Intestinal flora transplantation-based intestinal sleep disorder mouse model construction method and sleep box

Abstract

The invention discloses an intestinal flora transplantation-based intestinal sleep disorder mouse model construction method and a sleep box, and the method comprises the following steps: preparing intestinal flora of an insomnia patient based on a flora separation technology; the intestinal flora of the insomnia patient is implanted into the body of a sterile mouse by using a sterile animal flora transplantation technology; the sterile mouse implanted with the intestinal flora of the insomnia patient is fed in the sterile isolation bag for flora colonization; the construction of the intestinal sleep disorder mouse phenotype is verified by evaluating and analyzing the behavioristics, physiology and microbiomics of the mouse subjected to flora colonization. Through a flora transplantation technology, the flora of an insomnia patient is colonized in the intestinal tract of a sterile animal, evaluation analysis, verification and construction are performed through behavioristics, physiology and microbiomics to obtain the intestinal sleep disorder mouse model, and a model support is provided for exploration of sleep pathogenic bacteria and research and development of sleep probiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of constructing animal models, and specifically relates to a method for constructing an enterogenic sleep disorder mouse model based on intestinal flora transplantation and a sleep box. Background Art

[0002] Insomnia is a common sleep disorder, which refers to the problem that an individual is dissatisfied with the duration, quality or production of sleep, affecting the health and quality of life of a large number of people. Insomnia is not limited to difficulty falling asleep or interrupted sleep, but also includes waking up early and having difficulty falling asleep again. Long-term insomnia is closely related to health risks (such as cardiovascular disease, metabolic syndrome, etc.). Therefore, researchers are also exploring the relationship between insomnia and other chronic diseases, and trying to find prevention and intervention strategies.

[0003] Currently, there are limited means of diagnosis and treatment for sleep-related diseases. Ordinary drugs have serious side effects and often only treat the symptoms rather than the root cause. Using animal models of sleep disorders for basic research can provide more adequate means for the diagnosis and treatment of diseases. Establishing a stable and reliable animal model of insomnia can provide a key scientific basis for the clinical transformation of insomnia. By deeply understanding the physiological basis of insomnia, we can better develop personalized treatment methods and interventions to improve patients' treatment outcomes and quality of life.

[0004] Currently, researchers typically use one or more of the following animal models:

[0005] Sleep deprivation: By limiting the animal's sleep time or changing its sleep cycle, the animal will experience insomnia-like symptoms, such as difficulty falling asleep and shallow sleep.

[0006] Drug induction: Specific drugs, such as caffeine or sleeping pills, are administered to animals to simulate the symptoms of human insomnia. Genetic changes: Gene editing technology or breeding methods are used to change the genes or genetic background of animals, making them more likely to show insomnia-related characteristics. Environmental stimulation: Inducing insomnia-like reactions by exposing animals to different environmental stimuli, such as light, noise or social isolation.

[0007] However, these models cannot fully reflect the complexity and multifactorial influence of human insomnia. For example, drug induction is the use of specific drugs or chemical interventions, such as hypnotic drugs, antianxiety drugs or steroids, etc. It is difficult to eliminate the effects of the drugs themselves on mice in the model.

[0008] Environmental change is to change the environmental conditions of mice, such as irregular light cycles, noise exposure, etc., but environmental changes can easily cause stress in mice and lead to emotional problems.

[0009] It can be seen that these models are all passive insomnia induced by external stimuli, and cannot simulate the behavioral and pathological changes of insomnia well. In the past, enterogenic disease models mostly used antibiotics to eliminate animal flora for modeling. Since the current antibiotic modeling method cannot completely eliminate all intestinal flora of the recipient mice, it will affect the colonization efficiency after flora transplantation, and thus affect the success rate of modeling.

[0010] This shows that there is currently a lack of animal models of sleep disorders in basic scientific research, and the existing models are difficult to meet the needs of studying the pathogenesis of insomnia and drug development. Summary of the invention

[0011] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0012] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0013] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for constructing a mouse model of enteric sleep disorder based on intestinal flora transplantation.

[0014] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0015] Based on the bacterial flora separation technology, the intestinal flora of insomnia patients is prepared;

[0016] Using sterile animal microbiota transplantation technology, the intestinal microbiota of insomnia patients was implanted into sterile mice;

[0017] Germ-free mice implanted with intestinal flora from insomnia patients were raised in sterile isolation bags for bacterial colonization;

[0018] By evaluating and analyzing the behavior, physiology, and microbiome of mice after bacterial colonization, the construction of the phenotype of mice with enteric sleep disorders was verified.

