WO2023134203A1 - 脆弱拟杆菌及其两性离子荚膜多糖在制备用于治疗呼吸系统肿瘤药物中的应用 - Google Patents
脆弱拟杆菌及其两性离子荚膜多糖在制备用于治疗呼吸系统肿瘤药物中的应用 Download PDFInfo
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- bacteroides fragilis
- capsular polysaccharide
- zwitterionic
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Definitions
- Bacteroides fragilis ZY-312 Bacteroides fragilis ZY-312
- deposit number CGMCC No.10685 Bacteroides fragilis ZY-312 was isolated and obtained by the applicant unit of the present invention, and has been authorized for patent protection (patent number 201510459408.X). According to the provisions of the patent examination guidelines, the public can buy it from commercial channels or has been authorized without preservation, that is, No deposit certificate is required.
- the invention relates to the field of biomedicine, in particular to the application of Bacteroides fragilis and/or its zwitterionic capsular polysaccharide in the preparation of drugs for treating respiratory system tumors.
- Respiratory system neoplasm refers to any disease characterized by anatomically located malignant cells in the respiratory system, including lung cancer, nasopharyngeal cancer, and laryngeal cancer.
- lung cancer is a representative of respiratory system tumors.
- NSCLC non-small cell lung cancer
- SCLC small cell lung cancer
- the main histological 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), etc. Due to its unique biological behavior, SCLC is recommended to be divided into limited stage and extensive stage in various diagnosis and treatment guidelines.
- lung cancer A large number of epidemiological studies have shown that the main risk factors for lung cancer include: smoking and passive smoking, pollution caused by indoor fuel and cooking fumes, indoor radon exposure, outdoor air pollution, and genetic factors. Smoking is currently recognized as the most important risk factor for lung cancer. At the same time, due to the increasing air pollution caused by the development of industrialization in our country, fine particulate matter (PM2.5) in the air increases the risk of lung cancer death. There is also familial clustering in lung cancer patients. These indicate that genetic factors may play an important role in the population and/or individuals susceptible to environmental carcinogens.
- Nasopharyngeal carcinoma is the most common malignant tumor of the head and neck. Compared with other cancers, nasopharyngeal carcinoma is not common. According to IARC's survey, there will be about 129,000 new cases of nasopharyngeal carcinoma in 2020, accounting for 0.7% of all cancers.
- the histological classification of nasopharyngeal carcinoma adopts the WHO classification (2017 version), which is divided into keratinizing squamous cell carcinoma, nonkeratinizing squamous cell carcinoma, basaloid squamous cell carcinoma and other types of nasopharyngeal carcinoma.
- Current treatment methods for respiratory tumors include surgery, radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy.
- anatomic lung resection combined with platinum-containing double-drug regimen is the main treatment method for early and mid-stage lung cancer, and it is also an important method for clinically curing lung cancer.
- Most non-small cell lung cancers have been locally advanced or have distant metastases at the time of treatment, and cannot be surgically removed.
- Combined radiotherapy and chemotherapy are used.
- the chemotherapy regimen includes etoposide + cisplatin (EP) or carboplatin (EC), pemetrex Plug + cisplatin or carboplatin, paclitaxel or docetaxel + platinum.
- Nivolumab PD-1 inhibitor
- the first-line treatment of extensive-stage SCLC includes immunotherapy such as atezolizumab + EC regimen, programmed cell death-ligand 1 (PD-L1) monoclonal antibody durvalumab combined with other regimens.
- immunotherapy such as atezolizumab + EC regimen, programmed cell death-ligand 1 (PD-L1) monoclonal antibody durvalumab combined with other regimens.
- nasopharyngeal carcinoma is radiotherapy, and the combination of radiotherapy and platinum-based chemotherapy is the key to the treatment of locally advanced nasopharyngeal carcinoma.
- intensity-modulated radiotherapy in the treatment of nasopharyngeal carcinoma, the local control rate and overall survival rate of nasopharyngeal carcinoma have been significantly improved, but distant metastasis is the main failure mode in the treatment of nasopharyngeal carcinoma.
- Nasopharyngeal carcinoma can metastasize to multiple organs throughout the body through hematogenous metastasis, and the common metastatic sites are bone, lung and liver.
- nasopharyngeal carcinoma tissue is positive for EGFR and VEGFR
- EGFR monoclonal antibody cetuximab or nimotuzumab
- VEGFR monoclonal antibody bevacizumab
- tyrosine kinase inhibitor apatib
- Immunotherapy also plays an important role, such as camrelizumab + GP (gemcitabine + cisplatin), and toripalimab in the salvage regimen.
- Targeted therapy and immunotherapy play an important role in the treatment of metastatic and recurrent respiratory tumors, but their adverse reactions cannot be ignored, and can occur in the skin, neuroendocrine, gastrointestinal tract, liver, There may also be serious adverse reactions such as immune enteritis, pneumonia, hepatitis and myocarditis. Therefore, further improving the effect of combination therapy and reducing toxicity is a promising research direction for immunotherapy and targeted therapy.
- the object of the present invention is to provide an application of Bacteroides fragilis and its zwitterionic capsular polysaccharide in the treatment of respiratory system tumors.
- the present invention proves through a large number of experiments that Bacteroides fragilis, especially Bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685 and its zwitterionic capsular polysaccharide, especially capsular polysaccharide PSA, can increase anti-tumor factor IL-12 , IFN- ⁇ levels, inhibit the expression of tumor-promoting factor IL-1 ⁇ .
- the proportion of CD8 + CD45 + T cells that promote infiltration in tumors is upregulated and regulates the tumor tissue microenvironment. It can effectively inhibit the growth of lung transplanted tumors in mice, reduce the weight of tumors in situ, inhibit tumor metastasis, and effectively prevent and treat respiratory system tumors.
- Bacteroides fragilis and/or its zwitterionic capsular polysaccharide in the preparation of products for preventing and/or treating respiratory system tumors, said Bacteroides fragilis is a fragilis with a deposit number of CGMCC No.10685 Bacteroides ZY-312.
- the Bacteroides fragilis is a live bacterium or an inactivated bacterium; preferably, the inactivated bacterium is an inactivated bacterium with a complete morphology or structure or an inactivated bacterium with an incomplete morphological structure.
- the Bacteroides fragilis is live Bacteroides fragilis, Bacteroides fragilis that has undergone inactivation, genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation, Bacteroides fragilis Lysate, one or more of Bacteroides fragilis liquid culture supernatant.
- the respiratory system tumors include head and neck squamous cell carcinoma, non-small cell lung cancer, and small cell lung cancer.
- the head and neck squamous cell carcinoma includes nasopharyngeal carcinoma and laryngeal carcinoma.
- the zwitterionic capsular polysaccharide comprises capsular polysaccharide A.
- the structure of the capsular polysaccharide A is as follows:
- the weight-average molecular weight of the capsular polysaccharide A is 80-90KD, and the portion with a molecular weight distribution of 70KD-100KD accounts for 70-80% of the total.
- the content of the capsular polysaccharide A in the zwitterionic capsular polysaccharide exceeds 95wt%.
- the preparation method of the zwitterionic capsular polysaccharide comprises the following steps:
- the centrifugation in step (1) is centrifugation at 11000-13000 g for 8-12 minutes.
- the acid solution in step (1) may be one or more of organic acids, inorganic acids and acidic buffers.
- the inorganic acid may be hydrochloric acid, sulfuric acid, phosphoric acid, etc.
- the organic acid may be acetic acid, citric acid, etc.
- the molecular weight of the ultrafiltration membrane in step (2) may be 100, 50, 30, 10, 5, 3 KD or a range between any two molecular weight values.
- the ion exchange column described in step (3) is preferably 16mm ⁇ 200mm of DEAE Sepharose Fast Flow, the flow rate during chromatography is 15-25mL/min, and pH5.0-9.0 contains 0.2mol/L NaCl 20mmol/L Tris-HCl gradient elution 25 column volumes, section collection, 100mL/bottle (component); The molecular weight of described ultrafiltration membrane is 10KD.
- the product is food or medicine.
- the drug is the single application of Bacteroides fragilis or its zwitterionic capsular polysaccharide, or the combined application of Bacteroides fragilis and its zwitterionic capsular polysaccharide, or the separate application of Bacteroides fragilis or its zwitterionic capsular polysaccharide In combination with other drugs, or B. fragilis and its zwitterionic capsular polysaccharide in combination with other drugs.
- the dosage form of the medicine includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations.
- the medicine includes human medicine or animal medicine.
- the food product comprises milk powder, cheese, curd, yogurt, ice cream, or fermented cereal.
- the food can also be animal food, such as feed and the like.
- compositions for treating respiratory system tumors wherein the composition contains a pharmaceutically effective dose of Bacteroides fragilis with the preservation number CGMCC No. 10685 and/or its zwitterionic capsular polysaccharide.
- the pharmaceutically effective dose of the Bacteroides fragilis is 10 6 -10 10 CFU.
- the pharmaceutically effective dose of the zwitterionic capsular polysaccharide is 1-30 mg/kg.
- the Bacteroides fragilis is one or more of live bacteria, inactivated bacteria with complete morphology and structure, and inactivated bacteria with incomplete morphology and structure.
