Application of bacteroides ovale in diagnosis or prognosis of liver cancer
By detecting the content of Bacillus ovarice in the feces of liver cancer patients and its cancer-promoting effects in mice, a new target for diagnosis and treatment of liver cancer is provided, and the problem of lack of effective prevention and treatment methods for liver cancer targeting fecal bacteria in the prior art is solved, and a new idea of early diagnosis and potential treatment of liver cancer is realized.
Patent Information
- Application Number
- CN202510086195.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-05-06
AI Technical Summary
There is a lack of effective methods in the prior art for the prevention and treatment of liver cancer, especially in targeting fecal bacteria.
By detecting the significantly increased content of B. ovatus in the feces of patients with liver cancer and promoting liver cancer progression in mice, a new diagnostic and therapeutic target for liver cancer is provided. The method includes using metagenomic sequencing to detect Bacillus ovarice content in fecal samples and injecting mouse liver cancer cells into portal veins to induce liver cancer models.
The finding that Bacteroides ovariectomy is significantly increased in patients with liver cancer and promotes liver cancer progression in mouse models provides a potential target for the diagnosis and treatment of liver cancer, especially through the detection of metabolites of the bacteria such as iso-LCA.
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Abstract
Description
Technical Field
[0001] The present application relates to the field of biomedicine technology, and in particular to the application of Bacteroides ovatus in the diagnosis or prognosis of liver cancer. Background Art
[0002] The human small and large intestines are colonized by many different types of symbiotic bacteria, known as intestinal microorganisms or intestinal flora. According to statistics, the number of intestinal flora in each centimeter of colon reaches an astonishing 10 11 ~10 12 The composition of human colonic microorganisms is mainly Bacteroidetes and Firmicutes, with a small number of other bacterial genera, but there are also large differences between individuals, and the types and numbers of flora in different intestinal segments are also different. The shaping of the intestinal flora begins when the embryo communicates with the mother's flora. Once the flora is established after birth, tens of thousands of flora will colonize the intestine, and will then be affected by external environmental factors and lifestyle.
[0003] An important function of the intestinal flora is to digest dietary fiber, including polysaccharides contained in plant cell walls that are difficult for the human body to decompose, and other substances that can be decomposed by bacteria as nutrients. The short-chain fatty acids produced by bacteria decomposing fiber-derived polysaccharides can affect the homeostasis of intestinal epithelial cells and lymphocytes. In recent years, research on intestinal flora imbalance, enteritis, and colorectal cancer has also received attention. When the normal microbial community is destroyed, the number of pathogenic bacteria increases, and inflammatory-related diseases and even digestive tract tumors will occur. Therefore, exploring abnormally elevated or decreased intestinal microorganisms in the intestine plays an important role in the early diagnosis and timely treatment of cancer.
[0004] Currently, there are few reports on the prevention and treatment of liver cancer by targeting fecal microbiota in clinical practice. Summary of the invention
[0005] The purpose of this application is to overcome the shortcomings of the above-mentioned prior art and provide an application of Bacteroides ovatus in the diagnosis or prognosis of liver cancer. The content of Bacteroides ovatus (B. ovatus) in the present application is significantly increased in the feces of liver cancer patients, and promotes the progression of liver cancer in mice. It is further found that the bacteria can decompose the primary bile acid chenodeoxycholic acid (CDCA) under anaerobic conditions to produce the secondary bile acid isolithocholic acid (iso-LCA), which provides new ideas for the diagnosis and treatment of liver cancer.
[0006] To achieve the above purpose, the technical solution adopted by this application is:
[0007] The present application provides the use of a reagent for detecting the content of Bacteroides ovatus in the preparation of a product for liver cancer diagnosis or prognosis.
[0008] The inventors of the present application found that compared with healthy people, the content of Bacteroides ovatus (B. ovatus) in the feces of liver cancer patients was significantly increased, and it promoted the progression of liver cancer in mice, providing new ideas for new targets for the diagnosis and treatment of liver cancer.
[0009] As a preferred embodiment of the application described in this application, the product includes a chip, a kit or a test strip.
[0010] As a preferred embodiment of the application described in the present application, the reagent includes a reagent for detecting the content of Bacteroides ovatus in a stool sample using metagenomic sequencing.
[0011] The present application can also adopt other forms to detect the content of Bacteroides ovatus.
[0012] The present application uses metagenomic sequencing to detect the absolute or relative content of Bacteroides ovatus in fecal samples.
[0013] As a preferred embodiment of the application described in the present application, the content of the Bacteroides ovatus in the feces of liver cancer patients is higher than that in healthy people.
[0014] The present application also provides the use of Bacteroides ovatus in preparing a reagent for promoting an animal model of liver cancer.
[0015] As a preferred embodiment of the application described in the present application, the cell concentration of the Bacteroides ovatus is 1 to 5×10 8 CFU, preferably 10 8 CFU.
[0016] In this application, mice were gavaged with Bacteroides ovatus and liver cancer cells were injected into the portal vein to induce liver cancer. The liver / body weight ratio and tumor infiltration ratio of the same liver lobe of tumor-bearing mice treated with Bacteroides ovatus were higher than those of the PBS group and the control bacteria group, and the progression of liver cancer was more rapid, indicating that the Bacteroides ovatus of this application is a type of bacteria that can promote liver cancer.
