WO2023134200A1 - 脆弱拟杆菌或其两性离子荚膜多糖在制备防治消化系统肿瘤药物中的应用 - Google Patents
脆弱拟杆菌或其两性离子荚膜多糖在制备防治消化系统肿瘤药物中的应用 Download PDFInfo
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- bacteroides fragilis
- capsular polysaccharide
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Images
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Definitions
- the invention requires the patent application number 202210032994.X submitted to the State Intellectual Property Office of China on January 12, 2022, and the invention name is "Application of Bacteroides fragilis or its zwitterionic capsular polysaccharide in the preparation of drugs for preventing and treating digestive system tumors" Priority of earlier application. The entirety of this prior application is incorporated herein by reference.
- the invention relates to the field of biomedicine, in particular to the application of Bacteroides fragilis or its zwitterionic capsular polysaccharide in the preparation of drugs for preventing and treating digestive system tumors.
- Systemic drug therapy mainly includes chemotherapy drugs, molecular targeted drugs and immunotherapy drugs.
- chemotherapeutic drugs focus on cytotoxic aggressive drugs. Although these drugs are constantly updated, their properties are still drugs that cannot distinguish tumor cells from normal cells. Fatal, such as acute respiratory distress syndrome (ARDS) caused by bleomycin.
- ARDS acute respiratory distress syndrome
- Molecular-targeted drugs will specifically select oncogenic molecules to act in the body, so that the tumor can be programmed to reverse, or indirectly target the tumor angiogenesis, so that the tumor cells undergo ischemic apoptosis and necrosis without spreading to the surrounding tumor of normal tissue cells.
- molecularly targeted anti-tumor drugs have good specificity and high efficiency, and when exerting anti-tumor effects, they have less impact on normal organs of the human body and lower toxicity, which can alleviate the pain of treatment to a certain extent, and achieve good therapeutic effects.
- molecular targeted drugs include skin toxicity, cardiovascular toxicity, liver toxicity, and pulmonary toxicity. Among them, the mechanism of cardiovascular toxicity is complex, and once it occurs, it may cause serious injury to patients.
- Immunotherapy drugs include traditional antibody drugs targeting tumor tissue-specific antigens and emerging immune checkpoint inhibitors.
- the mechanism of action of traditional antibody drugs is to block tumor growth factor signaling pathways by targeting tumor cell surface antigens, or inhibit the formation of new blood vessels in the tumor microenvironment.
- Immune checkpoints are signaling factors necessary for the activation of effector cells in vivo. Immune checkpoint inhibitors can initiate the body's cellular immune therapy for malignant tumors by antagonizing co-inhibitory factors or activating co-stimulatory factors. Due to the large clinical dose (5-10mg/kg) of antibody drugs, the administration method is mostly through intravenous infusion, so the most common adverse drug reaction in clinical practice is "acute infusion reaction" caused by drug immunogenicity.
- antibody drugs will also have "on-target” toxicity in clinical practice.
- Agonistic antibodies targeting T cell co-stimulator receptors can also cause severe "cytokine storm” in clinical practice because they can fully activate T cells to release inflammatory factors; immune checkpoint proteins inhibit T cells by binding to their ligands Activation, maintaining the immune tolerance of normal tissues of the body.
- Antibody drugs targeting immune checkpoint proteins can also cause immune-related adverse events (Immune-related adverse events) in patients while activating immune cells, and the severity of adverse reactions is often positively correlated with the therapeutic effect, such as: Ipilimumab in Mild to moderate adverse reactions often occurred during the administration process, and some patients also experienced persistent diarrhea and fatal epidermal necrolysis.
- Ipilimumab in Mild to moderate adverse reactions often occurred during the administration process
- Some patients also experienced persistent diarrhea and fatal epidermal necrolysis.
- the research and development of anti-tumor drugs with better effects and less adverse reactions has always been a hot spot in drug research and development.
- Bacteroides fragilis (B. fragilis) is Gram-negative, rod-shaped, blunt-rounded at both ends and densely stained, capsulated, non-spore-free, non-motive obligate anaerobic bacteria, divided into enterotoxigenic bacteria Enterotoxigenic type (ETBF) and non-enterotoxigenic type (NTBF), which are part of the normal intestinal flora of humans and animals, mainly exist in the colon, and can also colonize and grow in the mucous membranes of the respiratory tract, gastrointestinal tract, and urogenital tract.
- EBF enterotoxigenic type
- NTBF non-enterotoxigenic type
- NTBF non-enterotoxigenic Bacteroides fragilis
- B.fragilis CPS can inhibit IL-1 ⁇ -mediated IL-8 type infantile necrotizing colitis.
- Numerous studies have shown that non-enterotoxigenic Bacteroides fragilis and its zwitterionic capsular polysaccharides have good curative effects on the prevention and treatment of acute and chronic enteritis and the regulation of the development of the host immune system.
- the object of the present invention is to provide an application of Bacteroides fragilis and its zwitterionic capsular polysaccharide in the prevention and treatment of digestive 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. Cancer cell apoptosis, effective prevention and treatment of digestive system tumors.
- Bacteroides fragilis and/or its zwitterionic capsular polysaccharide in the preparation of products for preventing and treating digestive system tumors, said Bacteroides fragilis is Bacteroides fragilis ZY- 312.
- 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 digestive system tumors include gastric cancer, colon cancer, esophageal cancer, pancreatic cancer, and liver cancer.
- the zwitterionic capsular polysaccharide comprises capsular polysaccharide A.
- the structure of the capsular polysaccharide A is as follows:
- the weight average molecular weight (Mw) of the capsular polysaccharide A is 80-90kD, and the part with Mw distributed between 70KD-100KD accounts for 70-80% of the total amount, and the weight average molecular weight/number average molecular weight ( The ratio of Mw/Mn) is 1.0-1.3.
- the content of 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 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, segmental collection, 100mL/bottle (component);
- the ultrafiltration membrane is 10KD.
- the product is food or 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.
- 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.
- compositions for preventing and treating tumors of the digestive system wherein the composition contains Bacteroides fragilis and/or its zwitterionic capsular polysaccharide with the preservation number CGMCC No. 10685.
- 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 structure of the capsular polysaccharide A is as follows:
- the weight average molecular weight of the capsular polysaccharide A is 80-90kD, and the part with Mw distributed in 70-100KD accounts for 70-80% of the total amount, and the weight average molecular weight/number average molecular weight (Mw/Mn ) ratio is 1.0-1.3.
- the content of the capsular polysaccharide A 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 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, segmental collection, 100mL/bottle (component);
- the ultrafiltration membrane is 10KD.
- the composition is a drug.
- the dosage form of the drug includes pills, tablets, granules, capsules, oral liquids or tube feeding preparations.
