WO2024046168A1 - 一株短乳杆菌及其抗宫颈癌应用 - Google Patents

一株短乳杆菌及其抗宫颈癌应用 Download PDF

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WO2024046168A1
WO2024046168A1 PCT/CN2023/114171 CN2023114171W WO2024046168A1 WO 2024046168 A1 WO2024046168 A1 WO 2024046168A1 CN 2023114171 W CN2023114171 W CN 2023114171W WO 2024046168 A1 WO2024046168 A1 WO 2024046168A1
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ynh
lactobacillus brevis
cervical cancer
hela cells
lactobacillus
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French (fr)
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何亮
杨宏英
谭丁及
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昆明医科大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/24—Lactobacillus brevis
    • 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

Definitions

  • the invention belongs to the technical field of microorganisms, and in particular relates to Lactobacillus brevis with anti-cervical cancer effect and its application.
  • Cervical Cancer is the most common malignant tumor of the female reproductive tract.
  • the "Global Cancer Statistics 2020" report shows that cervical cancer ranks fourth in incidence and mortality among female tumors worldwide.
  • the World Health Organization actively promotes HPV vaccination has reduced the incidence of cervical cancer, but the incidence remains high in low- and middle-income countries. Therefore, cervical cancer will remain a heavy disease and health economic burden for women around the world for a long time to come, so the treatment of cervical cancer is particularly important. Radical surgery is used for early-stage cervical cancer, and the prognosis is good, with a 5-year survival rate of 90%.
  • the treatment effect for cervical cancer (locally advanced cervical cancer) with local tumors larger than 4cm, local metastasis, and local infiltration is not very good.
  • the 5-year survival rate is 30-80%, and the prognosis is poor.
  • treatment methods for locally advanced cervical cancer mainly combined with surgery, radiotherapy, and chemotherapy.
  • the incidence rate of lymphocele alone can be as high as 25%. %; side effects after radiotherapy and chemotherapy are also obvious, such as bone marrow suppression, vaginitis, cystitis, and enteritis.
  • Lactobacillus is a microorganism beneficial to human health and is widely found in the gastrointestinal and reproductive tracts of the human body. Previous studies have shown that Lactobacillus has a therapeutic effect on a variety of human diseases, such as maintaining intestinal homeostasis, preventing aerobic vaginitis, recurrent cystitis, and lowering blood sugar and blood lipids. However, the therapeutic effect of Lactobacillus on tumors has been ignored. It was not until recent years that Lactobacillus was discovered to have an inhibitory effect on a variety of tumors. Lactobacilli have inhibitory effects on colorectal cancer, breast cancer, bladder cancer, and cervical cancer.
  • Lactobacillus acidophilus alone or in combination with ammonite reduces the occurrence of chemically induced experimental colon cancer
  • Lactobacillus casei promotes apoptosis of colon cancer cells by activating the JNK pathway
  • Lactobacillus brevis MK05 upregulates the apoptosis genes of the Bcl-2 family to promote Apoptosis of breast cancer cells
  • Lactobacillus rhamnosus inhibits bladder tumors in mice, and supernatants of Lactobacillus crispatus, Lactobacillus gastroenteritis, and Lactobacillus jamesii block the cycle of cervical cancer cells and exert anti-cancer effects.
  • Lactobacillus is a potential anti-cancer bioactive substance and may provide an effective treatment with low toxic and side effects for the clinical treatment of cervical cancer.
  • the genus Lactobacillus contains hundreds of species of lactobacilli, only a few of which have anti-cervical cancer effects, such as Lactobacillus jianschii, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gastroenteritis, and Lactobacillus casei .
  • the autonomy rate of core anti-cervical cancer strains in my country is very insufficient, which restricts the clinical application of Lactobacillus anti-cancer. There is an urgent need to discover more anti-cervical cancer strains and solve the problem of "lack of cores and few nuclei" of clinically available anti-cancer Lactobacilli. .
  • Lactobacillus brevis YNH has better anti-cancer effects in vitro than Lactobacillus reuteri, Lactobacillus crispatus, and Lactobacillus gasseri. Animal experiments show that Lactobacillus brevis YNH has the same anti-cancer effect in vivo. Effectively inhibit cervical cancer tumor proliferation. Previous studies on Lactobacillus anti-cervical cancer have mostly observed the phenomenon of Lactobacillus inhibiting cervical cancer cells. researchers found that Lactobacillus blocks the cycle of cervical cancer cells in the G1 or S phase and inhibits proliferation; it downregulates matrix metalloproteinases 2 and 9 and upregulates matrix metalloproteinases 2 and 9 in cervical cancer cells.
  • E-cadherin inhibits the epithelial-mesenchymal transition of cervical cancer; it upregulates apoptosis genes and autophagy genes and promotes cervical cancer death.
  • the above studies all explored the anti-cancer effect of Lactobacillus at the cellular level, but did not conduct in-depth exploration of the mechanism of its anti-cancer effect. There is an urgent need to further elucidate the anti-cancer effect of Lactobacillus. Since the anti-cancer effect of Lactobacillus is highly strain-specific and cell-specific, that is, different strains have different effects on the same type of tumor cells, and the same strain of Lactobacillus has different effects on different tumor cells. Therefore, screening probiotics with anti-cervical cancer effects has very important application value and practical significance.
  • the object of the present invention is to provide a strain of Lactobacillus brevis derived from healthy female reproductive tract and its application.
  • a kind of Lactobacillus brevis ( Lactobacillus brevis ) YNH characterized in that the Lactobacillus brevis ( Lactobacillus brevis ) YNH can inhibit the proliferation of cervical cancer HeLa cells and promote the apoptosis of cervical cancer HeLa cells.
  • the Lactobacillus brevis ( Lactobacillus brevis ) YNH is deposited in the General Microbiology Center of China Committee for the Collection of Microorganisms (CGMCC) with the collection number CGMCC No. 24368.
  • a bacterial agent characterized in that the bacterial agent can inhibit the proliferation of cervical cancer HeLa cells and promote the apoptosis of cervical cancer HeLa cells.
  • the active ingredient of the bacterial agent is Bacillus brevis as described in [1] ( Lactobacillus brevis )YNH.
  • Lactobacillus brevis YNH as described in [1] the inoculant as described in [2]
  • the filtrate of the fermentation broth as described in [4] The use of the bacterial suspension or the culture solution described in [5] in the preparation of drugs for treating or preventing cervical cancer.
  • Lactobacillus brevis YNH as described in [1] the inoculant as described in [2] the fermentation broth or the filtrate of the fermentation broth as described in [3], or the filtrate of the fermentation broth as described in [4]
  • a pharmaceutical composition characterized in that the pharmaceutical composition can inhibit the proliferation of cervical cancer HeLa cells and promote the apoptosis of cervical cancer HeLa cells, and the pharmaceutical composition contains Lactobacillus brevis as described in [1] ( Lactobacillus brevis ) YNH, the bacterial agent described in [2], the fermentation broth or the filtrate of the fermentation broth described in [3], the bacterial suspension described in [4] or the culture broth described in [5] any, and a pharmaceutically acceptable carrier.
  • composition according to [8] characterized in that the pharmaceutical composition is a probiotic preparation.
  • composition as described in [8] or [9], wherein the pharmaceutically acceptable carrier is selected from commonly used pharmaceutical flavoring agents, lubricants, fillers, and disintegrants. any of the groups.
  • the fermentation broth is specifically the fermentation broth obtained by inoculating the Lactobacillus brevis YNH into MRS culture medium and cultivating it statically at 37°C. Among them, the best cultivation time is more than 15 hours.
  • the Lactobacillus brevis YNH can inhibit cervical cancer; and, after co-culture with Hela cells, the Lactobacillus brevis YNH can upregulate the apoptosis genes Caspase3 and Caspase8; the Lactobacillus brevis YNH can upregulate the apoptosis genes Caspase3 and Caspase8; brevis ) YNH was co-cultured with Hela cells, and the CCK8 experiment found that Lactobacillus brevis YNH at MOI1000:1 and MOI10000:1 had an inhibitory effect on Hela cell proliferation starting from 48h;
  • Lactobacillus brevis YNH blocks the HeLa cell cycle in the S phase; at the protein level, Lactobacillus brevis YNH upregulates the S phase-related proteins CDK2 and CyclinE1 of the HeLa cell cycle, indicating that Lactobacillus brevis YNH blocks the HeLa cell cycle in the S phase.
  • Bacillus YNH has the effect of inhibiting the proliferation of Hela cells;
  • Lactobacillus brevis YNH After co-culture of Lactobacillus brevis YNH with Hela cells, it was found at the protein level that Lactobacillus brevis YNH down-regulated the anti-apoptotic protein Bax of Hela cells and up-regulated the pro-apoptotic proteins Bcl-2, Cleave-caspasse3, Cleave-caspasse8, Cleave-caspass9;
  • Lactobacillus brevis YNH can inhibit the proliferation of cervical cancer tumors by administrating it to nude mice.
  • the present invention also provides a fermented food produced by fermentation of Lactobacillus brevis YNH as mentioned above, which has anti-cervical cancer effect.
  • the fermented food includes dairy products, soy products, and fruit and vegetable products.
  • the preservation information of Lactobacillus brevis YNH provided by the present invention is as follows:
  • Lactobacillus brevis Lactobacillus brevis
  • Collection center registration number CGMCC No.24368.
  • the Lactobacillus brevis YNH provided by the invention can upregulate the apoptosis genes Caspase3 and Caspase8 after co-culture with Hela cells.
  • the Lactobacillus brevis YNH at MOI1000:1 and MOI10000:1 concentrations from It started to have an inhibitory effect on Hela cell proliferation at 48h. It was further found through flow cytometry that the Hela cell cycle was blocked in the S phase and the Hela cell cycle S phase related proteins CDK2 and CyclinE1 were upregulated at the protein level, indicating that Lactobacillus brevis YNH It has the effect of inhibiting HeLa cell proliferation.
  • Lactobacillus brevis YNH promoted Hela cell apoptosis
  • JC1 experiments found that Lactobacillus brevis YNH at MOI1000:1 and MOI10000:1 reduced Hela cell mitochondrial membrane potential and promoted Hela cell apoptosis.