[0019] As a preferred embodiment of the method for constructing a gut-derived sleep disorder mouse model based on intestinal flora transplantation described in the present invention, the method for preparing the intestinal flora of insomnia patients comprises collecting fresh fecal samples from insomnia patients under sterile conditions, diluting and evenly mixing with sterile physiological saline to form a coarse fecal suspension, removing large particles by filtration to ensure that the suspension is uniform, and obtaining a fecal suspension of insomnia patients, that is, a fecal suspension containing the intestinal flora of insomnia patients.

[0020] As a preferred embodiment of the method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to the present invention, the crude feces suspension is subjected to centrifugation, filtration, and density gradient centrifugation.

[0021] As a preferred embodiment of the method for constructing a gut-derived sleep disorder mouse model based on intestinal flora transplantation according to the present invention, the germ-free mice are not treated with antibiotics during the breeding process.

[0022] As a preferred embodiment of the method for constructing a mouse model of enteric sleep disorder based on intestinal flora transplantation according to the present invention, the behavioral assessment includes the assessment of activity level and sleep pattern.

[0023] As a preferred embodiment of the method for constructing a gut-derived sleep disorder mouse model based on intestinal flora transplantation according to the present invention, the sleep pattern includes sleep / wake cycles, rapid eye movement sleep, and non-rapid eye movement sleep.

[0024] As a preferred embodiment of the method for constructing a gut-derived sleep disorder mouse model based on intestinal flora transplantation described in the present invention, the physiological evaluation includes EEG activity, muscle activity, and stress hormone levels.

[0025] The beneficial effects of this construction method are:

[0026] Currently, there is a lack of animal models of sleep disorders in basic scientific research. In view of the above, the present invention discloses a modeling method of a gut-derived sleep disorder mouse model based on intestinal flora transplantation, which utilizes the flora of insomnia patients and sterile mice raised in a completely sterile environment, and colonizes the flora of insomnia patients in the intestines of sterile animals through flora transplantation technology, and evaluates and analyzes through behavior, physiology and microbiome to verify and construct a gut-derived sleep disorder mouse model, providing a model support for the exploration of sleep pathogens and the development of sleep probiotics.

[0027] The present invention also proposes a sleeping box used in the above-mentioned method of constructing a mouse model of enteric sleep disorder based on intestinal flora transplantation, comprising a box body for providing a feeding space for mice; a waterer installed on the box body, the waterer being installed on a body on the box body, a feeding tube connected to the body and extending to the inner side of the box body, and a medicine cabin arranged on the body; a floating mechanism, which is movably arranged on the inner side of the waterer; a driving mechanism, which can drive the floating mechanism and the inner side of the waterer to move; the body is used to load drinking water for feeding mice, the medicine cabin is used to load medicines, and when the floating mechanism sinks into the drinking water, it can raise the liquid level of the drinking water inside the body.

[0028] As a preferred solution of the sleeping box described in the present invention, wherein: an inner shaft is arranged inside the water dispenser; the floating mechanism includes a slip ring which is slidable on the outside of the inner shaft, and an expansion body installed on the slip ring; the driving mechanism includes a power part installed on the box body, and a magnetic body connected to the power part; the slip ring can be adsorbed by the magnetic body.

[0029] As a preferred embodiment of the sleeping box of the present invention, it further comprises a switch mechanism, which comprises a lifting core installed on the inner side of the inner shaft; a plug extending to the outer side of the inner shaft and capable of plugging the connection between the device body and the feeding tube; an extension body arranged at the end of the lifting core and extending to the outer side of the inner shaft;

[0030] When the floating mechanism moves upward, it can push the extension body, and the lifting core drives the plug to separate from the connection between the device body and the feeding tube.

[0031] The beneficial effects of the sleeping box: by setting a floating mechanism and a driving mechanism, the water stored in the drinking fountain and the medicine can be mixed, thereby ensuring that the medicine in the drinking water when the mice are fed meets the experimental requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. 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 creative labor. Among them:

[0033] Figure 1 This is a schematic diagram of the intestinal flora colonization distribution of the recipient mice after the flora transplantation of the present invention.

[0034] Figure 2 This is a schematic diagram of the sleep monitoring results of recipient mice after the bacterial flora transplantation of the present invention, the activity of orexin neurons in the brain region, and the content of serum short-chain fatty acids.

[0035] Figure 3 It is a schematic diagram of the overall structure of the sleeping box in the present invention.

[0036] Figure 4 It is a schematic diagram of the internal structure of the sleeping box in the present invention.

[0037] Figure 5 It is a schematic diagram of the structure of the drinking fountain and the driving mechanism in the present invention.

[0038] Figure 6 It is a schematic diagram of the internal structure of the drinking fountain in the present invention.

