WO2023134206A1

WO2023134206A1 - Use of bacteroides fragilis and zwitterionic capsular polysaccharide thereof in preparation of drug for preventing and treating genitourinary system tumors

Info

Publication number: WO2023134206A1
Application number: PCT/CN2022/120024
Authority: WO
Inventors: 李平; 邝高波; 王从峰; 倪赛
Original assignee: 广州知易生物科技有限公司
Priority date: 2022-01-12
Filing date: 2022-09-20
Publication date: 2023-07-20
Also published as: CN114344325A; CN114344325B

Abstract

Provided is a use of Bacteroides fragilis and/or a zwitterionic capsular polysaccharide thereof in the preparation of a drug for preventing and treating genitourinary system cancer. Bacteroides fragilis, especially Bacteroides fragilis ZY-312 the accession number of which is CGMCC No.10685 and a zwitterionic capsular polysaccharide A thereof, can regulate T cell and immune factor levels, enhance body immunity, and effectively prevent and treat genitourinary system tumors such as kidney cancer, bladder cancer, prostate cancer, bladder urothelial carcinoma, ovarian cancer, breast cancer, and cervical cancer.

Description

Application of Bacteroides fragilis and its zwitterionic capsular polysaccharide in the preparation of drugs for preventing and treating genitourinary system tumors

This application claims the patent application number 202210034071.8 submitted to the State Intellectual Property Office of China on January 12, 2022, and the name of the invention is "Bacteroides fragilis and its zwitterionic capsular polysaccharide in the preparation of drugs for the prevention and treatment of genitourinary system tumors" application of the "priority benefit of the earlier application". The entirety of said prior application is incorporated by reference into this application.

The microbial strains used in the implementation of the present invention have been preserved on April 2, 2015 at the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Management Committee (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing). Classification name: 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.

technical field

The invention relates to the field of biomedicine, in particular to the application of Bacteroides fragilis and/or its zwitterionic capsular polysaccharide in the prevention and treatment of genitourinary system tumors.

Background technique

Urogenital system tumors refer to tumors that occur in the urinary system and/or reproductive system. These include female breast and reproductive organ tumors, male reproductive organ tumors, and urinary organ tumors such as kidney, bladder, urothelial, breast, ovarian, and prostate cancers.

Renal cancer is one of the common malignant tumors of the urinary system, accounting for about 2%-3% of adult malignant tumors and 80%-90% of adult renal malignant tumors. The incidence of kidney cancer in my country is increasing year by year. In 2008, it has become the 10th male malignant tumor in my country, and it has become one of the most important tumors threatening health.

In addition to the main surgical treatment, the treatment of kidney cancer also includes drug therapy. Over the past decade, drug therapy for RCC has transitioned from nonspecific immunological approaches, to targeted therapy against vascular endothelial growth factor, and now to novel immunotherapeutic agents. High-dose interleukin-2 and interferon-a treatment of advanced RCC has a low overall response rate and no significant improvement in progression-free survival. With the emergence of targeted drugs, great progress has been made in the treatment of metastatic RCC, which can significantly improve the objective response rate and prolong the progression-free survival and overall survival of patients with metastatic RCC. However, with the use of targeted drugs for renal cancer, most patients will develop drug resistance over time, and are often accompanied by side effects such as rash, diarrhea, edema, and weight gain, which restrict the use of targeted drugs. Emerging immunotherapy is another treatment strategy that can be selected for advanced RCC, but the cost of treatment is high, and biomarkers that can effectively predict the individualized efficacy of immunotherapy have yet to be found, which limits the clinical application of immunotherapy in RCC.

Bladder cancer is the most common malignant tumor of the urinary system, with approximately 430,000 new cases and 170,000 deaths worldwide each year. Bladder cancer has a high incidence in western countries, and its incidence has also shown an obvious upward trend in my country. By 2020, bladder cancer will rank the 10th in the number of new cancer cases in the world and the 13th in the number of new cancer cases in China.

70-80% of bladder cancer patients belong to superficial bladder cancer, which is treated by transurethral resection of bladder tumor, but the recurrence rate is very high. Postoperative intravesical infusion drug treatment can prevent recurrence. Bladder infusion drugs include Bacillus Calmette-Guerin (BGG) , there are some other anti-mitotic agents such as mitomycin, doxorubicin and epirubicin, among which BGG is the most effective. These drugs usually have serious complications, such as frequent urination, urgency, dysuria, hematuria, and immunosuppression, which can cause more or less painful cystitis. A single intravesical infusion of mitomycin after cystectomy can reduce the chance of bladder cancer recurrence. Mitomycin treatment may cause an allergic reaction if splashed on the skin. Drugs for intravesical infusion are expensive, and the course of treatment is long, which increases the burden on patients' lives. For invasive bladder cancer, total cystectomy is required, and urinary diversion combined with radiotherapy and chemotherapy is required. There are many complications after total cystectomy, which brings great pain to the patient.

Prostate cancer is currently the second most common malignancy in men worldwide and the fifth leading cause of cancer death in men. According to statistics from the International Agency for Research on Cancer (IARC), there were an estimated 1.276 million new cases of prostate cancer worldwide in 2018. At present, in addition to surgery, many new drugs have emerged to improve the prognosis of prostate cancer, such as endocrine therapy drug androgen synthesis inhibitor abiraterone acetate and androgen receptor (androgen receptor, AR) antagonist enzalutamide , radiation therapy drug radium-223 chloride, immunotherapy drug sipuleucel-T, chemotherapy drug docetaxel and cabazitaxel, etc. In a certain period of time, they can all play a role in inhibiting prostate cancer, however, long-term use will bring side effects, such as bone pain, acute urinary retention, liver damage or cardiovascular dysfunction.

Ovarian cancer is one of the three major malignant tumors in gynecology. The incidence of ovarian cancer in my country is only behind cervical cancer and uterine body cancer, while the mortality rate ranks first among female reproductive system tumors. Because the early symptoms of ovarian cancer are not obvious, the disease is often developed to the middle and late stage when the patient sees a doctor. The standard regimen for ovarian cancer is minimally cytoreductive surgery combined with platinum-based systemic chemotherapy. At present, the first-line chemotherapy for ovarian cancer in China is combined chemotherapy based on cisplatin. However, due to the obvious toxic and side effects of chemotherapy drugs, patients often cannot tolerate them, and ovarian cancer chemotherapy is prone to drug resistance, and it is very easy to produce tumor peritoneal metastasis and produce ascites, which further aggravates the pain of patients.

Cervical cancer (cervical carcinoma) is the most common gynecological tumor, and its incidence rate has obvious regional differences. The average age of onset of cervical cancer is 40-50 years old, 60-70 years old is the most, and it is relatively rare in young women (less than 20 years old). According to the report of the authoritative medical journal "The Lancet", nearly 500,000 cases of cervical cancer are discovered in the world every year, and more than 270,000 people die from it. Moreover, this mortality rate is even more pessimistic in developing countries, where the fatality rate is as high as 80%. Commonly used cervical cancer treatment methods include surgery, radiotherapy and chemotherapy. Surgical treatment is more effective for early-stage patients, and it will damage the fertility of patients and cause a heavier psychological and physical burden. For middle-advanced patients, although carcinoma in situ is surgically resected, it is often accompanied by a high degree of lymph node metastasis. As an adjuvant treatment, radiotherapy is difficult to completely eliminate tumor cells, and it is more harmful to the human body. Chemotherapy is often used as the last line of treatment for patients with cervical cancer. Compared with radiotherapy, chemotherapy has shown superior therapeutic effects. For example, platinum-based combination chemotherapy has achieved good results in patients with advanced and metastatic cervical cancer and is encouraged as the first-line treatment for patients with locally high recurrence tumors. However, due to the high toxicity and side effects of traditional chemotherapy, patients often suffer great pain.

Breast cancer is a common oncological disease. According to the survey data of the International Agency for Research on Cancer (IARC) in 2018, the incidence of female breast cancer in the world is as high as 24.2%, and there are more than 300,000 new cases of female breast cancer in my country every year. At present, clinical drugs for breast cancer are mainly divided into 6 categories: cyclophosphamide is the representative of alkylating agents; 5-fluorouracil is the representative of antimetabolites; doxorubicin is the representative of antibiotics; vincristine is the representative of alkaloids; Docetaxel, a representative drug of paclitaxel; Cisplatin, a new anticancer drug similar to a bifunctional alkylating agent. Research on pit cancer drugs has progressed rapidly, but there are few drugs with good curative effect, low toxicity, and long-term effects. Most chemotherapy drugs are prone to drug resistance and are expensive, which brings huge economic pressure and unavoidable to patients. suffering from illness. The curative effect is poor for patients with advanced stage and metastases.

