in example 3 of the present invention;
FIG. 9 is a graph showing the trend of tumor volume change of mice in vivo in the experiment of the living organism of Acremonium muciniphilum CCTCC NO: M2020950 in example 3 of the present invention.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. All reagents used in the present invention are commercially available, and the sources thereof are not particularly limited, and some of the sources of the reagents are shown in Table 1 below.
TABLE 1
Experimental example 1 isolation, culture and identification of Acremonium muciniphilum CCTCC NO: M2020950
1. And (3) separating and culturing: 1g of feces from fresh healthy volunteers was placed in 9mL of sterile anaerobic L-cysteine-containing PBS (0.3 g/L) and shaken for 1 minute. 0.1mL of the mixture was dropped onto a culture medium A (BHI 38.5 g/L+mucin 2 g/L+L-cysteine 0.3 g/L+agar powder 17 g/L), streaked, and then placed in an anaerobic incubator (model: LAI-3, shang-Hailong device Co., ltd.) and cultured at 37℃for 96 hours. Suspected colonies were picked up on medium C (Columbia plate), anaerobically cultured at 37℃for 72h, and gram stained. Observing the morphology under a microscope, selecting bacterial colonies of gram-negative bacteria, streaking and inoculating on a Columbia blood plate, and carrying out anaerobic culture for 72 hours. And judging whether to purify according to the morphological characteristics of the bacterial colonies on the flat plate and the dyeing characteristics, the size, the shape and the distribution of the bacterial colonies observed under the lens. If the bacteria are not pure, the steps are continued, and the separation and passage are repeated for a plurality of times until purified strains are obtained.
2. Colony characterization: after 72h incubation on Columbia plates, the plates showed rounded protrusions, opaque, white, smooth surface, no hemolysis, and very small colonies, as shown in FIG. 1.
3. And (3) dyeing and observing: gram staining was performed under a microscope to show a typical oval shape for gram-negative bacteria, with no staining in the middle of the cells, staining of the edge profile, single cells or presence in pairs, see fig. 2.
4. And (3) carrying out strain identification by full-length sequencing of the 16S rRNA gene: one colony was picked up in a 1.5mL EP tube under anaerobic conditions using a micropipette tip and labeled, and 20. Mu.L ddH2O was added and bathed in boiling water for 6min. And carrying out subsequent PCR experiments by taking the PCR product as a template, carrying out 16S rRNA sequencing on the amplified PCR product, and carrying out NCBI blast on the sequence.
The PCR reaction system is as follows: 25. Mu.L of Premix Taq, 1. Mu.L of each of the upstream and downstream primers, and 2. Mu. L, ddH of the template 2 O 19μL。
The PCR reaction conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30 s; 40 cycles of 50 ℃ 60s,72 ℃ 30s and 72 ℃ 7 min; 4 ℃ plus infinity.
The primer sequences used were: general primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1); 1429R:5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).
The full-length nucleic acid sequence of the 16S rRNA gene (SEQ ID NO: 3) is as follows:
AACGAACGCTGGCGGCGTGGATAAGACATGCAAGTCGAACGAGAGAATTGCTAGCTTGCTAATAATTCTCTAGTGGCGCACGGGTGAGTAACACGTGAGTAACCTGCCCCCGAGAGCGGGATAGCCCTGGGAAACTGGGATTAATACCGCATAGTATCGAAAGATTAAAGCAGCAATGCGCTTGGGGATGGGCTCGCGGCCTATTAGTTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGACGGGTAGCCGGTCTGAGAGGATGTCCGGCCACACTGGAACTGAGACACGGTCCAGACACCTACGGGTGGCAGCAGTCGAGAATCATTCACAATGGGGGAAACCCTGATGGTGCGACGCCGCGTGGGGAAATGAAGGTCTTCGGATTGTAAACCCCTGTCATGTGGGAGCAAATTAAAAAGATAGTACCACAAGAGGAAGAGACGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGTCTCAAGCGTTGTTCGGAATCACTGGGCGTAAAGCGTGCGTAGGCTGTTTCGTAAGTCGTGTGTGAAAGGCGCGGGCTCAACCCGCGGACGGCACATGATACTGCGAGACTAGAGTAATGGAGGGGGAACCGGAATTCTCGGTGTAGCAGTGAAATGCGTAGATATCGAGAGGAACACTCGTGGCGAAGGCGGGTTCCTGGACATTAACTGACGCTGAGGCACGAAGGCCAGGGGAGCGAAAGGGATTAGATACCCCTGTAGTCCTGGCAGTAAACGGTGCACGCTTGGTGTGCGGGGAATCGACCCCCTGCGTGCCGGAGCTAACGCGTTAAGCGTGCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGAAATTGACGGGGACCCGCACAAGCGGTGGAGTATGTGGCTTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTAATGAACAACATGTGAAAGCATGCGACTCTTCGGAGGCGTTACACAGGTGCTGCATGGGCCGTCGTCAGCTCGTGTCGTGAGATGTTTGGTTAAGTCCAGCAACGAGCGCAACCCCTGTTGCCAGTTACCAGCCACGTGAAGGTGGGGACTCTGGCGAGACTGCCCAGATCAACTGGGAGGAAGGTGGGGACGACGTCAGGTCAGTATGGCCCTTATGCCCAGGGCTGCACACGTACTACAATGCCCAGTACAGAGGGGGCCGAAGCCGCGAGGCGGAGGAAATCCTAAAAACTGGGCCCAGTTCGGACTGTAGGCTGCAACCCGCCTACACGAAGCCGGAATCGCTAGTAATGGCGCATCAGCTACGGCGCCGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACATCATGGAAGCCGGTCGCACCCGAAGTATCTGAAGCCAACCGCAAGGAGGCAGGGTCCTAAGGTGAGACTGGTAACTGGGATG
the full-length sequence of the 16S rRNA gene of the strain obtained above was analyzed by comparison using NCBI database, and the strain was identified as a novel human Akkermansia muciniphila strain and designated Akkermansia muciniphila SZ.
Example 1 Acremonium muciniphilum CCTCC NO: M2020950
The culture medium is preserved in China center for type culture collection (CCTCC NO: M2020950), the preservation date is 12 months and 21 days in 2020, and the preservation address is the eight 299 of Wuchang district in Wuhan, hubei province.
EXAMPLE 2 cultivation of Acremonium muciniphilum CCTCC NO: M2020950
Liquid medium: BHI (38.5 g/L) +mucin (2 g/L), wherein the BHI medium composition (g/L) is: 10g of tryptone, 17.5g of beef heart infusion powder, 5g of sodium chloride, 2g of glucose and disodium hydrogen phosphate (12H) 2 O)2.5g。
Culture conditions: strictly anaerobic culture at 37 ℃; the inoculation amount is 1:100, and the culture is carried out for 72-96 hours.
The growth curve is shown in figure 3.
EXAMPLE 3 viable bacteria of Acremonium muciniphilum CCTCC NO: M2020950
Collecting thallus from 100mL culture obtained in culture example 2 (8000 rpm,20 min), adding PBS containing 2.5% glycerol, suspending, and adjusting viable bacteria concentration to about 5×10 8 CFU/mL。
EXAMPLE 4 dead bacteria of Acremonium muciniphilum CCTCC NO: M2020950
Collecting thallus from 100mL culture obtained in culture example 2 (8000 rpm,20 min), adding PBS containing 2.5% glycerol, suspending, and adjusting viable bacteria concentration to about 5×10 8 CFU/mL, pasteurized in a water bath at 75deg.C for 10 min.
EXAMPLE 5 fermentation broth of Acremonium muciniphilum CCTCC NO: M2020950
100mL of the culture obtained in culture example 2 (8000 rpm,20 min) was taken and the fermentation supernatant was collected by high-speed centrifugation and filtered sterilization was performed with a 0.22 μm pore size bacterial filter.
Experimental example 2
Inhibition of colon cancer cells HT29 and HCT116 by Acremonium muciniphilum CCTCC NO: M2020950
The experimental method comprises the following steps:
1. cell culture
Human colon cancer cells HT29 and HCT116 were cultured in 90% McCoy's 5a (Gibco) +10% FBS (Gibco), respectively, cells in exponential growth phase were collected, and PBS was resuspended to a suitable concentration (number of viable cells about 5X 10) 4 Per ml) were inoculated into 96-well plates, each well was inoculated with 100. Mu.L, and placed in an incubator at 37℃with 5% CO 2 Culturing for 12-24 hr until the cell attachment rate is about 75%, and adding medicine.