  • Apoptosis after co-culture of Lactobacillus brevis YNH and Hela cells, it was found at the protein level that Lactobacillus brevis YNH down-regulated the anti-apoptotic protein Bax and up-regulated the pro-apoptotic proteins Bcl-2 and Cleave in Hela cells.
  • Animal experiments found that Lactobacillus brevis YNH can inhibit the proliferation of cervical cancer tumors by administrating it to nude mice.
  • a in Figure 1 shows the specific strain types contained in the vaginal flora of healthy people (people without HPV infection) and the vaginal flora of patients with LSIL and HSIL (people with HPV infection).
  • B in Figure 1 shows the types of strains contained in the vaginal flora of healthy people. Results of principal component analysis of vaginal flora (people without HPV infection) and vaginal microorganisms in patients with LSIL and HSIL (people with HPV infection).
  • a in Figure 2 shows the percentage of different strains of Lactobacillus isolated from vaginal secretions of healthy people, and B in Figure 2 shows the phylogenetic tree constructed by different strains of Lactobacillus.
  • a in Figure 3 shows the colony morphology of Lactobacillus 1
  • B in Figure 3 shows the microscopic morphology of Lactobacillus 1 after Gram staining.
  • a in Figure 4 shows that Lactobacillus brevis YNH at a concentration of MOI100:1 has no effect on Hela cell proliferation.
  • B and C in Figure 4 show that Lactobacillus brevis at a concentration of MOI1000:1 and MOI10000:1 respectively. Lactobacillus brevis YNH was observed to inhibit HeLa cell proliferation at 48h.
  • a in Figure 5 shows the proportion of each cell cycle of Hela cells in the blank control group.
  • B and C in Figure 5 show the concentrations of Lactobacillus brevis YNH and Hela at MOI1000:1 and MOI10000:1 respectively.
  • D in Figure 5 shows the histogram of the proportion of HeLa cells in each cell cycle in Figures A, B, and C.
  • a in Figure 6 shows that different concentrations of Lactobacillus brevis YNH down-regulate the expression of S phase-related proteins CDK2 and CyclinE1 in cervical cancer Hela cells.
  • B in Figure 6 shows the relative expression levels of CDK2 and CyclinE1 protein bands. histogram.
  • Figure 7 shows that Lactobacillus brevis YNH up-regulates the expression of apoptosis-related genes Caspase3 and Caspase8 after co-culture with Hela cells for 48 hours.
  • FIG. 8 shows the apoptosis ratio of Hela cells in the blank control group and Hela cells after the action of Lactobacillus brevis YNH at concentrations of MOI1000:1 and MOI10000:1.
  • Figure 8 D shows the histogram of the apoptosis ratio of HeLa cells in panels A, B, and C.
  • Figure 9 shows that the mitochondrial membrane potential of HeLa cells decreased after co-culture of Lactobacillus brevis YNH and HeLa cells for 48 hours.
  • a in Figure 10 shows the effect of different concentrations of Lactobacillus brevis YNH on the expression of apoptotic proteins in HeLa cells
  • B in Figure 10 shows a bar graph of the relative expression amounts of different apoptotic proteins.
  • Figure 11 shows the effect of co-culture of Lactobacillus brevis YNH and Hela cells on the expression of epithelial-mesenchymal transition-related gene E-cadherin.
  • a in Figure 12 shows the changes in body weight of nude mice with cervical cancer subcutaneous tumors in the Lactobacillus brevis YNH group and the blank control group.
  • B in Figure 12 shows the tumor bodies in the two groups of nude mice with cervical cancer subcutaneous tumors. Changes in size, C in Figure 12 shows the tumor body after killing two groups of nude mice with subcutaneous cervical cancer tumors, and D in Figure 12 shows a histogram of the tumor weight after killing two groups of nude mice with subcutaneous cervical cancer tumors.
  • the results in Figure 12 are all the first animal experiments on the treatment of cervical cancer by Lactobacillus brevis YNH strain.
  • Figure 13 shows the HE staining results of the hearts, livers and kidneys of nude mice with cervical cancer subcutaneous tumors in the Lactobacillus brevis YNH group and the normal saline control group.
  • FIG 14 shows the tumor-bearing pictures of nude mice with cervical cancer subcutaneous tumors in four groups: the normal saline gavage group, the intraperitoneal injection of cisplatin group, the lactobacillus gavage group, and the lactobacillus combined with cisplatin intraperitoneal injection group.
  • Figure B in 14 shows the tumor bodies after the sacrifice of nude mice with cervical cancer subcutaneous tumors in four groups, including the saline gavage group, the cisplatin intraperitoneal injection group, the Lactobacillus gavage group, and the Lactobacillus combined with cisplatin intraperitoneal injection group.
  • the results in Figure 14 are all the second animal experiments on the treatment of cervical cancer by Lactobacillus brevis YNH strain.
  • the experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified.
  • the experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as Sambrook et al., Molecular Cloning: Experiment conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • vaginal secretions of healthy people people without HPV infection
  • low-grade cervical intraepithelial lesion LSIL
  • high-grade cervical intraepithelial lesion HSIL
  • H1046, H1052, and H1060 are vaginal secretions from HSIL patients; L1025, L1075, and L10102 are vaginal secretions from LSIL patients; N1072, N1074, and N1087 are vaginal secretions from healthy people;
  • Bacterial DNA was extracted using Tiangen's bacterial genomic DNA extraction kit (DP302). The specific experimental steps are as follows: Use a 15 ml centrifuge tube to collect 1.0 ⁇ 10 9 (OD 600 of 5 ml bacterial solution is 1-1.5) bacterial culture, centrifuge at 10000 rpm for 5 minutes and discard the supernatant. Add 200 ⁇ l buffer GA to the bacterial pellet and shake until the bacterial cells are suspended. Add 20 ⁇ l Proteinase solution, mix well and then add 220 ⁇ l buffer GB, shake for 15 seconds and place at 70°C for 10 minutes.
  • DP302 Tiangen's bacterial genomic DNA extraction kit
  • Transfer adsorption column CB3 to a clean centrifuge tube add 100 ⁇ l buffer TE to the middle of the adsorption membrane, leave it at room temperature for 5 minutes, and then centrifuge at 12,000 rpm for 2 minutes. Collect the solution into a centrifuge tube to obtain the bacterial DNA genome required in this example. .
  • Test results show that there are significant differences between the vaginal flora of healthy people (people without HPV infection) and the vaginal flora of patients with LSIL and HSIL (people with HPV infection).
  • the reproductive tract of healthy people is dominated by Lactobacillus iners and Lactobacillus crispatus.
  • Gardnerella species are the dominant species (see Figure 1A for details).
  • Principal Component Analysis (PCA) also showed that there were significant differences in the composition of vaginal microbiota in healthy people (people without HPV infection) and in patients with LSIL and HSIL (see Figure 1B for details).
  • Example 2 Collect and isolate healthy human vaginal Lactobacillus, analyze the specific Lactobacillus strains contained therein, and record the percentage of each strain (see Figure 2A for details), establish a Lactobacillus 16S rRNA gene database, and construct a Lactobacillus Bacillus phylogenetic tree, analyze and compare each evolutionary tree (see Figure 2B for details)
  • Gram staining microscopy shows that Gram-positive bacilli are Lactobacilli to be identified.
  • the strains to be identified were inoculated into 8 ml of sterilized MRS and cultured overnight in a 37°C incubator with 5% CO 2 .
  • Test results Lactobacilli were successfully isolated and identified, and the proportions of various Lactobacilli were calculated, as shown in Figure 2A, and a phylogenetic tree was constructed, as shown in Figure 2B.
  • Example 3 Isolation and identification of Lactobacillus brevis YNH strain
  • Lactobacillus brevis YNH was isolated from healthy female reproductive tracts.
  • Test method Place the reproductive tract swab in a test tube containing 1 mL of sterile PBS and shake for 30 seconds. Use this as the original solution to continuously dilute it ten times. Take 100 ⁇ L of the 10,000-fold diluted liquid and spread it on the MRS medium solid plate (1L MRS The medium formula is: 10g beef extract, 10g casein peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g diammonium hydrogen citrate, 1mL Tween 80, 2g K 2 HPO 4 , MgSO 4 .7H 2 O 0 .
  • Lactobacillus 1 was determined to be Lactobacillus brevis YNH (also referred to as the YNH strain in the following examples), and its classification was named Lactobacillus brevis .
  • This strain was deposited on January 24, 2022 The collection number is CGMCC No. 24368 at the General Microbiology Center of the Chinese Committee for the Collection of Microbial Cultures (referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101).
  • Blank control group add 200 ⁇ l of complete culture medium; MOI100:1 group: aspirate the culture medium in the plate, and add 200 ⁇ l of complete culture medium containing 2 ⁇ 10 5 bacteria to each well; MOI1000:1 group: aspirate the culture medium in the plate, and add 200 ⁇ l of complete culture medium containing 2 ⁇ 10 5 bacteria to each well; MOI1000:1 group: aspirate the culture medium in the plate, and add 200 ⁇ l of complete culture medium containing 2 Add 200 ⁇ l of complete medium containing 2 ⁇ 10 6 bacteria; MOI10000: Group 1: Aspirate the medium from the plate, and add 200 ⁇ l of complete medium containing 2 ⁇ 10 7 bacteria to each well. Continue to culture the 96-well plate in 5% CO 2 and 37°C incubator, and then measure the cell OD value every 24 hours every day.
  • Example 5 Flow cytometry to detect the effect of YNH strain on HeLa cell cycle
  • Test method Digest, centrifuge, and count cells in the logarithmic growth phase. Add 5 ⁇ 10 5 Hela cells to each T25 bottle. Remove the culture medium after the cells adhere to the wall. Add 4 ml of complete culture medium to the control group, MOI1000: Group 1 added 4 ml of complete culture medium containing 5 ⁇ 10 8 bacteria, MOI 10000: Group 1 added 4 ml of complete culture medium containing 5 ⁇ 10 9 bacteria, and continued to be cultured in a 5% CO 2 , 37°C incubator for 48 hours. After 48 hours, the cells were digested and centrifuged at 1000 rpm/min for 4 minutes to collect the cells in each group.