- the medicine includes human medicine or animal medicine.
- the drug is Bacteroides fragilis or its zwitterionic capsular polysaccharide used alone, or Bacteroides fragilis and its zwitterionic capsulated polysaccharide used in combination.
- the drug can be administered orally, enemaly or parenterally.
- the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.
- Bacteroides fragilis especially Bacteroides fragilis ZY-312 with the preservation number of CGMCC No. 10685 and its zwitterionic capsular polysaccharide can reduce inflammation in vivo, protect normal tissues, and improve the body's anti-tumor immune response , effectively prevent and treat digestive system tumors.
- 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 the Bacteroides fragilis ZY-312 of embodiment 1;
- Fig. 2 is the microscopic observation figure after Gram staining of Bacteroides fragilis ZY-312 of embodiment 1;
- Fig. 3 is the capsular polysaccharide A NMR spectrometer analysis figure of embodiment 2;
- A-E are the 1H spectrum, 13C spectrum, COZY spectrum, HSQC spectrum, HMBC spectrum analyzed by the capsular polysaccharide A nuclear magnetic resonance spectrometer of embodiment 2 respectively;
- Fig. 4 is the chemical structural formula of the structural unit of the capsular polysaccharide A of Bacteroides fragilis prepared in Example 2.
- 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 raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. All cells were purchased from ATCC; all cell culture materials were purchased from Gibco; all experimental animals were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. 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.
- the application of the Bacteroides fragilis and/or its zwitterionic capsular polysaccharide of the present invention in the preparation of a product for preventing and treating digestive system tumors, wherein the product can be medicine or food.
- the Bacteroides fragilis is Bacteroides fragilis ZY-312 with the preservation number CGMCC No.10685, the zwitterionic capsular polysaccharide is extracted from the Bacteroides fragilis ZY-312, and the zwitterionic capsular polysaccharide contains capsular polysaccharide A, the molecular weight of the capsular polysaccharide A is 80-90kD, Mw/Mn is 1.0-1.3, and the part with Mw distributed in 70-100KD accounts for 70-80% of the total; the content of the capsular polysaccharide A exceeds 95wt %.
- the Bacteroides fragilis can be one or more of live bacteria, inactivated bacteria with complete morphology and structure, and inactivated bacteria with incomplete
- the medicine for preventing and treating digestive system tumors of the present invention 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 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.
- Embodiment 1 the fermentation culture of Bacteroides fragilis
- 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.
- 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 weight average molecular weight of the prepared capsular polysaccharide A is 80-90kDa, Mw/Mn is 1.0-1.3, and the part with Mw distributed in 70KD-100KD accounts for 70-80% of the total; See Figure 4 for the chemical structure. In the following examples, it is referred to as "ZY-312 PSA".
- Example 3 Bacteroides fragilis and its capsular polysaccharide prevent orthotopic transplantation of MFC gastric cancer in mice
- mice of 615 inbred strain were randomly divided into 9 groups according to body weight interval, namely blank group, model group, 5-fluorouracil group (5-FU, Sigma Company, 20mg/kg), ZY-312 Viable bacteria low (10 6 CFU/monkey), medium (10 8 CFU/bird), high (10 10 CFU/bird) group, ZY-312 inactivated bacteria group (10 10 cell/bird), ZY-312 PSA group (1mg/kg), ZY-312+PSA (10 10 CFU/monkey, 1mg/kg) group, 10 in each group.
- body weight interval namely blank group, model group, 5-fluorouracil group (5-FU, Sigma Company, 20mg/kg), ZY-312 Viable bacteria low (10 6 CFU/monkey), medium (10 8 CFU/bird), high (10 10 CFU/bird) group, ZY-312 inactivated bacteria group (10 10 cell/bird), ZY-312 PSA group (1mg/kg), ZY-312+PSA (10 10 CFU/monkey, 1mg/kg)
- the tumor source was cut into small tumor pieces of about 1 mm 3 , implanted into the gastric tissue of mice in the model group and each drug-administered group, the stomach was solidified with OB gel, and the stomach was reset, and then the abdomen was closed layer by layer. Observed day by day, the appearance of a palpable mass is the sign of tumor formation. Mice in the blank group underwent the same laparotomy, but no tumor mass was implanted.
- mice in the blank group and model group were orally administered 0.2 mL of normal saline once a day; the 5-FU group was intraperitoneally injected with 5-FU once every two days according to body weight; each administration group was orally administered once a day. Corresponding dose of drug. A total of 12 days of administration. 24 hours after the last administration, all mice were plucked from the eyes to get blood, and then euthanized, and the tumor tissue, spleen and thymus were isolated under aseptic conditions and weighed. Tumors were divided into two parts, one for cryopreservation and one for immunohistochemical analysis.
- Serum cytokine detection Luminex technology detects the levels of IL-2, INF- ⁇ and TNF- ⁇ in mouse serum.
- SPSS statistical software 25.0 was used for statistical analysis.
- the model group had obvious tumor masses, and the modeling was successful.
- the tumor weights of each administration group decreased; there were significant differences between the 5-FU group and the high-dose Bacteroides fragilis group.
- the spleen and thymus indexes in the model group increased, and 5-FU down-regulated the spleen and thymus indexes of the mice, which may be due to the immunosuppressive toxicity of chemotherapy drugs; while ZY-312 and its PSA up-regulated the organ indexes, It shows that ZY-312 and its PSA can maintain and improve immune function.
- IFN- ⁇ , IL-2 and TNF- ⁇ are all known anti-tumor immune cytokines. Compared with the blank group, the levels of IFN- ⁇ , IL-2 and TNF- ⁇ in the model group were significantly increased; while compared with the model group, 5-FU down-regulated the expression levels of these three immune factors, and ZY-312 and its PSA up-regulated The expression levels of the above-mentioned immune factors improve the body's anti-tumor immune response.
- Bacteroides fragilis ZY-312 and its capsular polysaccharide can effectively prevent and treat gastric cancer by maintaining and enhancing the body's immunity.
- mice were given intraperitoneal injection of azomethane (AOM was purchased from Sigma, USA) at 10 mg/kg on Day 0; day 7 began to freely drink 2% dextran sodium sulfate (DSS was purchased from Sweden). TdB company), until the end of day14; day28-day35 and day49-day56 drink 2% DSS again.
- AOM azomethane
- DSS dextran sodium sulfate
- the blank group and the model group were intragastrically administered with 0.2 mL of normal saline once a day, the 5-FU group was intraperitoneally injected once a week, and the rest of the drug groups were intragastrically administered with corresponding doses of drugs once a day.