- the Bacteroides fragilis is live Bacteroides fragilis, Bacteroides fragilis that has undergone inactivation, genetic recombination, transformation or modification, attenuation, chemical treatment, physical treatment or inactivation, Bacteroides fragilis Lysate, one or more of Bacteroides fragilis liquid culture supernatant.
- the zwitterionic capsular polysaccharide comprises capsular polysaccharide A.
- the zwitterionic capsular polysaccharide is obtained from the Bacteroides fragilis ZY-312.
- the structure of the capsular polysaccharide A is as follows:
- the weight-average molecular weight of the capsular polysaccharide A is 80-90KD, and the portion with a molecular weight distribution of 70KD-100KD accounts for 70-80% of the total.
- the content of the capsular polysaccharide A in the zwitterionic capsular polysaccharide exceeds 95wt%.
- the preparation method of the zwitterionic capsular polysaccharide comprises the following steps:
- the centrifugation in step (1) is centrifugation at 11000-13000 g for 8-12 minutes.
- the acid solution in step (1) may be one or more of organic acids, inorganic acids and acidic buffers.
- the inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.
- the organic acid can be acetic acid, citric acid, etc.
- the molecular weight of the ultrafiltration membrane in step (2) may be 100, 50, 30, 10, 5, 3 KD or a range between any two molecular weight values.
- the ion exchange column described in step (3) is preferably 16mm ⁇ 200mm of DEAE Sepharose Fast Flow, the flow rate during chromatography is 15-25mL/min, and pH5.0-9.0 contains 0.2mol/L NaCl 20mmol/L Tris-HCl gradient elution 25 column volumes, section collection, 100mL/bottle (component); The molecular weight of described ultrafiltration membrane is 10KD.
- the composition is a probiotic composition, a health product composition or a pharmaceutical composition.
- the probiotic composition for preventing and treating respiratory system tumors of the present invention wherein the probiotic composition contains Bacteroides fragilis ZY-312 with the preservation number of CGMCC No. 10685 and/or is extracted from the Bacteroides fragilis ZY-312 312 zwitterionic capsular polysaccharides.
- the probiotic composition may also include one of probiotics or microorganisms from Saccharomyces spp., Lactobacillus spp. and normal human intestinal flora or more.
- the probiotics of the Saccharomyces genus may include Saccharomyces boulardii and/or Saccharomyces cerevisiae.
- the pharmaceutical composition for preventing and treating respiratory system tumors of the present invention contains Bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685 and/or zwitterionic capsular polysaccharide extracted from ZY-312.
- the pharmaceutical composition may further include a pharmaceutically acceptable carrier.
- the dosage form of the pharmaceutical composition can be pill, tablet, granule, capsule, powder, suspension, oral liquid or enema. It can be administered in the form of oral, enema or parenteral administration, and the administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.
- the health product composition for preventing and treating respiratory system tumors of the present invention contains Bacteroides fragilis ZY-312 with a preservation number of CGMCC No. 10685 and/or zwitterionic capsular polysaccharide extracted from ZY-312.
- the present invention also provides a method for preventing and/or treating respiratory system tumors, comprising administering therapeutically effective doses of Bacteroides fragilis, Bacteroides fragilis zwitterionic capsular polysaccharide, the above products and/or compositions to patients.
- prevention includes prevention and/or treatment.
- Bacteroides fragilis especially Bacteroides fragilis ZY-312 with the deposit number of CGMCC No.10685 and its zwitterionic capsular polysaccharide, especially capsular polysaccharide A (PSA)
- PSA capsular polysaccharide A
- the cells induce apoptosis of non-small cell lung cancer cells; in vivo, it can inhibit the expression of tumor-promoting factor IL-1 ⁇ by increasing the levels of anti-tumor factors IL-12 and IFN- ⁇ .
- the proportion of CD8 + CD45 + T cells that promote infiltration in tumors is upregulated and regulates the tumor tissue microenvironment.
- Bacteroides fragilis and its PSA reduce the weight of transplanted tumors in established mice with non-small cell lung cancer and small cell lung cancer, and can effectively inhibit the growth of lung transplanted tumors in mice.
- Bacteroides fragilis can reduce the number of tumor metastases in the nude mouse nasopharyngeal carcinoma metastasis model, effectively prevent the occurrence, development, recurrence and metastasis of respiratory tumors, and improve the quality of life of patients.
- Bacteroides fragilis ZY-312 that the present invention adopts does not contain BFT gene, is non-toxigenic bacterial strain, and acute toxicity proves, and this bacterial strain is all nonpathogenic to normal mouse and nude mouse (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. 2017 Mar 17; 8:435.).
- Fig. 1 is the colony characteristic figure of Bacteroides fragilis ZY-312 of the embodiment of the present invention 1;
- Fig. 2 is the microscopic observation diagram after Gram staining of Bacteroides fragilis ZY-312 in Example 1 of the present invention
- 3A-3E are the 1 H spectrum, 13 C spectrum, COZY spectrum, HSQC spectrum, and HMBC spectrum analyzed by the capsular polysaccharide A NMR spectrometer in Example 3 of the present invention, respectively;
- Fig. 4 is the chemical structural formula of the structural unit of Bacteroides fragilis capsular polysaccharide A prepared in Example 3 of the present invention.
- the raw materials and reagents used in the following examples are commercially available. All cells were purchased from ATCC; all cell culture materials and trypsin were purchased from Gibco; all experimental animals were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; or could be prepared by known methods.
- the experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions.
- Embodiment 1 the fermentation culture of Bacteroides fragilis
- Bacteroides fragilis ZY-312 Streak inoculation of Bacteroides fragilis ZY-312 strain on blood plate, anaerobic culture for 48h. Observe the colony morphological characteristics, staining characteristics, size, club shape and distribution, etc. Colony characteristics: Bacteroides fragilis ZY-312 was cultured on a blood plate for 48 hours, and it appeared round, slightly convex, translucent, white, smooth, non-hemolytic, and the diameter of the colony was between 1-3 mm, see Figure 1.
- Bacteroides fragilis ZY-312 was examined by Gram staining. It is a Gram-negative bacterium with a typical rod shape, blunt rounded ends and dense staining. The uncolored part in the middle of the bacteria is like a vacuole. figure 2.
- Embodiment 2 Bacteroides fragilis living bacteria liquid and the preparation of inactivated bacteria liquid
- Example 2 Select the single bacterium colony cultivated in Example 1 and inoculate it into the plant-derived peptone liquid medium for fermentation and cultivation for 8 hours (at a temperature of 37° C.) to obtain a live Bacteroides fragilis liquid; After 15 minutes, the supernatant was removed, and the precipitate was collected to obtain the Bacteroides fragilis ZY-312 sludge.
- the above bacterial liquid was taken and subjected to conventional heat inactivation treatment to obtain the inactivated bacterial liquid of Bacteroides fragilis ZY-312.
- the bacteria slime prepared in Example 1 was used to carry out the experiment.
- the prepared capsular polysaccharide A has a weight-average molecular weight of 80-90 KDa and a Mw/Mn of 1.0-1.2.
- the chemical structure is shown in FIG. 4 .
- Example 4 Drug efficacy experiment of Bacteroides fragilis and its zwitterionic capsular polysaccharide (PSA) promoting apoptosis of mouse lung cancer cell line Lewis cell strain through macrophages in vitro
- PSA zwitterionic capsular polysaccharide
- Mouse lung cancer cells Lewis cells and RAW264.7 cells were grown in DMEM medium containing 10% FBS, added 1% penicillin/streptomycin, cultured in an incubator at 37°C, 5% CO2 concentration, and saturated humidity . Medium was changed every two days.
- the cell line was taken out from liquid nitrogen, and rapidly melted in a constant temperature water bath at 37°C. Open the freezing tube under sterile conditions, transfer the liquid to a 15 mL centrifuge tube, resuspend the cells with 2-3 mL of DMEM complete medium, centrifuge at 1000 rpm for 5 min. After the supernatant was discarded, 5 mL of DMEM complete medium was added, the cells were inoculated in a culture flask, and cultured in an incubator with 5% CO 2 concentration for 48 hours at a temperature of 37°C.
- Cell resuspension Add 100 ⁇ l of 1 ⁇ Binding Buffer, and blow gently to obtain a single-cell suspension.
- Cell apoptosis Calculate the early apoptotic cells with Annexin V-FITC single positive (Annexin V-FITC+/PI+) and the late apoptotic cells with Annexin V-FITC and PI double positive (Annexin V-FITC+/PI+) The sum of the proportion of apoptotic cells.
- the apoptosis rate of the negative group was significantly lower than that of the positive control.
- Bacteroides fragilis ZY-312 and ZY-312 inactivated bacterial solution and ZY-312PSA can promote Lewis cell apoptosis through RAW264.7 cells.
- Example 5 Drug efficacy test of Bacteroides fragilis and its zwitterionic capsular polysaccharides in the treatment of non-small cell lung cancer xenografts in mice
- Lewis lung cancer tumor-derived mouse cell suspension was inoculated subcutaneously in the axilla of the right forelimb of each mouse at about 0.2 mL to establish the model.