Urothelial carcinoma is one of the most common malignant tumors of the genitourinary system. It can occur in all transitional epithelium of the urinary tract, including the renal pelvis, ureter, bladder, and urethra. Urothelial carcinoma of the bladder is the most common, accounting for more than 90%. Urothelial cancer is divided into three types according to the degree of tumor invasion and the presence or absence of distant metastasis: non-muscle-invasive urothelial cancer (UMIBC), muscle-invasive urothelial cancer (muscle-invasive urothelial cancer). , MIBC), locally advanced or metastatic disease. The diagnosis and treatment of urothelial carcinoma are divided into two categories: bladder urothelial carcinoma and upper tract urothelial carcinoma. The approach to treatment of bladder urothelial carcinoma is the same as that described previously for bladder cancer. The treatment for upper urothelial carcinoma is ureterectomy (radical resection with preservation of renal function is selected according to the severity) combined with postoperative intravesical infusion chemotherapy; the first-line treatment for metastatic upper urothelial carcinoma is a combination of chemotherapy drugs containing cisplatin and Monoclonal antibody therapy; T3-4 upper tract urothelial carcinoma can be treated with radiotherapy. Patients with urothelial carcinoma often have incomplete renal function. Surgery combined with bladder infusion therapy may damage the renal function of the patient, and at the same time there is a possibility of recurrence; chemotherapy will bring toxic reactions to the patient, and adjuvant radiotherapy alone has not shown a significant impact on overall survival. benefits.

At present, surgery is still the main treatment for malignant tumors. Surgical treatment of genitourinary system tumors will seriously affect the quality of life of patients, and the chemoradiotherapy drugs used in clinical practice lack selectivity and targeting, which will cause serious adverse reactions to cancer patients. Therefore, finding chemotherapeutic drugs with high efficiency, low toxicity, targeted and selective anticancer effects is an important direction and urgent task of anticancer drug research. , there is an urgent need for a new type of anticancer drug to prolong its survival.

Contents of the invention

In order to overcome the above-mentioned defects existing in the prior art, 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 genitourinary 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 A can regulate the level of T cells and immune factors, enhance the immunity of the body, and effectively prevent and treat Kidney cancer, bladder cancer, prostate cancer, bladder urothelial cancer, ovarian cancer and other genitourinary system tumors.

In order to achieve the above object, the present invention adopts the following technical solutions:

In the first aspect, an application of Bacteroides fragilis and/or its zwitterionic capsular polysaccharide in the preparation of products for preventing and/or treating tumors of the genitourinary system is provided, and the Bacteroides fragilis is a preservation number of CGMCC No. Bacteroides fragilis ZY-312, the zwitterionic capsular polysaccharide is extracted from Bacteroides fragilis ZY-312.

In some of these embodiments, 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.

In some of these embodiments, the zwitterionic capsular polysaccharide comprises capsular polysaccharide A. Wherein, the structure of the capsular polysaccharide A is as follows:

According to the present invention, the weight-average molecular weight of the capsular polysaccharide A is 80-90KD, the part with Mw distributed in 70-100KD accounts for 70-80% of the total, and the ratio of weight-average molecular weight/number-average molecular weight (Mw/Mn) 1.0-1.3.

In some of the embodiments, the content of capsular polysaccharide A in the zwitterionic capsular polysaccharide exceeds 95wt%.

In some of these embodiments, the preparation method of the zwitterionic capsular polysaccharide comprises the following steps:

(1) Centrifuge the fermented Bacteroides fragilis bacteria liquid to collect the sediment to obtain the Bacteroides fragilis bacteria sludge; take the bacteria sludge, add purified water 3 to 10 times the weight of the bacteria sludge to suspend the bacteria, and adjust with acid solution The pH is 2.0-4.5, extracted at 50-120°C for 0.5-3.0 hours, cooled to room temperature, centrifuged at room temperature, and the supernatant is taken to obtain a crude sugar solution;

(2) The crude sugar solution is concentrated by ultrafiltration membrane ultrafiltration to remove small molecular impurities until the conductivity is stable, and the reflux liquid is collected;

(3) Add an equal volume of 40mmol/L Tris-HCl to the reflux liquid to convert the salt; ion exchange column chromatography, gradient elution, segmented collection, SEC-HPLC tracking monitoring, and merge the 206nm absorption peak into a single, symmetrical peak component , ultrafiltration membrane ultrafiltration, adding purified water and repeated ultrafiltration until the conductivity is stable, collecting the reflux liquid and freeze-drying to obtain the Bacteroides fragilis zwitterionic capsular polysaccharide.

In some of these embodiments, the centrifugation in step (1) is centrifugation at 11000-13000 g for 8-12 minutes.

In some of these embodiments, the acid solution in step (1) may be one or more of organic acids, inorganic acids and acidic buffers. Wherein, the inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.; the organic acid can be acetic acid, citric acid, etc.

In some of these embodiments, 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.

In some of these embodiments, 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.

In some of these embodiments, the product is food or medicine.

In some of these embodiments, 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.

In some of these embodiments, 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.

In some of the embodiments, the medicine is B. fragilis or its zwitterionic capsular polysaccharide used alone, or B. fragilis and its zwitterionic capsulated polysaccharide used in combination.

According to an embodiment of the present invention, the genitourinary system tumor refers to a tumor occurring in the urinary system and/or reproductive system. These include female breast and reproductive organ tumors, male reproductive organ tumors, and urinary organ cancers such as kidney, bladder, urothelial, breast, ovarian, cervical, and prostate cancers.

In a second aspect, the present invention provides a composition for preventing and treating genitourinary system tumors, wherein the composition contains Bacteroides fragilis ZY-312 and/or its zwitterionic capsular polysaccharide with the preservation number CGMCC No.10685 .

In some of these embodiments, the zwitterionic capsular polysaccharide comprises capsular polysaccharide A. The structure of the capsular polysaccharide A is as follows:

In some of these embodiments, 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.

In some of these embodiments, the composition is a drug.

In some of these embodiments, the drug can be administered orally, enemaly or parenterally.

In some of these embodiments, the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.

According to the present invention, the genitourinary system tumor refers to a tumor that occurs in the urinary system and (or) reproductive system. These include female breast and reproductive organ tumors, male reproductive organ tumors, and urinary organ cancers such as kidney, bladder, urothelial, breast, ovarian, cervical, and prostate cancers.

The present invention also provides a method for preventing and/or treating genitourinary system tumors, comprising administering a therapeutically effective amount of the above product to a patient.

Wherein, the "prevention" includes prevention and/or treatment.

Beneficial effects of the present invention:

The present invention unexpectedly finds that the Bacteroides fragilis of the present invention, especially the Bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685 and its zwitterionic capsular polysaccharide A can regulate the levels of T cells and immune factors, and enhance the body Immunization, effective prevention and treatment of kidney cancer, bladder cancer, prostate cancer, bladder urothelial cancer, ovarian cancer, breast cancer, cervical cancer and other genitourinary 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.). According to patent ZL201510459408.X and scientific literature Xu W, Su P, Zheng L, Fan H, Wang Y, Liu Y, Lin Y, Zhi F.In vivo Imaging of a Novel Strain of Bacteroides fragilis via Metabolic Labeling.Front Microbiol.2018 Oct 1; 9:2298. reported that the strain has good tolerance to gastric acid and bile salts, which can ensure its survival and effective colonization in the stomach.

Description of drawings

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 respectively ^{the 1} H spectrum, ¹³ C spectrum, COZY spectrum, HSQC spectrum, and HMBC spectrum analyzed by the capsular polysaccharide A NMR spectrometer of Example 2 of the present invention;

Fig. 4 is the chemical structural formula of the structural unit of Bacteroides fragilis capsular polysaccharide A prepared in Example 2 of the present invention.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies realized based on the above contents of the present invention are covered within the scope of protection intended by the present invention.

Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available, 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.; Or it can 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.

Unless otherwise defined or clearly indicated by background, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Embodiment 1 Fermentation culture of Bacteroides fragilis

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.

Morphology under the microscope: 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.

Select a single colony and inoculate it in plant-derived peptone liquid medium for fermentation and culture for 8 hours (at a temperature of 37°C) to obtain a bacterial liquid of Bacteroides fragilis ZY-312; the obtained bacterial liquid is centrifuged and precipitated at a speed of 3000r/min for 15 minutes, and then removed Clear and collect the sediment 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.