- mice On Day 91, all mice were euthanized, the colon was dissected under aseptic conditions, and the weight and size of the tumor were recorded; the colon tissue with the tumor was divided into three parts, one part was frozen in liquid nitrogen, one part was used for cytokine detection, and one part was used for cytokine detection. Parts were fixed with 10% neutral formaldehyde solution, routinely dehydrated, embedded in paraffin, sectioned, stained with eosin-hematoxylin (HE), and examined under a microscope.
- HE eosin-hematoxylin
- Cytokine detection Luminex technology was used to detect the contents of IL-10 and IL-17 in mouse colon tissue.
- Histopathology Microscopic examination after HE staining, observation of lymph, crypt structure and inflammatory changes, according to no/light/moderate/severe scoring, respectively 0/1/2/3.
- SPSS statistical software 25.0 was used for statistical analysis.
- the model group had obvious tumor mass, and the modeling was successful.
- IL-6 and IL-17 are pro-inflammatory factors, and IL-10 is an anti-inflammatory factor.
- IL-6 and IL-17 in the model group increased significantly, while the level of IL-10 decreased significantly.
- 5-FU down-regulated the levels of IL-6 and IL-17, but not significantly; ZY-312 and its PSA significantly down-regulated the level of IL-6, and the down-regulation range of IL-17 was greater than 5-FU.
- the levels of IL-10 in each administration group were significantly higher than those in the model group, but the levels of ZY-312 and its PSA were higher than those of 5-FU. This shows that ZY-312 and its PSA can reduce colonic inflammation.
- the histopathological score of the model group increased significantly.
- 5-FU down-regulated the histopathological score of the mouse colon, but it was not significant;
- ZY-312 and its PSA also down-regulated the histopathological score of the mouse colon, and had a positive effect on lymphoid hyperplasia, local invasion, and structural changes in crypts.
- Significant which shows that ZY-312 and its PSA have protective effects on tissues.
- B. fragilis ZY-312 and its capsular polysaccharide can inhibit tumor growth, reduce colonic inflammation, protect colonic tissue, and effectively prevent and treat colorectal cancer.
- mice 72 6-8 week-old ICR male mice were randomly divided into 9 groups according to body weight, 8 in each group: blank group, model group (normal saline), cisplatin group (DDP, Shandong Qilu Pharmaceutical Co., Ltd., 2mg/kg ), ZY-312 live bacteria low (10 6 CFU/bird), medium (10 8 CFU/bird), high dose group (10 10 CFU/bird).
- ZY-312 inactivated bacteria group (10 10 cells/monkey
- ZY-312 PSA group (1 mg/kg
- ZY-312+PSA 10 CFU/monkey, 1 mg/kg
- Eca109 esophageal cancer cells were cultured in DMEM medium containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL) under conventional conditions (37°C, saturated humidity, 5% CO 2 ) to the right During the growth phase, the cell concentration was adjusted to 1 ⁇ 10 7 cells/mL. Except for the blank group, 0.2 mL of cell suspension was subcutaneously injected into the armpit of each mouse, and 0.2 mL of normal saline was subcutaneously injected into the armpit of the blank group.
- Treatment started 24 hours after seeding the cells.
- the blank group and the model group were intragastrically administered 0.2 mL of normal saline once a day; the DDP group was intraperitoneally injected once every two days; except the DDP group, the other drug groups were intragastrically administered the corresponding drugs once a day. Continuous administration for 14 days.
- mice 24 hours after the last administration, all the mice were plucked from the eyeballs to collect blood, and then euthanized.
- the tumor tissue and spleen were isolated under aseptic conditions and weighed. Tumors were divided into two parts, one for cryopreservation and one for immunohistochemical analysis.
- Serum cytokine detection Luminex technology detects the levels of IL-2, INF- ⁇ and TNF- ⁇ in mouse serum.
- SPSS statistical software 25.0 was used for statistical analysis.
- the model group had obvious tumor masses, and the modeling was successful.
- the tumor weights of all groups decreased; the tumor weights of the DDP group and the ZY-312 combined with PSA group decreased significantly. This shows that B. fragilis and its PSA can effectively inhibit tumor growth; this effect is further enhanced when B. fragilis ZY-312 and its capsular polysaccharide are co-administered.
- the spleen index in the model group increased, and DDP down-regulated the spleen index of mice, which may be due to the immunosuppressive toxicity of chemotherapy drugs; while ZY-312 and its PSA up-regulated the organ index, indicating that ZY-312 and its PSA can maintain and improve immune function.
- the levels of IFN- ⁇ , IL-2 and TNF- ⁇ in the model group increased significantly; compared with the model group, DDP down-regulated the expression levels of these three immune factors, and ZY-312 and its PSA up-regulated the expression levels of IFN- ⁇ .
- the expression levels of ⁇ and IL-2 significantly up-regulated the expression level of TNF- ⁇ and improved the body's anti-tumor immune response.
- Bacteroides fragilis and its capsular polysaccharide can promote anti-tumor immune response and effectively prevent and treat esophageal cancer.
- Example 6 Experimental exploration of Bacteroides fragilis and its capsular polysaccharides preventing and treating transplanted tumors of mouse MPC-83 pancreatic cancer
- MPC-83 pancreas was cultured in RPMI-1640 medium containing 10% calf serum, penicillin (100 U/mL) and streptomycin (100 U/mL) under conventional conditions (37°C, saturated humidity, 5% CO 2 ).
- the cell concentration was adjusted to 5 ⁇ 10 7 cells/mL.
- 0.2 mL of cell suspension was subcutaneously injected into the back of each mouse, and 0.2 mL of normal saline was subcutaneously injected into the back of the blank group.
- mice 24 hours after the last administration, all mice were plucked from the eyeballs to collect blood, and then euthanized, and the tumor tissues were isolated and weighed under aseptic conditions. Tumors were divided into two parts, one for cryopreservation and one for immunohistochemical analysis.
- Serum cytokine detection Luminex technology detects the content of TGF- ⁇ 1 and VEGF in mouse serum.
- SPSS statistical software 25.0 was used for statistical analysis.
- the model group had obvious tumor mass, and the modeling was successful.
- TGF- ⁇ 1 and VEGF are inhibitory cytokines that regulate T cell function in the body.
- the levels of TGF- ⁇ 1 and VEGF in the model group were significantly increased; compared with the model group, 5-FU down-regulated the levels of TGF- ⁇ 1 and VEGF, and ZY-312 and its PSA also down-regulated the levels of the above cytokines.
- the level of regulation of VEGF is equivalent to that of 5-FU.
- ZY-312 and its PSA can improve the body's anti-tumor response by down-regulating immunosuppressive factors.
- Bacteroides fragilis ZY-312 and its capsular polysaccharide can promote the body's anti-tumor immune response, and can effectively prevent and treat pancreatic cancer.