[0039] Figure 7 It is a schematic diagram of the floating mechanism structure in the present invention.

[0040] Figure 8 For the present invention Figure 7 Enlarged view of point A in the middle.

[0041] Fig. 9 It is a schematic diagram of the switch mechanism structure in the present invention.

[0042] Fig.10 For the present invention Fig. 9 Enlarged view of point B in the middle.

[0043] Fig.11 It is a schematic diagram of the slip ring structure in the present invention. DETAILED DESCRIPTION

[0044] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0045] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0046] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

[0047] Materials and Equipment:

[0048] Experimental mice: germ-free mice

[0049] Stool samples: Stool samples from insomnia patients and healthy donors.

[0050] Aseptic processing equipment: such as clean bench.

[0051] Fecal transplant equipment: gavage needle, syringe.

[0052] Example 1. This example provides a method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation, specifically:

[0053] S1: Based on the bacterial flora isolation technology, the intestinal flora of insomnia patients and healthy donors were prepared;

[0054] Patients with insomnia diagnosed clinically according to the diagnostic criteria for insomnia in the Diagnostic and Statistical Manual of Mental Disorders or the International Classification of Diseases were included;

[0055] Fresh stool samples were collected from insomnia patients and healthy donors, diluted with sterile saline and evenly mixed to form a crude stool suspension, which was then subjected to centrifugation, filtration, and density gradient centrifugation to remove large particles and ensure a uniform suspension. The stool suspensions of insomnia patients and healthy donors, i.e., intestinal flora of insomnia patients and healthy donors, were obtained, respectively, and stored at low temperature for later use.

[0056] S2: Using the sterile animal microbiota transplantation technology, the intestinal microbiota was implanted into the germ-free mice;

[0057] Use a gavage needle or syringe to orally or rectally transplant the fecal suspension of the insomnia patient obtained in step S1 into the body of sterile recipient SPF mice raised in a sterile isolation bag to obtain the experimental group, while the control group is transplanted with fecal suspension of healthy donors.

[0058] S3: The mice in the experimental and control groups were raised under sterile conditions for bacterial colonization;

[0059] The transplanted mice were housed in sterile isolation bags for two weeks for bacterial colonization.

[0060] S4: Validation of the construction of the gut-derived sleep disorder mouse phenotype;

[0061] This was verified by evaluating and analyzing the behavior, physiology, and microbiome of mice after bacterial colonization.

[0062] A1: behavioral and physiological monitoring;

[0063] Three weeks after transplantation, the mice were subjected to behavioral tests (such as elevated plus maze experiments), and electroencephalography (EEG), electromyography (EMG), polysomnography (PSG) and other techniques were used to evaluate the sleep and wakefulness of the recipient mice colonized with the insomnia patient's microbiota.

[0064] Specifically, brain sleep monitoring and muscle testing require electrode surgery to implant electrodes. The surgical method refers to the following steps:

[0065] Mice need to be acclimatized to the experimental environment and handling procedures beforehand.

[0066] Anesthetize the mouse before surgery and ensure that it maintains a stable body temperature and anesthesia during the surgery.

[0067] EEG electrode implantation:

[0068] EEG electrodes are precisely implanted into specific brain regions, such as the frontal lobe, parietal lobe or temporal lobe, using micromanipulators to record brain electrical activity. The position and depth of the EEG electrodes need to be precisely controlled to ensure that clear signals are recorded and to avoid damaging brain tissue.

[0069] EMG electrode implantation:

[0070] EMG electrodes are implanted into the neck or other muscle areas of mice to record muscle electrical activity. These electrodes can be used to monitor the muscle tone state of mice at different sleep stages, especially muscle relaxation during rapid eye movement (REM) sleep.

[0071] Electrode fixation: After implantation, the electrodes are fixed to the mouse's skull or muscle using a specific fixation substance (such as clinical-grade medical glue or bone cement) to prevent the electrodes from shifting or falling off.

[0072] Postoperative Care: After surgery, the recovery of the mice needs to be monitored and appropriate care and analgesics need to be provided. There is usually an adaptation period during which the mice can return to normal behavior and sleep patterns for subsequent experimental recording and analysis.

[0073] A2: Collect mouse fecal samples and perform 16S sequencing on human donor samples and mouse fecal samples;

[0074] The intestinal microbes of human donors and recipient mice after transplantation were sequenced by 16S RNA sequencing to verify the effectiveness of intestinal microbial colonization after flora transplantation. The specific steps are as follows:

[0075] Sample collection: Microbial DNA was collected from fecal samples of mice. Prior to collection, samples were kept cool and dry to prevent alteration of the microbial community.

