CN114404455B - Application of bacteroides fragilis and zwitterionic capsular polysaccharide thereof in preparation of medicines for treating respiratory system tumors - Google Patents

Application of bacteroides fragilis and zwitterionic capsular polysaccharide thereof in preparation of medicines for treating respiratory system tumors Download PDF

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CN114404455B
CN114404455B CN202210034079.4A CN202210034079A CN114404455B CN 114404455 B CN114404455 B CN 114404455B CN 202210034079 A CN202210034079 A CN 202210034079A CN 114404455 B CN114404455 B CN 114404455B
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bacteroides fragilis
capsular polysaccharide
tumor
zwitterionic
use according
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CN114404455A (en
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王薇
蔡琰
郑丽君
梁德宝
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Guangzhou Zhiyi Biotechnology Co Ltd
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Guangzhou Zhiyi Biotechnology Co Ltd
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Abstract

The invention discloses application of bacteroides fragilis and/or zwitterionic capsular polysaccharide thereof in preparing medicaments for treating respiratory system tumors, and particularly relates to bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685 and zwitterionic capsular polysaccharide thereof, which can inhibit the expression of tumor promoting factors IL-1 beta by increasing the level of antitumor factors IL-12 and IFN-gamma, promote the up-regulation of the proportion of infiltrated CD8+CD45+T cells in tumors, regulate the microenvironment of tumor tissues, reduce the weight of in-situ tumors, inhibit the number of tumor metastases, effectively prevent and treat respiratory system tumors, be singly used for cancer treatment, be combined with other microorganism, operation, radiotherapy, chemotherapy and other treatment means, obviously improve the comprehensive curative effect, relieve the injury of radiotherapy and chemotherapy to organisms, effectively prevent the occurrence and development of respiratory system tumors and recurrence and metastasis of the tumors and improve the life quality of patients.

Description

Application of bacteroides fragilis and zwitterionic capsular polysaccharide thereof in preparation of medicines for treating respiratory system tumors
The microbial strain used in the implementation process of the invention is preserved in China general microbiological culture Collection center (CGMCC) (No. 3 of North Chen West Lu 1 of the Korean area of Beijing city) in China general microbiological culture Collection center (CGMCC) of 4 months and 2 days of 2015. Classification naming: bacteroides fragilis ZY-312 (bacteroides fragilis ZY-312) with the preservation number of CGMCC No.10685. Bacteroides fragilis ZY-312 was isolated by the applicant's entity and has been under the authority of the patent protection (patent No. 2015175508. X), and under the provisions of the patent prosecution guidelines, the public was able to buy from commercial sources or has been granted without preservation, i.e. without providing proof of preservation.
Technical Field
The invention relates to the field of biological medicine, in particular to application of bacteroides fragilis and/or zwitterionic capsular polysaccharide thereof in preparation of medicines for treating respiratory system tumors.
Background
Respiratory neoplasms refer to any disease characterized by anatomical localization of malignant cells in the respiratory system, including lung cancer, nasopharyngeal cancer, laryngeal cancer, and the like. Among them, lung cancer is the representative of respiratory system tumor, 180 tens of thousands of lung cancer deaths worldwide in 2020, far exceeding other cancer types, the first number of potential cancer deaths. From a pathological and therapeutic perspective, lung cancer can be broadly divided into two major categories, non-small cell lung cancer (non small celllung cancer, NSCLC) and small cell lung cancer (small celllung cancer, SCLC), wherein non-small cell lung cancer accounts for about 80% -85% and the remainder small cell lung cancer. According to the histological typing standard of lung cancer released by the World Health Organization (WHO) in 2015, the major tissue types of lung cancer are squamous cell carcinoma and adenocarcinoma, accounting for about 80% of all primary lung cancers. Other rare types of primary lung cancer include: adenosquamous carcinoma, large cell carcinoma, neuroendocrine carcinoma (carcinoid, atypical carcinoid, and small cell carcinoma), and the like. Wherein SCLC is classified into a limited period and a wide period according to the advice in each diagnosis and treatment guideline due to the unique biological behavior.
Numerous epidemiological studies have shown that the major risk factors for lung cancer development include: smoking and passive smoking, pollution by indoor fuel and cooking fumes, indoor radon exposure, outdoor air pollution, and genetic factors are all related. Smoking is currently recognized as the most important risk factor for lung cancer. Meanwhile, as the industrialized development of China causes increasingly serious air pollution, fine particulate matters (PM 2.5) in the air increase the death risk of lung cancer. Familial aggregation also occurs in lung cancer patients. These illustrative genetic factors may play an important role in people and/or individuals susceptible to environmental carcinogens.
Nasopharyngeal carcinoma is the most common malignancy of the head and neck, a malignancy that occurs from the epithelium of the nasopharyngeal cavity surface or nasopharyngeal crypt. Nasopharyngeal carcinoma is less common than other cancers, and according to IARC investigation, about 12.9 ten thousand new cases of nasopharyngeal carcinoma are observed in 2020, accounting for 0.7% of all cancers. The histological classification of nasopharyngeal carcinoma adopts WHO classification (2017 edition) standard, and is classified into keratinized squamous cell carcinoma, non-keratinized squamous cell carcinoma, basal cell-like squamous cell carcinoma, and other types of nasopharyngeal carcinoma. The occurrence of nasopharyngeal carcinoma is generally related to the combined action of a number of factors, of which the more definite is: genetic factors, EB virus infection, environmental factors (such as aromatic hydrocarbon, nitrosamine, nickel, etc.).
Current treatment methods for respiratory tumors include surgery, radiation therapy, chemotherapy, molecular targeted therapy, immunotherapy, and the like.
In the treatment of NSCLC, anatomical pneumonectomy supplemented with a platinum-containing double-dose regimen is the primary treatment for early-to-mid lung cancer, and is also an important method for clinically curing lung cancer at present. Most non-small cell lung cancers have been metastasized locally advanced or distant at the time of treatment, cannot be surgically resected, and use a combination of radiotherapy and chemotherapy, the chemotherapy regimen comprising Etoposide+cisplatin (EP) or carboplatin (EC), pemetrexed+cisplatin or carboplatin, paclitaxel or docetaxel+platins. Because SCLC has high malignancy and easy metastasis, but is sensitive to chemotherapy and radiotherapy, the comprehensive treatment of combining chemotherapy, radiotherapy and prophylactic brain radiotherapy is mainly adopted. Due to lack of clinical symptoms and effective screening procedures, most lung cancers are diagnosed as advanced, and the likelihood of local recurrence of NSCLC after resection remains high. Emerging targeted therapies and immunotherapies are mainly used for metastatic and recurrent tumors. In the treatment of NSCLC, for example, EGFR gene mutation in patients is used in the first line drug treatment of advanced NSCLC, and EGFR tyrosine kinase inhibitors including gefitinib, erlotinib, etc. can be selected. Patients with advanced NSCLC who are driving gene negative can select the PD-1 inhibitor, na Wu Liyou mab (Nivolumab) which was just marketed in bulk. First line treatment of extensive SCLC has incorporated immunotherapy such as the Abelimumab+EC regimen, apoptosis-ligand 1 (PD-L1) mab kovalli You Shan anti-combination regimen, and the like.