2. Sample processing
Acremonium muciniphilum CCTCC NO: M2020950 viable bacteria: same as in example 3;
acremonium muciniphilum CCTCC NO: M2020950 dead bacteria: same as in example 4;
acremonium muciniphilum CCTCC NO: M2020950 fermentation supernatant: same as in example 5.
And (3) dilution treatment: the live bacteria (concentration of 5×10) 8 CFU/mL) was 10-fold gradient diluted with PBS containing 2.5% glycerol; the dead bacteria (concentration 5×10) 8 CFU/mL) was 10-fold gradient diluted with PBS; the fermentation supernatant was diluted with PBS at 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% and 5% respectively.
4. Administration treatment
Adding 100 mu L of different concentrations of Alkermansia muciniphila CCTCC NO: M2020950 (Akkermansia muciniphila SZ for short in the application) viable bacteria and dead bacteria into the inoculated 96-well plate cell culture medium, incubating for 72 hours, setting a blank control group (McCoy's 5a culture solution+cell suspension) and a control group (PBS+cell suspension), and setting 5 repeated holes in each group (Akkermansia muciniphila SZ viable bacteria/dead bacteria+McCoy's 5a culture solution+cell suspension with different concentrations);
100. Mu.L of fermentation supernatants of different concentrations were added to the inoculated 96-well plate cell culture medium and incubated for 72h, a blank control group (McCoy's 5a culture medium), a control group (liquid culture medium), an experimental group (fermentation supernatants of different concentrations) were set, 5 replicate wells were set for each group, and the experiment was repeated three times.
The old medium was removed, 100. Mu.L of fresh medium+20. Mu.L of MTT solution was added and placed in an incubator to continue at 37℃with 5% CO 2 Culture 4 h.
The culture was terminated, 150. Mu.L of DMSO was added to each well, and the mixture was slowly shaken uniformly for about 10 minutes to dissolve the purple crystals sufficiently.
The OD values of each well were measured at 570nm/630nm using a microplate reader and absorbance values of each group were recorded.
Experimental results: see fig. 4-7.
As shown in FIGS. 4-7, akkermansia muciniphila SZ live bacteria and dead bacteria have weak inhibition effect on colorectal cancer tumor cells HT29 and HCT116, but fermentation supernatant has strong inhibition effect on proliferation of human colorectal cancer cells HT29 and HCT 116. It is presumed that Akkermansia muciniphila SZ bacteria produce various short chain fatty acids during the growth process, and further inhibit proliferation of colon cancer cells, and that the reason why viable bacteria are ineffective is probably that the oxygen tolerance of the bacteria is low, and a large number of viable bacteria die with time, and effective metabolites cannot be secreted. In conclusion, the in vitro fermentation product of the strain can effectively inhibit proliferation of colon cancer tumor cells.
Experimental example 3
Akkermansia muciniphila SZ the antitumor effect of the live bacteria medicine in the animal model of the mouse colon cancer cell strain MC38 xenograft C57BL/6 mouse.
Test animals: female C57BL/6 or mice, 6-7 weeks old (mice at tumor cell inoculation were expected to be week old), and mice at cell inoculation had a body weight range of 18-22g, purchased from Junkidae Biotechnology Co., ltd.
Environmental conditions of laboratory animal feeding room: the experimental animals are all fed in an independent ventilation box with constant temperature and constant humidity, and are adapted to the environment in advance for at least 7 days, the temperature of a feeding room is 20-26 ℃, the humidity is 40-70%, ventilation is carried out for 10-20 times per hour, the brightness is alternated between seven in the morning and seven in the evening, and the illumination is carried out between seven in the evening and seven in the morning and the darkness is carried out between seven in the evening and seven in the morning; the cobalt 60 radiation sterilized mouse complete pellet feed is continuously supplied, the mouse complete pellet feed is not limited to be taken freely, tap water is drunk (the mouse complete pellet feed is used after high-pressure steam sterilization), and a drinking bottle is supplied with water uninterruptedly and is taken freely. The feeding rat box is a polysulfone rat box, and is used after autoclaving, and the specification is 325 mm multiplied by 210 multiplied by mm multiplied by 180 mm; the padding is autoclaved corncob, 5 animals are contained in each box, and IACUC approval number, experiment starting time, subject responsible person, experimenter, animal source, group, animal number and the like are marked on the cage card; the experimental animals were marked with ear marks.