- ZY-312 low (10 6 CFU/body), high (10 10 CFU/body) dose group ZY-312 inactivated bacteria (10 10 cell/body), ZY-312PSA low ( 10mg/kg) and ZY-312PSA high (30mg/kg) for 3 consecutive weeks; DDP (cisplatin, Shandong Qilu Pharmaceutical Co., Ltd.) group was injected intraperitoneally once a week for 5 consecutive weeks.
- Lewis cells were cultured in DMEM medium containing 10% fetal bovine serum, the culture temperature was 37°C, the gas environment was 5% CO 2 , and the humidity was saturated humidity; the culture medium was replaced according to the cell growth speed and the color change of the culture medium. Digest and passage with 0.25% trypsin. According to the growth of the cells, the cells in the logarithmic growth phase were prepared into a single cell suspension, and the cell concentration was adjusted to 2 ⁇ 10 4 cells/mL.
- Well-grown Lewis lung cancer tissues were selected and sacrificed under aseptic conditions by removing the cervical spine.
- the tumor tissue was stripped from the armpit, chopped, homogenized, and ground, and the ratio of tumor mass (g) to normal saline (mL) was 1:3.
- mice were randomly divided into 7 groups, 10 mice in each group: normal saline group, DDP group, ZY-312 low (10 6 CFU/mouse), high (10 10 CFU/mouse) ) dose group, ZY-312 inactivated bacteria group (10 10 cell/only), ZY-312PSA low (10mg/kg) and ZY-312PSA high (30mg/kg) dose groups.
- Normal saline group 0.2mL/rat, once a day, for 3 consecutive weeks;
- DDP group 0.3 mL/time, 3 mg/kg intraperitoneal injection, once a week, continuous injection for 3 weeks;
- ZY-312 low- and high-dose groups, inactivated bacteria groups and ZY-312PSA low- and high-dose groups 0.2 mL/cattle, once a day, for 3 consecutive weeks.
- Tumor inhibition rate (%) (average tumor weight of normal saline group-average tumor weight of administration group)/average tumor weight of normal saline group ⁇ 100%;
- Table 2 The antitumor efficacy calculated based on the tumor weight on the 21st day after group administration
- Tumor inhibition rate (%) (average tumor weight of normal saline group-average tumor weight of administration group)/average tumor weight of normal saline group ⁇ 100%.
- CD8 + T cells play an important role in tumor immunity and are effector cells that directly kill tumor cells.
- Bacteroides fragilis ZY-312 and its zwitterionic capsular polysaccharide can regulate the tumor immune microenvironment in mice and effectively treat non-small cell lung cancer xenografts in mice.
- Example 6 Drug efficacy test of Bacteroides fragilis and its zwitterionic capsular polysaccharides in the treatment of transplanted tumors of small cell lung cancer
- the human small cell lung cancer cell line NCI-H526 was cultured, and the NCI-H526 cells in the logarithmic growth phase were adjusted to a single cell suspension with a cell concentration of 2 ⁇ 10 7 cells/mL. Under sterile conditions, Matrigel was diluted 1:1 with PBS and placed on ice for injection. NCI-H526 cells were diluted with Matrigel gel (BD, 356234) at a ratio of 1:1 to prepare a tumor cell suspension with a concentration of 1 ⁇ 10 7 cells/mL. The cell suspension was inoculated into 4-5 week-old nude mice with a body weight of (22 ⁇ 2) g at about 0.2 mL each. The right forelimb axilla of nude mice was subcutaneously constructed.
- Tumor inhibition rate% 100% ⁇ (average tumor weight of normal saline group-average tumor weight of administration group)/average tumor weight of normal saline group.
- Elisa was used to detect IL-12 (R&D Systems, M1270, the same below), IFN- ⁇ (R&D Systems, MIF00, the same below) and IL-1 ⁇ (R&D Systems, MLB00C, the same below) in tumors of lung cancer xenograft mouse models Cytokine levels.
- Table 4 The antitumor efficacy calculated based on the tumor weight on the 21st day after group administration
- Tumor inhibition rate% 100 ⁇ (average tumor weight of normal saline group-average tumor weight of administration group)/average tumor weight of normal saline group.
- IL-12 comes from activated lymphocytes, which can induce the cytotoxic activity of CTL and NK cells and promote the secretion of anti-tumor cytokines such as IFN- ⁇ and TNF- ⁇ ; Multiple tumor growth and metastasis in mice and humans.
- Bacteroides fragilis ZY-312 and its PSA can regulate the levels of tumor-related immune factors and effectively treat small cell lung cancer xenografts in mice.
- Example 7 Drug efficacy test of Bacteroides fragilis and its zwitterionic capsular polysaccharide in the treatment of nasopharyngeal carcinoma transplantation and metastases
- RPMI-1640 medium containing fetal bovine serum to culture human nasopharyngeal carcinoma cell line 5-8F, and adjust the cell concentration of 5-8F cells in the logarithmic growth phase to 2 ⁇ 10 6 cells/mL under sterile conditions
- Single cell suspension the cell suspension is inoculated into 5-7 week old female nude mice weighing 20-24g at about 0.2mL each.
- the nude mice were directly injected with 0.2 mL of 5-8F single cell suspension at 2 ⁇ 10 6 cells/mL through the tail vein.
- ZY-312 low (10 6 CFU/monkey), high (10 10 CFU/monkey) dose group
- ZY-312 inactivated bacteria (10 10 cells/monkey)
- ZY-312PSA low 10mg/kg
- ZY-312PSA high 30mg/kg
- DDP group cisplatin, Shandong Qilu Pharmaceutical Co., Ltd.
- mice When the experiment was over or when the tumor-bearing mice showed obvious signs of exhaustion such as weight loss, arched back, and listlessness, they were killed by cervical dislocation, and the transplanted tumor was taken to measure its weight and calculate the tumor inhibition rate. Liver and lung tissues were taken to observe the metastatic focus and tumor body Tissues were stored in a -80°C refrigerator for detection of cytokines, etc.
- Tumor inhibition rate% 100% ⁇ (average tumor weight of normal saline group-average tumor weight of administration group)/average tumor weight of normal saline group.
- grade I metastases the diameter is less than 0.15 mm
- grade II metastases are 0.15-1 mm in diameter
- grade III metastases are 1-2 mm in diameter
- grade IV metastases are larger than 2 mm in diameter.
- the total number of metastases the number of grade I metastases + the number of grade II metastases * 2 + the number of grade III metastases * 3 + the number of grade IV metastases * 4.
- Elisa was used to detect the levels of cytokines such as IL-12, IFN- ⁇ and IL-1 ⁇ in the serum of lung cancer xenograft mouse models.
- Table 6 The antitumor efficacy calculated based on the tumor weight on the 21st day after group administration
- Tumor inhibition rate% 100% ⁇ (average tumor weight of normal saline group-average tumor weight of administration group)/average tumor weight of normal saline group.
- the levels of IL-1 ⁇ in each administration group decreased significantly, and there were extremely significant differences between the Bacteroides fragilis ZY-312 low-dose, high-dose, inactivated bacterial liquid and PSA high-dose groups and the normal saline group, and the PSA low
- the level of dose group was lower than that of DDP group. It shows that Bacteroides fragilis ZY-312 and its capsular polysaccharide up-regulate the levels of anti-tumor factors IL-12 and IFN- ⁇ , and inhibit the expression of tumor-promoting factor IL-1 ⁇ .
- Bacteroides fragilis ZY-312 and zwitterionic capsular polysaccharide can regulate the level of tumor-related immune factors, and effectively treat transplanted and metastatic tumors of nasopharyngeal carcinoma in mice.