Currently, the accepted and effective radical treatment means of nasopharyngeal carcinoma are radiotherapy, and the combination of radiotherapy and platinum-based drug chemotherapy is a key for treating locally advanced nasopharyngeal carcinoma. With the wide application of intensity modulated radiotherapy and the like in nasopharyngeal carcinoma treatment, the local control rate and the total survival rate of the nasopharyngeal carcinoma are obviously improved, but the remote metastasis is the most main failure mode in the nasopharyngeal carcinoma treatment. Nasopharyngeal carcinoma can be transferred to multiple organs of the whole body through blood circulation, and common transfer sites are bones, lungs and liver. Generally, the weight of the drug is about 10% of the primary patients, and the patients who are not found to transfer during primary treatment still can transfer far after about 15% of the patients. For the treatment of recurrent or metastatic nasopharyngeal carcinoma, chemotherapy, immunotherapy, targeted therapy, etc. are often selected. If EGFR and VEGFR of nasopharyngeal carcinoma tissues are positive, EGFR monoclonal antibodies (cetuximab or Nituzumab), VEGFR monoclonal antibodies (bevacizumab), tyrosine kinase inhibitors (apatinib, an Luoti ni and the like) and recombinant human vascular endothelial inhibin and the like can be used for targeted treatment. Immunotherapy also plays an important role, such as carlizumab+gp (gemcitabine+cisplatin), and terlipressin Li Shan in rescue treatment regimens.
Targeting therapy and immunotherapy are emerging hot therapeutic methods, play an important role in the treatment of metastatic and recurrent respiratory tumors, but adverse reactions of the therapeutic methods cannot be ignored, and the therapeutic methods can occur in various systems such as skin, neuroendocrine, gastrointestinal tract, liver, lung, heart, kidney and the like, and serious adverse reactions such as immune enteritis, pneumonia, hepatitis, myocarditis and the like can also exist. Therefore, the further improvement of the combined treatment effect and the reduction of toxicity are research directions which have breakthrough prospects compared with the targeted treatment.
The important literature published in the Nature journal in 2017 proposes "intestinal-pulmonary axis microecology regulation", which proves that intestinal flora is closely related to tumors, however, no microecological product for treating respiratory tumors exists in the prior art.
Bacteroides fragilis (Bacteroides fragilis, b. Fragilis) is a gram-negative, rod-shaped, blunt-ended and dense-stained, capsular, non-spore, unpowered, obligate anaerobic bacterium, classified into Enterotoxigenic (ETBF) and non-enterotoxigenic (NTBF), which are part of the normal flora of the human and animal intestinal tract, mainly present in the colon, and the mucous membranes of the respiratory, gastrointestinal and genitourinary tracts can also colonize. Researches show that the non-enterotoxigenic bacteroides fragilis (NTBF) has important probiotic effect. The cytokine IL-10, which regulates T cell expansion and produces a cell that blocks the development of pathogenic Th17 cells, has an anti-inflammatory effect and is considered to be a potential new generation of probiotics. Several studies have shown that NTBF can secrete anti-inflammatory cytokine IL-10, promote Th1/Th2 cell balance, resist intestinal inflammation, and have therapeutic effects on colitis induced by dextran sodium sulfate DSS. The relationship of bacteroides fragilis to the host is largely dependent on its highly complex and dynamic capsular structure, b.fragilis zwitterionic capsular polysaccharide (capsular polysaccharide, CPS) is the first recognized commensal factor to regulate the development of the host immune system, reversing morphological, cellular and functional defects in sterile animals. Development of the application direction of the bacteroides fragilis or the extract zwitterionic capsular polysaccharide thereof for the medicaments for treating respiratory system tumors has prospect and is necessary.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide the bacteroides fragilis and the application of the zwitterionic capsular polysaccharide thereof in treating respiratory system tumors. A large number of experiments prove that the bacteroides fragilis, in particular to bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685 and zwitterionic capsular polysaccharide thereof, in particular to capsular polysaccharide PSA, can inhibit the expression of tumor promoting factors IL-1 beta by increasing the levels of anti-tumor factors IL-12 and IFN-gamma. The proportion of CD8+CD45+T cells which promote infiltration in the tumor is up-regulated, and the microenvironment of the tumor tissue is regulated. Can effectively inhibit the growth of lung transplanted tumor in mice, reduce the weight of in-situ tumor, inhibit tumor metastasis, and effectively prevent and treat respiratory system tumor.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, an application of bacteroides fragilis and/or zwitterionic capsular polysaccharide thereof in preparing a product for preventing and/or treating respiratory system tumors is provided, wherein the bacteroides fragilis is bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685.
In some embodiments, the bacteroides fragilis is one or more of a live bacterium, an inactivated bacterium with intact morphological structure, or an inactivated bacterium with incomplete morphological structure.
In some embodiments, the bacteroides fragilis is one or more of live bacteroides fragilis, inactivated, genetically recombined, engineered or modified, attenuated, chemically treated, physically treated or inactivated bacteroides fragilis, bacteroides fragilis lysate, bacteroides fragilis liquid culture supernatant.
In some of these embodiments, the respiratory tumor comprises head and neck squamous carcinoma, non-small cell lung carcinoma, small cell lung carcinoma.
In some of these embodiments, the head and neck squamous carcinoma includes nasopharyngeal carcinoma, laryngeal carcinoma.
In some of these embodiments, the zwitterionic capsular polysaccharide comprises capsular polysaccharide a. Wherein the structure of the capsular polysaccharide A is as follows:
according to the invention, the capsular polysaccharide A has a weight average molecular weight of 80-90kD, wherein the fraction having a molecular weight distribution of 70-100 kD accounts for 70-80% of the total amount.
In some of these embodiments, the capsular polysaccharide a is present in an amount in excess of 95wt%.
In some of these embodiments, the method of preparing the zwitterionic capsular polysaccharide comprises the steps of:
(1) Centrifuging the fermented and cultured bacteroides fragilis bacterial liquid to collect sediment, thus obtaining bacteroides fragilis bacterial mud; adding purified water with the mass 3-10 times of that of the bacterial sludge into the bacterial sludge to suspend the bacterial sludge again, adjusting the pH of the bacterial sludge to 2.0-4.5 by using an acid solution, extracting for 0.5-3.0 h at 50-120 ℃, cooling to room temperature, centrifuging at normal temperature, and taking the supernatant to obtain a crude sugar solution;
(2) Concentrating the crude sugar solution by ultrafiltration membrane ultrafiltration to remove small molecular impurities until the conductivity is stable, and collecting reflux liquid;
(3) Adding 40mmol/L Tris-HCl salt in equal volume into the reflux liquid; ion exchange column chromatography, gradient elution, sectional collection, SEC-HPLC tracking and monitoring, combining components with single and symmetrical peaks of 206nm absorption peak, ultrafiltering with ultrafiltration membrane, adding purified water, repeatedly ultrafiltering until the conductivity is stable, collecting reflux liquid, and lyophilizing to obtain Bacteroides fragilis zwitterionic capsular polysaccharide.
