Method for constructing coolah source elderly mouse model by using coprophilous fungus transplantation technology

Abstract

The invention discloses a method for constructing a coolah source elderly mouse model by using a coprophilous fungus transplantation technology, and belongs to the technical field of biomedicine. According to the method, C57BL / 6N mice are selected and normally fed in groups; the method comprises the following steps: freely drinking antibiotics to construct a sterile environment, and constructing a coolah source old-age mouse model by utilizing a coprophilous fungus transplantation technology; during the test, apparent states such as diet, activity, mental state and defecation of the mouse are observed, and after the test is finished, the expression quantity of senescence factors of each tissue of the mouse is detected by a qPCR (quantitative polymerase chain reaction) method. Test results show that the expression quantity of aging factors of mice can be increased successfully after 6-week intragastric administration of the colacobacterium colati suspension, and it is proved that the mice have aging characterization. The model has obvious advantages and wide application prospects in the aspects of clarification of the aging pathogenesis of the organism and drug research and development, can deeply study drug use of colaz, ensures that colaz is properly treated and potential side effects are minimized, and better improves the safety of drug use.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and provides a method for constructing an elderly mouse model sourced from koalas by using fecal microbiota transplantation technology. Background Art

[0002] In the rich ecosystems of the earth, wild animals are closely related to the natural environment. Their interactions not only shape the evolutionary process of organisms but also maintain the prosperity of global biodiversity. As a unique and rare species in Australia, the survival status of koalas directly affects the stability and integrity of the local ecosystem. However, in recent years, due to human activities, the living environment of koalas has faced severe challenges. Factors such as deforestation, urban expansion, and climate change have led to a decreasing habitat for koalas, limited food sources, and a sharp decline in the population. In this context, although traditional conservation measures have achieved certain results, they are still difficult to cope with the increasingly severe survival threats. Therefore, inventing conservation patents for this species has profound demonstration significance. It conveys a clear signal to the world that humans have the ability and responsibility to protect wild animals and the natural environment through scientific and technological innovation. This will stimulate more people's environmental awareness and innovative actions, and promote the development of global biodiversity conservation.

[0003] Koalas have special living habits and body structures, and many drugs may be unsafe for them. Therefore, we need to conduct in-depth research on the drug use of koalas to ensure that they receive appropriate treatment and minimize potential side effects. Through in-depth research on the drug use of koalas, we can better improve the safety of drug use. This not only helps to ensure that koalas receive timely and effective treatment but also avoids the negative impact of drugs on their health. In addition, this research will also help to raise people's awareness of the drug use risks of rare species and promote the protection and management of wild animals.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a method for constructing an elderly mouse model sourced from koalas by using fecal microbiota transplantation technology, conduct in-depth in vitro research on the drug use of koalas, ensure that koalas receive appropriate treatment and minimize potential side effects, and better improve the safety of drug use.

[0006] The present invention first transplants the intestinal microbiota of healthy elderly koalas into mice by means of a suspension, aiming to expand the scope of monitoring, and more deeply and comprehensively explore the functional changes of various organs of mice caused by the intestinal microbiota of adult and aging captive koalas, so as to provide a more accurate monitoring method for subsequent drug administration.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A method for constructing an elderly mouse model of koala origin using fecal microbiota transplantation technology, comprising the following steps:

[0009] (1) Preparation of fecal bacteria suspension: The feces from elderly koalas are made into a fecal bacteria suspension;

[0010] (2) The mice are administered an antibiotic mixture by gavage and / or drinking water, and the antibiotics include vancomycin, ampicillin, neomycin, and metronidazole;

[0011] (3) Model establishment: The mice in step (2) are transplanted with the fecal bacteria suspension prepared in step (1) to obtain an elderly mouse model of koala origin.

[0012] Further, step (1) specifically includes the following steps: The fresh feces of elderly koalas are dissolved in sterile physiological saline to make a suspension, and through filtration and centrifugation to remove the supernatant, the obtained precipitate is the bacterial solution, and the bacterial solution is dissolved to obtain a fecal bacteria suspension.

[0013] Preferably, the elderly koalas refer to koalas over 8 years old;

[0014] Preferably, the concentration of the suspension is 0.15 - 0.25 g / mL; more preferably 0.2 g / mL.