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Abstract
公开了脆弱拟杆菌和/或其两性离子荚膜多糖在制备用于治疗呼吸系统肿瘤的药物中的应用。脆弱拟杆菌,特别是保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312及其两性离子荚膜多糖,可通过增加抗肿瘤因子IL-12、IFN-γ的水平,抑制肿瘤促进因子IL-1β的表达,促进肿瘤中浸润的CD8 +CD45 +T细胞比例有上调,调节肿瘤组织微环境,降低原位肿瘤的重量,抑制肿瘤转移灶数量,可有效地防治呼吸系统肿瘤,不仅能够单独用于癌症治疗,还可以与其他微生物、手术、放疗、化疗等治疗手段联合应用,显著提高综合疗效,减轻放化疗对机体的伤害,有效预防呼吸系统肿瘤的发生发展及其复发转移,提高患者的生活质量。
Description
本申请要求享有2022年1月12日向中国国家知识产权局提交的,专利申请号为202210034079.4,发明名称为“脆弱拟杆菌及其两性离子荚膜多糖在制备用于治疗呼吸系统肿瘤药物中的应用”的在先申请的优先权权益。所述在先申请的全文通过引用的方式结合于本申请中。
本发明在实施过程中所使用的微生物菌种已于2015年4月2日在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)(北京市朝阳区北辰西路1号院3号)保藏。分类命名:脆弱拟杆菌ZY-312(bacteroides fragilis ZY-312),保藏编号CGMCC No.10685。脆弱拟杆菌ZY-312由本发明申请单位自行分离获得,并且已经在授权专利保护(专利号201510459408.X),按照专利审查指南的规定,公众能够从商业渠道买到或已经授权,不用保藏,即不用提供保藏证明。
本发明涉及生物医药领域,具体涉及一种脆弱拟杆菌和/或其两性离子荚膜多糖在制备治疗呼系统肿瘤药物中的应用。
呼吸系统肿瘤是指任何特征为在呼吸系统中解剖学定位为恶性细胞的疾病,包括肺癌、鼻咽癌、喉癌等。其中,肺癌作为呼吸系统肿瘤的代表,2020年全球肺癌死亡180万例,远超其他癌症类型,位居癌症死亡人数第一。从病理和治疗角度,肺癌大致可以分为非小细胞肺癌(non small celllung cancer,NSCLC)和小细胞肺癌(small celllung cancer,SCLC)两大类,其中非小细胞肺癌约占80%~85%,其余为小细胞肺癌。依据世界卫生组织(WHO)2015年发布的肺癌组织学分型标准,肺癌主要组织类型为鳞状细胞癌和腺癌,约占全部原发性肺癌的80%左右。其他少见类型原发性肺癌包括:腺鳞癌、大细胞癌、神经内分泌癌(类癌、不典型类癌和小细胞癌)等。其中SCLC由于其独特的生物学行为,各个诊疗指南中建议分为局限期与广泛期。
大量的流行病学研究表明,肺癌发生的主要危险因素包括:吸烟和被动吸烟、室内燃料和烹调油烟所致污染、室内氡暴露、室外空气污染和遗传因素等。吸烟是目前公认的肺癌最主要的危险因素。同时,由于我国工业化发展导致空气污染日益加重,空气中细颗粒物(PM2.5)增加肺癌死亡风险。肺癌患者中也存在家族聚集现象。这些说明遗传因素可能在对环境致癌物易感的人群和(或)个体中起重要作用。
鼻咽癌是头颈部最常见的恶性肿瘤,是由鼻咽腔表面或鼻咽隐窝的上皮发生的恶性肿瘤。 鼻咽癌相对于其他癌症来说并不算常见,根据IARC的调查,在2020年约有12.9万鼻咽癌新发病例,在所有癌症中占比0.7%。鼻咽癌组织学分型采用WHO分类(2017版)标准,分为角化型鳞状细胞癌、非角化型鳞状细胞癌、基底细胞样鳞状细胞癌和其他类型鼻咽癌。
目前针对呼吸系统肿瘤的治疗方法包括手术、放疗、化疗、分子靶向治疗和免疫治疗等。
在NSCLC的治疗中,解剖性肺切除术辅以含铂双药方案是早中期肺癌的主要治疗手段,也是目前临床治愈肺癌的重要方法。大多数非小细胞肺癌在治疗时已经局部晚期或远处转移,不能手术切除,使用放疗、化疗联合疗法,化疗方案包括依托泊苷+顺铂(EP)或卡铂(EC)、培美曲塞+顺铂或卡铂、紫杉醇或多西紫杉醇+铂类。由于SCLC恶性程度高、易转移,但对化疗、放疗较敏感,主要采用化疗、放疗和预防性脑放疗结合的综合治疗。由于缺乏临床症状和有效的筛查程序,大多数肺癌被诊断已为晚期,进行切除术后NSCLC局部复发的可能性依然较高。新兴的靶向治疗和免疫疗法主要用于转移、复发肿瘤。在NSCLC的治疗中,晚期NSCLC的一线药物治疗中如患者EGFR基因突变,可选择表皮生长因子受体酪氨酸激酶抑制剂,包括吉非替尼、厄罗替尼等。驱动基因阴性的晚期NSCLC患者可选择刚刚获批上市的PD-1抑制剂纳武利尤单抗(Nivolumab)。广泛期SCLC的一线治疗加入了免疫治疗如阿替利珠单抗+EC方案,细胞程式死亡-配体1(PD-L1)单抗度伐利尤单抗联用等方案。
目前鼻咽癌公认和有效的根治性治疗手段为放射治疗,放疗与铂类药物化疗相结合是治疗局部晚期鼻咽癌的关键。随着调强放疗等在鼻咽癌治疗中广泛应用,鼻咽癌的局部控制率和总生存率得到显著提高,但远端转移是鼻咽癌治疗中最主要的失败模式。鼻咽癌可通过血行转移至全身多处器官,常见的转移部位有骨、肺和肝。一般占初治患者的10%左右,初治时未发现转移的患者中,治疗后15%左右仍会发生远处转移。对于复发或转移性鼻咽癌的治疗多选用化疗、免疫治疗、靶向治疗等。若鼻咽癌组织EGFR和VEGFR阳性,可用EGFR单克隆抗体(西妥昔单抗或尼妥珠单抗)、VEGFR单克隆抗体(贝伐单抗)、酪氨酸激酶抑制剂(阿帕替尼、安罗替尼等)及重组人血管内皮抑制素等靶向治疗。免疫疗法也发挥重要作用,如卡瑞利珠单抗+GP(吉西他滨+顺铂),以及在挽救治疗方案中的特瑞普利单抗。
靶向治疗和免疫疗法作为新兴的热门治疗方法,在转移、复发呼吸系统肿瘤的治疗中起到重要作用,但其不良反应却不容忽视,可发生在皮肤、神经内分泌、胃肠道、肝、肺、心脏、肾脏等各个系统,还可能存在免疫性肠炎、肺炎、肝炎和心肌炎等严重不良反应。所以进一步提高联合治疗效果和减少毒性是免疫治疗与靶向治疗比较有突破前景的研究方向。
2017年Nature杂志发表的重要文献提出“肠-肺轴微生态调控”,证实肠道菌群与肿瘤密切相关,然而现有技术还没有用于治疗呼吸系统肿瘤的微生态产品。
发明内容
为克服现有技术中所存在的上述缺陷,本发明的目的是提供一种脆弱拟杆菌及其两性离子荚膜多糖在治疗呼吸系统肿瘤中的应用。本发明通过大量实验证明,脆弱拟杆菌特别是保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312及其两性离子荚膜多糖,特别是荚膜多糖PSA,可通过增加抗肿瘤因子IL-12、IFN-γ的水平,抑制肿瘤促进因子IL-1β的表达。促进肿瘤中浸润的CD8
+CD45
+T细胞比例有上调,调节肿瘤组织微环境。能有效抑制小鼠体内肺部移植肿瘤的生长,降低原位肿瘤的重量,抑制肿瘤转移,可有效地防治呼吸系统肿瘤。
为了实现上述目的,本发明采用如下技术方案:
第一方面,提供一种脆弱拟杆菌和/或其两性离子荚膜多糖在制备预防和/或治疗呼吸系统肿瘤的产品中的应用,所述脆弱拟杆菌为保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
在其中一些实施例中,所述脆弱拟杆菌是活菌或灭活菌;优选地,所述灭活菌为形态结构完整的灭活菌或形态结构不完整的灭活菌。
在其中一些实施例中,所述脆弱拟杆菌是脆弱拟杆菌活菌体,经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌,脆弱拟杆菌裂解物,脆弱拟杆菌液体培养上清液中的一种或多种。
在其中一些实施例中,所述呼吸系统肿瘤包括头颈部鳞癌、非小细胞肺癌、小细胞肺癌。
在其中一些实施例中,所述头颈部鳞癌包括鼻咽癌、喉癌。
在其中一些实施例中,所述两性离子荚膜多糖含有荚膜多糖A。其中,所述荚膜多糖A的结构如下所示:
根据本发明的实施方案,所述荚膜多糖A的重均分子量为80-90KD,其中分子量分布于70KD-100KD的部分占总量的70-80%。
在其中一些实施例中,所述荚膜多糖A在所述两性离子荚膜多糖中的含量超过95wt%。
在其中一些实施例中,所述两性离子荚膜多糖的制备方法包括以下步骤:
(1)将发酵培养后的脆弱拟杆菌菌液离心收集沉淀物,即得脆弱拟杆菌菌泥;取菌泥,加入菌泥质量3~10倍的纯化水使菌体重悬,用酸溶液调节其pH至2.0~4.5,50~120℃提取0.5~3.0h,冷却至室温,常温离心,取上清,得到粗糖溶液;