  • Preparation of propidium iodide staining solution Add 0.5 ml of staining buffer, 25 ⁇ l of 20 ⁇ propidium iodide staining solution, and 10 ⁇ l of 50 ⁇ RNase A to each tube. The preparation is shown in the table below.
  • Example 6 Western blot detection of changes in cell cycle proteins after YNH strain acts on Hela
  • control group in this experiment is HeLa cells co-cultured without YNH strain
  • experimental groups are HeLa cells co-cultured with YNH strains at MOI1000:1 and MOI10000:1 concentrations.
  • the specific test steps are as follows:
  • Configure BCA working fluid Configure the working fluid in a ratio of 50:1 between liquid A and liquid B.
  • Protein standard (1 mg/ml) was added to the 96-well plate at 0, 1, 2, 4, 6, 8, and 10 ⁇ l, and each well was filled up to 10 ⁇ l with deionized water.
  • Three secondary wells were set for each concentration. Take 2 ⁇ l of the protein solution to be tested and add 8 ⁇ l of deionized water. Set 3 secondary wells for each sample. Add 200 ⁇ l BCA working solution to each well, 5% CO 2 , and incubate in a 37°C incubator for 30 minutes. Use a microplate reader to measure the OD value at a wavelength of 562nm. Based on the measured OD value of the protein standard and the known concentration of the protein standard, calculate the equation of absorbance and protein concentration. Substitute the OD value of the sample to be tested into the equation to obtain the protein concentration of the sample to be tested.
  • Protein denaturation Mix the protein solution and Buffer at a ratio of 1:4 by volume. After preparation, the protein concentration is 80% of the original concentration. Boil the protein solution for 10 minutes, cool and place at -20°C for later use.
  • Electrophoresis Electrophoresis at a constant voltage of 80V until the sample reaches the lower edge of the compression gel, and then at a constant voltage of 120V until the sample reaches the lower edge of the separation gel.
  • Film transfer Activate the PVDF membrane with methanol and cover it with glue. Fix it with the film transfer clamp and transfer it to the film transfer tank. Add the transfer liquid, place the ice box in the transfer tank, place the tank in a foam box filled with ice-water mixture, and use a constant current of 300mA for 2 hours.
  • Antibody incubation The blocked PVDF membrane was washed three times with 1 ⁇ TBST for 10 minutes each time. Determine the band based on the marker position and the molecular weight of the target protein, cut it, and incubate it in primary antibody at 4°C overnight. After the incubation is completed, take it out, wash the membrane 3 times with 1 ⁇ TBST, 10 min each time, add secondary antibody and incubate at room temperature for 2 h.
  • Test results As shown in A in Figure 6, compared with the control group, the YNH strains of MOI1000:1 and MOI10000:1 down-regulated the expression of cell cycle S phase-related proteins CDK2 and CyclinE1.
  • Example 7 YNH strain upregulates the expression of Hela cell apoptosis genes Caspase3 and Caspase8
  • the Lactobacillus group consists of YNH strains and Hela cells co-cultured at 37°C and 5% CO 2 for 24 hours.
  • the control group consists of Hela cells co-cultured at 37°C and 5% CO 2 for 24 hours.
  • RNA from the two groups of cells is extracted. PCR experiments were used to detect changes in the mRNA levels of apoptosis-related genes Caspase3 and Caspase8 compared with those in Hela cells that were not co-cultured.
  • Co-culture Lactobacillus with Hela cells at a concentration of MOI 1000:1 for 24 hours Discard the medium and add 1 ml Trizol to lyse the cells. Mix evenly by pipetting and transfer to an EP tube. Lyse at room temperature for 15 minutes. Add 0.2ml chloroform, mix by inverting, and let stand at room temperature for 5 minutes. Centrifuge at 12000g for 15 minutes at 4°C. After centrifugation, it can be seen that the tube is divided into three layers: the uppermost colorless transparent water layer, the middle white film layer, and the lower pink liquid layer. Carefully absorb the upper colorless water layer, do not inhale the white film layer, and transfer the liquid to a new EP tube.
  • RNA purity OD 260 / OD 280 is in the range of 1.8-2.0, and the ratio of OD 260 / OD 230 is greater than 2.0, ensuring the purity and quality of RNA for subsequent experiments.
  • Thermo Scientific RevertAid RT kit K1691
  • add 1 ⁇ l of Random Hexamer primer add the volume of RNA solution, and add water to make up the volume to 12 ⁇ l.
  • add 4 ⁇ l of 5 ⁇ Reaction Buffer 1 ⁇ l of RiboLock RNase Inhibitor, 2 ⁇ l of 10mM dNTPMix, and 1 ⁇ l of RevertAid M-M ⁇ lV RT, for a total volume of 20 ⁇ l.
  • the entire system was incubated at 25°C for 5 minutes, 42°C for 60 minutes, and heated at 70°C for 5 minutes for reverse transcription. Then perform the qRT-PCR experiment according to Roche's instructions.
  • the primer sequences used during the experiment are shown in the table below.
  • Test results It was found that compared with the control group, co-culture of YNH strain and HeLa cells up-regulated the expression of apoptosis genes Caspase3 and Caspase8 in cervical cancer HeLa cells (see Figure 7 for details).
  • Example 8 Flow cytometry to detect the effect of YNH strain on Hela cell apoptosis
  • Test method The control group in the test is Hela cells co-cultured without adding YNH strain, and the experimental groups are Hela cells co-cultured with YNH strains added at MOI1000:1 and MOI10000:1 concentrations.
  • the specific test steps are as follows:
  • test results Through flow cytometry, we observed that YNH strain promoted apoptosis of cervical cancer Hela cells after co-cultured with Hela cells for 48 hours. As shown in A in Figure 8, the apoptosis rate in the control group was 6.61%, as shown in B in Figure 8, the apoptosis rate in the MOI1000:1 group was 9.75%, and as shown in C in Figure 8, the apoptosis rate in the MOI10000:1 group was 14.858%.
  • Example 9 JC-1 experiment detects that after the YNH strain acts on Hela cells, the cell mitochondrial membrane potential decreases
  • control group in this experiment is HeLa cells co-cultured without YNH strain
  • experimental groups are HeLa cells co-cultured with YNH strains at MOI1000:1 and MOI10000:1 concentrations.
  • the specific test steps are as follows:
  • JC-1 Staining Buffer Add 2ml JC-1 Staining Buffer (5 ⁇ ) to 8ml distilled water to prepare, and place on ice after completion. Add 10 ⁇ M CCCP to the positive control well and incubate at 37°C for 20 min. Aspirate the culture medium from each 6-well plate, add PBS to wash the cells twice, and wash away the residual culture medium and non-adherent bacteria. Add 1 ml of JC-1 staining working solution to each well, mix well and incubate for 20 minutes in a 37°C incubator with 5% CO 2 . After the incubation, discard the supernatant, wash the cells twice with JC-1 staining buffer (1 ⁇ ), add 2 ml of complete culture medium, and observe the fluorescence under a fluorescence microscope.
  • Test method For specific test methods and groupings, please refer to the above Western blot to detect changes in cell cycle proteins after YNH strain acts on Hela.
  • Test results As shown in A in Figure 10, compared with the control group, YNH strains of MOI1000:1 and MOI10000:1 down-regulated the anti-apoptotic protein Bax and up-regulated the pro-apoptotic proteins Bcl-2 and Cleave after acting on Hela cells. -caspass3, Cleave-caspasse8, Cleave-caspasse9.
  • Test method The test group is HeLa cells co-cultured with YNH strain, and the control group is HeLa cells not co-cultured with YNH strain.
  • the YNH strain was co-cultured with Hela cells, and cellular RNA was extracted and PCR experiments were performed to detect changes in the mRNA level of the epithelial-to-mesenchymal transition-related gene E-cadherin compared to those in Hela cells that were not co-cultured.
  • YNH strain upregulates the expression of Hela cell apoptosis genes Caspase3 and Caspase8".
  • Test results We found that the YNH strain upregulated the expression of the epithelial-mesenchymal transition-related gene E-cadherin in cervical cancer Hela cells (see Figure 11 for details).
  • mice 6-7 weeks old female BALB/c nude mice, body weight (18 ⁇ 2) g;
  • mice were fed a normal diet for 1 week and then randomly divided into two groups: a saline group and a YNH group, with 3 mice in each group.
  • the YNH group began to receive lactobacilli (bacterial solution concentration 1 ⁇ 10 9 /200 ⁇ L, only, every day) by gavage for 14 days from the second week, and the normal saline group was given the same amount of normal saline every day for 14 consecutive days;
  • a count of 5 ⁇ 10 6 Hela cells/200 ⁇ l was inoculated into the armpit of the right forelimb of nude mice. 1-2 weeks later, the tumor volume reached about 130-150 mm 3 for follow-up experiments. During the tumor formation period, intragastric administration was still performed daily. The YNH group was administered daily. The rats in the normal saline group were given 0.2 ml of Lactobacillus brevis daily, and the normal saline group was given the same amount of normal saline every day. After tumor formation, administration was continued for 14 days, and the body weight and tumor size of the mice were observed.
  • Test results As shown in A in Figure 11, the body weight of nude mice in both the YNH group and the normal saline group increased over time. As shown in B in Figure 12, the tumor volume in the YNH group and the normal saline group increased over time, and the tumor volume in the YNH group was smaller than that in the normal saline group. As shown in Figure 12 C and D, after the nude mice were killed at the mercy endpoint, the tumor volume of the YNH group was smaller than that of the normal saline group, and the difference was statistically significant. (See Figure 12 for details).
  • Test method Soak the fixed tissue in alcohol from low concentration to high concentration for dehydration, place it in xylene to replace the alcohol in the tissue, and then embed it in paraffin.
  • the paraffin blocks were sectioned and dried in a 45°C incubator, soaked in xylene for 5 minutes, dewaxed with alcohol from high to low concentrations, and placed in distilled water after completion. Stain the sections in hematoxylin aqueous solution for 3-4 minutes, hydrochloric acid differentiation solution for 10-20 seconds, and return to blue in PBS. Rinse the film under running water and dehydrate it in 70% and 90% alcohol for 10 minutes each. Stain in alcohol-eosin staining solution for 3 minutes. After staining is completed, the slides are mounted with neutral resin.