[0015] Preferably, the filtration is through two - stage filtration. Specifically, it is first filtered with 2 layers of sterile gauze to remove large - particle substances, and then secondarily filtered with 4 layers of sterile gauze;

[0016] Preferably, the centrifugation conditions are 2 - 8 °C, 5000 - 8000 r / min, and centrifugation for 10 - 20 min; more preferably 4 °C, 6000 r / min, and centrifugation for 15 min.

[0017] Preferably, after the bacterial solution is resuspended with physiological saline, sterile glycerol is added such that the ratio of bacterial solution: physiological saline: sterile glycerol = 7 - 9:7 - 9:2 - 6; more preferably 9:9:2.

[0018] Further, in step (2),

[0019] The mice are 8 - week - old C57BL / 6N mice; preferably 8 - week - old C57BL / 6N male mice. C57BL / 6J mice are nicotinamide nucleotide transhydrogenase mutants and will show changes such as redox - related imbalances, so C57BL / 6N mice are selected for aging research;

[0020] The antibiotic dosage is vancomycin 0.1 - 0.15 mg per mouse, ampicillin 0.2 - 0.3 mg per mouse, neomycin 0.2 - 0.3 mg per mouse, and metronidazole 0.2 - 0.3 mg per mouse; for 2 weeks.

[0021] Further preferably, the antibiotic dosage is vancomycin 0.1 mg per mouse, ampicillin 0.2 mg per mouse, neomycin 0.2 mg per mouse, and metronidazole 0.2 mg per mouse; for 2 weeks.

[0022] The antibiotic mixture is 0.5 mg / mL vancomycin, 1 mg / mL ampicillin, 1 mg / mL neomycin, and 1 mg / mL metronidazole.

[0023] Furthermore, in step (3),

[0024] The transplantation method is gavage;

[0025] The volume of the transplanted fecal microbiota suspension is 200 - 300 μL per mouse per time, 3 times a week, for 4 - 6 weeks (preferably 6 weeks); further preferably, the volume of the transplanted fecal microbiota suspension is 200 μL per mouse per time, 3 times a week, for 4 - 6 weeks (preferably 6 weeks);

[0026] Evaluate the relevant phenotypes of koala - sourced aged mice, and the method includes: evaluating through the expression levels of senescence factors in the cerebral cortex and jejunal tissue.

[0027] Preferably, the senescence factors include TNF - α, IL - 6, CLEC4E, etc.;

[0028] In the present invention, mice are selected as experimental animals, gavage with normal saline is used as the blank control group, and gavage with koala fecal microbiota suspension is used as the model group to explore the effects of transplanting aged koala fecal microbiota on the mouse body.

[0029] When transplanting mice in the present invention, the detection of successful establishment of the model is mainly reflected in the following aspects:

[0030] (1) The expression levels of senescence - related secreted phenotypic factors in the model mice are significantly increased;

[0031] (2) The model mice show obvious koala - type immune senescence. Senescence is related to changes in inflammation and immune homeostasis. The senescence of immune cells is related to the changes in the homeostasis of cytokine levels and the alterations of metabolic pathways. And cytokines are immune regulatory factors produced by cells of the innate immune system and the adaptive immune system.

[0032] The koala - sourced aged mouse model constructed by the above - mentioned method is used to clarify the pathogenesis of koala body senescence and / or develop anti - aging drugs for koalas.

[0033] The present invention has the following advantages and effects compared with the prior art:

[0034] C57BL / 6N mice were selected in the present invention and grouped for normal feeding; the daily water intake of the mice was 4 - 7 mL / d, and antibiotics were freely provided to the mice according to the water intake to construct a sterile environment, and then a koala - sourced aged mouse model was constructed using fecal microbiota transplantation technology; during the experiment, the apparent states of the mice such as diet, activity, mental state, defecation, etc. were observed. After the experiment ended, the expression levels of senescence factors in various tissues of the mice were detected by Real - time quantitative PCR (qPCR). The experimental results showed that intragastric administration of the fecal suspension of aged koalas for 6 weeks could successfully increase the expression levels of senescence factors in mice, proving that the mice showed senescence characteristics. This model has obvious advantages and broad application prospects in clarifying the pathogenesis of organism senescence and drug research and development, and can conduct in - depth research on the drug use of koalas, ensuring that koalas receive appropriate treatment and minimizing potential side effects, and better improving the safety of drug use. Brief Description of the Drawings

[0035] Figure 1 It is a histological section of the jejunum of a mouse.