(2)粗糖溶液经超滤膜超滤浓缩、除小分子杂质,至电导率稳定,收集回流液;
(3)回流液中加入等体积40mmol/L Tris-HCl转盐;离子交换柱层析,梯度洗脱,分段收集,SEC-HPLC跟踪监测,合并206nm吸收峰为单一、对称峰的组分,超滤膜超滤,加入纯化水反复超滤,至电导率稳定,收集回流液,冻干,得到脆弱拟杆菌两性离子荚膜多糖。
在其中一些实施例中,步骤(1)中所述离心为11000~13000g离心8~12min。
在其中一些实施例中,步骤(1)中所述酸溶液可以是有机酸、无机酸和酸性缓冲液中的一种或多种。其中,所述无机酸可以是盐酸、硫酸、磷酸等;所述有机酸可以是乙酸、柠檬酸等。
在其中一些实施例中,步骤(2)中所述超滤膜的分子量可以为100、50、30、10、5、3KD或者任意两个分子量值之间的范围。
在其中一些实施例中,步骤(3)中所述离子交换柱优选为DEAE Sepharose Fast Flow的16mm×200mm,层析时的流速15~25mL/min,pH5.0~9.0含0.2mol/L NaCl 20mmol/L Tris-HCl梯度洗脱25个柱体积,分段收集,100mL/瓶(组分);所述超滤膜的分子量为10KD。
在其中一些实施例中,所述产品为食品或药品。
在其中一些实施例中,所述药品为脆弱拟杆菌或其两性离子荚膜多糖单独应用、或脆弱拟杆菌和其两性离子荚膜多糖联合应用、或脆弱拟杆菌或其两性离子荚膜多糖分别与其他药物联用、或脆弱拟杆菌和其两性离子荚膜多糖一起与其他药物联用。
在其中一些实施例中,所述药品的剂型包括丸剂、片剂、颗粒剂、胶囊、口服液或管饲制剂。所述药品包括人用药或动物用药。
在其中一些实施例中,所述食品包括奶粉、干酪、凝乳、酸奶酪、冰激凌或发酵谷类食品。所述食品还可以是动物食品,比如饲料等。
第二方面,提供一种治疗呼吸系统肿瘤的组合物,其中,所述组合物含有药学有效剂量的保藏编号为CGMCC No.10685的脆弱拟杆菌和/或其两性离子荚膜多糖。
在其中一些实施例中,所述脆弱拟杆菌的药学有效剂量为10
6-10
10CFU。
在其中一些实施例中,所述两性离子荚膜多糖的药学有效剂量为1-30mg/kg。
在其中一些实施例中,所述脆弱拟杆菌是活菌、形态结构完整的灭活菌、形态结构不完整的灭活菌中的一种或多种。
在其中一些实施例中,所述脆弱拟杆菌是脆弱拟杆菌活菌体,经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌,脆弱拟杆菌裂解物,脆弱拟杆菌液体培养上清液中的一种或多种。
在其中一些实施例中,所述两性离子荚膜多糖含有荚膜多糖A。
在其中一些实施例中,所述两性离子荚膜多糖取自所述脆弱拟杆菌ZY-312。
在其中一些实施例中,所述荚膜多糖A的结构如下所示:
根据本发明的实施方案,所述荚膜多糖A的重均分子量为80-90KD,其中分子量分布于70KD-100KD的部分占总量的70-80%。
在其中一些实施例中,其中,所述荚膜多糖A在所述两性离子荚膜多糖中的含量超过95wt%。
在其中一些实施例中,所述两性离子荚膜多糖的制备方法包括以下步骤:
(1)将发酵培养后的脆弱拟杆菌菌液离心收集沉淀物,即得脆弱拟杆菌菌泥;取菌泥,加入菌泥质量3~10倍的纯化水使菌体重悬,用酸溶液调节其pH至2.0~4.5,50~120℃提取0.5~3.0h,冷却至室温,常温离心,取上清,得到粗糖溶液;
(2)粗糖溶液经超滤膜超滤浓缩、除小分子杂质,至电导率稳定,收集回流液;
(3)回流液中加入等体积40mmol/L Tris-HCl转盐;离子交换柱层析,梯度洗脱,分段收集,SEC-HPLC跟踪监测,合并206nm吸收峰为单一、对称峰的组分,超滤膜超滤,加入纯化水反复超滤,至电导率稳定,收集回流液,冻干,得到脆弱拟杆菌两性离子荚膜多糖。
在其中一些实施例中,步骤(1)中所述离心为11000~13000g离心8~12min。
在其中一些实施例中,步骤(1)中所述酸溶液可以是有机酸、无机酸和酸性缓冲液中的一种或多种。其中,无机酸可以是盐酸、硫酸、磷酸等;有机酸可以是乙酸、柠檬酸等。
在其中一些实施例中,步骤(2)中所述超滤膜的分子量可以为100、50、30、10、5、3KD 或者任意两个分子量值之间的范围。
在其中一些实施例中,步骤(3)中所述离子交换柱优选为DEAE Sepharose Fast Flow的16mm×200mm,层析时的流速15~25mL/min,pH5.0~9.0含0.2mol/L NaCl 20mmol/L Tris-HCl梯度洗脱25个柱体积,分段收集,100mL/瓶(组分);所述超滤膜的分子量为10KD。
在其中一些实施例中,所述组合物为益生菌组合物、保健品组合物或药物组合物。
本发明的用于防治呼吸系统肿瘤的益生菌组合物,其中,所述益生菌组合物含有保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312和/或提取自所述脆弱拟杆菌ZY-312的两性离子荚膜多糖。根据本发明的实施方案,所述益生菌组合物还可包括来自酵母菌属(Saccharomyces spp.)、乳酸杆菌属(Lactobacillus spp.)及人肠道正常菌群的益生菌或微生物中的一种或多种。例如,所述酵母菌属的益生菌可包括布拉氏酵母(Saccharomyces boulardii)和/或酿酒酵母(Saccharomyces cerevisiae)。
本发明用于防治呼吸系统肿瘤的药物组合物中,含有保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312和/或提取自ZY-312的两性离子荚膜多糖。根据本发明的实施方案,所述药物组合物还可包括药学上可接受的载体。该药物组合物的剂型可为丸剂、片剂、颗粒剂、胶囊、散剂、混悬剂、口服液或灌肠剂。可通过口服、灌肠或肠胃外的形式给药,其给药周期可为间歇给药、周期性给药、持续给药或长期给药。
本发明用于防治呼吸系统肿瘤的保健品组合物中,含有保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312和/或提取自ZY-312的两性离子荚膜多糖。
本发明还提供一种预防和/或治疗呼吸系统肿瘤的方法,包括向患者施用治疗有效量的脆弱拟杆菌、脆弱拟杆菌两性离子荚膜多糖、上述产品和/或组合物。
其中,所述“防治”包括预防和/或治疗。
本发明的有益效果:
本发明通过大量实验证明,脆弱拟杆菌特别是保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312及其两性离子荚膜多糖,尤其是荚膜多糖A(PSA),在体外能够促进巨噬细胞诱导非小细胞肺癌细胞凋亡;在体内可通过增加抗肿瘤因子IL-12、IFN-γ的水平,抑制肿瘤促进因子IL-1β的表达。促进肿瘤中浸润的CD8
+CD45
+T细胞比例有上调,调节肿瘤组织微环境。脆弱拟杆菌及其PSA在建立的小鼠非小细胞肺癌和小细胞肺癌降低移植肿瘤的重量,能有效抑制小鼠体内肺部移植肿瘤的生长。同时,脆弱拟杆菌在构建的裸鼠鼻咽癌转移模型中能降低肿瘤转移灶数量,有效预防呼吸系统肿瘤的发生发展及其复发转移,提高患者的生活质量。
本发明采用的脆弱拟杆菌ZY-312不含BFT基因,是非产毒菌株,急性毒性证实,该菌株 对正常小鼠和裸鼠均无致病性(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.2017 Mar 17;8:435.)。根据专利ZL201510459408.X和科技文献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.2018 Oct 1;9:2298.的报道,该菌株对胃酸、胆盐有着较好的耐性,能够保证其在肠道中的存活和有效定植。
图1为本发明实施例1的脆弱拟杆菌ZY-312的菌落特征图;
图2为本发明实施例1的脆弱拟杆菌ZY-312进行革兰氏染色后的显微镜观察图;
图3A-3E分别为本发明实施例3的荚膜多糖A核磁共振波谱仪分析的
1H谱、
13C谱、COSY谱、HSQC谱、HMBC谱图;
图4为本发明实施例3制备得到的脆弱拟杆菌荚膜多糖A的结构单元的化学结构式。
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解,下列实施例仅为示例性地说明和解释本发明,而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明,以下实施例中使用的原料和试剂均为市售商品。所有细胞购自ATCC;所有细胞培养材料及胰酶购自Gibco;所有实验动物购自浙江维通利华实验动物技术有限公司;或者可以通过已知方法制备。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。