- Example 7 Experimental exploration of Bacteroides fragilis and its capsular polysaccharides preventing and treating H22 liver cancer xenografts in mice
- mice 72 5-6 week-old ICR male mice were randomly divided into 9 groups according to body weight, 8 in each group: blank group, model group (normal saline), cyclophosphamide group (CTX, Baxter Oncology GmbH company, 30mg/kg ), ZY-312 live bacteria low (10 6 CFU/bird), medium (10 8 CFU/bird), high dose group (10 10 CFU/bird).
- CTX cyclophosphamide group
- medium 10 8 CFU/bird
- high dose group 10 CFU/bird
- ZY-312 inactivated bacteria group (10 10 cells/monkey
- ZY-312 PSA group (1 mg/kg
- ZY-312+PSA 10 CFU/monkey, 1 mg/kg
- H22 ascites-type liver cancer was cultured in RPMI-1640 medium containing 10% calf serum, penicillin (100 U/mL) and streptomycin (100 U/mL) under conventional conditions (37°C, saturated humidity, 5% CO 2 ) When the cells reached the logarithmic growth phase, the cell concentration was adjusted to 5 ⁇ 10 6 cells/mL. Except for the blank group, 0.2 mL of cell suspension was subcutaneously injected into the right shoulder of each mouse in each group, and 0.2 mL of normal saline was subcutaneously injected into the right shoulder of the blank group.
- Treatment started the day after inoculation.
- the blank group and the model group were intragastrically administered 0.2 mL of normal saline once a day; the cyclophosphamide (CTX) group was intraperitoneally injected once a day; except the CTX group, the other drug groups were intragastrically administered the corresponding drugs once a day. Continuous administration for 15 days.
- CTX cyclophosphamide
- mice After the last administration, all mice were fasted without food and water. After 16 hours, all mice were plucked from the eyeballs to collect blood, and then euthanized. The tumor tissue, spleen, thymus, and liver were isolated and weighed under aseptic conditions. Tumors were divided into two parts, one for cryopreservation and one for immunohistochemical analysis.
- liver function test The microplate method was used to detect the liver function indexes in the serum.
- Serum cytokine detection Luminex technology detects the levels of IL-2, IFN- ⁇ , and TNF- ⁇ in mouse serum.
- SPSS statistical software 25.0 was used for statistical analysis.
- the model group had obvious tumor masses, and the modeling was successful.
- the tumor weights of all groups decreased; the tumor weights of the CTX, ZY-312 high-dose and ZY-312 combined with PSA groups decreased significantly. This shows that Bacteroides fragilis and its PSA can effectively inhibit tumor growth, and this tumor inhibitory effect is further enhanced when B. fragilis ZY-312 and its capsular polysaccharide are co-administered.
- the spleen and thymus indexes of the model group increased; compared with the model group, CTX down-regulated the spleen and thymus indexes of mice, which may be due to the immunosuppressive toxicity of chemotherapy drugs; while compared with CTX, ZY-312 and its PSA up-regulated organ index, indicating that ZY-312 and its PSA can maintain and improve immune function.
- mice in each group Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of mice in each group (mean ⁇ SD)
- ALT and AST are marker proteins for evaluating the degree of liver damage.
- ALT and AST in the model group increased significantly, showing that liver cancer cells have damaged normal liver tissues.
- CTX aggravated the damage;