[0036] Figure 2 It is the expression levels of senescence factors in the cerebral cortex of 4 groups of mice (n = 5).

[0037] Figure 3 It is the expression levels of senescence factors in the jejunum of 4 groups of mice (n = 5).

[0038] Note: * indicates significant difference (P < 0.05). Detailed Embodiments

[0039] The present invention will be further described in detail below in conjunction with embodiments and the drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0040] Common senescence - associated secretory phenotype factors in mice and koala immune - related markers are shown in Tables 1 and 2;

[0041] Table 1 Common senescence - associated secretory phenotype factors

[0042]

[0043] Table 2 List of currently published koala immune - related markers

[0044]

[0045] Example 1

[0046] I. Experimental methods

[0047] (1) Preparation of donor fecal microbiota suspension

[0048] 1. Take 4 g of fresh feces from 4 old male koalas, place the fecal samples in appropriate sterile EP tubes, dissolve them in sterile physiological saline (20 mL) according to the ratio (mass / volume, W / V = 1:5), vortex to make a suspension with a concentration of 0.2 g / mL, and pre-cool the centrifuge to 4 °C in advance;

[0049] 2. First, quickly filter with two layers of sterile gauze to remove large particulate matter, and then filter again with four layers of sterile gauze;

[0050] 3. Centrifuge the filtered sample at 4 °C, 6000 r / min for 15 min. After centrifugation, remove the supernatant. The obtained precipitate is the bacterial suspension, and add an equal mass of physiological saline and shake well;

[0051] 4. Add sterile glycerol with a final concentration of 10% w / w to the bacterial suspension (bacterial suspension 45% w / w: physiological saline 45% w / w: sterile glycerol 10% w / w), vortex and dispense into 2 mL EP tubes, and store at -80 °C for later use. This is the fecal microbiota suspension.

[0052] (2) Preparation of antibiotic mixture

[0053] Vancomycin at 0.5 mg / mL, ampicillin at 1 mg / mL, neomycin at 1 mg / mL, metronidazole at 1 mg / mL, 0.2 mL / rat, for 2 weeks.

[0054] (3) Koala-derived fecal microbiota transplantation

[0055] Select 8-week-old male C57BL / 6N mice and group them (Table 3) for normal feeding; after 7 days of adaptive feeding, the model group freely drinks the antibiotic mixture in step (2) (where the average daily water intake of mice is 4 - 7 mL / d, and the mice are given free access to antibiotics according to the water intake), for 14 days. Stop using antibiotics 1 day before transplantation, and administer the fecal microbiota suspension by gavage, 3 times a week. Observe the general signs of the mice daily and record the body weight, and record the water intake and food intake weekly. The transplantation grouping is shown in Table 3.

[0056] Table 3 Fecal microbiota transplantation grouping

[0057]

[0058] (4) Determination indexes and detection methods

[0059] 1. Physical sign examination

[0060] During the experiment, observe the apparent states of the mice such as diet, activity, mental state, defecation, etc., and weigh the body weight 3 times a week;

[0061] 2. Tissue section

[0062] After the experiment, fresh jejunum tissues were collected. After removing the intestinal contents, the collected tissues were washed in physiological saline and then immersed in 4% paraformaldehyde solution for fixation.

[0063] 3. Real-time quantitative PCR (qPCR)

[0064] The expression levels of senescence factors TNF-α, IL-6, and CLEC4E in the cerebral cortex and jejunum tissues of mice were detected by SYBR dye method.

[0065] 4. Data analysis

[0066] The experimental data were statistically analyzed using IBM SPSS Statistics 27 and GraphPad Prism 9.0 software. The comparison of two groups of data was analyzed by independent samples T-test, and the comparison of multiple groups of data was analyzed by one-way ANOVA to analyze the differences among groups. The multiple comparisons among groups were performed by Duncan's new multiple range test. A P value less than 0.05 was considered statistically significant. The Ct values of qPCR were analyzed using Quant Studio TM Design & Analysis Software.