除非另外定义或由背景清楚指示,否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
实施例1:脆弱拟杆菌的发酵培养
将脆弱拟杆菌ZY-312菌种划线接种于血平皿,厌氧培养48h。观察菌落形态特征、染色特性、大小、球杆状和分布情况等。菌落特征:脆弱拟杆菌ZY-312在血平皿上培养48h后,呈现圆形微凸、半透明、白色、表面光滑、不溶血,菌落直径在1-3mm之间,参见图1。
显微镜下形态:脆弱拟杆菌ZY-312进行革兰氏染色镜检,为革兰阴性细菌,呈现典型的杆状,两端钝圆而浓染,菌体中间不着色部分形如空泡,参见图2。
实施例2脆弱拟杆菌活菌液及灭活菌液的制备
1)活菌液制备
选取实施例1中培养的单个菌落接种于植物源蛋白胨液体培养基中进行发酵培养8小时(温度为37℃),得脆弱拟杆菌活菌液;所得菌液离心沉淀,转速3000r/min,离心15min,去上清,收集沉淀物,即得脆弱拟杆菌ZY-312菌泥。
2)灭活菌液的制备
取上述菌液,常规热灭活处理,得脆弱拟杆菌ZY-312灭活菌液。
实施例3:脆弱拟杆菌荚膜多糖的制备
采用实施例1制备的菌泥进行实验。
(1)取50g菌泥,加入300g纯化水使菌体重悬,用1mol/L盐酸溶液调节其pH至3.5,100℃提取1.5h,冷却至室温,12000g常温离心10min,取上清,得到粗糖溶液;
(2)粗糖溶液经10KD超滤膜超滤浓缩、除小分子杂质,至电导率稳定,收集回流液;
(3)回流液中加入等体积40mmol/L Tris-HCl(pH8.5)转盐;DEAE Sepharose Fast Flow离子交换柱层析(16mm×200mm),流速20mL/min,20mmol/L Tris-HCl(pH8.5,含0.2mol/L NaCl)梯度洗脱25个柱体积,分段收集,100mL/瓶(组分),SEC-HPLC跟踪监测,合并206nm吸收峰为单一、对称峰的组分,10KD超滤膜超滤,加入纯化水反复超滤,至电导率稳定,收集回流液,冻干,得到脆弱拟杆菌提取物;
(4)称量30mg步骤(3)所述的脆弱拟杆菌提取物,溶于0.5mL D
2O,加入1μl丙酮(
1H,2.22;
13C,30.89)定标。采用500MHz Bruker核磁共振波谱仪分析
1H、
13C、COSY、HSQC、HMBC谱(参见图3A-3E),确证步骤(3)收集的脆弱拟杆菌提取物为荚膜多糖A,结合脂质含量低于0.02%,蛋白残留低于1%,核酸残留低于0.05%。通过GPC(凝胶渗透色谱)分析,制得的荚膜多糖A重均分子量为80-90KDa,Mw/Mn为1.0-1.2,化学结构参见图4。
实施例4:脆弱拟杆菌及其两性离子荚膜多糖(PSA)在体外通过巨噬细胞促进小鼠肺癌细胞系Lewis细胞株凋亡的药效实验
1、实验设计
本实施例通过向小鼠巨噬细胞系RAW264.7加入低、中、高剂量的脆弱拟杆菌ZY-312活菌液,高剂量的ZY-312灭活菌及低、中、高剂量的ZY-312的PSA进行孵育,使用Transwell共培养小室(Corning,3412)与Lewis细胞共培养在24h收取细胞采用流式细胞仪(Beckman Coulter)检测肿瘤细胞凋亡情况。以PBS作为对照。上述ZY-312的灭活菌液由实施例2方法制备,PSA参照实施例3方法制备,下同。
2、培养方法
(1)小鼠Lewis细胞和RAW264.7细胞的复苏和传代
小鼠肺癌细胞Lewis细胞和RAW264.7细胞均生长于含10%FBS的DMEM培养基中,加入1%青霉素/链霉素,37℃温度下,5%CO
2浓度,饱和湿度的培养箱培养。每两天更换一次培养基。
a)液氮中取出细胞株,在37℃恒温水浴锅中迅速融化。无菌条件下打开冻存管,转移液体至15mL的离心管中,用DMEM完全培养基2-3mL重悬细胞,1000rpm,离心5min。弃上清后,加入DMEM完全培养基5mL,将细胞接种于培养瓶,37℃温度下,5%CO
2浓度的培养箱培养48h。
b)弃去原培养液,加入1mL 0.25%胰蛋白酶,消化5min,加入1mL含10%胎牛血清的DMEM培养基终止反应,并移至15mL离心管中,1000rpm离心5分钟;弃上清液,加入适宜的含10%胎牛血清的DMEM完全培养基,将细胞分接于培养瓶,37℃温度下,5%CO
2浓度的培养箱培养24h。
(2)脆弱拟杆菌菌液培养
a)取脆弱拟杆菌ZY-312菌种,加入200μL TSB培养基,复溶,划板,37℃、厌氧培养48h。
b)挑取单菌落接入10mL TSB培养基,加入5%(v/v)血清,37℃、厌氧培养12h。
c)取1瓶500mL TSB培养基,加入5%(v/v)血清,接入1%(v/v)b)培养后的脆弱拟杆菌,37℃、厌氧培养48h。
d)取上述菌液,常规热灭活处理,得灭活菌液。
e)按照实施例3制备ZY-312的PSA。
3、脆弱拟杆菌通过RAW264.7巨噬细胞对Lewis细胞凋亡的影响
(1)将500μL的RAW264.7细胞(10
5)分别接种于6孔Transwell板下室中,分别加入10μL 10
6、10
8、10
10CFU/mL的脆弱拟杆菌活菌液ZY-312、ZY-312灭活菌液(10
10cell/mL)及0.02、0.2、2mg/mL ZY-312的PSA,进行预处理6h,随后分别加入接种Lewis细胞的上室。设置仅加入等量RAW264.7细胞的孔作为阴性对照,加入等量肿瘤细胞和顺铂(DDP)(15μM)(山东齐鲁制药有限公司)的孔作为阳性对照组,每组复孔3个。于37℃温度,5%CO
2培养箱培养。
(2)使用Annexin V凋亡检测试剂盒(诺唯赞,A211-01)在24h收集检测Lewis细胞的凋亡情况:
a)用不含EDTA的胰酶消化细胞,终止消化后收集细胞,1000rpm、4℃离心5min,弃上清。
b)洗涤细胞:用预冷的PBS洗涤细胞两次,每次均在1000rpm、4℃离心5min,弃上清。
c)细胞重悬:加入100μl的1×Binding Buffer,轻轻吹匀至单细胞悬液。
d)细胞染色:加入5μl Annexin V-FITC和5μl PI Staining Solution,轻轻吹匀;避光、室温(20~25℃)孵育10min;加入400μl的1×Binding Buffer,轻轻混匀。染色后样品在1h内用流式细胞仪检测。
e)细胞凋亡情况:计算Annexin V-FITC单阳性(Annexin V-FITC﹢/PI﹣)的早期凋亡细胞和Annexin V-FITC和PI双阳性(Annexin V-FITC﹢/PI﹢)的晚期凋亡细胞比例之和。
4、实验结果:
表1 Lewis细胞凋亡率(%)(均值±标准差,n=3)
注:与阳性对照组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
如表1所示,阴性组(仅加入RAW264.7细胞)凋亡率显著低于阳性对照。脆弱拟杆菌ZY-312及ZY-312灭活菌液和ZY-312PSA均能够通过RAW264.7细胞促进Lewis细胞凋亡。脆弱拟杆菌ZY-312组内的差异不显著,无论是活菌、灭活菌还是PSA组,Lewis细胞凋亡情况与阳性对照组相比均有统计学差异。
因此,脆弱拟杆菌活菌ZY-312、ZY-312灭活菌和ZY-312PSA均能够通过RAW264.7促进Lewis细胞凋亡。
实施例5:脆弱拟杆菌及其两性离子荚膜多糖治疗小鼠非小细胞肺癌移植瘤的药效试验
将处于对数生长期的小鼠肺癌细胞系Lewis细胞调整细胞浓度至2×10
4个/mL的单细胞悬液,无菌条件下用注射器以0.2mL/只的量将Lewis细胞接种于SPF级C57BL/6小鼠右腋窝皮下,制备Lewis肺癌瘤源小鼠。第11天复种一次,第21天正式实验时,选取生长良好的Lewis肺癌组织,无菌条件下,脱颈椎处死,从腋下剥取瘤体组织,剪碎、匀浆、研磨,按肿瘤质量(g)与生理盐水(mL)1:3比例制成浓度为1×10
7个/mL的瘤细胞悬液。Lewis肺癌瘤源小鼠细胞悬液以每只0.2mL左右接种于小鼠右前肢腋窝皮下进行造模。造模后一周开始给药,ZY-312低(10
6CFU/只)、高(10
10CFU/只)剂量组、ZY-312灭活菌(10