  • Example 14 Second animal experimental observation on the treatment of cervical cancer with Lactobacillus YNH strain
  • mice 6-7 weeks old female BALB/c nude mice, body weight (18 ⁇ 2) g;
  • the first group was the normal saline gavage control group
  • the second group was the intraperitoneal injection of cisplatin group
  • the third group was the Lactobacillus gavage group
  • the fourth group was the lactobacilli gavage group.
  • Bacillus combined with cisplatin intraperitoneal injection group starting from the second week, the control group and cisplatin intraperitoneal injection group were intragastrically administered with normal saline for 14 days, the Lactobacillus intragastric injection group and the Lactobacillus combined with cisplatin intraperitoneal injection group were intragastrically administered YNH strain ( The concentration of bacterial solution is 1 ⁇ 10 9 /200 ⁇ L, only, every day) by intragastric administration for 14 days;
  • the first group normal saline gavage control group: fed with normal diet for 1 week; in the second week, normal saline gavage was started for 14 days, and then the tumor formation experiment was performed; after tumor formation, normal saline gavage was continued for 14 days;
  • Group 2 cisplatin intraperitoneal injection group: The concentration of cisplatin was 2 mg/(kg ⁇ d), dissolved in normal saline, and intraperitoneally injected 0.2 ml per animal for a total of 3 injections. They are the 7th, 14th, and 21st days after vaccination;
  • lactobacilli gavage group fed with normal diet for 1 week; from the second week, lactobacilli (bacterial solution concentration 1 ⁇ 10 9 /200 ⁇ L, only, daily) were gavaged for 14 days, and then the tumor formation experiment was carried out (adult Lactobacillus gavage was not stopped during the tumor experiment); gavage with normal saline was continued for 14 days after tumor formation;
  • Group 4 (Lactobacillus combined with cisplatin intraperitoneal injection group): The cisplatin dosage regimen is the same as the second group, and the Lactobacillus dosage regimen is the same as the third group;
  • mice diet mouse diet, body weight, tumor size.
  • Test results As shown in Figure 14A and B, compared with the normal saline gavage control group, the tumor volume of the cisplatin intraperitoneal injection group, Lactobacillus intragastric injection group, and Lactobacillus combined with cisplatin intraperitoneal injection group was smaller. The tumor volume in the intraperitoneal injection group of Bacillus combined with cisplatin was the smallest. It shows that gavage YNH strain can inhibit the proliferation of cervical cancer subcutaneous tumors (see Figure 14 for details).

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Abstract

提供了一株短乳杆菌及其抗宫颈癌应用。所提供的短乳杆菌具体为短乳杆菌（ Lactobacillus brevis）YNH，它在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.24368。实验证明，短乳杆菌（ Lactobacillus brevis）YNH具有降低宫颈癌Hela细胞增殖活力，阻滞Hela细胞周期于S期，降低Hela细胞线粒体膜电位，促进Hela细胞凋亡。短乳杆菌（ Lactobacillus brevis）YNH能通过抑制PI3K/AKT信号通路抑制Hela细胞增殖、促进凋亡，抑制裸鼠宫颈癌皮下瘤的增殖，对宫颈癌Hela细胞具有较好的抑制活性，可用于调节宫颈癌患者生殖道微生态平衡、预防及治疗宫颈癌、提升宫颈癌患者生存率，该菌用于制备抑制宫颈癌发生发展的发酵食品及相关药物，具有非常广泛的应用前景。

Description

一株短乳杆菌及其抗宫颈癌应用 技术领域
本发明属于微生物技术领域，尤其涉及一种具有抗宫颈癌作用的短乳杆菌及应用。
 背景技术
宫颈癌（Cervical Cancer）是最常见的女性生殖道恶性肿瘤，“2020年全球癌症统计数据”报告显示，宫颈癌在全球女性肿瘤中发病率和死亡率均位居第四，世界卫生组织积极推行HPV疫苗接种，降低宫颈癌发病率，但低中收入国家发病率仍然居高不下。因此，在将来很长一段时间内，宫颈癌仍将是全球女性沉重的疾病和卫生经济负担，那么宫颈癌的治疗显得尤为重要。早期宫颈癌采用根治性手术，预后好，5年生存率达到90%。但对于局部肿瘤大于4cm、局部转移、局部浸润的宫颈癌（局部晚期宫颈癌）治疗效果并不是很好，5年生存率为30-80%，预后差。局部晚期宫颈癌治疗方法众多，以手术、放疗、化疗多种方式联合治疗为主，但都存在其不足之处，术后并发症多，仅淋巴囊肿一项并发症的发生率即可高达25%；放化疗后副作用也明显，如骨髓抑制、阴道炎、膀胱炎、肠炎。寻找更好的治疗方法或药物，既提高晚期宫颈癌的治愈率，降低毒副反应，又提高患者的生活质量成为当务之急。
乳杆菌（lactobacillus）是对人体健康有益的微生物，其广泛存在于人体的胃肠道和生殖道。既往研究表明乳杆菌对人体多种疾病有治疗作用，如维持肠道稳态、预防需氧性阴道炎、复发性膀胱炎、降低血糖和血脂。但乳杆菌对肿瘤的治疗作用一直被忽视，直至近年才发现乳杆菌对多种肿瘤具有抑制作用。乳杆菌对结直肠癌、乳腺癌、膀胱癌、宫颈癌均有抑制作用。研究表明嗜酸乳杆菌单独或联合菊石运用减少化学诱导的实验性结肠癌发生；干酪乳杆菌通过激活JNK通路促进结肠癌细胞凋亡；短乳杆菌MK05上调Bcl-2家族的凋亡基因促进乳腺癌细胞的凋亡；鼠李糖乳杆菌抑制小鼠膀胱肿瘤，卷曲乳杆菌、加式乳杆菌、詹式乳杆菌上清液阻滞宫颈癌细胞周期，发挥抗癌作用。并且乳杆菌的抗癌作用具有选择性，只损伤肿瘤细胞，却对正常的细胞没有毒性作用。因此，乳杆菌是有潜能的抗癌生物活性物质，可能为临床治疗宫颈癌提供低毒副作用的有效治疗方式。
乳杆菌属包含上百种乳杆菌，仅有少数乳杆菌具有抗宫颈癌作用，如詹式乳杆菌、罗伊乳杆菌、鼠李糖乳杆菌、卷曲乳杆菌、加式乳酸杆菌、干酪乳杆菌。我国抗宫颈癌的核心菌株的自主率十分不足，制约了乳杆菌抗癌的临床运用，亟需挖掘更多抗宫颈癌作用的菌株，解决临床可用抗癌乳杆菌的“缺芯少核”问题。我们通过实验发现短乳杆菌（ Lactobacillus brevis）YNH在体外较罗伊乳杆菌、卷曲乳杆菌、格氏乳杆菌有更好的抗癌效果，动物实验显示短乳杆菌（ Lactobacillus brevis）YNH在体内同样有效抑制宫颈癌瘤体增殖。既往乳杆菌抗宫颈癌研究多观察乳杆菌抑制宫颈癌细胞的现象，研究者发现乳杆菌阻滞宫颈癌细胞周期于G1或S期，抑制增殖；下调宫颈癌细胞基质金属蛋白酶2和9，上调E-cadherin,抑制宫颈癌上皮间质转化；上调凋亡基因和自噬基因，促进宫颈癌死亡。以上研究均在细胞水平探究乳杆菌的抗癌作用，未对其抗癌作用的机制进行深入探究，亟需进一步阐明乳杆菌抗癌作用的机制。由于乳杆菌的抗癌作用是高度菌株特异性和细胞特异性，即不同菌株对同种肿瘤细胞的作用不同，乳杆菌同一菌株对不同肿瘤细胞的作用也不同。因此筛选具有抗宫颈癌作用的益生菌具有非常重要的应用价值和现实意义。
发明内容
本发明的目的是提供一株健康女性生殖道来源的短乳杆菌及其应用。
[1].一种短乳杆菌（ Lactobacillus brevis）YNH，其特征在于所述短乳杆菌（ Lactobacillus brevis）YNH可以抑制宫颈癌Hela细胞增殖、促进宫颈癌Hela细胞凋亡，所述短乳杆菌（ Lactobacillus brevis）YNH在中国微生物菌种保藏管理委员会普通微生物中心（CGMCC）的保藏编号为CGMCC No .24368。
[2].一种菌剂，其特征在于所述菌剂可以抑制宫颈癌Hela细胞增殖、促进宫颈癌Hela细胞凋亡，所述菌剂的活性成分为如[1]所述的短乳杆（ Lactobacillus brevis）YNH。
[3].包含如[1]所述的短乳杆菌（ Lactobacillus brevis）YNH的发酵液或发酵液的过滤液。
[4].包含如[1]所述的短乳杆菌（ Lactobacillus brevis）YNH的菌悬液。
[5].一种培养有如[1]所述的短乳杆菌（ Lactobacillus brevis）YNH的培养液。
[6].如[1]所述的短乳杆菌（ Lactobacillus brevis）YNH、[2]所述的菌剂、[3]所述的发酵液或发酵液的过滤液、[4]所述的菌悬液或[5]所述的培养液在制备用于治疗或预防宫颈癌的药物中的用途。
[7].如[1]所述的短乳杆菌（ Lactobacillus brevis）YNH、[2]所述的菌剂、[3]所述的发酵液或发酵液的过滤液、[4]所述的菌悬液或[5]所述的培养液在制备用于调节和/或改善妇女生殖道微生态平衡的药物或试剂中的用途。
[8].一种药物组合物，其特征在于所述药物组合物可以抑制宫颈癌Hela细胞增殖、促进宫颈癌Hela细胞凋亡，所述药物组合物包含如[1]所述的短乳杆菌（ Lactobacillus brevis）YNH、[2]所述的菌剂、[3]所述的发酵液或发酵液的过滤液、[4]所述的菌悬液或[5]所述的培养液中的任一种，以及药学上可接受的载体。
[9].如[8]所述的药物组合物，其特征在于所述药物组合物为益生菌制剂。
[10].如[8]或[9]所述的药物组合物，其中所述药学上可接受的载体选自由药学上通常使用的矫味剂、润滑剂、填充剂、崩解剂组成的组中的任一种。
在本发明的一个实施例中，所述发酵液具体为将所述短乳杆菌（ Lactobacillus brevis）YNH 接种于MRS培养基中，在37℃静置培养后得到的发酵液。其中，培养时间最好在15小时以上。
所述短乳杆菌（ Lactobacillus brevis）YNH能够抑制宫颈癌；并且，所述短乳杆菌（ Lactobacillus brevis）YNH与Hela细胞共培养后，可以上调凋亡基因Caspase3和Caspase8；所述短乳杆菌（ Lactobacillus brevis）YNH与Hela细胞共培养后，通过CCK8实验发现MOI1000:1及MOI10000:1浓度的短乳杆菌（ Lactobacillus brevis）YNH从48h开始对Hela细胞增殖有抑制作用；
通过流式细胞发现短乳杆菌（ Lactobacillus brevis）YNH将Hela细胞周期阻滞于S期 ；在蛋白水平上短乳杆菌（ Lactobacillus brevis）YNH上调Hela细胞周期S期相关蛋白CDK2、CyclinE1，说明短乳杆菌YNH具有抑制Hela细胞增殖的作用；
流式细胞术发现短乳杆菌（ Lactobacillus brevis）YNH促进Hela细胞凋亡；
JC1实验发现MOI1000:1及MOI10000:1浓度的短乳杆菌（ Lactobacillus brevis）YNH降低Hela细胞线粒体膜电位，促进Hela细胞凋亡；
短乳杆菌（ Lactobacillus brevis）YNH与Hela细胞共培养后，在蛋白水平上发现短乳杆菌YNH下调Hela细胞抗凋亡蛋白Bax,上调促凋亡蛋白Bcl-2、Cleave-caspasse3、Cleave-caspasse8、Cleave-caspasse9；
动物实验通过给裸鼠灌胃短乳杆菌（ Lactobacillus brevis）YNH发现其可以抑制宫颈癌瘤体增殖
在一些具体的实施方式中，本发明还提供了一种利用如上所述的具有抗宫颈癌作用的短乳杆菌（ Lactobacillus brevis）YNH发酵生产制得的发酵食品。
在一些优选的实施方式中，所述发酵食品包括乳制品、豆制品、果蔬制品。
所述短乳杆菌（ Lactobacillus brevis）YNH或所述菌剂或所述发酵液或发酵液的过滤液或代谢物或菌悬液或培养液在如下任一中的应用也属于本发明的保护范围：
(b1)调节妇女生殖道微生态平衡；
(b2)制备用于调节妇女生殖道微生态平衡的产品。
本发明提供的短乳杆菌（ Lactobacillus brevis）YNH的保藏信息如下：
菌种名称：短乳杆菌
拉丁名： Lactobacillus brevis 
菌株编号：YNH
保藏机构：中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称：CGMCC 
地址：北京市朝阳区北辰西路1号院3号
保藏日期：2022年01月24日
保藏中心登记入册编号：CGMCC No .24368.