[0017] As a preferred embodiment of the application described in the present application, the liver cancer animal model includes a liver cancer animal model induced by Hepa 1-6 or RIL-175.
[0018] The method for constructing the liver cancer animal model induced by Hepa 1-6 or RIL-175 comprises the following steps:
[0019] After anesthesia, a small incision of about 1 cm was made along the midline of the mouse abdomen using sterile surgical scissors. The portal vein was then found and 2-3×10 liver cancer cells were directly injected into the mouse. 6Hepa 1-6 or RIL-175 tumor cells were injected into the hepatic portal vein, and finally the inner and outer epithelial sutures of the mice were surgically sutured to obtain the Hepa 1-6 or RIL-175-induced liver cancer animal model.
[0020] The present application also provides the use of Bacteroides ovatus in decomposing primary bile acid into secondary bile acid.
[0021] As a preferred embodiment of the application described in the present application, the primary bile acid includes chenodeoxycholic acid; the secondary bile acid includes isolithocholic acid.
[0022] As a preferred embodiment of the application described in the present application, the Bacteroides ovatus is cultured in an anaerobic environment for 48 to 72 hours to decompose primary bile acid into secondary bile acid.
[0023] The present application further found that isolithocholic acid was detected in the bacterial supernatant to which chenodeoxycholic acid was added, indicating that Bacteroides ovatus can decompose the primary bile acid chenodeoxycholic acid (CDCA) to produce the secondary bile acid isolithocholic acid (iso-LCA) under anaerobic conditions, providing new ideas for new targets for the diagnosis and treatment of liver cancer.
[0024] Among them, the present application uses liquid phase mass spectrometry LC-MS to efficiently and sensitively detect dozens of common bile acids including iso-LCA.
[0025] Compared with the prior art, this application has the following beneficial effects:
[0026] The present application provides an application of Bacteroides ovatus in the diagnosis or prognosis of liver cancer. The present application finds through metagenomic sequencing analysis that the content of Bacteroides ovatus (B. ovatus) in the feces of liver cancer patients is significantly increased, and further proves the carcinogenic effect of Bacteroides ovatus through a mouse HCC model induced by the portal vein. Then, through liquid chromatography-mass spectrometry detection, it is found that the bacteria can decompose the primary bile acid chenodeoxycholic acid (CDCA) under anaerobic conditions to produce the secondary bile acid isolithocholic acid (iso-LCA), indicating the potential of Bacteroides ovatus and its metabolites in clinical HCC auxiliary diagnosis (tumor markers), treatment or prognosis evaluation. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is the result of analyzing the relative content of Bacteroides ovatus in feces of the control group and HCC patient group by metagenomic sequencing;
[0028] Figure 2 The results of Bacteroides ovatus promoting the progression of liver cancer in mice ( Figure 2 Middle A shows that mice were gavaged with Bacteroides ovatus or control bacteria two weeks in advance, and then RIL-175 was injected into the hepatic portal vein to induce HCC. Three weeks after induction, the mice were sacrificed and the differences in liver tumor load and photos of the mice liver were detected; Figure 2 Middle B is the liver / body weight ratio of tumor-bearing mice and the tumor infiltration ratio of the same liver lobe of tumor-bearing mice);
[0029] Figure 3 The results of Bacteroides ovatus decomposing the primary bile acid chenodeoxycholic acid (CDCA) to produce the secondary bile acid isolithocholic acid (iso-LCA) ( Figure 3 A in the figure is a flow chart; Figure 3 (B) is the result of three independent repeated experiments on the content of chenodeoxycholic acid (CDCA) and isolithocholic acid (iso-LCA). DETAILED DESCRIPTION
[0030] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.
[0031] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.
[0032] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.
[0033] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.
[0034] Example 1: The content of Bacteroides ovatus in the feces of HCC patients increased significantly
[0035] This application uses metagenomic sequencing to analyze the relative content of Bacteroides ovatus in the feces of clinical HCC patients and control populations.
[0036] Methods: Fresh stool samples were collected from HCC patients and control group (healthy) people, and the bacterial genome was extracted using a bacterial genome extraction kit after quick freezing in liquid nitrogen. Library construction and sequencing analysis were performed. Specific references: Library strategy: WGS, Library selection: Metagenomic, Library selection: Random, Library layout: paired, Platform: ILLUMINA, Instrument model: Illumina NovaSeq 6000.
[0037] Results: Figure 1 As shown in the figure, the content of Bacteroides ovatus in stool samples from HCC patients was significantly increased compared with that in stool samples from the control group.
[0038] Example 2: Bacteroides ovatus promotes the progression of liver cancer in mice
[0039] 1. In vitro anaerobic culture of Bacteroides ovatus:
[0040] Methods: Bacteroides ovatus was inoculated on blood agar plates and incubated in an anaerobic incubator at 37°C for 2-3 days. Bacterial colonies were collected with a sterile scraper and PBS was added at a concentration of 5.0×10 8 CFU / ml PBS.