In some of these embodiments, the centrifugation in step (1) is 11000 to 13000g centrifugation for 8 to 12 minutes.
In some of these embodiments, the acid solution in step (1) may be one or more of an organic acid, an inorganic acid, and an acidic buffer. Wherein the inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.; the organic acid may be acetic acid, citric acid, etc.
In some of these embodiments, the ultrafiltration membrane in step (2) can be 100, 50, 30, 10, 5, 3KD, or a range between any two molecular weight values.
In some of these embodiments, the ion exchange column in step (3) is preferably a column of DEAE Sepharose Fast Flow, 16mm×200mm, and the flow rate during chromatography is 15-25 mL/min, pH 5.0-9.0 contains 0.2mol/L NaCl 20mmol/L Tris-HCl gradient elution 25 column volumes, and the column volumes are collected in stages, 100 mL/bottle (component); the ultrafiltration membrane is 10KD.
In some embodiments, the product is a food or pharmaceutical product.
In some embodiments, the pharmaceutical product is bacteroides fragilis or its zwitterionic capsular polysaccharide alone, or in combination with its zwitterionic capsular polysaccharide, or in combination with other drugs, or in combination with its zwitterionic capsular polysaccharide, respectively.
In some embodiments, the dosage form of the pharmaceutical product comprises a pill, tablet, granule, capsule, oral liquid, or tube feeding formulation. The medicine comprises human medicine or animal medicine.
In some of these embodiments, the food product comprises milk powder, cheese, curd, yogurt, ice cream, or a fermented cereal. The food product may also be an animal food product, such as a feed or the like.
In a second aspect, a composition for treating respiratory system tumors is provided, wherein the composition contains a pharmaceutically effective dose of bacteroides fragilis and/or zwitterionic capsular polysaccharides thereof with the preservation number of CGMCC No. 10685.
In some embodiments, the pharmaceutically effective dose of bacteroides fragilis is 10 6 -10 10 CFU。
In some of these embodiments, the pharmaceutically effective dose of the zwitterionic capsular polysaccharide is 1-30mg/kg.
In some embodiments, the bacteroides fragilis is one or more of a live bacterium, an inactivated bacterium with intact morphological structure, and an inactivated bacterium with incomplete morphological structure.
In some embodiments, the bacteroides fragilis is one or more of live bacteroides fragilis, inactivated, genetically recombined, engineered or modified, attenuated, chemically treated, physically treated or inactivated bacteroides fragilis, bacteroides fragilis lysate, bacteroides fragilis liquid culture supernatant.
In some of these embodiments, the zwitterionic capsular polysaccharide comprises capsular polysaccharide a.
In some of these embodiments, the zwitterionic capsular polysaccharide is taken from the bacteroides fragilis ZY-312.
In some of these embodiments, the capsular polysaccharide a has the structure shown below:
according to the invention, the capsular polysaccharide A has a weight average molecular weight of 80-90kD, wherein the fraction having a molecular weight distribution of 70-100 kD accounts for 70-80% of the total amount.
In some of these embodiments, the capsular polysaccharide a is present in an amount in excess of 95wt%.
In some of these embodiments, the method of preparing the zwitterionic capsular polysaccharide comprises the steps of:
(1) Centrifuging the fermented and cultured bacteroides fragilis bacterial liquid to collect sediment, thus obtaining bacteroides fragilis bacterial mud; adding purified water with the mass 3-10 times of that of the bacterial sludge into the bacterial sludge to suspend the bacterial sludge again, adjusting the pH of the bacterial sludge to 2.0-4.5 by using an acid solution, extracting for 0.5-3.0 h at 50-120 ℃, cooling to room temperature, centrifuging at normal temperature, and taking the supernatant to obtain a crude sugar solution;
(2) Concentrating the crude sugar solution by ultrafiltration membrane ultrafiltration to remove small molecular impurities until the conductivity is stable, and collecting reflux liquid;
(3) Adding 40mmol/L Tris-HCl salt in equal volume into the reflux liquid; ion exchange column chromatography, gradient elution, sectional collection, SEC-HPLC tracking and monitoring, combining components with single and symmetrical peaks of 206nm absorption peak, ultrafiltering with ultrafiltration membrane, adding purified water, repeatedly ultrafiltering until the conductivity is stable, collecting reflux liquid, and lyophilizing to obtain Bacteroides fragilis zwitterionic capsular polysaccharide.
In some of these embodiments, the centrifugation in step (1) is 11000 to 13000g centrifugation for 8 to 12 minutes.
In some of these embodiments, the acid solution in step (1) may be one or more of an organic acid, an inorganic acid, and an acidic buffer. Wherein the inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.; the organic acid may be acetic acid, citric acid, etc.
In some of these embodiments, the ultrafiltration membrane in step (2) can be 100, 50, 30, 10, 5, 3KD, or a range between any two molecular weight values.
In some of these embodiments, the ion exchange column in step (3) is preferably a column of DEAE Sepharose Fast Flow, 16mm×200mm, and the flow rate during chromatography is 15-25 mL/min, pH 5.0-9.0 contains 0.2mol/L NaCl 20mmol/L Tris-HCl gradient elution 25 column volumes, and the column volumes are collected in stages, 100 mL/bottle (component); the ultrafiltration membrane is 10KD.
In some embodiments, the composition is a probiotic composition, a nutraceutical composition, or a pharmaceutical composition.
The invention relates to a probiotic composition for preventing and treating respiratory system tumors, wherein the probiotic composition contains bacteroides fragilis ZY-312 with a preservation number of CGMCC No.10685 and/or zwitterionic capsular polysaccharide extracted from the bacteroides fragilis ZY-312. The probiotic composition may further comprise one or more of probiotics or microorganisms from the genus Saccharomyces (Saccharomyces spp.), the genus Lactobacillus (Lactobacillus spp.), and the normal flora of the human intestinal tract. The probiotics of the genus Saccharomyces may include Saccharomyces boulardii (Saccharomyces boulardii) and/or Saccharomyces cerevisiae (Saccharomyces cerevisiae).
The pharmaceutical composition for preventing and treating respiratory system tumors contains bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685 and/or zwitterionic capsular polysaccharide extracted from ZY-312. The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The pharmaceutical composition can be in the form of pill, tablet, granule, capsule, powder, suspension, oral liquid or enema. The administration may be by oral, enema or parenteral forms, and the administration period may be intermittent, periodic, continuous or long-term.