- ZY-312 and its PSA down-regulated the levels of ALT and AST. This indicates that ZY-312 and its PSA can reduce the damage of cancer cells to the liver.
- mice in each group (mean ⁇ SD)
- the levels of IFN- ⁇ , IL-2 and TNF- ⁇ in the model group were significantly increased; compared with the model group, CTX down-regulated the expression levels of these three immune factors, and ZY-312 and its PSA significantly up-regulated the expression levels of IFN - ⁇ and TNF- ⁇ expression levels, up-regulate the expression level of IL-2, and improve the body's anti-tumor immune response.
- Bacteroides fragilis ZY-312 and its capsular polysaccharide can protect normal liver tissue, maintain and improve the body's anti-tumor immune response, and effectively prevent and treat liver cancer.
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Abstract
一种脆弱拟杆菌和/或其两性离子荚膜多糖在制备防治消化系统肿瘤药物中的应用。通过大量实验证明,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能够减轻炎症反应,保护正常组织,提高机体抗肿瘤免疫反应,有效防治消化系统肿瘤。
Description
本发明要求2022年01月12日向中国国家知识产权局提交的专利申请号为202210032994.X,发明名称为“脆弱拟杆菌或其两性离子荚膜多糖在制备防治消化系统肿瘤药物中的应用”的在先申请的优先权。该件在先申请的全文通过引用的方式结合于本发明中。
本发明涉及生物医药领域,具体涉及一种脆弱拟杆菌或其两性离子荚膜多糖在制备防治消化系统肿瘤药物中的应用。
据世界卫生组织2018年全球癌症统计,全球最常见的十种癌症中有一半是消化系统肿瘤,其中结直肠癌发病率和死亡率分别高居所有恶性肿瘤中的第三位和第二位,消化系统肿瘤和肝癌的死亡率紧随其后,Chen等2014年对中国癌症发病率和死亡率的研究表明,消化系统肿瘤、结直肠癌、肝癌的发病率均排在国内恶性肿瘤发病率的前五位,且肝癌、消化系统肿瘤、食管癌、结直肠癌、胰腺癌的死亡率紧跟致死率第一的肺癌之后。由此可见,消化系统恶性肿瘤已成为导致国内外癌症患者死亡的重要原因。随着人口老龄化、环境污染、食品安全和不健康生活方式等问题的加剧,消化系统肿瘤的发病率和死亡率仍有上升趋势。
目前,彻底的手术切除是治愈消化系统肿瘤的唯一手段。对于无手术根治机会或转移性肿瘤患者,目前应采取以全身药物治疗为主的综合治疗。全身药物治疗主要包括化疗药物、分子靶向药物及免疫治疗药物。
传统的化疗药物集中在细胞毒性攻击性药物上,尽管这类药物不断更新换代,但其性质仍然属于不能分辨肿瘤细胞和正常细胞的药物,其副作用遍及全身各个系统,轻重不一,严重时可致命,如博来霉素导致的急性呼吸窘迫综合征(ARDS)。
分子靶向药物在体内会特异性地选择致癌分子发生作用,使肿瘤发生程序化逆转,或间接靶向肿瘤心生血管,使肿瘤细胞发生缺血性凋亡、坏死,而不会波及肿瘤周围的正常组织细胞。与传统的化疗药物比较,分子靶向抗肿瘤药物具有良好的特异性和高效性,并且在发挥抗肿瘤作用时,对人体正常器官影响较小,毒性较低,可一定程度减轻治疗痛苦,并且实现良好的治疗效果。但是随着分子靶向药物的快速发展,药物种类多样化,呈现出的不良反应也逐渐增多,成为分子靶向药物发展和临床应用的主要影响因素。分子靶向药物常见的毒性包括皮肤毒性、心血管毒性、肝毒性和肺毒性,其中,心血管毒性作用机制复杂,一旦发生,可能造成患者的严重损伤。
免疫治疗药物包括传统的靶向肿瘤组织特异性抗原的抗体药物和新兴的免疫检查点抑制剂。传 统抗体药物作用机制为通过靶向肿瘤细胞表面抗原,阻断肿瘤生长因子信号通路,或遏制肿瘤微环境中新生血管的生成。免疫检查点是体内效应细胞激活所必须的信号因子,免疫检查点抑制剂通过拮抗共抑制因子或激活共刺激因子,可以启动机体的细胞免疫治疗恶性肿瘤。抗体药物由于临床剂量大(5-10mg/kg),给药方式多通过静脉滴注,因此临床上最为常见的药物不良反应是因药物免疫原性引起的“急性输液反应”。此外,由于药物靶点在正常组织上的非特异性表达,抗体药物还会在临床上出现“On-target”毒性。靶向T细胞共刺激因子受体的激动型抗体,由于可以充分激活T细胞释放炎症因子,在临床上也会引起严重的“细胞因子风暴”;免疫检查点蛋白通过与其配基结合抑制T细胞活化,维持着机体正常组织的免疫耐受。靶向免疫检查点蛋白的抗体药物在激活免疫细胞的同时,也会导致患者出现免疫相关不良反应(Immune-related adverse events),而且不良反应的严重程度往往和治疗效果正相关,如:Ipilimumab在给药过程中常出现轻度到中度不良反应,也有部分患者出现持续腹泻、致命的表皮坏死松解症等。研发效果更佳、不良反应更轻的抗肿瘤药物始终是药物研发的热点。
人体内共生着多种微生物。已有的研究表明,消化系统肿瘤与消化道微生物具有密切关系。在临床试验中,将婴儿双歧杆菌、嗜酸乳杆菌、粪肠球菌和蜡样芽胞杆菌的组合物用于减少胃切除引起的生理障碍,显著降低了白细胞,增加淋巴细胞增强免疫。益生菌组合物明显增加了拟杆菌属、粪杆菌属和阿克曼氏菌等益生菌的数量,并降低了链球菌的丰度。另外一项临床试验则表明嗜酸乳杆菌、植物乳杆菌、乳双歧杆菌和布拉氏酵母菌的组合制剂,显著降低了结直肠癌手术后的并发症风险。此外,临床研究也表明益生菌能明显改善消化系统肿瘤术后患者的炎症,降低IL-6、IL-8和TNF-α细胞因子的水平。
脆弱拟杆菌(Bacteroides fragilis,B.fragilis)为革兰氏染色阴性、杆状、两端钝圆而浓染、有荚膜、无芽胞、无动力的专性厌氧细菌,分为产肠毒素型(ETBF)和非产肠毒素型(NTBF),是人及动物肠道正常菌群的一部分,主要存在于结肠中,呼吸道、胃肠道及泌尿生殖道粘膜也可定植生长。研究发现非产肠毒型脆弱拟杆菌(NTBF)具有重要的益生作用,能够调控T细胞扩张和产生阻碍致病性Th17细胞发育的细胞因子IL-10,具有抗炎作用,被认为是具有潜力的新一代益生菌。已有多项研究表明NTBF能够分泌抑炎细胞因子IL-10,促进Th1/Th2细胞的平衡,抵抗肠道炎症,对葡聚糖硫酸钠DSS诱导的结肠炎具有治疗作用。脆弱拟杆菌与宿主的关系在很大程度上取决于其高度复杂和动态的荚膜结构,B.fragilis两性离子荚膜多糖(capsular polysaccharide,CPS)是首个公认的调节宿主免疫系统的发育,逆转无菌动物的形态、细胞和功能缺陷的共生因子。B.fragilis在小鼠肠道粘膜上的定植需要CPS介导抑制Th-17细胞的活性。Mazmanian等人证明CPS是B.fragilis修复结肠炎的关键物质,定植野生型B.fragilis或灌胃纯B.fragilis CPS的小鼠能避免幽门螺杆菌诱导后形成肠炎;相反,定植了CPS突变型的B.fragilis小鼠却不能免于形成肠炎。还有研究表明B.fragilis CPS能与CD4淋巴细胞上的TLR2受体分子相互作用,刺激调节型T细胞产生 IL-10,能保护机体免受由IL-17诱导形成的肠炎。在TLR2和TLR4受体协同作用下,B.fragilis CPS能抑制IL-1β介导的IL-8型婴儿坏死性结肠炎。众多研究表明,非产肠毒型脆弱拟杆菌及其两性离子荚膜多糖对防治急慢性肠炎、调节宿主免疫系统的发育等具有较好疗效。
