Application of the in vivo phenol-degrading strain Acinetobacter radioresistens CM38.2 in the prevention or adjuvant treatment of colon cancer

Abstract

The present invention provides an application of an in vivo phenol-degrading strain Acinetobacter radioresistens CM38.2 in preventing or assisting in the treatment of colon cancer, and belongs to the field of biomedical technology. The present invention first discovered that the strain Acinetobacter radioresistens CM38.2 can degrade phenol in the intestine of living animals, prevent or treat colon cancer caused by phenol or a high-tyrosine diet, reduce the probability of colon cancer and prolong survival; it can also significantly reduce the number of Ki67-positive and γH2AX-positive cells in colon cells and restore them to normal levels. In addition, the strain has no side effects on the human body, can be orally colonized in the intestine, maintains the concentration of toxic metabolites around intestinal stem cells at a low non-toxic level, greatly improves the safety of the drug, and reduces the damage of chemotherapy drugs to the body.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of an in vivo phenol-degrading strain Acinetobacterradioresistens CM38.2 in preventing or assisting in the treatment of colon cancer. Background Art

[0002] Colon cancer is a highly prevalent malignant tumor of the digestive tract, and its incidence and mortality rates have been increasing year by year in my country. Patients with early colon cancer may have no symptoms, but as the disease progresses and the lesions continue to grow, a series of symptoms may appear, such as increased frequency of bowel movements, blood and mucus in the stool, abdominal pain, diarrhea or constipation, intestinal obstruction, general fatigue, weight loss and anemia, which seriously affect the patient's normal life. The occurrence of colon cancer is closely related to the environment, living habits, etc., especially to the diet. A long-term high-protein diet will increase the probability of colon cancer.

[0003] Currently, the treatments for colon cancer include surgery, radiotherapy, chemotherapy, etc. However, the postoperative effects of patients are difficult to predict, and the recurrence rate is high, while chemotherapy is extremely harmful to the body. Therefore, a highly safe drug for the treatment of colon cancer is urgently needed.

[0004] Acinetobacter radioresistens is widely present in the natural environment and intestinal flora. It has strong adhesion and is widely used in the fields of bioremediation of nuclear contaminated environments and wastewater treatment, but its effect on cancer treatment has not yet been made public. Summary of the invention

[0005] The purpose of the present invention is to provide an application of an in vivo phenol-degrading strain Acinetobacter radioresistens CM38.2 in the prevention or auxiliary treatment of colon cancer, which can degrade phenol in the intestine of a living animal, intervene in and correct toxic metabolites caused by a high-protein diet, prevent or treat colon cancer caused by a phenol or high-tyrosine diet, reduce the probability of colon cancer and prolong survival.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides use of an in vivo phenol-degrading strain Acinetobacter radioresistens CM38.2 in preparing a drug for preventing or assisting in treating colon cancer. The registration number of the strain Acinetobacter radioresistens CM38.2 is CIP 111668.

[0008] Preferably, the colon cancer is phenol or high-tyrosine diet-related colon cancer.

[0009] Preferably, the strain Acinetobacter radioresistens CM38.2 can be used for preventing or assisting in treating colon cancer associated with phenol or high-tyrosine diet.

[0010] Preferably, it is characterized in that the strain Acinetobacter radioresistens CM38.2 can colonize in the intestine to degrade phenol in the intestine and reduce the incidence of colon cancer.

[0011] The present invention also provides a medicine for preventing or assisting in treating colon cancer, and the medicine comprises the strain Acinetobacter radioresistens CM38.2.

[0012] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0013] Preferably, the dosage form of the drug is one of a liquid preparation, a solid preparation, and a semisolid preparation.

[0014] The beneficial effects of the present invention compared with the prior art are:

[0015] (1) The present invention first discovered that the strain Acinetobacter radioresistens CM38.2 can degrade phenol in the intestines of living animals, intervene and correct toxic metabolites caused by a high-protein diet, and restore them to normal levels, prevent or treat colon cancer caused by a phenol or high-tyrosine diet, reduce the probability of colon cancer, and prolong survival. The strain Acinetobacter radioresistens CM38.2 can also significantly reduce the number of Ki67-positive and γHAX-positive cells in colon cells and restore them to normal levels.