Example 2 Preparation of Bacteroides fragilis capsular polysaccharide

The bacteria slime prepared in Example 1 was used to carry out the experiment.

(1) Take 50g of bacteria slime, add 300g of purified water to resuspend the bacteria, adjust its pH to 3.5 with 1mol/L hydrochloric acid solution, extract at 100°C for 1.5h, cool to room temperature, centrifuge at 12000g at room temperature for 10min, take the supernatant to obtain rough sugar solution;

(2) The crude sugar solution is concentrated by ultrafiltration through a 10KD ultrafiltration membrane to remove small molecular impurities until the conductivity is stable, and the reflux liquid is collected;

(3) Add an equal volume of 40mmol/L Tris-HCl (pH8.5) to the reflux liquid to convert salt; DEAE Sepharose Fast Flow ion exchange column chromatography (16mm×200mm), flow rate 20mL/min, 20mmol/L Tris-HCl ( pH8.5, containing 0.2mol/L NaCl) gradient elution for 25 column volumes, segmented collection, 100mL/bottle (component), SEC-HPLC tracking monitoring, combined 206nm absorption peak as a single, symmetrical peak component, 10KD ultrafiltration membrane ultrafiltration, add purified water and repeat ultrafiltration until the conductivity is stable, collect the reflux liquid, freeze-dry, and obtain the Bacteroides fragilis extract;

(4) Weigh 30 mg of the Bacteroides fragilis extract described in step (3), dissolve it in 0.5 mL D ₂ O, and add 1 μl of acetone ( ¹ H, 2.22; ¹³ C, 30.89) for calibration. ¹ H, ¹³ C, COZY, HSQC, and HMBC spectra were analyzed using a 500MHz Bruker NMR spectrometer (see Figures 3A-3E), and it was confirmed that the Bacteroides fragilis extract collected in step (3) was capsular polysaccharide A, and the combined lipid content Less than 0.02%, protein residues are less than 1%, and nucleic acid residues are less than 0.05%. According to GPC (gel permeation chromatography) analysis, the prepared capsular polysaccharide A has a weight average molecular weight of 80-90 KDa, an Mw/Mn of 1.0-1.3, and the chemical structure is shown in FIG. 4 .

Example 3 Drug efficacy test of Bacteroides fragilis and its zwitterionic capsular polysaccharides on mouse Renca renal carcinoma transplanted tumors

Adjust the cell concentration of kidney cancer Renca cells in the logarithmic growth phase to 1.5× ¹⁰⁷ cells/mL with PBS, and inoculate 100 μL of Renca cell suspension and 100 μL matrigel matrigel solution suspension in BALB under sterile conditions. /c mouse left flank subcutaneously. When the transplanted tumor grows to about 40-50mm ³ , start administration, divide into control group (normal saline), positive drug group (sunitinib, Pfizer, 50mg/kg), Bacteroides fragilis ZY-312 low (10 ⁶ CFU/piece), medium (10 ⁸ CFU/piece), high (10 ¹⁰ CFU/piece) dose group, Bacteroides fragilis ZY-312 inactivated bacteria (10 ¹⁰ cell/piece) group, Bacteroides fragilis ZY-312 pod Membrane polysaccharide A (PSA, 500 μg/mouse) was continuously gavaged for 4 weeks. After the last administration, the mice were euthanized, and the transplanted tumors were excised and weighed to calculate the tumor inhibition rate; peripheral blood, spleen MDSC, Treg cell ratio.

1. Establishment of transplanted tumor model of mouse renal carcinoma Renca cell line

(1) Preparation of BALB/c mice: 70 6-week-old male BALB/c mice were raised in the IVC animal room at room temperature of 20-22° C., free to eat and drink for 7 days.

(2) Kidney cancer Renca cells ready to be inoculated

1) Renca cells in the logarithmic growth phase were planted in 1640 culture medium (purchased from Gibco, the same below) culture flask containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL) In the medium, when the cell confluence reaches 90%-95% of the area of the bottom of the bottle, wash the cells with PBS, digest with 0.25% trypsin, transfer to a centrifuge tube, centrifuge at 800rpm for 5min, discard the supernatant; wash with PBS for 3 times to remove to remove residual medium.

2) The cell viability was detected by trypan blue staining, and the cells were used for animal inoculation only when the cell viability was greater than 90%.

3) Resuspend the cells with sterile PBS solution, adjust the cell density to 1.5×10 ⁷ cells/mL, transfer to a sterile Ep tube, and place in an ice box until use.

(3) Establishment of mouse kidney cancer xenograft tumor model

1) Gently pipette the cell suspension in the Ep tube to mix it evenly, and draw up the cell suspension with a 1mL syringe.

2) Disinfect the puncture point with povidone iodine solution, gently pick up the left rib skin of each mouse, inject 100 μL Renca cell suspension and 100 μL matrigel matrigel solution subcutaneously into each mouse, and use Carefully press the puncture point with a sterile cotton swab, and then disinfect the puncture point with povidone iodine solution to prevent infection.

3) After the cells were inoculated, the body weight and tumor volume of the mice in each group were monitored twice a week, and the tumor size was measured with a vernier caliper. When the transplanted tumor grew to about 40-50 mm ³ , the experimental mice were treated with drugs.

2. Grouping and administration

When the transplanted tumor grew to about ^40-50mm3 , the experimental mice were treated with drugs, and 70 mice were randomly divided into 7 groups, 10 in each group: control group (normal saline), positive drug group (sunitinib, 50mg/kg), ZY-312 low (10 ⁶ CFU/head), medium (10 ⁸ CFU/head), high (10 ¹⁰ CFU/head) dose group, ZY-312 inactivated bacteria group (10 ¹⁰ cell/head ), ZY-312PSA group (500μg/only).

Control group: 0.2mL normal saline per mouse, once a day, for 4 consecutive weeks;

Positive drug (sunitinib) group: 0.2mL/monkey, once every two days, for 4 consecutive weeks;

ZY-312 low, medium and high dose groups, inactivated bacteria group and PSA group: 0.2mL per mouse, once a day, for 4 consecutive weeks.

3. Detection indicators and statistical methods

(1) Tumor weight and tumor inhibition rate

At the end of the experiment, the transplanted tumors were excised and weighed, and the tumor inhibition rate was calculated. The tumor inhibition rate=100% (average tumor weight in the normal saline group-average tumor weight in the administration group)/average tumor weight in the normal saline group.

(2) Peripheral blood, spleen MDSC and Treg ratio measured by flow cytometry

After the administration, blood was collected from the eye sockets to collect 1 mL of peripheral blood from tumor-bearing mice. The mice were sacrificed and dissected, and the spleen was removed. Red blood cells were removed with 10 mL of erythrocyte lysate. The cells were washed twice by centrifugation at 500 g at room temperature for 5 min. The supernatant was removed and prepared with flow buffer. Make 5× ¹⁰⁷ cells/mL single cell suspension, add 0.5 μL Fc-Block antibody and mix evenly, let stand at 4°C for 15 minutes, centrifuge at 500×g for 5 minutes and remove the supernatant; add 0.5 μL corresponding fluorescently labeled antibody, Cool on ice for 45 minutes; wash the cells, centrifuge at 500×g for 5 minutes and remove the supernatant; resuspend the cells in 500 mL of flow cytometry buffer, and perform detection on a flow cytometer.

Statistical methods: SPSS statistical software 25.0 was used for statistical analysis.

4. Test results

(1) Tumor weight and tumor inhibition rate

Table 1 Tumor weight and tumor inhibition rate of mice in each group (mean±SD)

Note: Compared with the control group, * indicates significant difference p<0.05; ** indicates extremely significant difference p<0.01.

It can be seen from the above table that compared with the control group, the average weight of tumors in the positive drug group, ZY-312 low, medium and high dose, inactivated bacterial solution group, and ZY-312PSA group all decreased; There was no significant difference in tumor weight and tumor inhibition rate between the control group and the positive drug group.

(3) MDSC, Treg ratio

Table 2 Peripheral blood, spleen MDSC, Treg ratio of mice in each group (mean±SD)

MDSC and Treg are two important immune regulatory cells. MDSCs are derived from bone marrow and are heterogeneous cells with immunosuppressive functions. Various cytokines or growth factors in the tumor microenvironment can promote the expansion and activation of MDSCs by activating corresponding signaling pathways, and then inhibit TCR pathways, inhibit Various mechanisms, including T cell biological activity and Treg expansion, inhibit the function of immune cells and promote the occurrence of individual immune tolerance of tumors.

