Novel non-enterotoxin-producing bacteroides fragilis with colorectal cancer inhibiting effect

Abstract

The invention discloses a novel non-enterotoxin-producing bacteroides fragilis strain with a colorectal cancer inhibiting effect, belongs to the field of microorganisms, and is named as ZY0804, and the preservation number is CGMCC No.33783. The invention further discloses a preparation method of the novel non-enterotoxin-producing bacteroides fragilis strain. Experimental research proves that the novel NTBF derived from the para-carcinoma tissue of the CRC patient plays a remarkable role in inhibiting the development process of the colorectal cancer. Good application and medicine research values are realized.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a novel non-enterotoxin-producing Bacteroides fragilis strain having the function of inhibiting colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive tract, with the third highest incidence and the second highest mortality rate among malignant tumors. In recent years, the incidence and mortality of CRC have been on the rise worldwide. By 2030, it is expected that there will be more than 2.2 million new cases and more than 1.1 million deaths worldwide. Studies have shown that the occurrence of CRC is related to many factors such as diet, environment, genetics, and intestinal microorganisms. However, its specific causes and mechanisms are not clear, and there is currently no effective intervention plan. At present, studies have confirmed the existence of bacterial colonization in human tumors and adjacent tissues. These bacteria play an important biological role in the occurrence, development, metastasis, and immune escape of tumors. Some bacteria block the occurrence and development of tumors by secreting probiotic elements. The study of the probiotic mechanism of CRC-related bacteria has become a new direction for the prevention and treatment of CRC.

[0003] Bacteroides fragilis (BF) belongs to the genus Bacteroides and is a Gram-negative short rod. It is divided into enterotoxigenic bacteroides fragilis (ETBF) and nontoxigenic Bacteroides fragilis (NTBF) according to whether it secretes Bacteroides fragilis toxin. NTBF is considered to be one of the best probiotic candidates for the prevention and treatment of intestinal diseases such as CRC because it can produce beneficial factors such as polysaccharide A and short-chain fatty acids. The anaerobic nature of Bacteroides fragilis and its stringent requirements for the culture environment make the isolation and cultivation of this type of bacteria a major problem that plagues correlation studies. No NTBF strains isolated from CRC-related tissues have been reported. Summary of the invention

[0004] The applicant isolated a non-enterotoxin-producing Bacteroides fragilis from paracancerous tissues of CRC patients. The morphological, biochemical and genomic characteristics of the strain were analyzed and it was shown that the isolate was a new wild NTBF isolate, named ZY0804, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration. Its Latin name is Bacteroides fragilis, the deposit number is CGMCC No.33783, and the deposit date is March 10, 2025. Experimental studies have confirmed that this new NTBF derived from paracancerous tissues of CRC patients has a significant inhibitory effect on the development of colorectal cancer.

[0005] And the application of the NTBF strain of the present application in preparing drugs for treating colorectal cancer.

[0006] And using the NTBF strain of the present application to develop drugs for treating colorectal cancer.

[0007] The beneficial effects of the above scheme are as follows: the novel non-enterotoxigenic Bacteroides fragilis with colorectal cancer inhibitory effect of the present application has been identified as a new NTBF wild isolate. Through in vivo and in vitro experiments, it has been confirmed that it has a significant CRC inhibitory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The basic biological characteristics of the NTBF strain of the present invention; Figure 2 This is the isolation, culture and identification process of the NTBF strain of the present invention; Figure 3 This is the research process of the anti-cancer effect and mechanism of the NTBF strain of the present invention; Figure 4 This is a graph showing the CRC inhibition effect of the NTBF strain of the present invention; Figure 5 This is a Ki67 immunohistochemical staining image of a tumor tissue section of the NTBF strain of the present invention; Figure 6 This is a diagram showing the multi-angle inhibitory effect of the NTBF strain of the present invention on CRC; Figure 7 The expression of CDK2 and CyclinA2 in CRC cells mediated by the NTBF strain of the present invention. DETAILED DESCRIPTION

[0009] The technical scheme of the present invention is clearly and completely described below in conjunction with the specific embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0010] The applicant isolated a strain of non-enterotoxigenic Bacteroides fragilis NTBF (full genome Genebank accession number: CP139161) from adjacent tissues of CRC patients. Figure 1Through the analysis of the morphology, biochemical characteristics and genomic characteristics of the strain, according to the Bergey's Bacterial Identification Manual, the physiological and biochemical characteristics are consistent with the characteristics of B. fragilis; the complete map sequencing results showed that the bacterial genome consists of a closed chromosome with a genome size of 5174916bp, a G+C content of 43.19%, and 4369 genes. Based on the core gene system evolution analysis, the isolate was a new NTBF wild isolate, named ZY0804.