The health-care product composition for preventing and treating respiratory system tumors contains bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685 and/or zwitterionic capsular polysaccharide extracted from ZY-312.
The invention has the beneficial effects that:
a large number of experiments prove that the bacteroides fragilis, in particular to bacteroides fragilis ZY-312 with the preservation number of CGMCC No.10685And zwitterionic capsular polysaccharides thereof, particularly capsular polysaccharide A (PSA), which are capable of promoting macrophage-induced apoptosis of non-small cell lung cancer cells in vitro; the expression of the tumor promoting factor IL-1 beta can be inhibited by increasing the level of the anti-tumor factors IL-12 and IFN-gamma in vivo. CD8 promoting infiltration in tumors + CD45 + The proportion of T cells is up-regulated, and the microenvironment of tumor tissues is regulated. The bacteroides fragilis and the PSA thereof can reduce the weight of transplanted tumors in established mouse non-small cell lung cancer and small cell lung cancer, and can effectively inhibit the growth of lung transplanted tumors in mice. Meanwhile, the bacteroides fragilis can reduce the number of tumor metastasis in the constructed nude mouse nasopharyngeal carcinoma metastasis model, effectively prevent occurrence and development of respiratory system tumors and recurrence and metastasis thereof, and improve the life quality of patients.
The bacteroides fragilis ZY-312 adopted by the invention does not contain BFT genes, is a non-toxigenic strain, and has acute toxicity proved that the strain has no pathogenicity to normal mice and nude mice (Wang Y, deng H, li Z, tan Y, han Y, wang X, du Z, liu Y, yang R, bai Y, bi Y, zhi F.safety Evaluation of a Novel Strain of Bacteroides fragilis.front Microbiol.2017Mar17; 8:435.). According to patent zl2015175508. X and scientific literature Xu W, su P, zheng L, fan H, wang Y, liu Y, lin Y, zhi f.in vivo Imaging of a Novel Strain of Bacteroides fragilis via Metabolic labeling. Front microbiol.2018oct 1;9:2298. The strain has better tolerance to gastric acid and bile salts, and can ensure survival and effective colonization in intestinal tracts.
Drawings
FIG. 1 is a graph showing colony characteristics of Bacteroides fragilis ZY-312 in example 1 of the present invention;
FIG. 2 is a view of the gram-stained Bacteroides fragilis ZY-312 according to example 1 of the present invention;
FIG. 3 is a chart of the analysis of the capsular polysaccharide A nuclear magnetic resonance spectrometer of example 3 of the present invention;
A-E are respectively 1H spectrum, 13C spectrum, COSY spectrum, HSQC spectrum and HMBC spectrum analyzed by the capsular polysaccharide A nuclear magnetic resonance spectrometer in the embodiment 3;
FIG. 4 shows the chemical structural formula of the structural unit of the Bacteroides fragilis capsular polysaccharide A prepared in example 3 of the invention;
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples were all commercially available. All cells were purchased from ATCC; all cell culture materials and pancreatin were purchased from Gibco; all experimental animals were purchased from Zhejiang Veitz laboratory animal technologies Co., ltd; or may be prepared by known methods. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
Example 1: fermentation culture of bacteroides fragilis
The bacteroides fragilis ZY-312 strain is streaked and inoculated on a blood plate for anaerobic culture for 48 hours. Colony morphology, staining characteristics, size, sphere shape, distribution, etc. were observed. Colony characteristics: after the bacteroides fragilis ZY-312 is cultured on a blood plate for 48 hours, the bacteroides fragilis ZY-312 is slightly convex, semitransparent, white, smooth in surface and free from hemolysis, and the colony diameter is between 1 and 3mm, as shown in figure 1.
Morphology under microscope: the bacteroides fragilis ZY-312 was subjected to gram-stain microscopic examination to show a typical rod shape for gram-negative bacteria, and was rounded at both ends to be densely stained, and the non-colored part in the middle of the thallus was formed as a cavitation, see FIG. 2.
EXAMPLE 2 preparation of live and inactivated Bacteroides fragilis liquid
1) Preparation of living bacteria liquid
Inoculating the single colony cultured in the example 1 into a plant source peptone liquid culture medium for fermentation culture for 8 hours (the temperature is 37 ℃), and obtaining a live bacterial liquid of bacteroides fragilis; and (3) centrifuging the obtained bacterial liquid to precipitate at the rotating speed of 3000r/min, centrifuging for 15min, removing the supernatant, and collecting the precipitate to obtain the bacteroides fragilis ZY-312 bacterial sludge.
2) Preparation of inactivated bacterial liquid
Taking the bacterial liquid, and carrying out conventional heat inactivation treatment to obtain the Bacteroides fragilis ZY-312 inactivated bacterial liquid.
Example 3: preparation of Bacteroides fragilis capsular polysaccharide
Experiments were performed using the bacterial sludge prepared in example 1.
(1) Taking 50g of bacterial sludge, adding 300g of purified water to enable the bacterial sludge to be re-suspended, adjusting the pH of the bacterial sludge to 3.5 by using 1mol/L hydrochloric acid solution, extracting for 1.5h at 100 ℃, cooling to room temperature, centrifuging for 10min at 12000g of room temperature, and taking the supernatant to obtain a crude sugar solution;
(2) Ultrafiltering and concentrating the crude sugar solution with 10KD ultrafilter membrane to remove small molecular impurities until the conductivity is stable, and collecting the reflux;
(3) Adding an equal volume of 40mmol/L Tris-HCl (pH 8.5) salt to the reflux liquid; DEAE Sepharose Fast Flow ion exchange column chromatography (16 mm. Times.200 mm), gradient eluting with 20mL/min flow rate, 20mmol/L Tris-HCl (pH 8.5, containing 0.2mol/L NaCl) for 25 column volumes, collecting in sections, collecting 100 mL/bottle (component), tracking and monitoring by SEC-HPLC, combining components with 206nm absorption peak as single and symmetrical peak, ultrafiltering with 10KD ultrafiltration membrane, adding purified water, repeatedly ultrafiltering until conductivity is stable, collecting reflux liquid, and lyophilizing to obtain Bacteroides fragilis extract;
(4) Weighing 30mg of the Bacteroides fragilis extract in step (3), and dissolving in 0.5mL D 2 O, 1. Mu.l of acetone (1H, 2.22;13C, 30.89) was added for calibration. Analysis of the 1H and 13C, COSY, HSQC, HMBC spectra (see FIGS. 3A-E) using a 500MHz Bruker NMR spectrometer confirmed that the Bacteroides fragilis extract collected in step (3) was capsular polysaccharide A, the bound lipid content was less than 0.02%, the protein residue was less than 1%, and the nucleic acid residue was less than 0.05%. The capsular polysaccharide A obtained by GPC (gel permeation chromatography) analysis has weight averageThe molecular weight is 80-90kDa, mw/Mn is 1.0-1.2, and the chemical structure is shown in figure 4.