发明的效果
本发明提供的短乳杆菌（ Lactobacillus brevis）YNH与Hela细胞共培养后，可以上调凋亡基因Caspase3和Caspase8，通过CCK8实验发现MOI1000:1及MOI10000:1浓度的短乳杆菌（ Lactobacillus brevis）YNH从48h开始对Hela细胞增殖有抑制作用，进一步通过流式细胞发现将Hela细胞周期阻滞于S期，在蛋白水平上调Hela细胞周期S期相关蛋白CDK2、CyclinE1，说明短乳杆菌（Lactobacillus brevis）YNH具有抑制Hela细胞增殖的作用。流式细胞术发现短乳杆菌（ Lactobacillus brevis）YNH促进Hela细胞凋亡并且通过JC1实验发现MOI1000:1及MOI10000:1浓度的短乳杆菌（ Lactobacillus brevis）YNH降低Hela细胞线粒体膜电位，促进Hela细胞凋亡，短乳杆菌（ Lactobacillus brevis）YNH与Hela细胞共培养后，在蛋白水平上发现短乳杆菌（ Lactobacillus brevis）YNH下调Hela细胞抗凋亡蛋白Bax,上调促凋亡蛋白Bcl-2、Cleave-caspasse3、Cleave-caspasse8、Cleave-caspasse9。动物实验通过给裸鼠灌胃短乳杆菌（ Lactobacillus brevis）YNH发现其可以抑制宫颈癌瘤体增殖。
附图说明
图1中的A示出了健康人阴道菌群（无HPV感染人群）与 LSIL、HSIL（有HPV感染人群）患者阴道菌群中包含的具体菌株类型，图1中的B示出了健康人阴道菌群（无HPV感染人群）与 LSIL、HSIL（有HPV感染人群）患者阴道微生物主成成分分析结果。
图2中的A示出了从健康人阴道分泌物中分离的不同株乳杆菌所占百分比，图2中的B示出了不同株乳杆菌构建的系统发育树。
图3中的A示出了乳杆菌1的菌落形态，图3中的B示出了乳杆菌1革兰染色后的镜下形态。
图4中的A示出了MOI100：1浓度的短乳杆菌（ Lactobacillus brevis）YNH对Hela细胞增殖无影响，图4中的B、C分别示出了MOI1000：1、MOI10000:1浓度的短乳杆菌（ Lactobacillus brevis）YNH在48h观察到抑制Hela细胞增殖。
图5中的A示出了空白对照组的Hela细胞各细胞周期占比，图5中的B、C分别示出了MOI1000：1、MOI10000:1浓度的短乳杆菌（ Lactobacillus brevis）YNH与Hela细胞共培养48h后Hela细胞各周期占比，图5中的D示出了图A、B、C中的Hela细胞各细胞周期所占比例的柱状图。
图6中的A示出了不同浓度的短乳杆菌（ Lactobacillus brevis）YNH下调宫颈癌Hela细胞S期相关蛋白CDK2、CyclinE1表达，图6中的B示出了CDK2、CyclinE1蛋白条带相对表达量的柱状图。
图7示出了短乳杆菌（Lactobacillus brevis）YNH与Hela细胞共培养48h后上调凋亡相关基因Caspase3和Caspase8的表达量。
图8中的A、B、C分别示出了空白对照组Hela细胞、MOI1000:1、MOI10000:1浓度的短乳杆菌（ Lactobacillus brevis）YNH作用后的Hela细胞的细胞凋亡比例，图8中的D示出了图A、B、C中的Hela细胞凋亡比例的柱状图。
图9示出了短乳杆菌（ Lactobacillus brevis）YNH与Hela细胞共培养48h后使Hela细胞线粒体膜电位下降。
图10中的A示出了不同浓度的短乳杆菌（ Lactobacillus brevis）YNH对Hela细胞凋亡蛋白表达的影响，图10中的B示出了不同凋亡蛋白相对表达量的柱状图。
图11示出了短乳杆菌（ Lactobacillus brevis）YNH与Hela细胞共培养后影响上皮间质转化相关基因E-cadherin的表达量。
图12中的A示出了短乳杆菌（ Lactobacillus brevis）YNH组和空白对照组宫颈癌皮下瘤裸鼠的体重变化，图12中的B示出了两组宫颈癌皮下瘤裸鼠的瘤体大小变化，图12中的C示出了处死两组宫颈癌皮下瘤裸鼠后的瘤体，图12中的D示出了处死两组宫颈癌皮下瘤裸鼠的瘤体重量的柱状图。图12结果均为短乳杆菌YNH菌株治疗宫颈癌的第一次动物实验。
图13示出了短乳杆菌（ Lactobacillus brevis）YNH组和生理盐水的对照组宫颈癌皮下瘤裸鼠的心、肝、肾的HE染色结果。
图14中的A示出了生理盐水灌胃组、顺铂腹腔注射组、乳杆菌灌胃组、乳杆菌联合顺铂腹腔注射组等4组的宫颈癌皮下瘤裸鼠的荷瘤图，图14中的B示出了处死生理盐水灌胃组、顺铂腹腔注射组、乳杆菌灌胃组、乳杆菌联合顺铂腹腔注射组等4组宫颈癌皮下瘤裸鼠后的瘤体。图14的结果均为短乳杆菌YNH菌株治疗宫颈癌的第二次动物实验。
 实施方式 实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例（虽然表示本公开的具体实施方式）仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法，例如下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册（New York： Cold Spring Harbor Laboratory Press，1989）中所述的条件，或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。
实施例
分别收集健康人群（无HPV感染人群）、低级别宫颈上皮内瘤变（low-grade squamous intraepithelial lesion, LSIL）、高级别宫颈上皮内瘤变（high-grade squamous intraepithelial lesion, HSIL）患者的阴道分泌物各30份 ，进行总DNA的提取 ，MiSeq高通量测序。
分组情况：H1046、H1052、H1060为HSIL患者阴道分泌物；L1025、L1075、L10102为LSIL患者阴道分泌物；N1072、N1074、N1087为健康人群阴道分泌物；
试验步骤：
提取细菌DNA的具体方法如下所述：
细菌DNA的提取使用天根的细菌基因组DNA提取试剂盒（DP302）完成。具体的实验步骤如下：用15 ml 离心管收集1.0×10 9（5 ml 菌液OD 600 为1-1.5）的细菌培养物，10000rpm离心5分钟后弃上清。加入200μl缓冲液GA到菌体沉淀中，震荡至菌体悬浮。加入20μl Proteinase 溶液，混匀后再加入220μl缓冲液GB，震荡15秒后70℃放置10分钟。溶液澄清后加入220μl CH 3CH 2OH，震荡混匀15秒，将沉淀和上清液体转移到吸附柱CB3，12000rpm离心30秒，弃废液，将吸附柱CB3放入收集管中，加入500μl缓冲液GD，12000rpm离心30秒，弃废液。将吸附柱放入收集管中加入600μl漂洗液PW，12000rpm离心30秒，重复漂洗两次。将吸附柱CB3转移到干净的离心管中，向吸附膜中间部位加入100μl缓冲液TE，室温放置5分钟后12000rpm离心2分钟，将溶液收集到离心管中，得到本实施例需要的细菌DNA基因组。
试验结果：测序结果表明，健康人阴道菌群（无HPV感染人群）与LSIL、HSIL（有HPV感染人群）患者阴道菌群有明显差异，健康人生殖道以惰性乳杆菌及卷曲乳杆菌为主，而在LSIL、HSIL患者阴道菌群中，主要以加德纳菌为主（具体参见图1A）。主成分分析 (Principal Component Analysis，PCA)也表明健康人阴道菌群（无HPV感染人群）与 LSIL、HSIL患者阴道微生物组成有明显差异（具体参见图1B）。
实施例2：收集并分离健康人阴道乳杆菌，分析其中包含的具体的乳杆菌的菌株，并记录各个菌株所占的百分比（具体见图2A），建立乳杆菌16S rRNA基因数据库，并构建乳杆菌系统发育树，对各进化树进行分析比对（具体参见图2B）
试验方法：
阴道分泌物的收集及菌株鉴定
选取阴道健康的妇女，用无菌棉拭子于女性子宫颈口与粘膜交界处逆时针旋转3圈，停留10秒，取阴道分泌物置于试管中，依次编号后置于-80℃冷冻保存。取上述阴道拭子涂抹于血平板上，用接种环分区画线后置于5%CO 2，37℃的培养箱中培养。待菌落长出，挑取白色、半透明、光滑形凸起的菌进行分纯、涂片，涂片后革兰染色显微镜镜检为革兰阳性杆菌为待鉴定乳杆菌。将待鉴定菌株接种于8ml灭菌MRS中，5%CO 2，37℃的培养箱中培养过夜。使用细菌总DNA提取试剂盒提取DNA，利用PCR技术使用通用引物扩增细菌16sDNA片段并将产物进行序列测定。对获取的目的基因序列进行BLAST同源性比对分析。
试验结果：成功分离鉴定乳杆菌，并计算出各种乳杆菌所占比例，如图2A，并且构建系统发育树，如图2B所示。
实施例3：短乳杆菌（ Lactobacillus brevis）YNH菌株的分离与鉴定 
一、短乳杆菌（ Lactobacillus brevis）YNH菌株的分离 
短乳杆菌（ Lactobacillus brevis）YNH分离自健康的妇女生殖道。