[0016] (2) The present invention is based on the strain Acinetobacter radioresistens CM38.2 for treating colon cancer. The strain has no side effects on the human body and can be orally colonized in the intestine, maintaining the concentration of toxic metabolites around intestinal stem cells at a low non-toxic level. The safety of the drug is greatly improved, and the damage of chemotherapy drugs to the body is reduced, providing a new and effective auxiliary treatment drug for colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 This is the growth curve of the CM38.2 strain in Example 1 of the present invention in phenol as the single carbon source medium;

[0019] Figure 2 The change of phenol concentration in the culture medium when the CM38.2 strain in Example 1 of the present invention is grown in a phenol single carbon source culture medium;

[0020] Figure 3 The number of CM38.2 strains and the colonization of the strains in the feces of mice within one week after intragastric administration of the strains in Example 2 of the present invention;

[0021] Figure 4 is the phenol concentration in the colon contents of different groups of mice in Example 3 of the present invention;

[0022] Figure 5 The number of Ki67-positive cells and γH2AX-positive cells in the tissue sections of different groups of mice in Example 3 of the present invention;

[0023] Figure 6 The growth of colon tumors in different groups of apc- / + mice in Example 4 of the present invention. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1

[0030] Example 1 of the present invention detected the growth of strain Acinetobacter radioresistens CM38.2 on a basic medium with phenol as the single carbon source (Na2HPO4 6.78 g, KH2PO4 3.0 g, NaCl 0.5 g, NH4Cl 1.0 g, MgSO4 0.241 g, CaCl2 0.011 g, the above reagents were weighed and dissolved in 1 L of deionized water for sterilization, and phenol was added to make the final concentration 2 mmol / L), and the specific steps were as follows:

[0031] The strain Acinetobacterradioresistens CM38.2 with registration number CIP 111668 purchased from Paris, France was inoculated into a basic medium with phenol as the single carbon source (Na2HPO4 6.78 g, KH2PO4 3.0 g, NaCl 0.5 g, NH4Cl 1.0 g, MgSO4 0.241 g, CaCl2 0.011 g, the above reagents were weighed and dissolved in 1 L of deionized water for sterilization, and phenol was added to make the final concentration of 2 mmol / L), cultured at 37°C for 10 h, and the number of strains and phenol concentration were detected every two hours. The results are as follows: Figure 1 , 2 shown.

[0032] Figure 1 , 2 The results showed that the strain Acinetobacter radioresistens CM38.2 had the ability to degrade phenol.

[0033] Example 2

[0034] Example 2 of the present invention detected the colonization ability of the strain Acinetobacter radioresistens CM38.2 in the intestine, and the specific steps were as follows:

[0035] (1) Preparation of bacterial solution: The strain Acinetobacter radioresistens CM38.2 was inoculated into LB medium and cultured at 37°C for 12 h to obtain a concentration of 10 9 CFU / mL of bacterial solution.

[0036] (2) Ten SPF mice (purchased from Vital River) were selected. All mice were caged and adaptively raised in an SPF animal room for 1 week at room temperature (24±2)°C, with day and night alternation every 12 hours, and free access to water. After adaptive feeding, the mice were gavaged with 0.1 ml of 10 9 CFU / mL CM38.2 strain, gavage for one week, and detect the number of CM38.2 strains in mouse feces every day. The results are as follows Figure 3 shown.

[0037] (3) One week after the end of gavage, the colon tissue of the mice was obtained, and the colon tissue was embedded in paraffin and cut into slices with a thickness of 7 μm. After the tissue was hydrated, it was incubated at 37°C for 20 minutes with a PBS solution containing 10 mg / ml lysozyme, and then washed twice with PBS. The slices were then incubated overnight at 52°C with a mixture of 50 mmol / L bacterial-specific probes (the sequence of probe 1 was GATCACGGGTCAGCATACGGTAACGGTCTTTCGC-SEQ ID NO.1, and the sequence of probe 2 was GAGACGGCTTTGCATCAGCGTTGAAATAGCAGCAT-SEQ ID NO.2). After incubation, the slices were washed twice with SSC reagent, then stained with DAPI, and sealed after staining.

[0038] Figure 3 Shown, intragastrically administered 0.1 ml 10 9 CFU / ml of the strain, the strain can still maintain a good colon colonization ability ( Figure 3 ), and in situ hybridization of colon tissue revealed that the strain was able to colonize in the colonic crypts.

[0039] Example 3

[0040] Example 3 of the present invention detected the effect of strain Acinetobacter radioresistens CM38.2 on colon cancer in mice fed a high-tyrosine diet, and the specific steps were as follows:

[0041] (1) Fifteen adaptively reared SPF mice were divided into three groups: the blank group was fed a normal diet for 8 weeks and then gavaged with 0.1 ml LB medium for 8 weeks (ND+LB); the control group was fed a high-tyrosine diet for 8 weeks and then gavaged with 0.1 ml LB medium for 8 weeks (HT+LB); the experimental group was fed a tyrosine diet for 8 weeks and then gavaged with 0.1 ml 10 9 CFU / mL of the strain Acinetobacter radioresistens CM38.2 was gavaged for 8 weeks (HT+CM38.2).