但是目前尚未见有关脆弱拟杆菌或其两性离子荚膜多糖做为微生态制剂用于治疗消化系统肿瘤的报道。
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防治消化系统肿瘤中的应用。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能够减轻炎症反应,保护正常组织,提高机体抗肿瘤免疫反应,促进癌细胞凋亡,有效防治消化系统肿瘤。
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防治消化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,所述消化系统肿瘤包括胃癌、结肠癌、食管癌、胰腺癌、肝癌。
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:
在其中一些实施例中,所述荚膜多糖A的重均分子量(Mw)为80-90kD,Mw分布于70KD-100KD的部分占总量的70-80%,重均分子量/数均分子量(Mw/Mn)的比值为1.0-1.3。
在其中一些实施例中,所述两性离子荚膜多糖中荚膜多糖A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。
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。
第二方面,提供一种用于防治消化系统肿瘤的组合物,其中,所述组合物含有保藏编号为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。其中,所述荚膜多糖A的结构如下所示:
在其中一些实施例中,所述荚膜多糖A的重均分子量为80-90kD,Mw分布于70-100KD的部分占总量的70-80%,重均分子量/数均分子量(Mw/Mn)的比值为1.0-1.3。
在其中一些实施例中,其中,所述荚膜多糖A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);所述超滤膜为10KD。
在其中一些实施例中,所述组合物为药物。
在其中一些实施例中,所述药物的剂型包括丸剂、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,所述药物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。
在其中一些实施例中,所述药物可通过口服、灌肠或肠胃外的形式给药。
在其中一些实施例中,所述药物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在体内能够减轻炎症反应,保护正常组织,提高机体抗肿瘤免疫反应,有效防治消化系统肿瘤。
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.2017Mar 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.2018Oct 1;9:2298.的报道,该菌株对胃酸、胆盐有着较好的耐性,能够保证其在胃中的存活和有效定植。
图1为实施例1的脆弱拟杆菌ZY-312的菌落特征图;
图2为实施例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3为实施例2的荚膜多糖A核磁共振波谱仪分析图;
A-E分别为实施例2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为实施例2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。
菌种保藏信息:
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。
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,或者可以通过已知方法制备。 所有细胞购自ATCC;所有细胞培养材料购自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。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。
本发明的脆弱拟杆菌和/或其两性离子荚膜多糖在制备防治消化系统肿瘤的产品中的应用,其中,所述产品可为药品或食品。所述脆弱拟杆菌为保藏编号为CGMCC No.10685的脆弱拟杆菌ZY-312,所述两性离子荚膜多糖提取自所述脆弱拟杆菌ZY-312,所述两性离子荚膜多糖含有荚膜多糖A,所述荚膜多糖A的分子量为80-90kD,Mw/Mn为1.0-1.3,Mw分布于70-100KD的部分占总量的70-80%;所述荚膜多糖A的含量超过95wt%。所述脆弱拟杆菌可以是活菌、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。
本发明用于防治消化系统肿瘤的药物中,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。该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。
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。
选取单个菌落接种于植物源蛋白胨肉汤中进行发酵培养8小时(温度为37℃),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。
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2: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-E),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1.0-1.3,Mw分布于70KD-100KD的部分占总量的70-80%;化学结构参见图4。以下实施例中简称“ZY-312 PSA”。
实施例3脆弱拟杆菌及其荚膜多糖防治小鼠MFC胃癌原位移植瘤的实验探究
1、试验设计及流程
胃癌原位移植瘤模型制备:
(1)采用含10%小牛血清的RPMI-1640培养液在常规条件(37℃、饱和湿度、5%CO
2)下培养MFC消化系统肿瘤细胞至对数生长期,调整细胞密度为1×10
7个/mL。615近交系雄性小鼠5只,无菌条件下右侧腋下皮下注射0.2mLMFC细胞悬液,待肿瘤长至直径为1cm时处死荷瘤小鼠,作为原位移植用瘤源。
(2)用615近交系雄性小鼠90只,按体重区间随机分为9组,即空白组、模型组、5-氟尿嘧啶组(5-FU,Sigma公司,20mg/kg)、ZY-312活菌低(10
6CFU/只)、中(10
8CFU/只)、高(10
10CFU/只)组、ZY-312灭活菌组(10
10cell/只)、ZY-312 PSA组(1mg/kg),ZY-312+PSA(10
10CFU/只、1mg/kg)组,每组10只。将瘤源剪成约1mm
3的小瘤块,植入模型组及各给药组小鼠胃组织,OB胶凝固胃复位,然后分层关腹。逐日观察,以出现可触及的包块为成瘤标志。空白组小鼠行相同开腹手术,但不植入瘤块。
从术后第4天开始,空白组、模型组动物每日1次口服0.2mL生理盐水;5-FU组根据体重每两天1次腹腔注射5-FU;各给药组每日1次口服对应剂量药物。共给药12d。末次给药24h后,所有小鼠摘眼球取血,而后安乐死,无菌条件下分离瘤组织、脾脏及胸腺,并称重。肿瘤分为两份,一份冻存,一份用于免疫组化分析。