The experimental method comprises the following steps:
1. test group
The number of animals per group and detailed route of administration, dosages and schedule are shown in the table below:
TABLE 2
Note that: the dose was 10. Mu.L/g, g being the weight of the mice.
2. Cell culture
MC38 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Collection of MC38 cells in exponential growth phase, PBS was resuspended to 1X 10 7 The individual/mL was used for subcutaneous tumor inoculation in mice.
3. Construction of colon cancer mouse model
0.1mL of MC38 cells were inoculated subcutaneously on the right back of experimental mice, tumor growth was observed periodically, and when tumors grew to an average volume of 96mm3, the animals were randomly dosed according to tumor size and mouse weight, and tumor inoculation was recorded as day 0.
4. Random grouping
All animals were weighed before dosing began and tumor volumes were measured with vernier calipers. Since tumor volume affects the effectiveness of treatment, the mice are grouped according to their tumor volume by a random grouping design method to ensure that the tumor volumes are similar among the different groups.
5. Preparation of pharmaceutical solutions
The preparation of the compound solution is carried out in a biosafety cabinet or an ultra-clean workbench, and the compound solution is mixed uniformly before use, so that the medicinal preparation is ensured to be uniform.
The formulation is shown in Table 3 below.
TABLE 3 Table 3
6. Administration of drugs
The tumor inoculation day 9 was followed by the start of dosing according to the experimental design.
7. Experimental observations and data collection
After tumor cell inoculation, conventional monitoring includes the effect of tumor growth and treatment on normal animal behavior, specifically including activity, ingestion and drinking conditions, weight gain or reduction, eyes, hair and other abnormal conditions of experimental animals. Clinical symptoms observed during the trial are recorded in the raw data.After starting the administration, the body weight and the tumor size of the mice were measured on days 6, 8, 10, 12, 14, 16, and 19, respectively, of the administration. Tumor size calculation formula: tumor volume (mm) 3 ) =0.5× (tumor long diameter×tumor short diameter 2 )。
Experimental results
1. Weight change in mice: as shown in table 4 and fig. 8.
TABLE 4 Table 4
As can be seen from table 4 and fig. 8: none of the Akkermansia muciniphila SZ bacteria administered groups (experimental groups) showed a decrease in weight or death of the mice, no apparent drug toxicity, and well tolerated during the treatment period.
2. Mouse tumor size change conditions: as shown in table 5 and fig. 9.
TABLE 5
As can be seen from table 5 and fig. 9: the tumor volume was significantly reduced by about 1 fold after administration of the Akkermansia muciniphila SZ viable bacteria compared to the control group, and the reduction was statistically significantly different, indicating that the Akkermansia muciniphila SZ viable bacteria could significantly inhibit colon cancer growth. And the tumor-bearing mice are well tolerated by Akkermansia muciniphila SZ bacteria under the dosage of administration.
At present, the research of Akkermansia muciniphila SZ bacteria in the field of colon cancer has not been reported at home and abroad, but only some researches on the aspect of relieving colon inflammation are carried out, but no direct and deep research on whether Akkermansia muciniphila bacteria have a direct inhibition effect on colon cancer is carried out.
Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.
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Claims (5)
1. Acremonium muciniphilum strainAkkermansia muciniphila) The method is characterized by being preserved in China Center for Type Culture Collection (CCTCC) No. M2020950, the preservation date is 12 months and 21 days in 2020, and the preservation address is the eight 299 of Wuhan, wuchang district, hubei province.
2. A pharmaceutical formulation comprising the live, dead or fermentation supernatant of the akkermansia muciniphila of claim 1.
3. The pharmaceutical formulation according to claim 2, wherein the process for preparing the fermentation supernatant comprises the steps of:
s1, inoculating mucin-philic Acremonium CCTCC NO: M2020950;
s2, anaerobic culture;
s3, removing thalli to obtain the microbial inoculum.
4. The method for culturing akkermansia muciniphila according to claim 1, comprising the steps of:
s1, inoculating mucin-philic Acremonium CCTCC NO: M2020950;
s2, anaerobic culture.
5. Use of akkermansia muciniphila according to claim 1 for the preparation of a medicament for the prevention and/or treatment of colon tumours.
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CN113862193B (en) * | 2021-10-28 | 2023-12-22 | 江西普瑞森基因科技有限公司 | Acremonium muciniphilum and application thereof in preparation of antitumor drugs |
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