[0042] (2) After intragastric administration, the phenol content in the intestinal contents of mice was detected. The results were as follows: Figure 4 shown.

[0043] Depend on Figure 4 It can be seen that compared with the normal diet, the phenol concentration in the colon contents of mice fed a high-tyrosine diet increased, while the phenol concentration in the colon contents decreased after gavage with the CM38.2 strain. This shows that the CM38.2 strain can reduce the probability of colon cancer in mice by degrading phenol.

[0044] (3) The mice were killed and the colon tissues of the mice were embedded in paraffin and sectioned. After the sections were hydrated, they were incubated with Ki67 rabbit antibody or γH2AX rabbit antibody at 4°C overnight, washed twice with TBST buffer, incubated with HRP secondary antibody at room temperature for 1 hour, and then incubated with DAB, followed by washing twice with TBST buffer. The sections were then stained with hematoxylin and sealed. The results are shown in Figure 2. Figure 5 shown.

[0045] Figure 5 The results showed that the number of Ki67-positive and γH2AX-positive cells in the intestines of mice fed a high-tyrosine diet increased significantly, while the number of Ki67-positive and γH2AX-positive cells in the colon cells of mice after oral administration of the strain decreased significantly and returned to normal levels.

[0046] Example 4

[0047] Example 4 of the present invention detected the effect of strain Acinetobacter radioresistens CM38.2 on apc - / + The specific steps for the effect on mice are as follows:

[0048] 15 apc after adaptive feeding - / + The mice were divided into three groups on average. The blank group was fed with a normal diet for 15 weeks and gavaged with 0.1 ml LB medium every 3 days for 15 weeks (ND+LB); the control group was fed with a high-tyrosine diet for 15 weeks and gavaged with 0.1 ml LB medium every 3 days for 15 weeks (HT+LB); the experimental group was fed with a tyrosine diet for 15 weeks and gavaged with 0.1 ml 10 9CFU / mL of strain Acinetobacter radioresistens CM38.2 continued for 15 weeks (HT+CM38.2). After reaching the end of the experiment, the colon of the mice was taken for photography and measurement.

[0049] Depend on Figure 6 It can be seen that after intragastric administration of Acinetobacter radioresistens CM38.2, the high-tyrosine diet apc - / + The number of tumor cells in mice was significantly lower than that in the control group, indicating that intragastric administration of the strain Acinetobacterradioresistens CM38.2 of the present invention can prevent the occurrence of colon cancer and can be used as an auxiliary therapeutic drug for colon cancer.

[0050] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of the in vivo phenol-degrading strain Acinetobacter radioresistens CM38.2 in the preparation of a drug for preventing or assisting in the treatment of colon cancer, characterized in that: The registration number of the strain Acinetobacter radioresistens CM38.2 is CIP 111668.

2. The use of the in vivo phenol-degrading strain Acinetobacter radioresistens CM38.2 according to claim 1 in the preparation of a drug for preventing or assisting in the treatment of colon cancer, characterized in that: The colon cancer is phenol or high-tyrosine diet-related colon cancer.

3. The use of the in vivo phenol-degrading strain Acinetobacter radioresistens CM38.2 according to claim 1 in the preparation of a drug for preventing or assisting in the treatment of colon cancer, characterized in that: The strain Acinetobacterradioresistens CM38.2 can be used to prevent or treat colon cancer associated with phenol or high-tyrosine diet.

4. The use of the in vivo phenol-degrading strain Acinetobacter radioresistens CM38.2 in the preparation of a drug for preventing or assisting in the treatment of colon cancer according to claim 1, characterized in that: The strain Acinetobacterradioresistens CM38.2 can colonize in the intestine to degrade phenol in the intestine and reduce the incidence of colon cancer.

5. A drug for preventing or assisting in the treatment of colon cancer, characterized in that: The drug comprises strain Acinetobacterradioresistens CM38.2 and / or its metabolites.

6. The drug for preventing or assisting in treating colon cancer according to claim 5, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

7. The drug for preventing or assisting in treating colon cancer according to claim 6, characterized in that: The dosage form of the drug is one of a liquid preparation, a solid preparation, and a semisolid preparation.

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