Example 4: drug effect experiment of in vitro promotion of apoptosis of Lewis cell strain of mouse lung cancer cell line by macrophage by Bacteroides fragilis and zwitterionic capsular polysaccharide thereof
1. Materials and Experimental design (grouping)
In this example, bacterial cells were harvested at 24h by adding low, medium and high doses of live bacterial liquid of Bacteroides fragilis ZY-312 to the mouse macrophage cell line RAW264.7, incubating the bacterial liquid with high doses of inactivated bacteria of ZY-312 and low, medium and high doses of PSA of ZY-312, and detecting apoptosis of the tumor cells using a flow cytometer (Beckman Coulter) using a Transwell co-culture chamber (Corning, 3412). PBS was used as a control. The inactivated bacterial liquid of ZY-312 is prepared by a method of example 2, and the PSA is prepared by a method of example 3, as follows.
2. Culture method
(1) Resuscitating and passaging of mouse Lewis cells and RAW264.7 cells
Both Lewis cells and RAW264.7 cells of the lung cancer cells of the mice were grown in DMEM medium containing 10% FBS, 1% penicillin/streptomycin was added, and 5% CO was added at 37 ℃ 2 Culturing in an incubator with concentration and saturated humidity. The medium was changed every two days.
a) The cell lines were removed from the liquid nitrogen and rapidly thawed in a 37℃thermostat water bath. The freezing tube was opened under sterile conditions, the liquid was transferred to a 15mL centrifuge tube, the cells were resuspended in 2-3mL of DMEM complete medium, and centrifuged at 1000rpm for 5min. After discarding the supernatant, 5mL of DMEM complete medium was added, and the cells were inoculated into a flask at 37℃in 5% CO 2 The culture was performed in a concentration incubator for 48 hours.
b) The stock culture was discarded, 1mL of 0.25% trypsin was added, digested for 5min, 1mL of DMEM medium containing 10% fetal bovine serum was added to stop the reaction, and the mixture was transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 5 min; removing supernatant, adding appropriate DMEM complete medium containing 10% foetal calf serum, tapping cells into culture flask, and extracting with 5% CO at 37deg.C 2 The culture was performed in a concentration incubator for 24 hours.
(2) Bacteroides fragilis bacterial liquid culture
a) Taking Bacteroides fragilis ZY-312 strain, adding 200 mu L of TSB culture medium, re-dissolving, drawing a plate, and carrying out anaerobic culture for 48h at 37 ℃.
b) Single colonies were picked and inoculated with 10mL of TSB medium, 5% (v/v) serum was added, and anaerobic culture was performed at 37℃for 12h.
c) Taking 1 bottle of 500mL TSB culture medium, adding 5% (v/v) serum, inoculating 1% (v/v) b) cultured bacteroides fragilis, and performing anaerobic culture at 37 ℃ for 48 hours.
d) Taking the bacterial liquid, and carrying out conventional heat inactivation treatment to obtain an inactivated bacterial liquid.
e) A PSA for ZY-312 was prepared as in example 3.
3. Effect of Bacteroides fragilis on Lewis apoptosis by RAW264.7 macrophages
(1) mu.L of RAW264.7 cells (10 5 ) Respectively inoculating into 6-well Transwell plate lower chamber, respectively adding 10 μL 10 6 、10 8 、10 10 CFU/mL of Bacteroides fragilis bacterial solution ZY-312, ZY-312 inactivated bacterial solution (10) 10 cell/mL) and 0.02, 0.2, 2mg/mL of ZY-312, were pretreated for 6h, and then added to the upper chamber, respectively, where Lewis cells were seeded. Wells with only equal amounts of RAW264.7 cells added were set as negative controls, wells with equal amounts of tumor cells and cisplatin (DDP) (15 μm) (shandong zilu pharmaceutical limited) were set as positive control, 3 wells per group. At 37℃temperature, 5% CO 2 Culturing in an incubator.
(2) The apoptosis of Lewis cells was detected using an Annexin V apoptosis detection kit (Northenan, A211-01) at 24h collection:
a) Cells were digested with pancreatin without EDTA, harvested after termination of digestion, centrifuged at 1000rpm at 4℃for 5min and the supernatant discarded.
b) Washing the cells: the cells were washed twice with pre-chilled PBS, centrifuged at 1000rpm at 4℃for 5min each, and the supernatant was discarded.
c) Cell resuspension: mu.l of 1 Xbinding Buffer was added and gently swirled to a single cell suspension.
d) Cell staining: mu.l Annexin V-FITC and 5 mu l PI Staining Solution were added and gently swirled; incubating for 10min at room temperature (20-25 ℃) in the dark; 400 μl of 1 Xbinding Buffer was added and gently mixed. The stained samples were examined with a flow cytometer over 1 hour.
e) Apoptosis conditions: calculating the sum of early apoptotic cells of Annexin V-FITC single positive (Annexin V-FITC plus/PI minus) and late apoptotic cell proportion of Annexin V-FITC and PI double positive (Annexin V-FITC plus/PI plus).
4. Experimental results:
TABLE 1 Lewis apoptosis rate (%) (mean.+ -. Standard deviation, n=3)
Note that: compared to the positive control group, x represents a significant difference p <0.05; * Represents a very significant difference p <0.01.
As shown in table 1, the rate of apoptosis was significantly lower in the negative group (RAW 264.7 cells alone added) than in the positive control. The Bacteroides fragilis ZY-312 and the ZY-312 inactivated bacterial liquid and the ZY-312PSA can promote Lewis apoptosis through RAW264.7 cells. The difference in the Bacteroides fragilis ZY-312 group is not obvious, whether the bacterium is a live bacterium, an inactivated bacterium or a PSA group, and the Lewis apoptosis situation has statistical difference compared with the positive control group.
Therefore, bacteroides fragilis ZY-312, ZY-312 inactivated bacteria and ZY-312PSA can promote Lewis apoptosis through RAW 264.7.
Example 5: bacteroides fragilis and drug effect test of zwitterionic capsular polysaccharide for treating mouse non-small cell lung cancer transplantation tumor
Regulating the cell concentration of Lewis cell of lung cancer cell line of mouse in logarithmic growth phase to 2×10 4 Lewis lung carcinoma-derived mice were prepared by inoculating Lewis cells subcutaneously in the right armpit of SPF-grade C57BL/6 mice with a single cell suspension at 0.2 mL/mL using a syringe under aseptic conditions. Repeating the process once on the 11 th day, and in the 21 st day formal experiment, selecting a Lewis lung cancer tissue with good growth, removing cervical vertebra and killing under a sterile condition, peeling tumor tissue from the armpit, shearing, homogenizing and grinding, and obtaining the tumor tissue according to the mass (g) of the tumor and physiological saline (mL) 1:3 proportion to make the concentration 1X 10 7 individual/mL of tumor cell suspension. The Lewis lung cancer tumor source mouse cell suspension is inoculated into the right forelimb armpit of the mouse at about 0.2mL for molding. One week after molding, ZY-312 was low (10 6 CFU/only), high (10 10 CFU/dose group, ZY-312 inactivated bacteria (10) 10 cell/patient), ZY-312PSA low (10 mg/kg) and ZY-213PSA high (30 mg/kg) for 3 weeks of continuous gavage; the DDP (cisplatin, shandong Qilu pharmaceutical Co., ltd.) group was injected intraperitoneally 1 time per week for 5 weeks. When the experiment is finished or the tumor-bearing mice have obvious wasting signs such as emaciation, bow back, listlessness and the like, cervical vertebra is removed, the transplanted tumor is taken to measure the weight of the transplanted tumor, the tumor inhibition rate is calculated, fresh tumor body tissues are taken to detect the tumor microenvironment condition, and serum is taken to be stored in a refrigerator at the temperature of minus 80 ℃ for detecting cytokines and the like.