[0011] 1 Isolation and culture of NTBF 1.1 Bacterial enrichment Separate the tissue under a sterile environment, use a disposable sterile inoculation loop to pick up the cut surface of the sample, inoculate it into brain heart infusion broth for bacterial growth, place the plate upside down in an anaerobic culture box, and culture it anaerobically at 37°C for 18-24 hours. Observe the turbidity of the broth and use this broth as the selective culture inoculation material.

[0012] 1.2 Selective culture Use a disposable sterile inoculation loop to take a loop of enrichment solution, streak it onto a Bacteroides-bile-esculin agar (BBE) blood plate using the three-line method, place the plate upside down in an anaerobic culture box, and culture it anaerobically at 37 °C for 24-72 h. Observe the colony morphology, color, size, and surface properties of the colonies on the plate. Pick a single colony suspected of Bacteroides fragilis for an oxygen tolerance test.

[0013] 1.3 Oxygen resistance test Pick more than three characteristic colonies that meet the BF standard from the BBE blood agar plate and inoculate them onto the non-selective culture medium CDC anaerobic blood agar plate. Pick the same colony onto three plates and culture them in an anaerobic environment, a microaerobic environment, and an aerobic environment at 37°C incubator for 24-48 hours, respectively, and observe the growth of the colonies in the three environments.

[0014] 1.4 Purification and preservation of strains According to the growth conditions of the colonies in three environments (colonies that only grow in anaerobic environments are selected), colony morphology and microscopic observation results, the target colonies are picked and streaked on the BBE blood plate. After anaerobic culture at 37 ° C for 24-48 hours, a disposable sterile inoculation loop is used to pick and purify single colonies and inoculate them into 10 mL brain heart infusion broth. After anaerobic culture at 37 ° C for 18-24 hours, 0.25 mL sterile glycerol, 0.75 mL PBS and 0.5 mL bacterial solution are mixed thoroughly to make glycerol bacteria, and the strains are stored at -20 ° C and liquid nitrogen for standby use, and marked. The morphology of the colonies of Bacteroides fragilis on the BBE blood plate is slightly convex, smooth and shiny, with neat edges, brown, and the esculin in the culture medium can be decomposed to make the colonies black.

[0015] 2 Identification of NTBF isolates 2.1 Gram staining and microscopy Preparation: In the clean bench, use a disposable sterile inoculation loop to dip a loop of liquid and drop it in the center of the slide. After it dries, pass the slide back and forth over the flame of an alcohol lamp 2-3 times to dry and fix. The temperature of the slide should not be too hot. Primary staining: Add crystal violet staining solution to cover, stain for 1 min, then wash with distilled water. Mordant staining: Add Gram iodine solution to cover, act for 1 min, then wash with distilled water. Decolorization: Add decolorizing alcohol to cover, act for 10-30 s. Restaining: Add Tsar restaining solution to cover, restain for 1 min, wash with distilled water, and wait to dry. Microscopic examination: After the specimen is dry, add cedar oil to the slide, and observe the staining results with a 100× objective lens to observe the microscopic morphology of the bacteria.

[0016] 2.2 Biochemical properties determination The activated bacterial liquid of the purified and preserved strains was inoculated into the biochemical identification tubes respectively, and the operation was carried out according to the instructions for use of the bacterial microbiochemical identification tubes. Biochemical identification indicators of Bacteroides fragilis: esculin, galactose, cellobiose, melezitose, fructose, rhamnose, arabinose, mannitol, nitrate (reduced), mannose, sorbose, melibiose, muscarinose, sucrose, salicin, lacto-mannitol, glucose, inositol, purple milk, D-ribose, lactose, maltose, gelatin test and nitrate (gas production); catalase test.

[0017] 2.3 Complete bacterial genome sequencing Bacterial genomic DNA was extracted and the concentration and purity of the extracted DNA were measured using a micro-spectrophotometer. When the concentration was ≥60 ng / μL, OD260 / 280: 1.6-2.2, OD260 / OD230: 1.6-2.2, the DNA was sent to BGI for genome sequencing.

[0018] like Figure 2 This is the isolation, culture and identification process of NTBF.

[0019] 3 Identification of the tumor suppressor effect of NTBF 3.1 In vitro experiments Extraction of Bacteroides fragilis cell-free supernatant ( Bacteroides fragilisThe cell-free supernatant (BfCFS) was used to analyze its effect on the proliferation of CRC cells HT29 and normal colon epithelial cells NCM460 by CCK8 method, and the optimal concentration and action time were analyzed. Then, the effect of BfCFS on the proliferation of CRC cells (HT29, SW480, LoVo) was further verified by cell colony formation and EdU staining experiments. The effect of BfCFS on the apoptosis and cell cycle of HT29 cells was analyzed by flow cytometry, and the expression levels of apoptosis (Bax, Bcl-2, Caspase-3) and cell cycle (Cyclin A2, CDK2) related proteins were detected by Western Blot. The effect of BfCFS on the migration of CRC cells was explored by cell scratch assay. The stability of active substances in BfCFS was analyzed by adjusting the pH value of BfCFS with NaOH and HCl, heating in a metal bath at 100 ℃ for 10 min, and protease treatment.