试验方法：将生殖道拭子置于含1mL无菌的PBS的试管中振荡30s，以此为原液连续十倍稀释，取100μL稀释10000倍的液体涂布于MRS培养基固体平板(1L的MRS培养基配方为：牛肉膏10g，酪蛋白胨10g，酵母提取物5g，葡萄糖20g，乙酸钠5g，柠檬酸氢二铵2g，Tween 80 1mL，K 2HPO 4 2g，MgSO 4 .7H 2O 0 .58g，MnSO 4 .H 2O 0 .25g，琼脂粉15g，双蒸水定容到l L，高压灭菌)，置于厌氧培养箱中37℃，培养48h。然后挑取单克隆在MRS液体培养基中培养，将细菌涂布于血平板进行单个菌落的培养，挑取疑似格兰阳性的细菌进行镜检，将革兰阳性菌进行增菌提取RNA后送测序。本专利中的菌的菌落形态和革兰染色涂片见参见图3中的A和B，将该乳杆菌命名为乳杆菌1。
二、乳杆菌1菌株的鉴定 
将乳杆菌1菌体培养后收集，提取基因组 DNA，利用引物 (27F：5'-AGAGTTTGATCCTGGCTCAG-3 '（SEQ ID NO：2）和1492R：5 '- GGTTACCTTGTTACGACTT-3 '（SEQ ID NO：3）)对16S rRNA序列进行扩增，得到PCR产物。并对PCR产物进行测序。 
测序结果表明：PCR产物的序列（SEQ ID NO：1）如下所示：
ccccgtgcga tgtctatact gcaagtcgaa cgagcttccg ttgaatgacg tgcttgcact
gattttaaca atgaagcgag tggcgaactg gtgagtaaca cgtgggaaat ctgcccagaa
gcaggggata acacttggaa acaggtgcta ataccgtata acaacaaaat ccgcatggat
tttgtttgaa aggtggcttc ggctatcact tctggatgat cccgcggcgt attagttagt
tggtgaggta aaggcccacc aagacgatga tacgtagccg acctgagagg gtaatcggcc
acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac
aatggacgaa agtctgatgg agcaatgccg cgtgagtgaa gaagggtttc ggctcgtaaa
actctgttgt taaagaagaa cacctttgag agtaactgtt caagggttga cggtatttaa
ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt
gtccggattt attgggcgta aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc
ttcggcttaa ccggagaagt gcatcggaaa ctgggagact tgagtgcaga agaggacagt
ggaactccat gtgtagcggt ggaatgcgta gatatatgga agaacaccag tggcgaaggc
ggctgtctag tctgtaactg acgctgaggc tcgaaagcat gggtagcgaa caggattaga
taccctggta gtccatgccg taaacgatga gtgctaagtg ttggagggtt tccgcccttc
agtgctgcag ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact
caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagctacg
cgaagaacct taccaggtct tgacatcttc tgccaatctt agagataaga cgttcccttc
ggggacagaa tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt
aagtcccgca acgagcgcaa cccttattat cagttgccag cattcagttg ggcactctgg
tgagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt
atgacctggg ctacacacgt gctacaatgg acggtacaac gagtcgcgaa gtcgtgaggc
taagctaatc tcttaaagcc gttctcagtt cggattgtag gctgcaactc gcctacatga
agttggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg
tacacaccgc ccgtcacacc atgagagttt gtaacaccca aagccggtga gataaccttc
gggagtcagc cgtctaaggt cacaga
将此序列经过在NCBI网站(https://blast .ncbi .nlm .nih .gov/Blast .cgi)比对分析，鉴定为短乳杆菌。 
经过上述鉴定结果，确定乳杆菌1名称为短乳杆菌YNH（以下实施例中也称为YNH菌株），其分类命名为短乳杆菌 ( Lactobacillus brevis)，该菌株已于2022年01月24日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，保藏编号为CGMCC No .24368。
试验方法：短乳杆菌YNH置于5%CO 2，37℃的培养箱中，培养48h。培养结束后，8000rpm/min离心10min,弃上清液。PBS清洗细菌2次并离心，加入适量细胞培养基重悬，用完全培养基调零分光光度计后测量细菌浓度。根据需要的细菌数量，代入公式v=n/c（n为细菌数目，c为菌液浓度，v为菌液体积），计算需要取的细菌菌液体积。
取对数生长期Hela细胞进行消化、离心，加入完全培养基重悬细胞后行细胞计数。96孔板每孔加入200μl完全培养基，种2000个细胞，每个实验组设置3个副孔。96孔板周围一圈孔加入PBS,避免边缘效应。待细胞贴壁后，各个组第一个时间点的每孔加入10μl CCK8试剂，避光孵育1小时，用酶标仪测450nm波长下的OD值。各个组OD值接近时反应各组种板细胞数目接近，进一步加入处理因素。空白对照组：加入200μl完全培养基；MOI100:1组：吸出板中培养基，每孔加入含2×10 5个细菌的完全培养基200μl；MOI1000：1组：吸出板中培养基，每孔加入含2×10 6个细菌的完全培养基200μl；MOI10000：1组：吸出板中培养基，每孔加入含2×10 7个细菌的完全培养基200μl。将96孔板继续置于5%CO 2，37℃培养箱中培养，以后每天均间隔24h测量细胞OD值。
试验结果：与对照组相比，MOI100：1的短乳杆菌YNH作用下Hela细胞增殖不受影响，MOI1000:1和MOI10000:1的短乳杆菌YNH作用下Hela细胞增殖在48h开始受到抑制，差异具有统计学意义(P<0.05) ，说明短乳杆菌YNH对Hela细胞的增殖具有抑制作用（具体参见图4）。
实施例5： 流式细胞术检测YNH菌株对Hela细胞周期的影响
试验方法：取对数生长期细胞进行消化、离心、计数，于每个T25瓶中加入5×10 5个Hela细胞，待细胞贴壁后去除培养基，对照组加入4ml完全培养基，MOI1000：1组加入含5×10 8个细菌的完全培养基4ml，MOI10000：1组加入含5×10 9个细菌的完全培养基4ml，继续置于5%CO 2，37℃培养箱中培养48h。48h后消化细胞，1000rpm/min离心4分钟收集各组细胞，弃上清后加入1ml预冷的PBS重悬细胞，离心后弃上清。每管加入1ml 4℃预冷的70%乙醇充分重悬细胞，保证单个细胞成单个，充分的固定过夜。染色时去上清，预冷的PBS洗细胞两次，每管加入0.535ml碘化丙啶染色液，重悬细胞后37ºC避光温浴30分钟，4℃避光保存至上机检测。碘化丙啶染液的配置：每管加入0.5ml染色缓冲液，25μl的20×碘化丙啶染色液，10μl的50× RNase A，配制见下表。
试验结果：MOI1000:1、MOI10000:1的YNH菌株与宫颈癌Hela细胞共培养48h后，流式细胞仪检测各组细胞周期占比情况。图5中的A中对照组G1期细胞56.77%，图5中的B中MOI1000:1组G1期细胞52.16%，图5中的C中MOI10000:1组G1期细胞45.07%。与对照组相比，MOI1000：1组（t=3.849， P=0.018）和MOI10000:1组（t=10.8， P<0.01）G1期细胞占比减少，差异均有统计学意义。图5中的A中对照组S期细胞34.23%，图5中的B中MOI1000:1组S期细胞36.55%，图5中的C中MOI10000:1组S期细胞42.44%。与对照组相比，MOI1000：1组（t= -3.304, P=0.003）和MOI10000:1组（t= -14.11, P<0.01）S期细胞占比增加，差异具有统计学意义。图5中的A中对照组G2期细胞9.0%，图5中的B中MOI1000:1组G2期细胞11.28%，图5中的C中MOI10000:1组G2期细胞12.49%，与对照组相比，MOI1000:1组（t=-1.08， P=0.341）、MOI10000:1组（t=-1.42， P=0.228）细胞占比差异无统计学意义。以上结果提示乳杆菌1将Hela细胞周期阻滞于S期(具体参见图5)。
实施例6： Western blot检测YNH菌株作用于Hela后细胞周期蛋白的变化