[0067] II. Experimental results

[0068] 1. Tissue section

[0069] The collected tissues were washed in physiological saline and then immersed in 4% paraformaldehyde solution for fixation, and the specimens were sent for examination. The results are as Figure 1 shown. Under a 20× microscope, compared with the blank group, there were varying degrees of inflammatory cell infiltration in the 6-week group, and O6 was more obvious. It is speculated that the jejunum of elderly koalas is damaged.

[0070] 2. The GraphPad Prism 9.0 software was used to analyze the significance of differences among multiple groups of the experimental data. The expression levels of senescence factors TNF-α, IL-6, and CLEC4E in the cerebral cortex and jejunum of mice are as Figure 2 , Figure 3 shown.

[0071] The one-way ANOVA in the statistical software IBM SPSS Statistics 27 was used to analyze the significance of differences among multiple groups of the experimental data. All data were expressed as "mean ± standard deviation". The expression levels of senescence factors in the cerebral cortex and jejunum of mice are shown in Tables 4 and 5.

[0072] Table 4 Expression levels of senescence factors in the cerebral cortex of 4 groups of mice (n = 5)

[0073]

[0074] Note: Different superscript letters indicate significant differences (P < 0.05).

[0075] Table 5 Expression levels of senescence factors in the jejunum of 4 groups of mice (n = 5)

[0076]

[0077] Note: Different superscript letters indicate significant differences (P < 0.05).

[0078] As can be seen from the above charts, the expression levels of senescence factors TNF-α, IL-6, and CLEC4E in the cerebral cortex and jejunum of mice gavaged for 6 weeks are significantly better than those in the group gavaged for 4 weeks, and the brain-gut immune senescence expressed by aged koalas can be reproduced.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for constructing a koala-derived aged mouse model using fecal microbiota transplantation technology, characterized in that: The steps include: (1) Preparing a fecal bacteria suspension: preparing a fecal bacteria suspension from feces of elderly koalas; (2) administering an antibiotic mixture to mice by gavage and / or drinking water, wherein the antibiotics include vancomycin, ampicillin, neomycin and metronidazole; (3) Model establishment: The fecal microbiota suspension prepared in step (1) was transplanted into the mice in step (2) to obtain a koala-derived aged mouse model.

2. The method according to claim 1, characterized in that: Step (1) specifically comprises the following steps: dissolving fresh feces of old koalas in sterile physiological saline to prepare a suspension, filtering and centrifuging to remove the supernatant, the obtained precipitate is the bacterial liquid, and dissolving the bacterial liquid to obtain a fecal bacteria suspension.

3. The method according to claim 2, characterized in that: After the bacterial solution was resuspended with physiological saline, sterile glycerol was added so that the ratio of bacterial solution: physiological saline: sterile glycerol = 7-9:7-9:2-6.

4. The method according to claim 2 or 3, characterized in that: The concentration of the suspension is 0.15 to 0.25 g / mL; The filtration is performed by filtering twice, specifically filtering with 2 layers of sterile gauze first, and then filtering again with 4 layers of sterile gauze; The centrifugal conditions are 2-8° C., 5000-8000 r / min, and centrifugation for 10-20 min.

5. The method according to claim 1, characterized in that: In step (2), the mice are 8-week-old C57BL / 6N mice.

6. The method according to claim 1 or 5, characterized in that: The dosage of antibiotics was vancomycin 0.1-0.15 mg / animal, ampicillin 0.2-0.3 mg / animal, neomycin 0.2-0.3 mg / animal, and metronidazole 0.2-0.3 mg / animal; it lasted for 2 weeks.

7. The method according to claim 1, characterized in that: In step (3), the transplantation is performed by gavage; The volume of fecal microbiota suspension transplanted is 200-300 μL / animal / time, 3 times a week, for 4-6 weeks.

8. The method according to claim 1, characterized in that: The method for evaluating phenotypes related to koala-derived aged mice comprises: evaluating the expression levels of aging factors in cerebral cortex and jejunum tissues.

9. The method according to claim 8, characterized in that: The aging factors include TNF-α, IL-6, and CLEC4E.

10. Use of the koala-derived aged mouse model constructed by the method according to any one of claims 1 to 9 for clarifying the pathogenesis of koala aging and / or developing anti-aging drugs for koalas.

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