[0076] DNA extraction: Total microbial DNA is extracted from stool samples using an appropriate DNA extraction method. This step requires special attention to ensure that the extracted DNA is of sufficient purity and quality for subsequent sequencing analysis.

[0077] PCR amplification: Specific regions of the 16S rRNA gene are amplified using specific primers. These primers are usually designed to cover variable regions in the 16S rRNA gene (such as V1-V9) in order to obtain sufficient sequence information in sequencing.

[0078] Sequencing: Use high-throughput sequencing technology (such as the Illumina sequencing platform) to sequence the amplified 16SrRNA gene fragments. This method can sequence a large number of samples simultaneously and provide high-resolution information on the structure of the microbial community.

[0079] Data analysis: Bioinformatics analysis of the sequencing data includes sequence processing, OTU (operational taxonomic unit) clustering, species diversity assessment, comparison of microbial community structure, and function prediction.

[0080] This step can reveal the composition, diversity and dynamic changes of the mouse intestinal microbial community, study the effects of different factors (such as diet, drugs, disease models, etc.) on the intestinal microbial community, and explore the relationship between intestinal microorganisms and host health status (such as immune regulation, intestinal diseases, etc.).

[0081] A3: Data analysis and validation;

[0082] The data from sleep EEG monitoring were analyzed and presented to compare whether there were differences in the sleep characteristics of the microbiota mice of insomnia patients and those of healthy human mice, thereby verifying the construction of the sleep disorder phenotype.

[0083] Example 2: This example refers to Example 1 and constructs a mouse model of intestinal sleep disorder based on intestinal flora transplantation through scientific experimental methods. Specifically:

[0084] S1: Based on the bacterial flora isolation technology, the intestinal flora of insomnia patients and healthy donors were prepared;

[0085] Patients with insomnia diagnosed clinically according to the diagnostic criteria for insomnia in the Diagnostic and Statistical Manual of Mental Disorders or the International Classification of Diseases were included;

[0086] Fresh stool samples were collected from insomnia patients and healthy donors, diluted with sterile saline and evenly mixed to form a coarse stool suspension, and large particles were removed by filtration to ensure that the suspension was uniform, and the stool suspensions of insomnia patients and healthy donors, i.e., the intestinal flora of insomnia patients and healthy donors, were obtained respectively and stored at low temperature for later use.

[0087] S2: Using the sterile animal microbiota transplantation technology, the intestinal microbiota was implanted into the germ-free mice;

[0088] The fecal suspension of the insomnia patient obtained in step S1 is transplanted into the body of sterile recipient SPF mice housed in a sterile isolation bag via oral or rectal routes using a gavage needle or syringe to obtain an experimental group, while the fecal suspension of a healthy donor is transplanted into the control group.

[0089] S3: The mice in the experimental and control groups were raised under sterile conditions for bacterial colonization;

[0090] The transplanted mice were housed in a sterile isolation bag for two weeks to prevent interference from external microorganisms.

[0091] S4: Validation of the construction of the gut-derived sleep disorder mouse phenotype;

[0092] This was verified by evaluating and analyzing the behavior, physiology, and microbiome of mice after bacterial colonization.

[0093] A1: behavioral and physiological monitoring;

[0094] Three weeks after transplantation, the mice were subjected to behavioral tests (such as elevated plus maze experiments), and electroencephalography (EEG), electromyography (EMG) and other techniques were used to evaluate the sleep and wakefulness of the recipient mice colonized with the insomnia patient's microbiota.

[0095] Specifically, brain sleep monitoring and muscle testing require electrode surgery to implant electrodes. The surgical method refers to the following steps:

[0096] Mice need to be acclimatized to the experimental environment and handling procedures beforehand.

[0097] Anesthetize the mouse before surgery and ensure that it maintains a stable body temperature and anesthesia during the surgery.

[0098] EEG electrode implantation:

[0099] EEG electrodes are precisely implanted into specific brain regions, such as the frontal lobe, parietal lobe or temporal lobe, using micromanipulators to record brain electrical activity. The position and depth of the EEG electrodes need to be precisely controlled to ensure that clear signals are recorded and to avoid damaging brain tissue.

[0100] EMG electrode implantation:

[0101] EMG electrodes are implanted into the neck or other muscle areas of mice to record muscle electrical activity. These electrodes can be used to monitor the muscle tone state of mice at different sleep stages, especially muscle relaxation during rapid eye movement (REM) sleep.

[0102] Electrode fixation: After implantation, the electrodes are fixed to the mouse's skull or muscle using a specific fixation substance (such as clinical-grade medical glue or bone cement) to prevent the electrodes from shifting or falling off.