试验方法：本试验中的对照组为未加YNH菌株共培养的Hela细胞，实验组分别为加入MOI1000：1和MOI10000:1浓度YNH菌株共培养的Hela细胞。具体试验步骤如下：
取对数生长期细胞进行消化、离心、计数，于每个T25瓶中加入5 ×10 5个Hela细胞，待细胞贴壁后去除培养基，对照组加入4ml完全培养基，MOI1000：1组加入含5×10 8个细菌的完全培养基4ml，MOI10000：1组加入含5×10 9个细菌的完全培养基4ml，继续置于5%CO 2，37℃培养箱中培养48h。消化、离心各组细胞后，弃上清，预冷PBS重悬细胞3次，弃上清后加入含1%蛋白酶抑制剂的RIPA 100μl，重悬细胞后转移至1.5mlEP管中，冰上裂解20min。裂解结束后超声破壁机破碎3次，每次10s，中间间隔10s。低温超速离心机4℃，12000rpm离心15min，取上清，保存于-20℃冰箱。
配置BCA工作液：按A液与B液50：1的比例配置工作液。蛋白标准品（1mg/ml）按0、1、2、4、6、8、10μl加入到96孔板中，每孔再用去离子水补足至10μl，每个浓度设置3个副孔。取待测蛋白溶液2μl加入8μl去离子水，每个样品设置3个副孔。每孔加入200μl BCA工作液，5%CO 2，37℃培养箱中孵育30min。酶标仪测562nm波长下的OD值。根据蛋白标准品测得的OD值及已知蛋白标准品浓度，计算吸光度与蛋白浓度的方程式，将待测样品OD值代入方程，求得待测样品蛋白浓度。
(1)蛋白变性：将蛋白溶液和Buffer按体积1：4配制混匀，配制后蛋白浓度为原浓度的80%，将蛋白溶液煮沸10min，冷却后放-20℃备用。
（2）配胶：根据碧云天SDS-PAGE凝胶配制试剂盒说明书配制10%浓度的分离胶和压缩胶。加入4.1ml蒸馏水，3.3ml的30%Acr-Bis(29:1)，2.5ml的下层胶缓冲液（4×），最后加入0.1ml的10%凝胶聚合催化剂和0.004ml的TEMED（加入过早会使胶凝固），配制成2块下层胶。加入1.75ml蒸馏水，0.5ml的30%Acr-Bis(29:1)，0.75ml上层胶缓冲液（4×），最后加入0.03ml的10%凝胶聚合催化剂和0.003ml的TEMED，配制2块上层胶。
（3）灌胶：将玻璃板擦净，固定。将配好的分离胶缓缓加入两玻璃板之间缝隙，加至距板口3cm的位置，再加入无水乙醇至板上缘。待分离胶凝固后，倒掉无水乙醇并擦干玻片，加入压缩胶，加入过程中避免产生气泡，插入梳子，等待凝固。
（4）上样：压缩胶凝固后，将玻片固定于电泳槽，加满电泳液，缓慢拔出梳子。确定上样的蛋白质量后，根据v=m/c公式，计算蛋白上样体积。
（5）电泳： 80V恒压电泳至样品到压缩胶下缘，再120V恒压至样品到分离胶下缘。
（6）转膜：将PVDF膜甲醇活化后覆盖胶上，转膜夹将其固定后转移至转膜槽。加转膜液，放冰盒于转膜槽中，槽置于加满冰水混合物的泡沫箱中，300mA恒流2h。
（7）封闭：取出PVDF膜后，1× TBST洗3min，将膜放入5%浓度的奶粉中封闭2h。
（8）抗体孵育：封闭完成的PVDF膜置于1× TBST洗3次，每次10min。根据marker位置和目的蛋白分子量确定条带并切割，置于一抗中4℃孵育过夜。孵育完成后取出，1× TBST洗膜3次，每次10min，加入二抗常温孵育2h。
（9）显影：弃二抗后，1× TBST洗膜3次，每次10min。膜放于1：1配制显影液中避光浸润30s，成像系统曝光并采集图像。
试验结果：如图6中的A所示，与对照组相比，MOI1000:1和MOI10000:1的YNH菌株下调细胞周期S期相关蛋白CDK2、CyclinE1的表达。图6中的B为蛋白灰度相对定量的柱状图。如图6中的B所示，与对照组相比，MOI1000:1组、MOI10000:1组的CDK2（t=14.11, P<0.01；t=18.96, P<0.01）、CyclinE1(t=5.31， P=0.01；t=6.84， P=0.11)的相对表达量下降，差异均有统计学意义(具体参见图6)。
实施例7： YNH菌株上调Hela细胞凋亡基因Caspase3、Caspase8的表达
试验方法：乳杆菌组为YNH菌株与Hela细胞在37℃、5%CO 2条件下共培养24h，对照组为Hela细胞在37℃、5%CO 2条件下共培养24h，提取两组细胞RNA利用PCR实验检测凋亡相关基因Caspase3、Caspase8在mRNA水平上相较于未共培养Hela细胞的变化。
将乳杆菌按MOI1000:1浓度与Hela细胞共培养24h，弃培养基，加入1ml Trizol裂解细胞，吹打混匀后转移至EP管中，室温裂解15min。加入0.2ml氯仿，颠倒混匀，室温静置5min。4℃，12000g离心15min。离心后可见管内分为三层，最上面的无色透明水层，中间白膜层，下层粉红色液体层。小心吸取上层的无色水层，切勿吸入白膜层，转移液体至新的EP管中。加入0.5ml异丙醇，颠倒混匀，室温静置10min。4℃，12000g离心10min。离心结束后可见管壁上附着白色沉淀即为RNA，小心吸去液体，加入1ml的75%乙醇，颠倒混匀，充分洗涤RNA。4℃，7500g离心5min，弃乙醇，将RNA沉淀自然干燥至半透明状。加入10-30μl无酶的DEPC水，吹打混匀至RNA溶解。DEPC水调零Nano-drop 2000C超微量分光光度后，吸取2μlRNA溶液测量RNA浓度。RNA纯度OD 260/OD 280的比值在1.8-2.0范围内，OD 260/OD 230的比值大于2.0，保证RNA的纯度和质量可进行后续实验。根据Thermo逆转录说明书，逆转录合成cDNA。RNA的质量4ug，根据v=m/c（v代表体积，m代表质量，c代表浓度）计算需要的RNA溶液体积，进行逆转率。逆转率使用Thermo Scientific RevertAid RT试剂盒（K1691）进行，具体如下：加入Random Hexamer primer 1μl，加入RNA溶液的体积，加水补足体积12μl。再加入5× Reaction Buffer 4μl，RiboLock Rnase Inhibitor 1μl，10mM dNTPMix 2μl，RevertAid M-MμlV RT 1μl，总共20μl体积。整个体系置于25℃孵育5分钟，42℃孵育60分钟，70℃加热5分钟进行逆转录。再根据Roche的说明书进行qRT-PCR实验。实验过程中所用到的引物序列见下表。
试验结果：发现与对照组相比，YNH菌株与Hela细胞共培养后上调了宫颈癌Hela细胞凋亡基因Caspase3、Caspase8的表达（具体参见图7）。
实施例8： 流式细胞术检测YNH菌株对Hela细胞凋亡的影响
试验方法：试验中的对照组为未加YNH菌株共培养的Hela细胞，实验组分别为加入MOI1000：1和MOI10000:1浓度YNH菌株共培养的Hela细胞。具体试验步骤如下：
取对数生长期细胞进行消化、离心、计数，于每个T25瓶中加入5×10 5个Hela细胞，待细胞贴壁后去除培养基，对照组加入4ml完全培养基，MOI1000：1组加入含5×10 8个细菌的完全培养基4ml，MOI10000：1组加入含5×10 9个细菌的完全培养基4ml，继续置于5%CO 2，37℃培养箱中培养48h。用0.25%不含EDTA的胰酶消化细胞，消化至晃动瓶身刚好可见细胞滑落为宜。2000rpm离心10min，弃上清。用预冷1×PBS（4℃）重悬细胞两次，2000rpm离心10分钟，洗涤细胞。弃上清后，加入500μl 1×Binding Buffer，轻柔重悬细胞，避免吹打过重导致细胞膜的磷脂酰丝氨酸外翻，造成假阳性结果。吸取100μl细胞悬液转移至流式管内，加入5μl Annexin V-FITC混匀后，加入5μl PI溶液，混匀，室温下避光孵育15min。上机检测前再加入400μl 1× Binding Buffer。
试验结果：通过流式细胞术我们观察到YNH菌株与Hela细胞共培养48h后，促进宫颈癌Hela细胞凋亡。如图8中的A所示对照组凋亡率6.61%，如图8中的B所示MOI1000：1组凋亡率9.75%，如图8中C所示MOI10000:1凋亡率14.858%。如图8中的D所示，与对照组相比，MOI1000:1组（t=-9.802， P<0.01）、MOI10000:1组（t=-26.947， P<0.01）凋亡率增加，差异有统计学意义(具体参见图8中的D)。
实施例9： JC-1实验检测YNH菌株作用于Hela细胞后，细胞线粒体膜电位下降
试验方法：本试验中的对照组为未加YNH菌株共培养的Hela细胞，实验组分别为加入MOI1000：1和MOI10000:1浓度YNH菌株共培养的Hela细胞。具体试验步骤如下：
取对数生长期细胞进行消化、离心、计数，6孔板每孔加入1×10 5个Hela细胞，对照组加入3ml完全培养基，MOI1000：1组加入含1×10 8个细菌的完全培养基3ml，MOI10000：1组加入含1×10 9个细菌的完全培养基3ml，5%CO 2，37℃培养箱中培养48h。实验前先配置JC-1染色液：取50μl JC-1(200×)加入8ml超纯水稀释JC-1，再加入2ml JC-1染色缓冲液(5×)混匀。JC-1染色缓冲液：2ml JC-1染色缓冲液(5×)加入8ml蒸馏水进行配置，完成后置于冰上。阳性对照孔加入10μM的CCCP37℃孵育20min后，将各个6孔板中培养基吸出，加入PBS洗涤细胞两次，将残余培养基和未黏附的细菌洗去。每孔加入1ml JC-1染色工作液，混匀后于5%CO 2，37℃培养箱中孵育20min。孵育结束后，弃上清，用JC-1染色缓冲液(1×)洗涤细胞2次后加入2ml完全培养基，荧光显微镜下观察荧光。