[0020] 3.2 In vivo experiments The human CRC cell line HT29 was used to construct a xenograft model to study the anti-CRC activity of BfCFS in vivo. HT29 cell suspension (0.2 mL / cell) was incubated at 3×10 6 The mice were randomly divided into a control group (BHI treatment group) and an experimental group (BfCFS treatment group), with 6 mice in each group. Peritumoral injection was used for intervention, once every 4 days, and the tumor size was measured before each injection until the experiment was ended on the 28th day and the tumor weight was weighed. Then, the expression of Ki67 in tumor tissue was detected by immunohistochemical staining, and the expression of apoptosis (Bax, Bcl-2, Caspase-3) and cycle (Cyclin A2, CDK2) related proteins in tumor tissue cells were detected by Western Blot.

[0021] Figure 3 This is the research process of the anti-cancer effect and mechanism of NTBF in the present invention.

[0022] The CCK8 assay was used to first verify the effects of different concentrations of NTBF cell-free supernatant (BfCFS) on cell proliferation at different times on HT29 cells, and finally 5×10 7 CFU / mL and 48 h were the optimal concentration and time, and were used as the reference concentration and time for subsequent cell experiments and in vivo experiments ( Figure 4 a); To further clarify the inhibitory effect of NTBF on cell proliferation, cell colony formation experiments were performed ( Figure 4 b) Experiment with EdU ( Figure 4c) The proliferation of HT29, SW480, and LoVo after BfCFS intervention was detected and analyzed. The results of in vitro experiments confirmed that this paracancerous NTBF had a significant inhibitory effect on CRC proliferation; a Balb / c nude mouse CRC model was established, and it was verified that NTBF could significantly inhibit tumor growth in vivo ( Figure 4 d); BfCFS treatment significantly reduced the volume and weight of xenograft tumors. To detect the proliferation activity of tumor cells, we used immunohistochemical staining to detect the expression of Ki67, a tumor cell nuclear proliferation marker, in the BfCFS treatment group and the BHI negative control group. The results showed that the peritumoral injection of BfCFS reduced the staining intensity of Ki67 compared with the injection of BHI, as shown in Figure 2. Figure 5 , the Ki67 positive cell rate decreased by 29.12% ( P <0.05). The results showed that peritumoral injection of BfCFS significantly reduced the proliferation activity of tumor tissue cells compared with injection of BHI.

[0023] like Figure 5 The Ki67 immunohistochemical staining images of tumor tissue sections of the NTBF strain of the present invention (n=3), wherein A. the tumor sections were immunohistochemically stained using anti-Ki67 antibodies, and representative images were shown (magnification: the left image is x200, and the right image is x400); B. the Ki67 positivity rate of tumor tissue cells in mice treated with BfCFS and BHI (n=3), * indicates P<0.05.

[0024] To further explore the tumor suppressor mechanism of NTBF strain, we studied cell apoptosis ( Figure 6 a1&a2)、cell cycle( Figure 6 b) Cell migration ( Figure 6 c) The inhibitory effect of NTBF on CRC was explored from three perspectives. The results showed that BfCFS had no effect on the apoptosis and migration ability of CRC cells in vitro, while BfCFS treatment of HT29 cells led to S phase arrest of the cell cycle.

[0025] CDK-centered cell cycle drive and regulation mechanisms play a key role in the occurrence and development of cancer. We conducted preliminary detection and analysis of the expression of CDK2 and CyclinA2 in CRC cells mediated by NTBF. The experimental results showed that the expression of CDK2 and CyclinA2 was significantly inhibited under NTBF (e.g. Figure 7 The experimental results further verified that NTBF-mediated cell cycle S phase arrest may be related to the CDK-centered cell cycle driving and regulatory mechanism.

[0026] On this basis, those skilled in the art can develop and utilize the NTBF strain of the present application for the preparation of drugs for treating colorectal cancer, and can develop drugs for treating colorectal cancer comprising the NTBF strain of the present application and its derivatives. The significant effects of the embodiments of the present application can be expected.

[0027] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0028] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A strain of Bacteroides fragilis characterized by: The Bacteroides fragilis is named ZY0804 and its deposit number is CGMCC No.33783.

2. The use of Bacteroides fragilis according to claim 1, characterized in that: Used to prepare drugs for treating colorectal cancer.

3. A medicament comprising the Bacteroides fragilis and its derivatives as claimed in claim 1.

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