[0103] Postoperative Care: After surgery, the recovery of the mice needs to be monitored and appropriate care and analgesics need to be provided. There is usually an adaptation period during which the mice can return to normal behavior and sleep patterns for subsequent experimental recording and analysis.

[0104] A2: Collect mouse fecal samples and perform 16S sequencing on human donor samples and mouse fecal samples;

[0105] The intestinal microbes of human donors and recipient mice after transplantation were sequenced by 16S RNA sequencing to verify the effectiveness of intestinal microbial colonization after flora transplantation. The specific steps are as follows:

[0106] Sample collection: Microbial DNA was collected from fecal samples of mice. Prior to collection, samples were kept cool and dry to prevent alteration of the microbial community.

[0107] DNA extraction: Total microbial DNA is extracted from stool samples using an appropriate DNA extraction method. This step requires special attention to ensure that the extracted DNA is of sufficient purity and quality for subsequent sequencing analysis.

[0108] PCR amplification: Specific regions of the 16S rRNA gene are amplified using specific primers. These primers are usually designed to cover variable regions in the 16S rRNA gene (such as V1-V9) in order to obtain sufficient sequence information in sequencing.

[0109] Sequencing: Use high-throughput sequencing technology (such as the Illumina sequencing platform) to sequence the amplified 16SrRNA gene fragments. This method can sequence a large number of samples simultaneously and provide high-resolution information on the structure of the microbial community.

[0110] Data analysis: Bioinformatics analysis of the sequencing data includes sequence processing, OTU (operational taxonomic unit) clustering, species diversity assessment, comparison of microbial community structure, and function prediction.

[0111] This step can reveal the composition, diversity and dynamic changes of the mouse intestinal microbial community, study the effects of different factors (such as diet, drugs, disease models, etc.) on the intestinal microbial community, and explore the relationship between intestinal microorganisms and host health status (such as immune regulation, intestinal diseases, etc.).

[0112] A3: Data analysis and validation;

[0113] The data from sleep EEG monitoring were analyzed and presented to compare whether there were differences in the sleep characteristics between the microbiota of insomniac mice and that of healthy human mice, thereby verifying the construction of the sleep disorder phenotype of germ-free animals (SPF).

[0114] As attached Figure 1 , Attachment Figure 2 As shown, it is the verification of microbial colonization after the flora transplantation in this embodiment, and Figure 1 The contribution of PCoA1 on the horizontal axis was 26.46%, and the contribution of PCoA2 on the vertical axis was 10.62%. The β diversity changed significantly after fecal microbiota transplantation. FMT_HE, receiving fecal microbiota from healthy people; FMT_IN, receiving fecal microbiota from insomnia patients (n=7-8).

[0115] The butyrate content in the blood of FMT_IN mice after fecal microbiota transplantation was significantly reduced. (a) The co-labeling of c-Fos and orexin neurons in recipient mice after fecal microbiota transplantation (scale: 500um, 50um; green: c-Fos protein; red: orexinA neurons); (b) The number of co-labeled c-Fos and orexin neurons in FMT_IN mice after fecal microbiota transplantation decreased. FMT_HE, receiving fecal microbiota from healthy people; FMT_IN, receiving fecal microbiota from insomnia patients. All data are expressed as mean ± standard error (n = 5), ** represents p < 0.01.

[0116] Attached Figure 2 (a) Diagram of fecal microbiota transplantation; (b) Time distribution of Wake, NREM sleep and REM sleep in recipient mice during 24 hours after fecal microbiota transplantation; (c) Time distribution of Wake, NREM sleep and REM sleep in recipient mice during 12 hours of light period after fecal microbiota transplantation; (d) Time distribution of Wake, NREM sleep and REM sleep in recipient mice during 12 hours of dark period after fecal microbiota transplantation. FMT_HE, recipients of fecal microbiota from healthy people; FMT_IN, recipients of fecal microbiota from insomnia patients. Data are expressed as mean ± standard error (n = 12), ** represents p < 0.01, *** represents p < 0.001.

[0117] Comparative Example 1: This comparative example is different from Example 2 in that the feeding conditions of the mice in step S3 are adjusted, specifically:

[0118] S3: The mice in the experimental and control groups were raised under sterile conditions for bacterial colonization;

[0119] The transplanted mice were housed in sterile isolation bags to prevent interference from external microorganisms, and drinking water was replaced with a mixed antibiotic solution for two consecutive weeks.

[0120] The remaining steps refer to Example 2 to construct the sleep disorder model based on antibiotics (AIMD) in this comparative example.

[0121] Embodiment 3 is different from the above embodiments in that: this embodiment also discloses a sleeping box applied to the above construction method, including a box body 100 for providing a feeding space for mice.