As shown in Table 2 above, compared with the control group, the positive drug group, ZY-312 low, middle and high dose group, ZY-312 inactivated bacteria group and ZY-312PSA group can all down-regulate the MDSC levels in the peripheral blood and spleen of mice, and the fragile There was no significant difference between Bacteroides and PSA groups and positive drugs.

Compared with the control group, the positive drug group, ZY-312 low, middle and high dose group, ZY-312 inactivated bacteria group and ZY-312PSA group could significantly down-regulate the Treg levels in the peripheral blood and spleen of mice, and the levels of Bacteroides fragilis and PSA were significantly lowered. There was no significant difference between the groups and the positive drug. This shows that Bacteroides fragilis and its capsular polysaccharide A can regulate the level of immune cells in mice and reduce tumor-related immune tolerance.

In summary, Bacteroides fragilis ZY-312 and its capsular polysaccharide A can effectively treat Renca renal cell carcinoma xenografts in mice.

Example 4 Drug effect test of Bacteroides fragilis on human bladder cancer cell T24 cell transplanted tumor in mice

Adjust the cell concentration of the human bladder cancer cell line T24 cells in the logarithmic growth phase to 5× ¹⁰⁷ cells/mL with PBS, and inject 50 μL of T24 cell suspension and 50 μL of matrigel matrigel solution into the suspension under sterile conditions Inoculated subcutaneously on the dorsal side of BALB/nu male nude mice. Administration started 1 week after T24 cell inoculation, control group (normal saline), positive drug group (cisplatin, Shandong Qilu Pharmaceutical Co., Ltd., 5 mg/kg), low Bacteroides fragilis ZY-312 (10 ⁶ CFU/monkey), The medium (10 ⁸ CFU/rat), high (10 ¹⁰ CFU/rat) group, and the inactivated Bacteroides fragilis ZY-312 (10 ¹⁰ cell/rat) group were given continuous gavage for 4 weeks. After the last administration, the mice were sacrificed by cervical dislocation and the intact tumor was removed, the tumor mass was recorded and the tumor inhibition rate was calculated; the peritoneal macrophages of mice in each group were collected, and the expressions of IL-1 and TNF-α were detected by ELISA.

1. Establishment of human bladder cancer T24 cell xenograft model

(1) Preparation of BALB/nu nude mice: 60 6-week-old male BALB/nu nude mice were bred in the IVC animal room at a room temperature of 20-22° C. and had free access to feed for 7 days.

(2) Human bladder cancer T24 cells to be inoculated.

1) Human bladder cancer T24 cells in the logarithmic growth phase were planted in a culture flask containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL) in 1640 culture medium, and the cells were confluent. When the area of the bottom of the bottle reaches 90%-95%, wash the cells with PBS, digest with 0.25% trypsin, transfer to a centrifuge tube, centrifuge at 800rpm for 5min, discard the supernatant; wash with PBS for 3 times to remove residual culture base.

2) Resuspend the cells with sterile PBS solution, adjust the cell density to 5×10 ⁷ cells/mL, transfer to a sterile Ep tube, and place in an ice box until use.

(3) Establishment of mouse bladder cancer xenograft tumor model

2) Disinfect the puncture point with povidone iodine solution, gently lift the back skin of each mouse, inject 100 μL of human bladder cancer T24 cell suspension subcutaneously into each mouse, press the puncture point carefully with a sterile cotton swab, and then use iodine Volt solution to disinfect the puncture site to prevent infection.

3) One week after cell inoculation, the experimental mice were treated with drugs.

2. Grouping and administration

Eight weeks after inoculating the cells, 60 mice were randomly divided into 6 groups, 10 in each group: control group (normal saline), positive drug (cisplatin, 5 mg/kg), ZY-312 low (10 ⁶ CFU/mouse ), medium (10 ⁸ CFU/monkey), high (10 ¹⁰ CFU/bird) dose group, ZY-312 inactivated bacteria group (10 ¹⁰ cell/bird).

Control group (normal saline): 0.2mL/rat, once a day, for 4 consecutive weeks.

Positive drug group (cisplatin: 0.2 mL/time, 5 mg/kg intraperitoneal injection, once a week.

ZY-312 low-, medium-, and high-dose groups, and inactivated bacteria group: 0.2 mL per mouse, once a day, for 4 consecutive weeks.

3. Detection indicators and statistical methods

(1) Measure the tumor mass of mice in each group, and calculate the tumor inhibition rate

After the last administration, the mice were sacrificed by cervical dislocation, and the intact tumor tissues were peeled off, rinsed with 0.9% sodium chloride solution, blotted with absorbent paper, weighed, and the tumor mass was recorded and the tumor inhibition rate was calculated. Tumor inhibition rate (%) = (tumor weight of the control group - tumor weight of the drug-administered group)/tumor weight of the control group × 100%

(2) ELISA to measure the expression levels of IL-1 and TNF-α in mouse peritoneal macrophages

1) After the mice were euthanized, 5 mL of Hanks solution was injected into the peritoneal cavity, the accumulated abdomen was lightly rubbed, and the lavage fluid was sucked out. Centrifuge the perfusate at 1300r/min for 5min, discard the supernatant, wash 3 times with Hanks solution, combine the mouse cells, and resuspend the cells in RPMI 1640 culture medium containing 10% fetal bovine serum to prepare a concentration of 2×10 ⁶ cells/mL, add the cell suspension into a 24-well culture plate, 1 mL/well, incubate for 3 hours in a 5% CO ₂ incubator at 37°C to make the macrophages adhere to the wall, discard the supernatant, and use 10% The RPMI 1640 culture medium of fetal bovine serum was repeatedly washed 3 times to remove non-adherent cells, namely macrophages.

2) Add 500 μL of macrophages to be tested to each well of a 24-well plate, adjust the cell concentration to 2×10 ⁶ cells/mL, and add RPMI 1640 culture solution containing 10% fetal bovine serum to supplement to 1000 μL/well, each Set up 3 duplicate wells for the final concentration, and set up a blank control well (only containing RPMI 1640 culture solution). After cultured at 37°C and 5% CO ₂ for 2 days, the supernatant was aspirated, and the expression levels of IL-1 and TNF-α in macrophages were detected according to the instructions of the mouse ELISA kit.

4. Test results

(1) Tumor weight and tumor inhibition rate

Table 3 Tumor weight and tumor inhibition rate of mice in each group (mean±SD)

As shown in the above table, compared with the control group, the tumor weights of the mice in each administration group decreased, and the tumor weight and tumor inhibition rate of the Bacteroides fragilis groups were similar to those of the positive drug group. This shows that Bacteroides fragilis can effectively inhibit the growth of transplanted tumors of bladder cancer in mice, and this inhibitory effect is similar to that of positive drugs.

(2) Expression levels of IL-1 and TNF-α

Table 4 The expression levels of IL-1 and TNF-α in peritoneal macrophages of mice in each group (mean ± SD)

Both IL-1 and TNF-α are immune factors released by macrophages, which can improve the body's immunity and kill tumor cells.

As shown in the table above, compared with the control group, the level of IL-1 in the peritoneal macrophages of mice in the positive drug group decreased; Bacillus group, there is no dose dependence.

Compared with the control group, the level of TNF-α in peritoneal macrophages of mice in the positive drug group decreased; B. fragilis ZY-312 up-regulated the expression of TNF-α in peritoneal macrophages of mice. It is dose-dependent. This shows that Bacteroides fragilis ZY-312 can enhance the anti-tumor immune response of mouse peritoneal macrophages, which is different from the immunosuppression of positive drugs.

In summary, Bacteroides fragilis ZY-312 can enhance the anti-tumor immune response in mice, inhibit tumor growth, and effectively prevent and treat bladder cancer in mice.

Example 5 Drug efficacy test of Bacteroides fragilis zwitterionic capsular polysaccharide A on human bladder cancer T24 cell transplanted tumor in mice

Adjust the cell concentration of the human bladder cancer cell line T24 cells in the logarithmic growth phase to 5× ¹⁰⁷ cells/mL with PBS, and inject 50 μL of T24 cell suspension and 50 μL of matrigel matrigel solution into the suspension under sterile conditions Inoculated subcutaneously on the dorsal side of BALB/nu male nude mice. One week after inoculation of T24 cells, administration began, the control group (normal saline), positive drug group (cisplatin, Shandong Qilu Pharmaceutical Co., Ltd., 5 mg/kg), Bacteroides fragilis ZY-312 PSA low (100 μg/monkey), medium ( 300μg/rat), high (500ug/rat) dose group were administered by gavage continuously for 4 weeks. After the last administration, the mice were sacrificed by cervical dislocation and the intact tumor was removed, the tumor mass was recorded and the tumor inhibition rate was calculated; the peritoneal macrophages of mice in each group were collected, and the expressions of IL-1 and TNF-α were detected by ELISA.