1. Lewis cell culture
Lewis cells were cultured in DMEM medium containing 10% fetal bovine serum at 37℃under a gaseous atmosphere of 5% CO 2 The humidity is saturated humidity; the culture medium was changed according to the growth rate of cells and the color change of the culture medium, and passaging was digested with 0.25% trypsin. According to the growth condition of cells, preparing cells in logarithmic growth phase into single cell suspension, and regulating cell concentration to 2×10 4 And each mL.
2. Preparation of Lewis lung cancer tumor source mice
Taking 10 mice with mass of about (20+ -2) g and SPF grade C57BL/6 at 6-8 weeks of age, regulating cell concentration of Lewis cells of mouse lung cancer cell line in logarithmic growth phase to 2×10 4 The Lewis cells were inoculated subcutaneously into the right armpit of the mouse in an amount of 0.2 mL/mL using a syringe under aseptic conditions, and the tumors were excised when the subcutaneous tumors had grown to a diameter of about 1 cm.
3. Establishment of lung cancer transplantation tumor model
Selecting a Lewis lung cancer tissue with good growth, removing cervical vertebra, killing, peeling tumor tissue from armpit, shearing, homogenizing, grinding, and mixing tumor mass (g) with normal saline (mL) 1:3 proportion to make the concentration 1X 10 6 individual/mL of tumor cell suspension. Under aseptic conditions, subjecting the above The prepared cell suspension was inoculated subcutaneously in the right forelimb axilla of mice at about 0.2mL each.
4. Grouping and administration
After 1 week of establishment of the tumor transplantation model, 70 mice were randomly divided into 7 groups of 10: physiological saline group, DDP group, ZY-312 low (10) 6 CFU/only), high (10 10 CFU/dose group, ZY-312 inactivated bacteria group (10) 10 cell/alone), ZY-312PSA low (10 mg/kg) and ZY-213PSA high (30 mg/kg).
Physiological saline group: 0.2 mL/dose, 1 time per day, and continuous gastric lavage for 3 weeks;
DDP group: intraperitoneal injections were performed in an amount of 0.3 mL/dose, 3mg/kg, 1 time per week for 3 weeks;
low and high dose ZY-312 and inactivated bacteria and low and high ZY-312 PSA: the stomach was continuously irrigated for 3 weeks at 0.2 mL/dose, 1 time per day.
4. Detection index and method
(1) Tumor weight and tumor inhibition rate
In the administration process, if the mice have obvious wasting, bow back, listlessness and other signs, and after the administration is finished, cervical vertebra is removed for sacrifice, the weight of the transplanted tumor is measured, the tumor inhibition rate is calculated, and the tumor inhibition rate calculation formula is as follows: tumor inhibition rate (%) = (mean tumor weight in saline group-mean tumor weight in dosing group)/mean tumor weight in saline group x 100;
(2) Intratumoral T cell subpopulations
Freshly collected tumor samples were collected and minced into small pieces, and the minced tissue was transferred to a C-tube containing a digestive enzyme cocktail. After completion of tissue digestion and homogenization, single cell pellet was obtained by filtration through 70 μm sieve, and the cells obtained were counted and then extracted 1X 10 6 Individual living cells were antibody stained. CD45+ (Biolegend, 103151), CD3+CD8+CD45+ (CD 3 antibody: BD, 56344; CD8 antibody: BD, 563786) expression was detected using a flow cytometer.
5. Test results
(1) Tumor weight and tumor inhibition rate
TABLE 2 tumor suppression efficacy calculated based on tumor weight at day 21 after group administration
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Note that:
a. mean ± SD.
b. Tumor suppression ratio (%) = (average tumor weight in saline group-average tumor weight in administration group)/average tumor weight in saline group×100%.
c. The p-value between the two groups is calculated according to the unpaired t-test (two-charged) method.
As can be seen from table 2, the tumor weight was reduced in each of the administration groups compared to the physiological saline group, and the DDP group had a significant difference; bacteroides fragilis and capsular polysaccharide A groups did not have significant differences from the DDP group. It is demonstrated that Bacteroides fragilis ZY-312 and its zwitterionic capsular polysaccharide can effectively inhibit tumor growth in a mouse model of non-small cell lung cancer transplantation tumor, and the inhibition effect is similar to DDP.
(2) Tumor tissue microenvironment T cell subset
TABLE 3 tumor microenvironment T cell subsets (mean+ -SD) for groups of mice
Note that: compared to saline group, x represents significant difference p <0.05; * Represents a very significant difference p <0.01.
Cd8+ T cells play an important role in tumor immunity and are effector cells that directly kill tumor cells.
As can be seen from table 3, the DDP group mice had decreased cd8+cd45+ T cell levels compared to the saline group; the level of infiltrated CD8+CD45+T cells in the tumors of mice in the low-dose, high-dose, low-PSA, high-dose and inactivated bacteria liquid groups of Bacteroides fragilis ZY-312 is obviously increased. This indicates that the infiltration of tumor endolymphocytes is increased after the experimental animals are given bacteroides fragilis ZY-312 and the zwitterionic capsular polysaccharide thereof by lavage.
In conclusion, the bacteroides fragilis ZY-312 and the zwitterionic capsular polysaccharide thereof can regulate the tumor immunity microenvironment of the mice and effectively treat the non-small cell lung cancer transplantation tumor of the mice.