[0122] The sleeping box includes a drinking fountain 200 installed on the box body 100, a body 201 installed on the box body 100, a feeding tube 202 connected to the body 201 and extending to the inside of the box body 100, and a medicine cabin 203 provided on the body 201. The drinking fountain 200 can be installed on the side wall of the box body 100, one end of the feeding tube 202 is connected to the internal space of the body 201, and the liquid inside the body 201 can enter the inner side of the feeding tube 202. The feeding tube 202 is located in the box body 100. A ball is arranged at the inner end of the body 100, and the ball blocks the feeding tube 202. The implementation method is that the end of the feeding tube 202 located on the inner side of the box body 100 is a contraction opening, that is, the inner diameter gradually decreases, and the ball is located inside the contraction opening. Under the action of gravity in normal conditions, the ball can block the contraction opening. When the mouse licks the ball, the ball moves upward inside the contraction opening, so that a gap appears between the ball and the inner wall of the contraction opening, and the liquid inside the body 201 can flow out of the feeding tube 202 for the mouse to drink.

[0123] The medicine compartment 203 is located on the side wall of the device body 201, with one end of the medicine compartment 203 connected to the internal space of the device body 201 and the other end having a cover. After removing the cover, the medicine can be placed inside the medicine compartment 203. In order to improve the sealing effect, the cover is preferably made of rubber or silicone material. Multiple medicine compartments 203 are arranged on the side wall of the same device body 201 for loading different medicines, which can be loaded inside different medicine compartments 203.

[0124] The sleeping box includes a floating mechanism 300, which is movably arranged on the inner side of the drinking fountain 200; the sleeping box includes a driving mechanism 400, which can drive the floating mechanism 300 and the inner side of the drinking fountain 200 to move; the body 201 is used to load drinking water for feeding mice, and the medicine cabin 203 is used to load medicines. When the floating mechanism 300 sinks into the drinking water, it can raise the liquid level of the drinking water inside the body 201.

[0125] Furthermore, the floating mechanism 300 can move up and down on the inner side of the vessel body 201, so the floating mechanism 300 has two states, one is a higher position and does not contact the water inside the vessel body 201, and the other is a lower position and can sink into the water inside the vessel body 201. The driving mechanism 400 can adopt various powered structures, preferably an electric telescopic rod, which can drive the floating mechanism 300 to move up and down and adjust the position of the floating mechanism 300 inside the vessel body 201.

[0126] In this embodiment, three water dispensers 200 are installed on the entire box body 100. When in use, the medicine and water in one of the water dispensers 200 are in a mixed state, and the water and medicine inside the other two water dispensers 200 are separated. As time goes by, some medicines will completely fail in the water. At this time, the driving mechanism 400 corresponding to the two spare water dispensers 200 drives the floating mechanism 300 to move downward and sink into the water. The water level rises, overflowing the medicine cabin 203, and mixing with the medicine inside the medicine cabin 203.

[0127] Therefore, when applied to the above-mentioned construction method, the experimenter can mix the medicine and water at one time, and when the medicine inside the drinking fountain 200 in use fails, the spare drinking fountain 200 can be quickly replaced.

[0128] Embodiment 4 is different from the above-mentioned embodiment 3 in that: an inner shaft 204 is arranged inside the water dispenser 200; the floating mechanism 300 includes a slip ring 301 which is slidable on the outside of the inner shaft 204, and an expansion body 302 installed on the slip ring 301; the driving mechanism 400 includes a power part 401 installed on the box body 100, and a magnetic body 402 connected to the power part 401; the slip ring 301 can be adsorbed by the magnetic body 402.

[0129] Among them, the power part 401 is preferably an electric push rod, which can drive the magnetic body 402 to move up and down, and drive the slip ring 301 to move up and down on the outside of the inner shaft 204 through the adsorption force of the magnetic body 402 on the slip ring 301. Therefore, the slip ring 301 can drive the expansion body 302 to move up and down, thereby completing the driving of the entire floating mechanism 300. In this way, because there is no direct connection between the driving mechanism 400 and the floating mechanism 300, the same driving mechanism 400 can be applied to different drinking fountains 200 to drive the floating mechanisms 300 inside different drinking fountains 200.

[0130] For example, a slide rail and an electric slider structure are provided on the box body 100 , and the power member 401 is installed on the electric slider, which can drive the power member 401 to move. The power member 401 can move to the corresponding position of any one of the three drinking fountains 200 to drive the floating mechanism 300 inside the body 201 .