1. (1) Preparation of BALB/nu nude mice: 50 6-week-old male BALB/nu nude mice were raised in the IVC animal room at room temperature of 20-22° C., free to eat feed for 7 days.

(2) Human bladder cancer T24 cells to be inoculated.

1) Human bladder cancer T24 cells in the logarithmic growth phase were planted in a culture flask containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL) in 1640 culture medium, and the cells were confluent. When the area of the bottle bottom reaches 90%-95%, wash the cells with PBS, digest with 0.25% trypsin, transfer to a centrifuge tube, centrifuge at 800rpm for 5min, discard the supernatant; wash with PBS for 3 times to remove the residual medium .

(3) Establishment of mouse bladder cancer xenograft tumor model

2. Grouping and administration

After 8 weeks of cell inoculation, 50 mice were randomly divided into 5 groups, 10 in each group: control group (normal saline), positive drug group (cisplatin, 5mg/kg), Bacteroides fragilis ZY-312PSA low (100μg /rat), medium (300μg/rat), high (500ug/rat) dose groups.

Control group (normal saline): 0.2mL/rat, once a day, for 4 consecutive weeks;

Positive drug group (cisplatin): 0.2 mL/time, 5 mg/kg intraperitoneal injection, once a week, continuous injection for 4 weeks.

PSA low, medium and high dose groups: 0.2 mL per mouse, once a day, for 4 consecutive weeks.

3. Detection indicators and statistical methods

After the last administration, the mice were sacrificed by cervical dislocation, and the intact tumor tissues were peeled off, rinsed with 0.9% sodium chloride solution, blotted with absorbent paper, weighed, and the tumor mass was recorded and the tumor inhibition rate was calculated. Tumor inhibition rate (%) = (tumor weight of model group - tumor weight of observation group) / tumor weight of model group × 100%

4. Test results

(1) Tumor weight and tumor inhibition rate

Table 5 Tumor weight and tumor inhibition rate of mice in each group (mean ± SD)

As shown in the above table, compared with the control group, the tumor weights of the mice in each administration group decreased; there was no obvious dose difference in the Bacteroides fragilis capsular polysaccharide A group. This shows that Bacteroides fragilis capsular polysaccharide A can inhibit the growth of mouse bladder cancer xenografts in a dose-dependent manner.

(2) Expression levels of IL-1 and TNF-α

Table 6 The expression levels of IL-1 and TNF-α in peritoneal macrophages of mice in each group (mean ± SD)

As shown in the table above, compared with the control group, the level of IL-1 in peritoneal macrophages of mice in the positive drug group decreased; Bacteroides fragilis ZY-312 capsular polysaccharide A can up-regulate the expression of IL-1 in mouse peritoneal macrophages The amount of capsular polysaccharide in group A has no obvious dose dependence.

Compared with the control group, the TNF-α level of peritoneal macrophages in the positive drug group decreased; Bacteroides fragilis ZY-312 capsular polysaccharide A could up-regulate the expression of TNF-α in mouse peritoneal macrophages, and the capsular polysaccharide In group A, there was no obvious dose dependence. This shows that Bacteroides fragilis ZY-312 capsular polysaccharide A can dose-dependently enhance the anti-tumor immune response of mouse peritoneal macrophages, which is different from the immunosuppression of positive drugs.

In summary, Bacteroides fragilis capsular polysaccharide A can dose-dependently enhance the anti-tumor immune response in mice and inhibit the growth of human bladder cancer cell line T24 cell transplanted tumors in mice.

Example 6 Study on the efficacy of Bacteroides fragilis and its zwitterionic capsular polysaccharides on mouse RM1 prostate cancer xenografts

Adjust the cell concentration of RM1 cells in the logarithmic growth phase to 2× ¹⁰⁶ cells/mL with PBS, inoculate 100 μL of RM1 cell suspension into C57BL/6 mice with a syringe under sterile conditions and subcutaneously in the right back of the mouse Modeling of subcutaneous prostate cancer xenografts. When the tumor volume reached ^50mm3 , administration began, the control group (normal saline), the positive drug group (paclitaxel, Thermo Fisher Scientific (China) Co., Ltd., 20mg/kg), the Bacteroides fragilis ZY-312 low (10 ⁶ CFU/head), medium (10 ⁸ CFU/head), high (10 ¹⁰ CFU/head) dose group, Bacteroides fragilis ZY-312 inactivated bacteria (10 ¹⁰ cell/head) group, Bacteroides fragilis ZY-312 The capsular polysaccharide A (PSA, 500 μg/rat) group was fed continuously for 4 weeks. After the last administration, the mice were sacrificed by cervical dislocation and the intact tumors were removed, the tumor mass was recorded and the tumor inhibition rate was calculated.

1. Establishment of RM1 cell xenograft mouse model

(1) Preparation of C57BL/6 mice: 60 male C57BL/6 mice were raised in the IVC animal room at a room temperature of 20-22° C., free to eat and drink for 7 days.

(2) RM1 cells to be inoculated

1) Take the RM1 cells in the logarithmic growth phase and plant them in a culture flask containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL) in 1640 culture medium, until the cell confluence reaches the bottom of the flask When the area is 90%-95%, the cells were washed with PBS, digested with 0.25% trypsin, transferred to a centrifuge tube, centrifuged at 800rpm for 5min, and the supernatant was discarded; then washed 3 times with PBS to remove the residual medium.

2) Resuspend the cells in sterile PBS, adjust the cell density to 1.5×10 ⁷ cells/mL, take 1×10 ⁶ cells and mix them in 100 μL PBS, then mix with 100 μL thawed Matrigel-RPMI1640 and store in sterile Epsilon Tubes, kept in ice box until use.

(3) Establishment of mouse prostate cancer xenograft tumor model

2) Disinfect the puncture point with povidone iodine solution, gently lift the skin on the right back of each mouse, and inject 100 μL RM1 cell suspension subcutaneously into each mouse. After the injection, carefully press the puncture point with a sterile cotton swab, and then use iodine Volt solution to disinfect the puncture site to prevent infection.

3) When the tumor volume reaches 50 mm ³ , start to treat the experimental mice with drugs.

2. Grouping and administration

When the transplanted tumor grew to about ^50mm3 , the experimental mice were treated with drugs, and 70 mice were randomly divided into 7 groups, 10 in each group: control group (normal saline), positive drug group (paclitaxel, 20mg/kg), ZY -312 low (10 ⁶ CFU/head), medium (10 ⁸ CFU/head), high (10 ¹⁰ CFU/head) dose group, ZY-312 inactivated bacteria group (10 ¹⁰ cell/head), ZY-312PSA group (500μg/piece).

Control group: 0.2mL/rat, once a day, for 4 consecutive weeks;

Positive drug group (paclitaxel): 0.2 mL/time, intraperitoneal injection, once a week, continuous injection for 4 weeks

ZY-312 low-, medium-, and high-dose groups, inactivated bacteria groups, and ZY-312PSA: 0.2 mL/body, once a day, for 4 consecutive weeks.

3. Detection indicators and statistical methods

(1) Tumor weight and tumor inhibition rate

After the last administration, the mice were sacrificed by cervical dislocation and the intact tumor tissues were peeled off, rinsed with 0.9% sodium chloride solution, blotted with absorbent paper and weighed, the tumor mass was recorded and the tumor inhibition rate was calculated. Tumor inhibition rate (%)=(tumor body weight in model group-tumor body weight in observation group)/tumor body weight in model group×100%.

(2) Intratumoral cytokines

The mouse tumor tissues of the model group and the experimental group were taken, ground and centrifuged, and the supernatant was removed for testing. The temperature of the centrifuge was 4°C, and the rotation speed was 5000rpm/min. After centrifugation for 5min, the supernatant was transferred to a In bacterial Ep tubes, the expression level of IL-6 was determined by ELISA kit.

4. Test results

(1) Tumor weight and tumor inhibition rate

Table 7 Tumor weight and tumor inhibition rate (mean ± SD) of mice in each group

As shown in the above table, compared with the control group, low, medium and high doses of ZY-312, ZY-312 inactivated bacteria and ZY-312PSA can effectively reduce the tumor weight of mice, and B. Drugs did not differ significantly. The results show that Bacteroides fragilis ZY-312, ZY-312 inactivated bacteria and ZY-312PSA can inhibit the growth of RM1 cell transplanted tumors in mice, and have the potential to treat prostate cancer.