Example 6: bacteroides fragilis and drug effect test of zwitterionic capsular polysaccharide for treating small cell lung cancer transplantation tumor
1. Test design and flow
Culturing human small cell lung cancer cell line NCI-H526, regulating cell concentration of NCI-H526 in logarithmic phase to 2×10 7 Single cell suspensions per mL. Under aseptic conditions, matrigel and PBS were diluted in a 1:1 ratio and placed on ice for injection. NCI-H526 cells with Matrigel gel (BD, 356234) dilution 1:1 proportion to prepare the concentration of 1 multiplied by 10 7 individual/mL of tumor cell suspension. The cell suspension was inoculated at about 0.2mL each into 4-5 week old nude mice with a body weight of 22.+ -.2 g. The right forelimb armpit of the nude mice was molded subcutaneously. One week after molding, ZY-312 was low (10 6 CFU/only), high (10 10 CFU/dose group, ZY-312 inactivated bacteria (10) 10 cell/patient), ZY-312PSA low (10 mg/kg) and ZY-213PSA high (30 mg/kg) for 3 weeks of continuous gavage; the DDP (cisplatin, shandong Qilu pharmaceutical Co., ltd.) group was injected intraperitoneally 1 time per week for 3 weeks. When the experiment is finished or the tumor-bearing mice have obvious wasting signs, such as emaciation, bow back, listlessness and the like, cervical vertebra is removed, the transplanted tumors are taken to measure the weight of the transplanted tumors, the tumor inhibition rate is calculated, and then the transplanted tumors are frozen in a refrigerator at the temperature of minus 80 ℃ for detecting cytokines and the like.
Detecting the index:
(1) Tumor weight and tumor inhibition rate
Tumor inhibition ratio% = 100% × (mean weight of saline group tumor-mean weight of administration group tumor)/mean weight of saline group tumor.
(2) Cytokines and methods of use
Elisa was used to detect levels of cytokines such as IL-12 (R & D Systems, M1270, supra), IFN-gamma (R & D Systems, MIF00, infra) and IL-1β (R & D Systems, MLB00C, infra) in lung cancer-engrafted tumor mice model tumors.
2. Test results
(1) Tumor weight and tumor inhibition rate
TABLE 4 tumor suppression efficacy calculated based on tumor weight at day 21 after group administration
Note that:
a. mean ± SD.
b. Tumor inhibition rate% = 100× (mean weight of saline group tumor-mean weight of administration group tumor)/mean weight of saline group tumor.
c. The p-value between the two groups is calculated according to the unpaired t-test (two-charged) method.
As is clear from Table 4, the tumor weights were decreased in each of the administration groups compared to the physiological saline group, and there was no significant difference between the DDP group and the Bacteroides fragilis and PSA group. It is demonstrated that Bacteroides fragilis ZY-312 and its PSA can effectively inhibit tumor growth in a mouse model of small cell lung cancer transplantation tumor.
(2) Cytokines and methods of use
Table 5 levels of tumor cytokines (mean+ -SD) in groups of mice
Note that: compared to saline group, x represents significant difference p <0.05; * Represents a very significant difference p <0.01.
IL-12 is derived from activated lymphocytes, and can induce cytotoxic activity of CTL and NK cells and promote secretion of anti-tumor cytokines such as IFN-gamma, TNF-alpha and the like; IL-1β and its receptors have been shown to promote the growth and metastasis of a variety of tumors in transplanted mice and humans.
As is clear from the above table, the IL-12 levels were significantly increased in each of the administration groups compared to the physiological saline group, and the ZY-312 live bacteria, inactivated bacteria and PSA levels were higher than those in the DDP group.
The IFN-gamma level was elevated in each of the dosing groups compared to the saline group; the ZY-312 live bacteria, the inactivated bacterial liquid and the PSA have significance, and the PSA has no obvious dose dependency.
The IL-1β level was significantly reduced in each of the administration groups compared to the physiological saline group; the ZY-312 low, high dose, inactivated bacteria fluid and PSA high dose groups have very significant differences, and the PSA low dose group level is lower than the high dose group. This suggests that Bacteroides fragilis and its PSA up-regulate the levels of anti-tumor factors IL-12 and IFN-gamma, and inhibit the expression of tumor promoting factor IL-1 beta.
In conclusion, the bacteroides fragilis ZY-312 and the PSA thereof can regulate the level of tumor-related immune factors and effectively treat the mouse small cell lung cancer transplantation tumor.
Example 7: bacteroides fragilis and drug effect test of zwitterionic capsular polysaccharide for treating nasopharyngeal carcinoma transplantation and metastasis
1. Test design and flow
Culturing human nasopharyngeal carcinoma cell line 5-8F with RPMI-1640 medium containing fetal bovine serum, and regulating cell concentration of 5-8F cells in logarithmic growth phase to 2×10 under aseptic condition 6 The single cell suspension of each mL is inoculated into female nude mice with the age of 5-7 weeks and the weight of 20-24g at the concentration of about 0.2 mL. One week after tumor inoculation of nude mice, direct injection of 2×10 mice by tail vein 6 Intervention was given in a method of 0.2mL of 5-8F single cell suspension per mL. One week after subcutaneous tumor inoculation, ZY-312 was low (10 6 CFU/only), high (10 10 CFU/dose group, ZY-312 inactivated bacteria (10) 10 cell/patient), ZY-312PSA low (10 mg/kg) and ZY-213PSA high (30 mg/kg) for 3 weeks of continuous gavage; the DDP group (cisplatin, shandong Qilu pharmaceutical Co., ltd.) was intraperitoneally injected 1 time per week for 3 weeks. When the experiment is finished or the tumor-bearing mice have obvious wasting signs, such as emaciation, bow backs, listlessness and the like, cervical vertebra is removed, the transplanted tumors are taken for measuring the weight of the transplanted tumors, the tumor inhibition rate is calculated, liver and lung tissues are taken, a metastasis is observed, and the tumor tissues are placed in a refrigerator at the temperature of minus 80 ℃ for freezing and storing, so that cytokines and the like are detected.
Detecting the index:
(1) Weight of transplanted tumor and tumor inhibition rate
Tumor inhibition ratio% = 100% × (mean weight of saline group tumor-mean weight of administration group tumor)/mean weight of saline group tumor.
(2) Transfer range size and number
After taking out the lung and liver tissue, the lung and liver tissue were fixed in Bouin's solution (5mL+40% formaldehyde 25 mL+75 mL of saturated picric acid) and after two days the lung metastasis was observed, the lung metastasis was white, and the liver tissue was not seen as an obvious metastasis. The size of lung tissue metastasis was observed under an dissecting microscope and the number (graded by size) was recorded simultaneously, and the number of lung metastasis nodules was statistically analyzed for each group of nude mice. According to the method, the diameter of the grade I transfer oven is smaller than 0.15mm, the diameter of the grade II transfer oven is 0.15-1 mm, the diameter of the grade III transfer oven is 1-2 mm, and the diameter of the grade IV transfer oven is larger than 2mm. Total number of metastases = number of stage i metastases + number of stage ii metastases 2+ number of stage iii metastases 3+ number of stage iv metastases 4.
(3) Cytokines and methods of use
Elisa is used for detecting the level of cytokines such as IL-12, IFN-gamma, IL-1 beta and the like in the serum of a lung cancer transplantation tumor mouse model.