[0131] In order to improve the mixing effect of medicine and water, the floating mechanism 300 also includes a bottom cabin 303 installed below the slip ring 301, and a buoyancy body 304 arranged inside the bottom cabin 303. The buoyancy body 304 has a certain deformation ability. The top of the bottom cabin 303 is connected to the inside of the expansion body 302 through the slip ring 301. The top of the expansion body 302 is also provided with a water inlet groove 305, and the radial outer wall of the bottom cabin 303 is provided with a water outlet groove 306. The connection position between the top of the bottom cabin 303 and the slip ring 301 has a bell mouth 304. 7. Under normal conditions, the buoyancy body 304 can be raised and lowered on the inner side of the bottom cabin 303. When the driving mechanism 400 drives the slip ring 301 to move downward, the bottom cabin 303 will first contact the liquid inside the device body 201. The buoyancy makes the buoyancy body 304 move upward, blocking the water outlet trough 306 and the bell mouth 307, so that the liquid cannot directly enter the inner side of the expansion body 302 from the bottom cabin 303. Therefore, the ability to increase the liquid level when the expansion body 302 sinks into the water inside the device body 201 is guaranteed to the greatest extent.

[0132] As the expansion body 302 descends, the water level inside the body 201 overflows the medicine cabin 203, flushing the medicine, and the medicine enters the water. Then the water level reaches the water inlet groove 305, and enters the expansion body 302 through the water inlet groove 305. Then the driving mechanism 400 drives the slip ring 301 and drives the expansion body 302 to move upward. When leaving the water surface, the gravity of the water presses the buoyancy body 304 from the bottom, causing the buoyancy body 304 to move downward inside the bottom cabin 303, and the mixture of water and medicine can flow out from the water outlet groove 306, allowing the water and medicine to collide and mix, thereby improving the mixing effect of water and medicine.

[0133] When the magnetic body 402 is used to drive the slip ring 301 , in order to enhance the adsorption force, the outer diameter of the slip ring 301 can be enlarged so that the radial outer wall of the slip ring 301 is close to the inner wall of the device body 201 , thereby enhancing the adsorption capacity of the magnetic body 402 on the slip ring 301 .

[0134] When three drinking fountains 200 use the same driving mechanism 400, when the magnetic body 402 in the driving mechanism 400 absorbs the slip ring 301 inside one of the drinking fountains 200, it is also necessary to consider maintaining the positions of the slip rings 301 inside the other two drinking fountains 200 to avoid the floating mechanism 300 dropping into the water under the action of gravity and causing the medicine to contact with water prematurely.

[0135] Therefore, an arc-shaped spring piece 308 and a positioning protrusion 309 are provided on the radial inner wall of the slip ring 301, wherein both ends of the arc-shaped spring piece 308 are connected to the radial inner wall of the slip ring 301, and the arc-shaped outer wall contacts the inner shaft 204. A positioning groove 204a is provided on the radial outer wall of the inner shaft 204. When the slip ring 301 is not attracted by the magnetic body 402, the elastic force of the arc-shaped spring piece 308 will contact the slip ring 301, so that the positioning protrusion 309 is located on the inner side of the positioning groove 204a. When there is no magnetic body 402 adsorbed, the positioning protrusion 309 is located on the inner side of the positioning groove 204a to maintain the position of the entire floating mechanism 300, thereby preventing the floating mechanism 300 from descending into the water under the action of gravity, causing the problem of premature contact between the medicine and the water.

[0136] The position where the magnetic body 402 adsorbs the slip ring 301 is close to the positioning protrusion 309. At this time, the slip ring 301 will slightly move toward the magnetic body 402, the arc-shaped spring piece 308 will be deformed, and the positioning protrusion 309 will be separated from the positioning groove 204a, so that the positioning of the slip ring 301 can be released, allowing the slip ring 301 to move.

[0137] In order to allow the magnetic body 402 to attract the slip ring 301 from the side close to the positioning protrusion 309 each time, it is necessary to prevent the floating mechanism 300 from rotating. This can be achieved by setting a guide block and a guide groove between the inner shaft 204 and the floating mechanism 300, which will not be repeated here.

[0138] Embodiment 5 is different from the above embodiments in that: the sleeping box also includes a switch mechanism 500; it includes a lifting core 501, which is installed on the inner side of the inner shaft 204; a plug 502, which extends to the outside of the inner shaft 204 and can seal the connection between the device body 201 and the feeding tube 202; an extension body 503, which is arranged at the end of the lifting core 501 and extends to the outside of the inner shaft 204; when the floating mechanism 300 moves upward, it can push the extension body 503, and the lifting core 501 drives the plug 502 to separate from the connection between the device body 201 and the feeding tube 202.