检测项目与方法:
肿瘤重量与肿瘤生长抑制率:抑瘤率=100%(模型组肿瘤平均重量-给药组肿瘤平均重量)/模型组肿瘤平均重量。
脏器指数:脏器指数=脏器质量(mg)/体质量(g)。
血清细胞因子检测:Luminex技术检测小鼠血清IL-2、INF-γ和TNF-α的含量。
数据统计与分析:使用SPSS统计软件25.0进行统计学分析。
2、试验结果
(1)肿瘤重量、抑瘤率、脏器指数:
表1各组小鼠肿瘤重量、抑瘤率、脏器重量和脏器指数(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
相对于空白组,模型组出现明显肿瘤瘤块,造模成功。
相对于模型组,各给药组肿瘤重量均有所下降;5-FU组和脆弱拟杆菌高剂量组具有显著性差异。
相对于空白组,模型组脾脏,胸腺指数上升,5-FU下调了小鼠的脾脏和胸腺指数,这可能是由于化疗药的免疫抑制毒性;而ZY-312及其PSA上调了脏器指数,说明ZY-312及其PSA能够维持和提高免疫功能。
(2)血清细胞因子
表2各组小鼠血清细胞因子(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
IFN-γ、IL-2和TNF-α均是已知的抗肿瘤免疫细胞因子。相对于空白组,模型组IFN-γ、IL-2和TNF-α水平菌显著上升;而相对于模型组,5-FU下调了这三种免疫因子表达水平,ZY-312及其 PSA上调了上述免疫因子表达水平,提高了机体的抗肿瘤免疫反应。
综上,脆弱拟杆菌ZY-312及其荚膜多糖可以通过维持和增强机体免疫,而有效防治胃癌。
实施例4脆弱拟杆菌及其荚膜多糖防治AOM/DSS诱导小鼠结肠炎相关结肠癌的实验探究
1、试验设计及流程
6-8周龄C57BL/6雄性小鼠72只,根据体重随机分为9组,每组8只:空白组、模型组(生理盐水)、5-FU组(Sigma公司,20mg/kg)、ZY-312活菌低(10
6CFU/只)、中(10
8CFU/只)、高剂量组(10
10CFU/只)。另设ZY-312灭活菌组(10
10cell/只)、ZY-312 PSA组(1mg/kg),ZY-312+PSA(10
10CFU/只、1mg/kg)组。
除空白组外,所有小鼠于Day0,根据10mg/kg给予所有小鼠腹腔注射氧化偶氮甲烷(AOM购自美国Sigma公司);day7开始自由饮用2%葡聚糖硫酸钠(DSS购自瑞典TdB公司),至day14结束;day28-day35和day49-day56再次饮用2%DSS。
Day56起,空白组、模型组每天1次给予0.2mL生理盐水灌胃,5-FU组每周1次腹腔注射,其余给药组每天1次灌胃相应剂量药物。
Day91所有小鼠安乐死,无菌条件下剖取结肠,记录肿瘤重量和尺寸;将带有肿瘤的结肠组织分作三份,一份冻存于液氮中,一份用于细胞因子检测,一份以10%中性甲醛溶液固定,常规脱水,石蜡包埋,切片,伊红-苏木素(HE)染色,镜检。
检测项目与方法:
肿瘤重量与肿瘤生长抑制率:抑瘤率=100%(模型组肿瘤平均重量-给药组肿瘤平均重量)/模型组肿瘤平均重量。
细胞因子检测:Luminex技术检测小鼠结肠组织IL-10、IL-17的含量。
组织病理学:HE染色后镜检,观察淋巴、隐窝结构及炎性改变,按无/轻/中/重打分,分别为0/1/2/3。
数据统计与分析:使用SPSS统计软件25.0进行统计学分析。
2、试验结果
(1)肿瘤重量及抑瘤率
表4各组小鼠肿瘤重量及抑瘤率(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
与空白组相比,模型组出现明显瘤块,造模成功。
与模型组相比,各给药组肿瘤重量均有所下降,5-FU组和ZY-312联用PSA组肿瘤重量显著下降,这说明ZY-312及其PSA能够有效抑制小鼠肿瘤生长。在脆弱拟杆菌ZY-312及其荚膜多糖共同用药时,这种抑瘤效果进一步提升。
(2)细胞因子
表5各组小鼠结肠组织细胞因子(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
IL-6、IL-17为促炎因子,IL-10为抗炎因子。与空白组相比,模型组IL-6、IL-17水平显著上升,IL-10水平显著下降。而相对于模型组,5-FU下调了IL-6和IL-17的水平,但不具有显著性;ZY-312及其PSA显著下调了IL-6的水平,对IL-17的下调幅度大于5-FU。在IL-10的调节中,各给药组IL-10水平均显著大于模型组,但ZY-312及其PSA水平高于5-FU。这说明ZY-312及其PSA能够减轻结肠炎症。
(3)组织病理学
表6各组小鼠结肠组织病理学评分(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
与空白组相比,模型组组织病理学评分显著上升。5-FU下调了小鼠结肠组织病理学评分,但 不具有显著性;ZY-312及其PSA也下调了小鼠结肠组织病理学评分,且在淋巴增生和局部浸润、隐窝结构改变中具有显著性,这说明ZY-312及其PSA对组织具有保护作用。
综上,脆弱拟杆菌ZY-312及其荚膜多糖能够抑制肿瘤生长,减轻结肠炎症,保护结肠组织,有效防治结直肠癌。
实施例5脆弱拟杆菌及其荚膜多糖防治小鼠Eca109食管癌移植瘤的实验探究
1、试验设计及流程
6-8周龄ICR雄性小鼠72只,根据体重随机分为9组,每组8只:空白组、模型组(生理盐水)、顺铂组(DDP,山东齐鲁制药有限公司,2mg/kg)、ZY-312活菌低(10
6CFU/只)、中(10
8CFU/只)、高剂量组(10
10CFU/只)。另设ZY-312灭活菌组(10
10cell/只)、ZY-312 PSA组(1mg/kg)、ZY-312+PSA(10
10CFU/只、1mg/kg)组。
采用含10%小牛血清、青霉素(100U/mL)及链霉素(100U/mL)的DMEM培养液在常规条件(37℃、饱和湿度、5%CO
2)下培养Eca109食管癌细胞至对数生长期,调整细胞浓度为1×10
7个/mL,除空白组外,每组各小鼠腋下皮下注射0.2mL细胞悬液,空白组腋下皮下注射0.2mL生理盐水。
接种细胞24小时后开始治疗。空白组、模型组每日1次灌胃0.2mL生理盐水;DDP组每两天1次腹腔注射;除DDP组外,其余各给药组每日1次灌胃对应药物。连续给药14天。
末次给药24h后所有小鼠摘眼球取血,而后安乐死,无菌条件下分离瘤组织和脾脏,并称重。肿瘤分为两份,一份冻存,一份用于免疫组化分析。
检测项目与方法:
肿瘤重量与肿瘤生长抑制率:抑瘤率=100%(模型组肿瘤平均重量-给药组肿瘤平均重量)/模型组肿瘤平均重量。
脏器指数:脏器指数=脏器质量(mg)/体质量(g)。
血清细胞因子检测:Luminex技术检测小鼠血清IL-2、INF-γ和TNF-α的含量。
数据统计与分析:使用SPSS统计软件25.0进行统计学分析。
2、试验结果
(1)肿瘤重量、抑瘤率和脏器指数
表7各组小鼠肿瘤重量、抑瘤率和脏器指数(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
相对于空白组,模型组出现明显肿瘤瘤块,造模成功。
相对于模型组,各组肿瘤重量均有所下降;DDP组和ZY-312联用PSA组肿瘤重量显著下降。这说明脆弱拟杆菌及其PSA能够有效抑制肿瘤生长;在脆弱拟杆菌ZY-312及其荚膜多糖共同用药时,这种效果进一步提升。
相对于空白组,模型组脾脏指数上升,DDP下调了小鼠的脾脏指数,这可能是由于化疗药的免疫抑制毒性;而ZY-312及其PSA上调了脏器指数,说明ZY-312及其PSA能够维持和提高免疫功能。
(2)血清细胞因子
表8各组小鼠血清细胞因子(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
相对于空白组,模型组IFN-γ、IL-2和TNF-α水平菌显著上升;而相对于模型组,DDP下调了这三种免疫因子表达水平,ZY-312及其PSA上调了IFN-γ和IL-2的表达水平,显著上调了TNF-α的表达水平,提高了机体的抗肿瘤免疫反应。