试验结果：YNH菌株与宫颈癌Hela细胞共培养48h后，与对照组相比，MOI1000:1组、MOI10000:1组红色荧光随YNH菌株作用浓度的增加，荧光强度减低，绿色荧光随YNH菌株作用浓度的增加，荧光强度增加，绿色荧光占红绿色荧光的比例也增加，说明YNH菌株降低宫颈癌Hela细胞线粒体膜电位，促进宫颈癌Hela细胞凋亡(具体参见图9)。
试验方法：具体的试验方法和分组参见上述Western blot检测YNH菌株作用于Hela后细胞周期蛋白的变化。
试验结果：如图10中的A所示，与对照组相比，MOI1000：1、MOI10000:1的YNH菌株作用于Hela细胞后下调抗凋亡蛋白Bax,上调促凋亡蛋白Bcl-2、Cleave-caspasse3、Cleave-caspasse8、Cleave-caspasse9。如图10中的B所示，与对照组相比，MOI1000:1组、MOI10000:1组的PARP（t=14.90，P<0.01;t= 32.67，P<0.01）、Caspase3（t= 26.38，P=0.001；t=26.75，P<0.01）、Bcl-2（t=28.26，P<0.01；t= 28.32，P<0.01）随YNH菌株作用浓度增加相对表达量下降，差异具有统计学意义。与对照组相比，MOI10000:1组的Caspase8（t=17.04；P<0.01）、Caspase9（t=23.90，P<0.01）相对表达量下降，差异具有统计学意义。与对照组相比，MOI1000:1组、MOI10000:1组的Cleave-parp（t=-3.02、P=0.043；t=44.27、P<0.01）、Cleave-caspase8（t=-8.44、P=0.012；t=-11.66、P=0.005）、Cleave-caspase3（t=-15.14、P<0.01；t=-15.43、P=0.002）、Bax（t=-15.41、P<0.01；t=-21.55、P=0.001）随YNH菌株作用浓度增加，相对表达量增加，差异具有统计学意义。与对照组相比，MOI10000:1组的Cleave-caspase9（t=-7.29、P=0.002）相对表达量增加，差异具有统计学意义。以上蛋白结果表明YNH菌株促进Hela细胞亡(具体参见图10中的B)。
试验方法：试验组为与YNH菌株共培养的Hela细胞，对照组为未与YNH菌株共培养的Hela细胞。将YNH菌株与Hela细胞共培养，提取细胞RNA利用PCR实验检测上皮间质转化相关基因E-cadherin在mRNA水平上相较于未共培养Hela细胞的变化。具体PCR步骤参见上述实施例4“YNH菌株上调Hela细胞凋亡基因Caspase3、Caspase8的表达”中的相关步骤。
试验结果：我们发现YNH菌株上调宫颈癌Hela细胞上皮间质转化相关基因E-cadherin的表达(具体参见图11)。
试验用鼠：6-7 周龄雌性 BALB/c 裸鼠，体重(18±2 )g；
小鼠正常饮食喂养1周；后随机分为两组：生理盐水组和YNH组，每组3只。YNH组第二周开始进行乳杆菌（菌液浓度1×10 9/200μL，只，每天）灌胃14天，生理盐水组每日灌服等量的生理盐水，连续14天；
成瘤试验：
将计数为5×10 6Hela细胞/200μl接种于裸鼠右前肢腋下1-2周后，肿瘤体积达130～150mm 3左右进行后续试验，成瘤期间仍然每日进行灌胃，YNH组每日灌服短乳杆菌0 .2ml，生理盐水组每日灌服等量的生理盐水。成瘤后再连续给药14天，观察小鼠的体重、瘤块大小。
试验结果：如图11中的A所示，YNH组和生理盐水组的裸鼠体重均随时间延长体重增加。如图12中的B所示，YNH组和生理盐水组随时间延长，瘤体体积增大，YNH组较生理盐水组的瘤体体积更小。如图12中的C、D所示，仁慈终点处死裸鼠后YNH组瘤体体积较生理盐水组瘤体体积小，差异具有统计学意义。(具体参见图12）。
试验方法：将完成固定的组织从低浓度浸泡到高浓度的酒精中进行脱水，置于二甲苯中替换出组织中的酒精，进行石蜡包埋。将石蜡块切片后置于45℃恒温箱中烘干，二甲苯中浸泡5分钟，由高浓度到低浓度的酒精对组织片进行脱蜡，完成后置于蒸馏水中。将切片置于苏木精水溶液中染色3-4min，盐酸分化液10-20s，PBS返蓝。流水冲洗片子后置于70%和90%的酒精中脱水各10分钟。置于酒精伊红染色液中染色3分钟。染色完成后，用中性树脂进行封片。
试验结果：如图13所示，对两组裸鼠的心脏、肝脏、肾脏进一步做HE染色发现两组均无炎性细胞浸润，也未观察到形态学的变化，说明YNH的使用对裸鼠的心脏、肝脏、肾脏无毒性作用。
实施例14： 乳杆菌YNH菌株治疗宫颈癌的第二次动物实验观察
试验用鼠：6-7周龄雌性 BALB/c 裸鼠，体重(18±2 )g；
正常饮食喂养1周后随机分组；分为4组，第一组为生理盐水灌胃对照组、第二组为顺铂腹腔注射组、第三组为乳杆菌灌胃组、第四组为乳杆菌联合顺铂腹腔注射组；第二周开始生理盐水灌胃对照组、顺铂腹腔注射组灌胃生理盐水14天，乳杆菌灌胃组、乳杆菌联合顺铂腹腔注射组灌胃YNH菌株（菌液浓度1×10 9/200μL，只，每天）灌胃14天；
成瘤试验：
将计数为5×10 6 Hela细胞/200μl接种于裸鼠右前肢腋下1-2周（文献报道多在7天内成瘤，我们预实验的结果有两只小鼠补种瘤后才成功）后时肿瘤体积达130～150mm 3左右进行后续试验。
各组治疗方案：
第一组（生理盐水灌胃对照组）：正常饮食喂养1周；第二周开始进行生理盐水灌胃14天，后进行成瘤实验；成瘤后继续生理盐水灌胃14天；
第二组（顺铂腹腔注射组）：顺铂浓度为2 mg／(kg·d)，用生理盐水溶解，按照每只0.2 ml进行腹腔注射，共注射3次．分别为接种后第7、14、21天；
第三组（乳杆菌灌胃组）：正常饮食喂养1周；第二周开始乳杆菌（菌液浓度1×10 9/200μL，只，每天）灌胃14天，后进行成瘤实验（成瘤实验期间乳杆菌灌胃不停止）；成瘤后继续生理盐水灌胃14天；
第四组（乳杆菌联合顺铂腹腔注射组）：顺铂给药方案同第二组，乳杆菌给药方案同第三组；
观察指标：小鼠饮食、体重、瘤块大小。
试验结果：如图14A、B所示，与生理盐水灌胃对照组相比，顺铂腹腔注射组、乳杆菌灌胃组、乳杆菌联合顺铂腹腔注射组的瘤体体积更小，以乳杆菌联合顺铂腹腔注射组瘤块体积为最小。说明灌胃YNH菌株可以抑制宫颈癌皮下瘤的增殖 (具体参见图14）。
尽管为说明目的公开了本发明的实施例，但是本领域的技术人员可以理解：在不脱离本发明及所附权利要求的精神和范围内，各种替换、变化和修改都是可能的，因此，本发明的范围不局限于实施例所公开的内容。

Claims (10)

  1. 一种短乳杆菌（ Lactobacillus brevis）YNH，其特征在于所述短乳杆菌（ Lactobacillus brevis）YNH可以抑制宫颈癌Hela细胞增殖、促进宫颈癌Hela细胞凋亡，所述短乳杆菌（ Lactobacillus brevis）YNH在中国微生物菌种保藏管理委员会普通微生物中心（CGMCC）的保藏编号为CGMCC No .24368。
  2. 一种菌剂，其特征在于所述菌剂可以抑制宫颈癌Hela细胞增殖、促进宫颈癌Hela细胞凋亡，所述菌剂的活性成分为如权利要求1所述的短乳杆（ Lactobacillus brevis）YNH。
  3. 包含如权利要求1所述的短乳杆菌（ Lactobacillus brevis）YNH的发酵液或发酵液的过滤液。
  4. 包含如权利要求1所述的短乳杆菌（ Lactobacillus brevis）YNH的菌悬液。
  5. 一种培养有如权利要求1所述的短乳杆菌（ Lactobacillus brevis）YNH的培养液。
  6. 如权利要求1所述的短乳杆菌（ Lactobacillus brevis）YNH、权利要求2所述的菌剂、权利要求3所述的发酵液或发酵液的过滤液、权利要求4所述的菌悬液或权利要求5所述的培养液在制备用于治疗或预防宫颈癌的药物中的用途。
  7. 如权利要求1所述的短乳杆菌（ Lactobacillus brevis）YNH、权利要求2所述的菌剂、权利要求3所述的发酵液或发酵液的过滤液、权利要求4所述的菌悬液或权利要求5所述的培养液在制备用于调节和/或改善妇女生殖道微生态平衡的药物或试剂中的用途。
  8. 一种药物组合物，其特征在于所述药物组合物可以抑制宫颈癌Hela细胞增殖、促进宫颈癌Hela细胞凋亡，所述药物组合物包含如权利要求1所述的短乳杆菌（ Lactobacillus brevis）YNH、权利要求2所述的菌剂、权利要求3所述的发酵液或发酵液的过滤液、权利要求4所述的菌悬液或权利要求5所述的培养液中的任一种，以及药学上可接受的载体。
  9. 如权利要求8所述的药物组合物，其特征在于所述药物组合物为益生菌制剂。
  10. 如权利要求8或9所述的药物组合物，其中所述药学上可接受的载体选自由药学上通常使用的矫味剂、润滑剂、填充剂、崩解剂组成的组中的任一种。
PCT/CN2023/114171 2022-09-01 2023-08-22 一株短乳杆菌及其抗宫颈癌应用 WO2024046168A1 (zh)

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