(2) Intratumoral cytokines

Table 8 IL-6 expression levels in tumors of mice in each group (mean ± SD)

IL-6 signaling in cancer cells promotes tumor growth, progression and recurrence. As shown in the table above, after treatment, compared with the control group, low, medium and high doses of ZY-312, ZY-312 inactivated bacteria and ZY-312PSA can all significantly inhibit the secretion of IL-6 (P<0.01) . There was no significant difference between Bacteroides fragilis and PSA groups and the positive drug.

In summary, Bacteroides fragilis ZY-312 can regulate the level of immune factors in mice, inhibit tumor growth, and effectively prevent and treat prostate cancer in mice.

Example 7 Drug efficacy experiment of Bacteroides fragilis and its zwitterionic capsular polysaccharide A on mouse SKOV-3 ovarian cancer xenografts

Adjust the cell concentration of SKOV-3 cells in the logarithmic growth phase to 1× ¹⁰⁷ cells/mL with PBS, and inoculate 200 μL of SKOV-3 cell suspension with a syringe under the sterile condition into the subcutaneous groin of the left lower limb of nude mice for subcutaneous ovary Establishment of xenograft tumor model. After the volume ^of subcutaneous transplanted tumor in nude mice reached 50mm ³ , administration began. cells), ZY-312 inactivated bacteria group (10 ¹⁰ cells/cell), Bacteroides fragilis ZY-312 PSA low (100μg/cell), medium (300μg/cell), high (500μg/cell) dose groups 2 weeks. The next day after drug withdrawal, the mice were sacrificed by cervical dislocation and the intact tumor was removed, the tumor mass was recorded and the tumor inhibition rate was calculated; at the same time, ELISA test was performed on the tumor tissue to detect the expression of immune factors IL-2 and IFN-γ in the tumor tissue.

1. Establishment of SKOV-3 cell xenograft model

(1) Preparation of mice: 60 5-6-week-old female BALB/c nude mice were raised in an SPF grade animal room at a room temperature of 20-22°C, free to eat and drink for one week.

(2) SKOV-3 cells to be inoculated.

1) Take the SKOV-3 cells in the logarithmic growth phase and plant them in the culture flask of 1640 culture medium containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL), until the degree of cell fusion reaches When the area of the bottom of the flask reaches 90%, wash the cells with PBS, digest with 0.25% trypsin, transfer to a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant; wash twice with PBS to remove the residual medium.

2) Resuspend the cells with sterile PBS, adjust the cell density to 1×10 ⁷ cells/mL, put them in a sterile Ep tube, and place them in an ice box for later use.

(3) Establishment of mouse ovarian cancer xenograft tumor model

2) Disinfect the puncture point with povidone iodine solution, gently lift the groin skin of the left lower limb of each nude mouse, inject 200 μL of SKOV-3 cell suspension subcutaneously into each nude mouse, carefully press the puncture point with a sterile cotton swab after the injection, and then Disinfect the puncture site with povidone iodine solution to prevent infection.

3) When the volume of the subcutaneously transplanted tumor reaches 50 mm ³ , start to treat the experimental mice with drugs.

2. Grouping and administration

70 tumor-infected nude mice were randomly divided into 7 groups, 10 in each group: control group (normal saline), positive drug group (cisplatin, 2mg/kg), ZY-312 live bacteria group (10 ¹⁰ CFU/mouse) , ZY-312 inactivated bacteria group (10 ¹⁰ cells/body), ZY-312PSA low (100μg/body), medium (300μg/body), high (500μg/body) dose groups.

Control group (normal saline): 0.2mL/rat, once a day, for 4 consecutive weeks;

Positive drug group (cisplatin): the chemotherapy drug cisplatin was selected and injected into the intraperitoneal cavity according to the dose according to the conventional treatment method of chemotherapy, once every other day for 4 weeks.

ZY-312 live bacteria, inactivated bacteria and ZY-312PSA low, medium and high dose groups: 0.2mL per mouse, once a day, for 4 consecutive weeks.

3. Detection indicators and statistical methods

(1) Tumor weight and tumor inhibition rate

After the last administration, the mice were sacrificed by cervical dislocation, and the intact tumor tissues were peeled off, rinsed with 0.9% sodium chloride solution, blotted with absorbent paper, weighed, and the tumor mass was recorded and the tumor inhibition rate was calculated. Tumor inhibition rate (%)=(tumor body weight in model group-tumor body weight in observation group)/tumor body weight in model group×100%.

(2) Detection of immune factors IL-2 and IFN-γ

24 hours after the last administration and before sacrifice by cervical dislocation, blood was collected by picking the eyeball, and centrifuged in a 1.5mL anticoagulant centrifuge tube at 3000r·min ^-1 for 10min with a centrifugal radius of 10.5cm. The levels of IL-2 and IFN-γ were operated according to the instructions of the kit.

4. Test results

(1) Tumor weight and tumor inhibition rate

Table 9 Tumor weight and tumor inhibition rate of mice in each group (mean±SD)

As shown in the above table, compared with the control group, the tumor weights of mice in each administration group decreased. The Bacteroides fragilis and PSA groups were not significantly different from the positive drug, and there was no obvious dose dependence in the PSA group. This shows that Bacteroides fragilis and its PSA can effectively inhibit the growth of mouse ovarian cancer xenografts.

(2) Serum immune factor levels

Table 10 Serum IL-2 and IFN-γ levels of mice in each group (mean ± SD)

Both IL-2 and IFN-γ are known anti-tumor immune factors. As shown in Table 10 above, compared with the control group, the levels of IL-2 and IFN-γ in the positive drug group decreased, which may be related to the immunosuppression of chemotherapeutic drugs; each group of Bacteroides fragilis and its PSA all regulated serum IL to varying degrees. -2. The level of IFN-γ has no obvious dose dependence in the PSA group. This shows that Bacteroides fragilis and its PSA can up-regulate the level of anti-tumor immune factors in mouse serum and enhance the body's anti-tumor immune response.

In conclusion, Bacteroides fragilis ZY-312 and its zwitterionic capsular polysaccharide can up-regulate the level of serum anti-tumor cytokines and effectively prevent and treat SKOV-3 ovarian cancer xenografts in nude mice.

Example 8 Drug efficacy test of Bacteroides fragilis and its zwitterionic capsular polysaccharide A on mouse U14 cervical cancer cell xenografts

The mouse cervical cancer U14 cells in the logarithmic growth phase were adjusted to 1× ¹⁰⁷ cells/mL with PBS, and 200 μL of the cell suspension was inoculated subcutaneously on the right back of Kunming mice with a syringe under sterile conditions. Administration started 7 days after inoculation, control group (normal saline), positive drug group (cisplatin, 3mg/kg), ZY-312 live bacteria group (10 ¹⁰ CFU/only), ZY-312 inactivated bacteria group (10 ¹⁰ cell/monkey), PSA low (100 μg/bird), medium (300 μg/bird), high (500 μg/bird) dose groups were administered by gavage continuously for 14 days. 24 hours after the last administration, the mice were sacrificed by cervical dislocation and the intact tumors were removed, the tumor mass was recorded and the tumor inhibition rate was calculated. Before sacrifice by cervical dislocation, the eyeballs were taken to collect blood, and the peripheral blood T lymphocyte subsets CD4 ⁺ , CD8 ⁺ T cells were analyzed by flow cytometry.

1. Establishment of mouse cervical cancer U14 cell xenograft model

(1) Preparation of Kunming mice: 70 6-8-week-old Kunming mice were raised in the IVC animal room at a room temperature of 20-22° C., free to eat and feed for 7 days.

(2) U14 cells to be inoculated.

1) Take the U14 cells in the logarithmic growth phase and plant them in a culture flask containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL) in 1640 culture medium, and wait until the cell fusion degree reaches the bottom of the bottle When the area is 90%-95%, the cells were washed with PBS, digested with 0.25% trypsin, transferred to a centrifuge tube, centrifuged at 800rpm for 5min, and the supernatant was discarded; then washed 3 times with PBS to remove the residual medium.

2) Resuspend the cells with sterile PBS solution, adjust the cell density to 1×10 ⁷ cells/mL, transfer to a sterile Ep tube, and place in an ice box until use.