[0139] The inner shaft 204 has a cavity 204b inside, and the bottom of the cavity 204b is in an open state. The end of the lifting core 501 equipped with a plug 502 extends out from here, and the plug 502 can plug the connecting port 205 between the device body 201 and the feeding tube 202. A top cavity 204c with a diameter larger than the cavity 204b is provided at the top of the inner shaft 204. A limiting disk 501a is provided at the end of the lifting core 501 located in the top cavity 204c, and the extension body 503 is installed on the limiting disk 501a. A through groove is opened at the bottom of the top cavity 204c. Under the action of gravity, the bottom end of the extension body 503 extends from the inside of the top cavity 204c through the through groove.

[0140] Therefore, when the floating mechanism 300 does not interfere with the extension body 503, under the influence of gravity, the plug 502 blocks the connection between the device body 201 and the feeding tube 202. Therefore, when one of the device bodies 201 is in use, the magnetic body 402 drives the slip ring 301, so that the entire floating mechanism 300 reaches a state of interference with the extension body 503. At this time, the plug 502 is separated from the connection port 205 between the device body 201 and the feeding tube 202.

[0141] When the medicine inside the device 201 loses its effectiveness and stops being used, the magnetic body 402 separates from the floating mechanism 300, the floating mechanism 300 cannot contact the extension body 503, the lifting core 501 moves downward, and the plug 502 can block the connection port 205 between the device body 201 and the feeding tube 202, so that the mouse can only take drinking water containing medicine.

[0142] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation, characterized in that: include, Based on the bacterial flora separation technology, the intestinal flora of insomnia patients is prepared; Using sterile animal microbiota transplantation technology, the intestinal microbiota of insomnia patients was implanted into sterile mice; Germ-free mice implanted with intestinal flora from insomnia patients were raised in sterile isolation bags for bacterial colonization; By evaluating and analyzing the behavior, physiology, and microbiome of mice after bacterial colonization, the construction of the phenotype of mice with enteric sleep disorders was verified.

2. The method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to claim 1, characterized in that: The method for preparing the intestinal flora of insomnia patients is as follows: Under sterile conditions, fresh stool samples are collected from insomnia patients, diluted with sterile physiological saline and evenly mixed to form a crude fecal suspension, and large particles are removed by filtration to ensure that the suspension is uniform, thereby obtaining a fecal suspension of insomnia patients, that is, a fecal suspension containing intestinal flora of insomnia patients.

3. The method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to claim 2, characterized in that: The crude feces suspension is centrifuged, filtered, and subjected to density gradient centrifugation.

4. The method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to claim 1, characterized in that: The germ-free mice were not treated with antibiotics during the breeding process.

5. The method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to claim 1, characterized in that: The behavioral assessment includes assessment of activity level and sleep patterns.

6. The method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to claim 5, characterized in that: The sleep patterns include sleep / wake cycles, rapid eye movement sleep, and non-rapid eye movement sleep.

7. The method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to claim 1, characterized in that: The physiological assessment includes electrical brain activity, muscle activity, and stress hormone levels.

8. A sleep box used in the method for constructing a mouse model of intestinal sleep disorder based on intestinal flora transplantation according to any one of claims 1 to 7, characterized in that: include, The box (100) is used to provide a breeding space for mice; A drinking fountain (200) is installed on the box (100), the drinking fountain (200) being installed on a body (201) on the box (100), a feeding tube (202) communicating with the body (201) and extending to the inside of the box (100), and a medicine cabin (203) provided on the body (201); A floating mechanism (300) movably disposed inside the drinking fountain (200); A driving mechanism (400) capable of driving the floating mechanism (300) and the inner side of the drinking fountain (200) to move; The device body (201) is used to load drinking water for feeding mice, the medicine compartment (203) is used to load medicine, and when the floating mechanism (300) sinks into the drinking water, it can raise the liquid level of the drinking water inside the device body (201).

9. The sleeping box according to claim 8, characterized in that: An inner shaft (204) is arranged inside the drinking fountain (200); The floating mechanism (300) comprises a slip ring (301) slidably disposed on the outside of the inner shaft (204), and an expansion body (302) mounted on the slip ring (301); The driving mechanism (400) comprises a power member (401) mounted on the box (100), and a magnetic body (402) connected to the power member (401); The slip ring (301) can be adsorbed by the magnetic body (402).

10. The sleeping box according to claim 8 or 9, characterized in that: Also included is a switch mechanism (500); These include, A lifting core (501) installed on the inner side of the inner shaft (204); A plug (502) extending to the outside of the inner shaft (204) and capable of sealing the connection between the device body (201) and the feeding tube (202); An extension body (503) is disposed at the end of the lifting core (501) and extends to the outside of the inner shaft (204); When the floating mechanism (300) moves upward, it can push the extension body (503), and the lifting core (501) drives the plug (502) to separate from the connection between the device body (201) and the feeding tube (202).

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