10cell/只)、 ZY-312PSA低(10mg/kg)和ZY-312PSA高(30mg/kg)连续灌胃3周;DDP(顺铂,山东齐鲁制药有限公司)组每周腹腔注射1次,连续注射5周。当实验结束或荷瘤鼠出现明显的消瘦、弓背、精神萎靡等衰竭体征时,脱颈椎处死,取移植肿瘤测定其重量并计算抑瘤率,取新鲜瘤体组织检测肿瘤微环境情况,取血清至于-80℃冰箱中冻存,用于检测细胞因子等。
1、Lewis细胞培养
Lewis细胞培养于含10%胎牛血清的DMEM培养基中,培养温度为37℃,气体环境为5%CO
2,湿度为饱和湿度;根据细胞生长的速度及培养液的颜色变化更换培养液,用0.25%的胰蛋白酶消化传代。根据细胞生长情况,将对数生长期的细胞制备成单细胞悬液,将细胞浓度调整至2×10
4个/mL。
2、Lewis肺癌瘤源小鼠的制备
取6~8周龄,体质量约(20±2)g,SPF级C57BL/6小鼠10只,将处于对数生长期的小鼠肺癌细胞系Lewis细胞调整细胞浓度至2×10
4个/mL的单细胞悬液,无菌条件下用注射器以0.2mL/只的量将Lewis细胞接种于小鼠右腋窝皮下,当皮下移植瘤长至直径约1cm左右时剥取移植瘤。
3、肺癌移植瘤模型的建立
选取生长良好的Lewis肺癌组织,无菌条件下,脱颈椎处死,从腋下剥取瘤体组织,剪碎、匀浆、研磨,按肿瘤质量(g)与生理盐水(mL)1:3比例制成浓度为1×10
6个/mL的瘤细胞悬液。在无菌条件下,将上述制备的细胞悬液,以每只0.2mL左右接种于小鼠右前肢腋窝皮下。
4、分组及给药
移植瘤模型建立1周后,将70只小鼠随机分为7组,每组10只:生理盐水组,DDP组,ZY-312低(10
6CFU/只)、高(10
10CFU/只)剂量组,ZY-312灭活菌组(10
10cell/只),ZY-312PSA低(10mg/kg)和ZY-312PSA高(30mg/kg)剂量组。
生理盐水组:0.2mL/只,每天1次,连续灌胃3周;
DDP组:按照0.3mL/次、3mg/kg的量进行腹腔注射,每周1次,连续注射3周;
ZY-312低、高剂量组和灭活菌组及ZY-312PSA低、高剂量组:按照0.2mL/只,每天1次,连续灌胃3周。
5、检测指标及方法
(1)肿瘤重量及抑瘤率
给药过程中如有小鼠出现明显的消瘦、弓背、精神萎靡等衰竭体征,及给药结束后,脱颈 椎处死,取移植肿瘤测其重量并计算抑瘤率,抑瘤率计算公式:抑瘤率(%)=(生理盐水组平均瘤重-给药组平均瘤重)/生理盐水组平均瘤重×100%;
(2)肿瘤内T细胞亚群
收集新鲜采集的肿瘤样本剪碎成小块,将剪碎的组织转移至装有消化酶混合液的C管中。组织消化、匀浆完成后,经70μm筛网过滤得到单细胞沉淀,对所得细胞进行计数,之后抽取1×10
6个活细胞进行抗体染色。使用流式细胞仪检测CD45
+(Biolegend,103151)、CD3
+CD8
+CD45
+(CD3抗体:BD,563024;CD8抗体:BD,563786)表达情况。
6、试验结果
(1)肿瘤重量及抑瘤率
表2基于分组给药后第21天肿瘤重量计算得出的抑瘤药效
注:
a.平均值±SD。
b.抑瘤率(%)=(生理盐水组平均瘤重-给药组平均瘤重)/生理盐水组平均瘤重×100%。
c.两组间p值按照unpaired t-test(two-tailed)方法计算。
由表2可知,与生理盐水组相比,各给药组的肿瘤重量均有所降低,DDP组具有显著性差异;脆弱拟杆菌及其荚膜多糖A各组与DDP组不具有显著性差异。说明在非小细胞肺癌移植瘤的小鼠模型中,脆弱拟杆菌ZY-312及其两性离子荚膜多糖能够有效抑制肿瘤生长,这种抑制效果与DDP相似。
(2)肿瘤组织微环境T细胞亚群
表3各组小鼠肿瘤微环境T细胞亚群(mean±SD)
注:与生理盐水组相比,*表示差异显著p<0.05;**表示差异极显著p<0.01。
CD8
+T细胞在肿瘤免疫中发挥重要作用,是直接杀伤肿瘤细胞的效应细胞。
由表3可知,与生理盐水组相比,DDP组小鼠CD8
+CD45
+T细胞水平下降;脆弱拟杆菌ZY-312低、高剂量,PSA低、高剂量组,以及灭活菌液组小鼠肿瘤中浸润的CD8
+CD45
+T细胞水平均显著上升。这表明实验动物经灌胃给予脆弱拟杆菌ZY-312及其两性离子荚膜多糖后肿瘤内淋巴细胞的浸润有所增加。
综上所述,脆弱拟杆菌ZY-312及其两性离子荚膜多糖能够调节小鼠肿瘤免疫微环境,有效治疗小鼠非小细胞肺癌移植瘤。
实施例6:脆弱拟杆菌及其两性离子荚膜多糖治疗小细胞肺癌移植瘤的药效试验
1、试验设计及流程
培养人小细胞肺癌细胞系NCI-H526,将处于对数生长期的NCI-H526细胞调整细胞浓度至2×10
7个/mL的单细胞悬液。无菌条件下,Matrigel与PBS按照1:1配比稀释并放于冰上待注射。NCI-H526细胞与Matrigel胶(BD,356234)稀释液1:1比例制成浓度为1×10
7个/mL的瘤细胞悬液。细胞悬液以每只0.2mL左右接种于4-5周龄,体重(22±2)g的裸鼠。裸鼠右前肢腋窝皮下进行造模。造模后一周开始给药,ZY-312低(10
6CFU/只)、高(10
10CFU/只)剂量组、ZY-312灭活菌(10
10cell/只)、ZY-312PSA低(10mg/kg)和ZY-312PSA高(30mg/kg)连续灌胃3周;DDP(顺铂,山东齐鲁制药有限公司)组每周腹腔注射1次,连续注射3周。当实验结束或荷瘤鼠出现明显的消瘦、弓背、精神萎靡等衰竭体征时,脱颈椎处死,取移植肿瘤测定其重量并计算抑瘤率,随后至于-80℃冰箱中冻存,用于检测细胞因子等。
检测指标:
(1)肿瘤重量和抑瘤率
抑瘤率%=100%×(生理盐水组肿瘤平均重量-给药组肿瘤平均重量)/生理盐水组肿瘤平均重量。
(2)细胞因子
采用Elisa检测肺癌移植瘤小鼠模型肿瘤中IL-12(R&D Systems,M1270,下同)、IFN-γ(R&D Systems,MIF00,下同)和IL-1β(R&D Systems,MLB00C,下同)等细胞因子的水平。
2、试验结果
(1)肿瘤重量和抑瘤率
表4基于分组给药后第21天肿瘤重量计算得出的抑瘤药效
注:
a.平均值±SD。
b.抑瘤率%=100×(生理盐水组肿瘤平均重量-给药组肿瘤平均重量)/生理盐水组肿瘤平均重量。
c.两组间p值按照unpaired t-test(two-tailed)方法计算。
由表4可知,与生理盐水组相比,各给药组肿瘤重量均有所下降,DDP组与脆弱拟杆菌及其PSA各组不具有显著性差异。说明在小细胞肺癌移植瘤的小鼠模型中,脆弱拟杆菌ZY-312及其PSA能够有效抑制肿瘤生长。
(2)细胞因子
表5各组小鼠肿瘤细胞因子水平(mean±SD)
注:与生理盐水组相比,*表示差异显著p<0.05;**表示差异极显著p<0.01。
IL-12来自活化的淋巴细胞,能够诱导CTL和NK细胞的细胞毒活性并促进其分泌IFN-γ、TNF-α等抗肿瘤细胞因子;IL-1β及其受体已被证明可以促进移植小鼠和人类的多种肿瘤生长和转移。
由表5可知,与生理盐水组相比,各给药组IL-12水平均显著上升,ZY-312活菌、灭活菌及PSA各组水平高于DDP组。
与生理盐水组相比,各给药组IFN-γ水平均有所上升;ZY-312活菌、灭活菌液及PSA各组 具有显著性,PSA组内具有不明显的剂量依赖性。
与生理盐水组相比,各给药组IL-1β水平均显著下降;ZY-312低、高剂量、灭活菌液及PSA高剂量组具有极显著差异,PSA低剂量组水平低于高剂量组。这说明脆弱拟杆菌及其PSA上调了抗肿瘤因子IL-12、IFN-γ的水平,抑制肿瘤促进因子IL-1β的表达。
综上所述,脆弱拟杆菌ZY-312及其PSA能够调节肿瘤相关免疫因子水平,有效治疗小鼠小细胞肺癌移植瘤。
实施例7:脆弱拟杆菌及其两性离子荚膜多糖治疗鼻咽癌移植和转移瘤的药效试验
1、试验设计及流程
使用含胎牛血清的RPMI-1640培养基培养人鼻咽癌细胞系5-8F,无菌条件下,将处于对数生长期的5-8F细胞调整细胞浓度至2×10
6个/mL的单细胞悬液,细胞悬液以每只0.2mL左右接种于5-7周龄,体重20-24g的雌性裸鼠。裸鼠接种肿瘤后一周,采用裸鼠尾静脉直接注射2×10
6个/mL的5-8F单细胞悬液0.2mL的方法给予干预。皮下接种肿瘤后一周开始给药,ZY-312低(10
6CFU/只)、高(10
10CFU/只)剂量组、ZY-312灭活菌(10
10cell/只)、ZY-312PSA低(10mg/kg)和ZY-312PSA高(30mg/kg)剂量组连续灌胃3周;DDP组(顺铂,山东齐鲁制药有限公司)组每周腹腔注射1次,连续注射3周。当实验结束或荷瘤鼠出现明显的消瘦、弓背、精神萎靡等衰竭体征时,脱颈椎处死,取移植肿瘤测定其重量并计算抑瘤率,取肝脏及肺组织,观察转移灶,瘤体组织置于-80℃冰箱中冻存,用于检测细胞因子等。