(3) Establishment of mouse cervical cancer xenograft tumor model

2) Disinfect the puncture point with povidone iodine solution, gently lift the skin on the right side of each mouse, and inject 200 μL of U14 cell suspension subcutaneously into each mouse. After the injection, carefully press the puncture point with a sterile cotton swab, and then use iodine Volt solution to disinfect the puncture site to prevent infection.

3) 7 days after the cells were inoculated, the experimental mice were treated with drugs.

2. Grouping and administration

Seven days after cell inoculation, 70 mice were randomly divided into 7 groups, 10 in each group: control group (normal saline), positive drug group (cisplatin, 3 mg/kg), ZY-312 live bacteria group (10 ¹⁰ CFU /bird), ZY-312 inactivated bacteria group (1010cell/head), PSA low (100μg/head), medium (300μg/head), high (500μg/head) dose groups.

Control group (normal saline): 0.2mL/rat, once a day, for 14 consecutive days;

Positive drug group (cisplatin): 0.2 mL/time, 3 mg/kg intraperitoneal injection, once every three days, 5 times in total.

ZY-312 live bacteria, inactivated bacteria and ZY-312PSA low, medium and high dose groups: 0.2mL per mouse, once a day, for 14 consecutive days.

3. Detection indicators and statistical methods

(1) Tumor weight and tumor inhibition rate

24 hours after the last administration, the mice were sacrificed by cervical dislocation and the intact tumor tissues were peeled off, rinsed with 0.9% sodium chloride solution, blotted with absorbent paper and weighed, the tumor mass was recorded and the tumor inhibition rate was calculated. Tumor inhibition rate (%) = (tumor weight of model group - tumor weight of observation group) / tumor weight of model group × 100%

(2) Peripheral blood T cell subsets

24 hours after the last administration and before sacrifice by cervical dislocation, blood was collected by picking the eyeball, and 10 μL CD4 ⁺ , CD8 ⁺ T monoclonal antibody and 50 μL EDTA anticoagulant blood were added, mixed evenly, and incubated in the dark for 20 minutes, and 2 mL hemolysin was added to each tube to avoid Incubate with light for 20min, centrifuge at 2000r·min ^-1 for 10min with a centrifugal radius of 10.5cm, discard the supernatant, add 2mL PBS to each tube to mix, centrifuge to discard the supernatant, repeat washing twice, discard the supernatant, add 2mL PBS to re- The peripheral blood T lymphocyte subsets CD4 ⁺ , CD8 ⁺ T cells were analyzed by flow cytometry after suspension.

4. Test results

(1) Tumor weight and tumor inhibition rate

Table 11 Tumor weight and tumor inhibition rate (mean ± SD) of mice in each group

As shown in the table above, compared with the control group, each administration group effectively reduced the weight of transplanted tumors in mice, and the positive drug, ZY-312 inactivated bacteria group and PSA high-dose group had significant differences; Bacteroides fragilis ZY-312 There is no significant difference between each group and PSA group and the positive drug, and there is no obvious dose dependence in the PSA group. It shows that Bacteroides fragilis and its zwitterionic capsular polysaccharide A can inhibit the growth of cervical cancer xenografts in mice.

(2) Peripheral blood T cell subsets

Table 12 Ratio of peripheral blood CD4 ⁺ T cells and CD8 ⁺ T cells of mice in each group (mean±SD)

As shown in the table above, compared with the control group, the proportion of CD4 ⁺ T cells in the positive drug group increased slightly, while the proportion of CD8 ⁺ T cells decreased, which may be related to the immunosuppression of chemotherapy drugs. Compared with the control group, B. fragilis and its PSA groups up-regulated the proportions of CD4 ⁺ and CD8 ⁺ T cells to varying degrees, and there was no obvious dose-dependence in the PSA group. This shows that Bacteroides fragilis and its PSA can regulate the proportion of peripheral blood T lymphocytes and enhance the body's anti-tumor immune response.

In summary, Bacteroides fragilis and its zwitterionic capsular polysaccharide can enhance the body's anti-tumor immune response and inhibit the growth of cervical cancer U14 xenografts in mice.

Example 9 Drug efficacy test of Bacteroides fragilis and its zwitterionic capsular polysaccharide on transplanted tumor of mouse breast cancer 4T1 cells

The breast cancer 4T1 cells in the logarithmic growth phase were adjusted to a cell concentration of 5×10 ⁶ cells/mL with PBS, and 200 μL of the cell suspension was inoculated subcutaneously in the right armpit of BALB/c mice with a syringe under sterile conditions. After 5-7 days of inoculation, when the tumor diameter reached 2-5mm, administration began. ¹⁰ CFU/body), ZY-312 inactivated bacteria group (10 ¹⁰ cells/piece), ZY-312PSA low (100μg/piece), medium (300μg/piece), high (500μg/piece) dose groups were administered continuously for 3 week. After the last administration, the mice were sacrificed by cervical dislocation and the intact tumors were removed, the tumor mass was recorded and the tumor inhibition rate was calculated. Mouse cancer tissues were taken, and the levels of IL-2 and IFN-γ in the tissues were measured by ELISA.

1. Establishment of mouse 4T1 breast cancer cell xenograft model

(1) Preparation of BALB/c mice: 70 6-8-week-old female BALB/c mice were raised in the IVC animal room at a room temperature of 20-22° C., free to eat and drink for 7 days.

(2) 4T1 cells to be inoculated

1) Plant the 4T1 cells in the logarithmic growth phase in a culture flask containing 10% calf serum, penicillin (100U/mL) and streptomycin (100U/mL) in DMEM culture medium, and wait until the cell confluence reaches the bottom of the flask When the area is 90%-95%, the cells were washed with PBS, digested with 0.25% trypsin, transferred to a centrifuge tube, centrifuged at 800rpm for 5min, and the supernatant was discarded; then washed 3 times with PBS to remove the residual medium.

2) Resuspend the cells with sterile PBS solution, adjust the cell density to 5×10 ⁶ cells/mL, transfer to a sterile Ep tube, and place in an ice box until use.

(3) Establishment of mouse breast cancer xenograft tumor model

2) Anesthetize the mice, gently lift the skin of the right armpit of each mouse, and inject 200 μL of 4T1 cell suspension subcutaneously into each mouse. After the injection, carefully press the puncture point with a sterile cotton swab, and then disinfect the puncture point with iodophor solution to prevent infection.

3) After 5-7 days of inoculation, when the tumor diameter reaches 2-5 mm, the experimental mice are treated with drugs.

2. Grouping and administration

70 mice were randomly divided into 7 groups, 10 in each group: control group (normal saline), positive drug group (doxorubicin, 2mg/kg), ZY-312 live bacteria group (10 ¹⁰ CFU/mouse), ZY -312 inactivated bacteria group (10 ¹⁰ cells/monkey), PSA low (100 μg/bird), medium (300 μg/bird), high (500 μg/bird) dose groups.

Control group (normal saline): 0.2mL/rat, once a day, for 3 consecutive weeks;

Positive drug group (doxorubicin): 0.2 mL/time, 2 mg/kg intraperitoneal injection, once every other day, for 3 consecutive weeks.

ZY-312 bacteria and PSA low-, medium-, and high-dose groups: 0.2 mL/body, once a day, for 3 consecutive weeks.

3. Detection indicators and statistical methods

(1) Tumor weight and tumor inhibition rate

(2) Levels of immune factors

Collect freshly collected tumor samples and chop them into small pieces, and transfer the chopped tissues into tubes with pre-cooled PBS. Homogenize using a tissue homogenizer to make a 10% tissue homogenate. Centrifuge at 3000 rpm for 20 min, take the supernatant, and detect the levels of IL-2 and IFN-γ in the tumor tissue according to the instructions of the ELISA test kit.

4. Test results

(1) Tumor weight and tumor inhibition rate

Table 13 Tumor weight and tumor inhibition rate of mice in each group (mean±SD)

As shown in the table above, compared with the control group, the tumor weights of the mice in each administration group decreased, and there was a significant difference between the positive drug and the high-dose PSA group; there was no significant difference between the Bacteroides fragilis and PSA groups and the positive drug, and the PSA There was no obvious dose dependence within the group. This shows that Bacteroides fragilis and its zwitterionic capsular polysaccharide can inhibit the growth of 4T1 breast cancer xenografts in mice.

(2) Levels of immune factors

Table 14 Levels of tumor IL-2 and IFN-γ in each group of mice (mean ± SD)

As shown in the table above, compared with the control group, each administration group up-regulated the IL-2 level in the mouse tumors, and the high-dose PSA group had a significant difference; the PSA group had a certain dose-dependence.