[0041] 2. Bacteroides ovatus promotes HCC progression in mice:
[0042] Methods: For the Hepa 1-6 or RIL-175-induced hepatocellular carcinoma (HCC) model, a small incision of about 1 cm was made along the midline of the abdomen of anesthetized mice using sterile surgical scissors. Then the portal vein was found and 2-3×10 HCC cells were directly injected into the hepatic portal vein. 6 Hepa1-6 or RIL-175 tumor cells were injected into the portal vein of the liver, and the inner and outer epithelial sutures of the mice were surgically sutured to obtain the Hepa1-6 or RIL-175-induced hepatocellular carcinoma (HCC) model. 8 CFU Bacteroides ovatus (purchased from Guangdong Provincial Microbial Culture Collection Center, with the collection number of GDMCC NO.: 1.970) or control bacteria (Lactobacillus, Lactoballlusacidophilus purchased from China Industrial Microbial Culture Collection Management Center, CICC 6085), twice a week for 2 consecutive weeks.
[0043] The results are as follows Figure 2 shown.
[0044] in, Figure 2A shows that mice were gavaged with Bacteroides ovale or control bacteria two weeks in advance, and then RIL-175 was injected into the portal vein to induce HCC. Three weeks after induction, the mice were sacrificed and the differences in liver tumor load and liver photos were detected.
[0045] Figure 2 Middle B shows the liver / body weight ratio of tumor-bearing mice and the tumor infiltration ratio of the same liver lobe of tumor-bearing mice.
[0046] The results showed that after HCC was induced in mice that were gavaged with Bacteroides ovatus and Lactobacillus (control bacteria) in advance, liver cancer progressed more rapidly in mice treated with Bacteroides ovatus, indicating that Bacteroides ovatus is a type of bacteria that promotes liver cancer.
[0047] Example 3: Bacteroides ovatus decomposes primary bile acid chenodeoxycholic acid (CDCA) to produce secondary bile acid isolithocholic acid (iso-LCA)
[0048] In this example, bile acids in bacterial culture supernatant were detected by LC-MS:
[0049] Methods: Bacteroides ovatus was cultured in a medium (blood agar plate, Huankai Biotechnology Cat#024070) in the presence or absence of the primary bile acid chenodeoxycholic acid (CDCA) (1 mM) in an anaerobic culture device (Modular Incubator Chamber / Hypoxia Chamber Billups-Rothenberg, Inc Cat#(MIC-101)) for 48 hours.
[0050] The culture supernatant was collected and the bile acid content was detected by mass spectrometry, and the experiment was repeated three times independently (B0-72-1, B0-72-2, B0-72-3).
[0051] The results are as follows Figure 3 shown.
[0052] in, Figure 3 Figure A is a flow chart of the process by which Bacteroides ovatus decomposes the primary bile acid chenodeoxycholic acid (CDCA) to produce the secondary bile acid isolithocholic acid (iso-LCA).
[0053] Figure 3 Middle B is the result of three independent repeated experiments on the content of chenodeoxycholic acid (CDCA) and isolithocholic acid (iso-LCA).
[0054] The results showed that iso-LCA was detected in the bacterial supernatant of the group with added primary bile acid chenodeoxycholic acid (CDCA), indicating that Bacteroides ovatus can decompose primary bile acid CDCA into isolithocholic acid iso-LCA.
[0055] The present application provides an application of Bacteroides ovatus in the diagnosis or prognosis of liver cancer. The content of Bacteroides ovatus (B. ovatus) in the present application is significantly increased in the feces of liver cancer patients, and promotes the progression of liver cancer in mice. It is further found that the bacteria can decompose the primary bile acid chenodeoxycholic acid (CDCA) under anaerobic conditions to produce the secondary bile acid isolithocholic acid (iso-LCA), which provides new ideas for new targets for the diagnosis and treatment of liver cancer.
[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.
Claims
1. Application of a reagent for detecting the content of Bacteroides ovatus in the preparation of a product for liver cancer diagnosis or prognosis.
2. The use according to claim 1, characterized in that The product includes a chip, a test kit or a test strip.
3. The use according to claim 1, characterized in that The reagent includes a reagent for detecting the content of Bacteroides ovatus in a stool sample by using metagenomic sequencing.
4. The use according to claim 1, characterized in that The content of the Bacteroides ovatus in the feces of liver cancer patients is higher than that in healthy people.
5. Application of Bacteroides ovatus in the preparation of reagents to promote animal models of liver cancer.
6. The use according to claim 5, characterized in that The cell concentration of the Bacteroides ovatus is 1 to 5×10 8 CFU.
7. The use according to claim 5, characterized in that The liver cancer animal model includes a liver cancer animal model induced by Hepa 1-6 or RIL-175.
8. Application of Bacteroides ovatus in decomposing primary bile acid into secondary bile acid.
9. The use according to claim 8, characterized in that The primary bile acid includes chenodeoxycholic acid; the secondary bile acid includes isolithocholic acid.
10. The use according to claim 8, characterized in that The ovatus Bacteroides is cultured in an anaerobic environment for 48 to 72 hours to decompose primary bile acid into secondary bile acid.