检测指标:
(1)移植瘤重量及抑瘤率
抑瘤率%=100%×(生理盐水组肿瘤平均重量-给药组肿瘤平均重量)/生理盐水组肿瘤平均重量。
(2)转移灶大小与数量
将肺及肝组织取出后先放置Bouin氏液(冰醋酸5mL+40%甲醛25mL+饱和苦味酸75mL)中固定,待两天后观察肺部转移灶,已显示为白色,而肝组织未见明显转移灶。在解剖显微镜下观察肺组织转移化的大小,并同时记录数目(按大小分级),对各组裸鼠肺转移结节的数目进行统计学分析。按照Ⅰ级转移灶为直径小于0.15mm,Ⅱ级转移灶为直径0.15~1mm,Ⅲ级转移灶为直径1~2mm,Ⅳ级转移灶为直径大于2mm。总转移灶数=Ⅰ级转移灶数+Ⅱ级转移化数*2+Ⅲ级转移灶数*3+Ⅳ级转移化数*4。
(3)细胞因子
采用Elisa检测肺癌移植瘤小鼠模型血清中IL-12、IFN-γ和IL-1β等细胞因子的水平。
2、实验结果
(1)移植瘤重量及抑瘤率
表6基于分组给药后第21天肿瘤重量计算得出的抑瘤药效
注:
a.平均值±SD。
b.抑瘤率%=100%×(生理盐水组肿瘤平均重量-给药组肿瘤平均重量)/生理盐水组肿瘤平均重量。
c.两组间p值按照unpaired t-test(two-tailed)方法计算。
由表6可知,与生理盐水组相比,各给药组的肿瘤重量均有所下降;脆弱拟杆菌ZY-312活菌、灭活菌及PSA各组与DDP组无明显差异。说明在鼻咽癌移植瘤的小鼠模型中,脆弱拟杆菌ZY-312及其PSA能够抑制肿瘤生长,这种抑制效果与DDP类似。
(2)转移灶大小与数量
表7各组小鼠总转移灶数(mean±SD)
注:与生理盐水组相比,*表示差异显著p<0.05;**表示差异极显著p<0.01。
由表7可知,与生理盐水组相比,各给药组的肿瘤转移灶数量均有所下降;DDP组、脆弱拟杆菌ZY-312高剂量组、灭活菌组及PSA高剂量组具有显著性差异。说明在小鼠鼻咽癌转移模型中,脆弱拟杆菌ZY-312及其PSA能够有效抑制肿瘤转移。
(3)细胞因子
表8各组小鼠血清细胞因子(mean±SD)
注:与生理盐水组相比,*表示差异显著p<0.05;**表示差异极显著p<0.01。
由表8可知,与生理盐水组相比,各给药组IL-12水平均显著上升;脆弱拟杆菌活菌、灭活菌及PSA高剂量组水平高于DDP组,PSA低剂量组水平与DDP组相似。
与生理盐水组相比,各给药组的IFN-γ的水平均有所上升;ZY-312活菌组、灭活菌液组及PSA各组与生理盐水组具有显著性差异,PSA组内具有不明显的剂量依赖性。
与生理盐水组相比,各给药组IL-1β水平均显著下降,脆弱拟杆菌ZY-312低、高剂量、灭活菌液及PSA高剂量组与生理盐水组具有极显著差异,PSA低剂量组水平低于DDP组。说明脆弱拟杆菌ZY-312及其荚膜多糖上调了抗肿瘤因子IL-12和IFN-γ的水平,抑制肿瘤促进因子IL-1β的表达。
综上所述,脆弱拟杆菌ZY-312及两性离子荚膜多糖能够调节肿瘤相关免疫因子水平,有效治疗小鼠鼻咽癌移植瘤和转移瘤。
当然,本发明还可有其它多种实施例,在不背离本发明精神及其实质的情况下,熟悉本领域的技术人员当可根据本发明作出各种相应的改变和变形,但这些相应的改变和变形都应属于本发明所附的权利要求的保护范围。
Claims (10)
- 一种脆弱拟杆菌和/或其两性离子荚膜多糖在制备预防和/或治疗呼吸系统肿瘤的产品中的应用,其特征在于,所述脆弱拟杆菌为保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312。
- 根据权利要求1所述的应用,其特征在于,所述脆弱拟杆菌是活菌、形态结构完整的灭活菌、形态结构不完整的灭活菌中的一种以上;和/或,所述脆弱拟杆菌是脆弱拟杆菌活菌体,经过灭活、基因重组、改造或修饰、减毒、化学处理、物理处理或灭活的脆弱拟杆菌,脆弱拟杆菌裂解物,脆弱拟杆菌液体培养上清液中的一种或多种。
- 根据权利要求1或2所述的应用,其特征在于,所述呼吸系统肿瘤包括头颈部癌、非小细胞肺癌、小细胞肺癌。优选地,所述头颈部癌还包括鼻咽癌、喉癌。
- 根据权利要求1-4任一项所述的应用,其特征在于,所述两性离子荚膜多糖的制备方法包括以下步骤:(1)将发酵培养后的脆弱拟杆菌菌液离心收集沉淀物,即得脆弱拟杆菌菌泥;取菌泥,加入菌泥质量3~10倍的纯化水使菌体重悬,用酸溶液调节其pH至2.0~4.5,50~120℃提取0.5~3.0h,冷却至室温,常温离心,取上清,得到粗糖溶液;(2)粗糖溶液经超滤膜超滤浓缩、除小分子杂质,至电导率稳定,收集回流液;(3)回流液中加入等体积40mmol/L Tris-HCl转盐;离子交换柱层析,梯度洗脱,分段收集,SEC-HPLC跟踪监测,合并206nm吸收峰为单一、对称峰的组分,超滤膜超滤,加入纯化水反复超滤,至电导率稳定,收集回流液,冻干,得到脆弱拟杆菌两性离子荚膜多糖。优选地,步骤(1)中所述离心为11000~13000g离心8~12min;优选地,步骤(1)中所述酸溶液可以是有机酸、无机酸和酸性缓冲液中的一种或多种;其中,无机酸可以是盐酸、硫酸、磷酸等;有机酸可以是乙酸、柠檬酸等;优选地,步骤(2)中所述超滤膜的分子量可以为100、50、30、10、5、3KD或者任意两个分子量值之间的范围;优选地,步骤(3)中所述离子交换柱为DEAE Sepharose Fast Flow的16mm×200mm,层析时的流速15~25mL/min,pH5.0~9.0含0.2mol/L NaCl 20mmol/L Tris-HCl梯度洗脱25个柱体积,分段收集,100mL/瓶(组分);所述超滤膜的分子量为10KD。
- 根据权利要求1-5任一项所述的应用,其特征在于,所述产品为食品或药品;优选地,所述药品为脆弱拟杆菌或其两性离子荚膜多糖单独应用、或脆弱拟杆菌和其两性离子荚膜多糖联合应用、或脆弱拟杆菌或其两性离子荚膜多糖分别与其他药物联用、或脆弱拟杆菌和其两性离子荚膜多糖一起与其他药物联用;优选地,所述其他药物为乳酸菌;进一步优选地,所述乳酸菌为ATCC 53103乳酸菌;优选地,所述药品为人用药或动物用药;优选地,所述药品的剂型包括丸剂、片剂、颗粒剂、胶囊、口服液或管饲制剂;优选地,所述食品为奶粉、干酪、凝乳、酸奶酪、冰激凌或发酵谷类食品;优选地,所述食品还可以是动物食品,比如饲料。
- 一种用于治疗呼吸系统肿瘤的药物组合物,其中,所述药物组合物含有药学有效剂量的保藏编号为CGMCC No.10685的脆弱拟杆菌和/或其两性离子荚膜多糖。
- 根据权利要求7所述的药物组合物,其特征在于,所述脆弱拟杆菌的药学有效剂量为10 6-10 10CFU;和/或,所述两性离子荚膜多糖的药学有效剂量为1-30mg/kg。
- 根据权利要求7所述的药物组合物,其特征在于,所述两性离子荚膜多糖含夹膜多糖A,所述夹膜多糖A的重均分子量为80-90kD;优选地,所述两性离子荚膜多糖中夹膜多糖A的含量超过95wt%。
- 根据权利要求7-9任一项所述的药物组合物,其特征在于,所述两性离子荚膜多糖的制备方法包括以下步骤:(1)将发酵培养后的脆弱拟杆菌菌液离心收集沉淀物,即得脆弱拟杆菌菌泥;取菌泥,加 入菌泥质量3~10倍的纯化水使菌体重悬,用酸溶液调节其pH至2.0~4.5,50~120℃提取0.5~3.0h,冷却至室温,常温离心,取上清,得到粗糖溶液;(2)粗糖溶液经超滤膜超滤浓缩、除小分子杂质,至电导率稳定,收集回流液;(3)回流液中加入等体积40mmol/L Tris-HCl转盐;离子交换柱层析,梯度洗脱,分段收集,SEC-HPLC跟踪监测,合并206nm吸收峰为单一、对称峰的组分,超滤膜超滤,加入纯化水反复超滤,至电导率稳定,收集回流液,冻干,得到脆弱拟杆菌提取物。优选地,步骤(1)中所述离心为11000~13000g离心8~12min;优选地,步骤(1)中所述酸溶液可以是有机酸、无机酸和酸性缓冲液中的一种或多种;其中,无机酸可以是盐酸、硫酸、磷酸等;有机酸可以是乙酸、柠檬酸等;优选地,步骤(2)中所述超滤膜的分子量可以为100、50、30、10、5、3KD或者任意两个分子量值之间的范围;优选地,步骤(3)中所述离子交换柱为DEAE Sepharose Fast Flow的16mm×200mm,层析时的流速15~25mL/min,pH5.0~9.0含0.2mol/L NaCl 20mmol/L Tris-HCl梯度洗脱25个柱体积,分段收集,100mL/瓶(组分);所述超滤膜的分子量为10KD。
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