Compared with the control group, each drug administration group up-regulated the level of IFN-γ in the mouse tumors; the up-regulation range of the Bacteroides fragilis and PSA groups was greater than that of the positive drug group. There is a certain dose dependence in the PSA group. This shows that Bacteroides fragilis and its PSA can regulate the level of immune factors in the mouse tumor and enhance the body's anti-tumor immune response.

In summary, Bacteroides fragilis and its zwitterionic capsular polysaccharide can inhibit the growth of 4T1 breast cancer xenografts in mice.

Certainly, the present invention also can have other multiple embodiments, without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and deformations according to the present invention, but these corresponding Changes and deformations should belong to the scope of protection of the appended claims of the present invention.

Claims

An application of Bacteroides fragilis and/or its zwitterionic capsular polysaccharide in the preparation of products for the prevention and/or treatment of genitourinary system tumors, characterized in that the Bacteroides fragilis is the fragilis with the preservation number CGMCC No.10685 Bacteroides ZY-312, the zwitterionic capsular polysaccharide is extracted from Bacteroides fragilis ZY-312.
The application according to claim 1, characterized in that, the Bacteroides fragilis is a live Bacteroides fragilis cell, and the fragilis that has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, physically treated or inactivated One or more of Bacteroides, Bacteroides fragilis lysate, and Bacteroides fragilis liquid culture supernatant.
The use according to claim 1 or 2, characterized in that the zwitterionic capsular polysaccharide contains capsular polysaccharide A. Wherein, the structure of the capsular polysaccharide A is as follows:

Preferably, the weight average molecular weight of the capsular polysaccharide A is 80-90KD, the part with Mw distributed in 70-100KD accounts for 70-80% of the total, and the ratio of weight average molecular weight/number average molecular weight (Mw/Mn) is 1.0-1.3. More preferably, the content of capsular polysaccharide A in the zwitterionic capsular polysaccharide exceeds 95wt%.

Preferably, the preparation method of the zwitterionic capsular polysaccharide comprises the following steps:

(1) Centrifuge the fermented Bacteroides fragilis bacteria liquid to collect the sediment to obtain the Bacteroides fragilis bacteria sludge; take the bacteria sludge, add purified water 3 to 10 times the weight of the bacteria sludge to suspend the bacteria, and adjust with acid solution The pH is 2.0-4.5, extracted at 50-120°C for 0.5-3.0 hours, cooled to room temperature, centrifuged at room temperature, and the supernatant is taken to obtain a crude sugar solution;

(2) The crude sugar solution is concentrated by ultrafiltration membrane ultrafiltration to remove small molecular impurities until the conductivity is stable, and the reflux liquid is collected;

(3) Add an equal volume of 40mmol/L Tris-HCl to the reflux liquid to convert the salt; ion exchange column chromatography, gradient elution, segmented collection, SEC-HPLC tracking monitoring, and merge the 206nm absorption peak into a single, symmetrical peak component , ultrafiltration membrane ultrafiltration, adding purified water and repeated ultrafiltration until the conductivity is stable, collecting the reflux liquid and freeze-drying to obtain the Bacteroides fragilis zwitterionic capsular polysaccharide.

Preferably, the centrifugation in step (1) is centrifugation at 11000-13000 g for 8-12 minutes.

Preferably, the acid solution in step (1) can be one or more of organic acid, inorganic acid and acid buffer. Wherein, the inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.; the organic acid can be acetic acid, citric acid, etc.

Preferably, the molecular weight of the ultrafiltration membrane in step (2) can be 100, 50, 30, 10, 5, 3KD or a range between any two molecular weight values.

Preferably, 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, pH5.0-9.0 contains 0.2mol/L NaCl 20mmol/L Tris -HCl gradient elution for 25 column volumes, collected in sections, 100mL/bottle (component); the molecular weight of the ultrafiltration membrane is 10KD.
The use according to any one of claims 1-3, characterized in that the product is food or medicine.

Preferably, the food product comprises milk powder, cheese, curd, yogurt, ice cream or fermented cereals. Preferably, the food can also be animal food, such as feed and the like.

Preferably, 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.

Preferably, the drug is Bacteroides fragilis or its zwitterionic capsular polysaccharide used alone, or Bacteroides fragilis and its zwitterionic capsulated polysaccharide used in combination.
The use according to any one of claims 1-4, characterized in that the genitourinary system tumors include female breast and reproductive organ tumors, male reproductive organ tumors, and urinary organ tumors.

Preferably, the genitourinary system tumors include renal cancer, bladder cancer, urothelial cancer, breast cancer, ovarian cancer, cervical cancer and prostate cancer.
A composition for preventing and treating tumors of the genitourinary system is characterized in that the composition contains Bacteroides fragilis ZY-312 with the preservation number CGMCC No. 10685 and/or its zwitterionic capsular polysaccharide.
The composition according to claim 6, characterized in that, the Bacteroides fragilis is a live Bacteroides fragilis cell, which has been inactivated, genetically recombined, transformed or modified, attenuated, chemically treated, physically treated or inactivated One or more of Bacteroides fragilis, lysate of Bacteroides fragilis, liquid culture supernatant of Bacteroides fragilis.
The composition according to claim 6 or 7, wherein the zwitterionic capsular polysaccharide contains capsular polysaccharide A. Wherein, the structure of the capsular polysaccharide A is as follows:

Preferably, the weight average molecular weight of the capsular polysaccharide A is 80-90KD, the part with Mw distributed in 70-100KD accounts for 70-80% of the total, and the ratio of weight average molecular weight/number average molecular weight (Mw/Mn) is 1.0-1.3. More preferably, the content of the capsular polysaccharide A exceeds 95wt%.

Preferably, the preparation method of the zwitterionic capsular polysaccharide comprises the following steps:

(1) Centrifuge the fermented Bacteroides fragilis bacteria liquid to collect the sediment to obtain the Bacteroides fragilis bacteria sludge; take the bacteria sludge, add purified water 3 to 10 times the weight of the bacteria sludge to suspend the bacteria, and adjust with acid solution The pH is 2.0-4.5, extracted at 50-120°C for 0.5-3.0 hours, cooled to room temperature, centrifuged at room temperature, and the supernatant is taken to obtain a crude sugar solution;

(2) The crude sugar solution is concentrated by ultrafiltration membrane ultrafiltration to remove small molecular impurities until the conductivity is stable, and the reflux liquid is collected;

(3) Add an equal volume of 40mmol/L Tris-HCl to the reflux liquid to convert the salt; ion exchange column chromatography, gradient elution, segmented collection, SEC-HPLC tracking monitoring, and merge the 206nm absorption peak into a single, symmetrical peak component , ultrafiltration membrane ultrafiltration, adding purified water and repeated ultrafiltration until the conductivity is stable, collecting the reflux liquid and freeze-drying to obtain Bacteroides fragilis.

Preferably, the centrifugation in step (1) is centrifugation at 11000-13000 g for 8-12 minutes.

Preferably, the acid solution in step (1) can be one or more of organic acid, inorganic acid and acid buffer. Wherein, the inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.; the organic acid can be acetic acid, citric acid, etc.

Preferably, the molecular weight of the ultrafiltration membrane in step (2) can be 100, 50, 30, 10, 5, 3KD or a range between any two molecular weight values.

Preferably, 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, pH5.0-9.0 contains 0.2mol/L NaCl 20mmol/L Tris -HCl gradient elution for 25 column volumes, collected in sections, 100mL/bottle (component); the molecular weight of the ultrafiltration membrane is 10KD.
The composition according to any one of claims 6-8, wherein the composition is a medicine. Preferably, the food product comprises milk powder, cheese, curd, yogurt, ice cream or fermented cereals. Preferably, the food can also be animal food, such as feed and the like.

Preferably, 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.

Preferably, the drug is Bacteroides fragilis or its zwitterionic capsular polysaccharide used alone, or Bacteroides fragilis and its zwitterionic capsulated polysaccharide used in combination.

Preferably, the drug can be administered orally, enemaly or parenterally.

Preferably, the drug administration cycle can be intermittent administration, periodic administration, continuous administration or long-term administration.
The composition according to any one of claims 6-9, characterized in that, the genitourinary system tumors include female breast and reproductive organ tumors, male reproductive organ tumors and urinary organ tumors.

Preferably, the genitourinary system tumors include renal cancer, bladder cancer, urothelial cancer, breast cancer, ovarian cancer, cervical cancer and prostate cancer.