2. Experimental results
(1) Weight of transplanted tumor and tumor inhibition rate
TABLE 6 tumor suppression efficacy calculated based on tumor weight at day 21 after group administration
Note that:
a. mean ± SD.
b. Tumor inhibition ratio% = 100% × (mean weight of saline group tumor-mean weight of administration group tumor)/mean weight of saline group tumor.
c. The p-value between the two groups is calculated according to the unpaired t-test (two-charged) method.
As can be seen from table 6, the tumor weight was decreased in each of the administration groups compared to the physiological saline group; the Bacteroides fragilis ZY-312 live bacteria, inactivated bacteria and PSA groups have no obvious difference from the DDP groups. It is demonstrated that Bacteroides fragilis ZY-312 and its PSA can inhibit tumor growth in a mouse model of nasopharyngeal carcinoma, and this inhibition effect is similar to DDP.
(2) Transfer range size and number
TABLE 7 total number of metastases (mean+ -SD) for each group of mice
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Note that: compared to saline group, x represents significant difference p <0.05; * Represents a very significant difference p <0.01.
As can be seen from table 7, the number of tumor metastases was reduced in each of the administration groups compared to the physiological saline group; the DDP group, the Bacteroides fragilis ZY-312 high dose group, the inactivated bacterium group and the PSA high dose group have significant differences. It is demonstrated that Bacteroides fragilis ZY-312 and its PSA can effectively inhibit tumor metastasis in a mouse nasopharyngeal carcinoma metastasis model.
(3) Cytokines and methods of use
Table 8 serum cytokines (mean+ -SD) of mice in each group
Note that: compared to saline group, x represents significant difference p <0.05; * Represents a very significant difference p <0.01.
From the above table, the IL-12 levels were significantly increased in each of the administration groups compared to the physiological saline group; the live bacteria, inactivated bacteria and PSA high dose group level of Bacteroides fragilis are higher than that of DDP group, and the PSA low dose group level is similar to that of DDP group.
The IFN-gamma levels were elevated in each of the dosing groups compared to the saline group; the ZY-312 live bacteria group, the inactivated bacteria liquid group and the PSA groups have obvious differences from the physiological saline group, and the PSA group has no obvious dose dependency.
Compared with the normal saline group, the IL-1 beta level of each administration group is obviously reduced, the low and high dose of the bacteroides fragilis ZY-312, the inactivated bacteria liquid and the PSA high dose group have extremely obvious differences from the normal saline group, and the PSA low dose group has lower level than the DDP group. It shows that the bacteroides fragilis ZY-312 and capsular polysaccharide thereof up-regulate the level of anti-tumor factors IL-12 and IFN-gamma and inhibit the expression of tumor promoting factors IL-1 beta.
In conclusion, the bacteroides fragilis ZY-312 and the zwitterionic capsular polysaccharide can regulate the level of tumor-related immune factors, and can effectively treat the nasopharyngeal carcinoma transplantable tumor and metastatic tumor of mice.
Of course, the present invention is capable of other various embodiments and its several details are capable of modification and variation in light of the present invention, as will be apparent to those skilled in the art, without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (11)

1. The application of bacteroides fragilis and/or zwitterionic capsular polysaccharide thereof in preparing products for preventing and/or treating respiratory system tumors is characterized in that bacteroides fragilis ZY-312 with the preservation number of CGMCC No. 10685;
the respiratory system tumor is non-small cell lung cancer, small cell lung cancer and nasopharyngeal carcinoma;
the zwitterionic capsular polysaccharide contains capsular polysaccharide A, and the structure of the capsular polysaccharide A is shown as follows:
the weight average molecular weight of the capsular polysaccharide A is 80-90kD.
2. The use according to claim 1, wherein the bacteroides fragilis is one or more of live bacteria, inactivated bacteria with complete morphological structure, inactivated bacteria with incomplete morphological structure;
and/or the bacteroides fragilis is one or more of live bacteroides fragilis and inactivated bacteroides fragilis.
3. Use according to claim 1, characterized in that the content of capsular polysaccharide a in the zwitterionic capsular polysaccharide is more than 95wt%.
4. Use according to any one of claims 1 to 3, characterized in that the preparation method of the zwitterionic capsular polysaccharide comprises the following steps:
(1) Centrifuging the fermented and cultured bacteroides fragilis bacterial liquid to collect sediment, thus obtaining bacteroides fragilis bacterial mud; adding purified water with the mass 3-10 times of that of the bacterial sludge into the bacterial sludge to suspend the bacterial sludge again, adjusting the pH of the bacterial sludge to 2.0-4.5 by using an acid solution, extracting for 0.5-3.0 h at 50-120 ℃, cooling to room temperature, centrifuging at normal temperature, and taking the supernatant to obtain a crude sugar solution;
(2) Concentrating the crude sugar solution by ultrafiltration membrane ultrafiltration to remove small molecular impurities until the conductivity is stable, and collecting reflux liquid;
(3) Adding 40mmol/L Tris-HCl salt in equal volume into the reflux liquid; ion exchange column chromatography, gradient elution, sectional collection, SEC-HPLC tracking and monitoring, combining components with single and symmetrical peaks of 206nm absorption peak, ultrafiltering with ultrafiltration membrane, adding purified water, repeatedly ultrafiltering until the conductivity is stable, collecting reflux liquid, and lyophilizing to obtain Bacteroides fragilis zwitterionic capsular polysaccharide.
5. The use according to claim 4, wherein in the process for the preparation of the zwitterionic capsular polysaccharide,
The centrifugation in the step (1) is 11000-13000 g for 8-12 min;
and/or the acid solution in step (1) is one or more of an organic acid, an inorganic acid and an acidic buffer;
and/or the ultrafiltration membrane in step (2) is 100, 50, 30, 10, 5, 3KD or a range between any two of the molecular weight values mentioned above;
and/or, the ion exchange column in the step (3) is DEAE Sepharose Fast Flow of 16mm multiplied by 200mm, the flow rate during chromatography is 15-25 mL/min, the pH is 5.0-9.0, the gradient elution is carried out on 25 column volumes by 0.2mol/L NaCl20mmol/L Tris-HCl, and the column volumes are collected in sections and 100 mL/bottle; the ultrafiltration membrane is 10KD.
6. The use according to claim 5, wherein in the preparation of the zwitterionic capsular polysaccharide, the mineral acid is hydrochloric acid, sulfuric acid, phosphoric acid; the organic acid is acetic acid or citric acid.
7. The use according to claim 1, wherein the product is a pharmaceutical product.
8. The use according to claim 7, wherein,
the medicine is prepared from bacteroides fragilis or zwitterionic capsular polysaccharide thereof which are used singly or combined with other medicines together.
9. The use according to claim 8, wherein,
the other medicine is lactobacillus.
10. The use according to claim 9, wherein,
the lactobacillus is ATCC 53103 lactobacillus.
11. The use according to claim 7 or 8, characterized in that,
the medicine is a human medicine or an animal medicine;
and/or the dosage form of the medicine comprises pill, tablet, granule, capsule, oral liquid or tube feeding preparation.
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