综上,脆弱拟杆菌及其荚膜多糖能够促进抗肿瘤免疫反应,可有效防治食管癌。
实施例6脆弱拟杆菌及其荚膜多糖防治小鼠MPC-83胰腺癌移植瘤的实验探究
1、试验设计及流程
4-5周龄昆明小鼠72只,雌雄各半,根据体重随机分为9组,每组8只:空白组、模型组(生理盐水)、5-FU组(Simga公司,20mg/kg)、ZY-312活菌低(10
6CFU/只)、中(10
8CFU/只)、高剂量组(10
10CFU/只)。另设ZY-312灭活菌组(10
10cell/只)、ZY-312 PSA组(1mg/kg)、ZY-312+PSA (10
10CFU/只、1mg/kg)组。
采用含10%小牛血清、青霉素(100U/mL)及链霉素(100U/mL)的RPMI-1640培养液在常规条件(37℃、饱和湿度、5%CO
2)下培养MPC-83胰腺癌细胞至对数生长期,调整细胞浓度为5×10
7个/mL,除空白组外,每组各小鼠背部皮下注射0.2mL细胞悬液,空白组背部皮下注射0.2mL生理盐水。
当荷瘤小鼠皮下肿瘤刚可触及时开始治疗。空白组、模型组每日1次灌胃0.2mL生理盐水;5-FU组每天1次腹腔注射;除5-FU组外,其余各给药组每日1次灌胃对应药物。连续给药29天。
末次给药24h后所有小鼠摘眼球取血,而后安乐死,无菌条件下分离瘤组织并称重。肿瘤分为两份,一份冻存,一份用于免疫组化分析。
检测项目与方法:
肿瘤重量与肿瘤生长抑制率:抑瘤率=100%(模型组肿瘤平均重量-给药组肿瘤平均重量)/模型组肿瘤平均重量。
血清细胞因子检测:Luminex技术检测小鼠血清TGF-β1、VEGF的含量。
数据统计与分析:使用SPSS统计软件25.0进行统计学分析。
2、试验结果
(1)肿瘤重量和抑瘤率
表9各组小鼠肿瘤重量和抑瘤率(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
与空白组相比,模型组出现明显瘤块,造模成功。
与模型组相比,各组肿瘤重量均有所下降,ZY-312高剂量组和ZY-312与PSA联用组肿瘤重量显著下降,这说明ZY-312及其PSA能够有效抑制小鼠肿瘤生长。在脆弱拟杆菌ZY-312及其荚膜多糖共同用药时,这种抑瘤效果进一步提升。
(2)血清细胞因子
表10各组小鼠血清细胞因子(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
TGF-β1、VEGF是机体内调节T细胞功能的抑制性细胞因子。与空白组相比,模型组TGF-β1、VEGF水平明显上升;而与模型组相比,5-FU下调了TGF-β1和VEGF的水平,ZY-312及其PSA也下调了上述细胞因子的水平,其对VEGF的调节幅度与5-FU相当。上述结果说明ZY-312及其PSA能够通过下调免疫抑制因子,提高机体的抗肿瘤反应。
综上,脆弱拟杆菌ZY-312及其荚膜多糖能够促进机体抗肿瘤免疫反应,可以有效防治胰腺癌。
实施例7脆弱拟杆菌及其荚膜多糖防治小鼠H22肝癌移植瘤的实验探究
1、试验设计及流程
5-6周龄ICR雄性小鼠72只,根据体重随机分为9组,每组8只:空白组、模型组(生理盐水)、环磷酰胺组(CTX,Baxter Oncology GmbH公司,30mg/kg)、ZY-312活菌低(10
6CFU/只)、中(10
8CFU/只)、高剂量组(10
10CFU/只)。另设ZY-312灭活菌组(10
10cell/只)、ZY-312 PSA组(1mg/kg)、ZY-312+PSA(10
10CFU/只、1mg/kg)组。
采用含10%小牛血清、青霉素(100U/mL)及链霉素(100U/mL)的RPMI-1640培养液在常规条件(37℃、饱和湿度、5%CO
2)下培养H22腹水型肝癌细胞至对数生长期,调整细胞浓度为5×10
6个/mL,除空白组外,每组各小鼠右肩皮下注射0.2mL细胞悬液,空白组右肩皮下注射0.2mL生理盐水。
接种后第二天开始治疗。空白组、模型组每日1次灌胃0.2mL生理盐水;对环磷酰胺(CTX)组每天1次腹腔注射;除CTX组外,其余各给药组每日1次灌胃对应药物。连续给药15天。
末次给药后禁食不禁水,16h后所有小鼠摘眼球取血,而后安乐死,无菌条件下分离瘤组织、脾脏、胸腺、肝脏并称重。肿瘤分为两份,一份冻存,一份用于免疫组化分析。
检测项目与方法:
肿瘤重量与肿瘤生长抑制率:抑瘤率=100%(模型组肿瘤平均重量-给药组肿瘤平均重量)/模型组肿瘤平均重量。
脏器指数:脏器指数=脏器质量(mg)/体质量(g)。
肝功能测定:微板法检测血清中肝功能指标。
血清细胞因子检测:Luminex技术检测小鼠血清IL-2、IFN-γ、TNF-α的含量。
数据统计与分析:使用SPSS统计软件25.0进行统计学分析。
2、试验结果
(1)肿瘤重量、抑瘤率和脏器指数
表12各组小鼠肿瘤重量、抑瘤率和脏器指数(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
相对于空白组,模型组出现明显肿瘤瘤块,造模成功。
相对于模型组,各组肿瘤重量均有所下降;CTX、ZY-312高剂量及ZY-312联用PSA组肿瘤重量显著下降。这说明脆弱拟杆菌及其PSA能够有效抑制肿瘤生长,在脆弱拟杆菌ZY-312及其荚膜多糖共同用药时,这种抑瘤效果进一步提升。
相对于空白组,模型组脾脏,胸腺指数上升;相对于模型组,CTX下调了小鼠的脾脏和胸腺指数,这可能是由于化疗药的免疫抑制毒性;而相对于CTX,ZY-312及其PSA上调了脏器指数,说明ZY-312及其PSA能够维持和提高免疫功能。
(2)肝功能指标
表13各组小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
谷丙转氨酶(ALT)、谷草转氨酶(AST)均是评价肝脏损害程度的标志性蛋白。与空白组相比,模型组ALT、AST明显上升,显示出肝癌细胞对肝脏正常组织的破坏。与模型组相比,CTX 加重了这种损伤;ZY-312及其PSA下调了ALT、AST的水平。这表明ZY-312及其PSA能够减轻癌细胞对肝脏的损害。
(3)血清细胞因子
表14各组小鼠血清细胞因子(mean±SD)
注:与模型组比较,*表示差异显著p<0.05;**表示差异极显著p<0.01。
相对于空白组,模型组IFN-γ、IL-2和TNF-α水平均显著上升;而相对于模型组,CTX下调了这三种免疫因子表达水平,ZY-312及其PSA显著上调了IFN-γ和TNF-α的表达水平,上调了IL-2的表达水平,提高了机体的抗肿瘤免疫反应。
综上,脆弱拟杆菌ZY-312及其荚膜多糖能够保护正常肝脏组织,维持和提高机体抗肿瘤免疫反应,有效防治肝癌。
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防治消化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,所述消化系统肿瘤包括胃癌、结肠癌、食管癌、胰腺癌、肝癌。
- 根据权利要求4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;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,所述药品的剂型包括丸剂、片剂、颗粒剂、胶囊、口服液或管饲制剂;进一步优选地,所述药品为人用药或动物用药。
- 一种用于防治消化系统肿瘤的组合物,其中,所述组合物含有保藏编号为CGMCC No.10685的脆弱拟杆菌和其两性离子荚膜多糖,或保藏编号为CGMCC No.10685的脆弱拟杆菌的两性离子荚膜多糖。
- 根据权利要求7所述的组合物,其特征在于,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9所述的组合物,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);所述超滤膜为10KD。
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