Construction method and application of rapid aging animal model

Abstract

The invention relates to a construction method and application of a rapid aging animal model. According to the invention, the C5a protein is injected into the hippocampus of an animal in a brain stereotactic manner, so that the rapid aging animal model is successfully constructed. The rapid aging animal model construction method provided by the invention has high repeatability, and the rapid aging animal model can be efficiently and stably prepared; moreover, the rapid aging animal model constructed according to the method disclosed by the invention shows obvious and stable learning and memory disorder and aging-related phenotypes, including reduction of learning and memory ability, increase of aging marker level, damage of synapse-related protein and the like, has high representativeness, and can be applied to the rapid aging animal model. Therefore, the method is not only suitable for pathological mechanism research of senescence and related diseases or symptoms, but also provides a powerful tool for drug development of senescence related diseases or symptoms (such as cognitive hypofunction caused by senescence), and shows a good industrial application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of animal model construction, and particularly relates to a method for constructing a rapidly aging animal model and its application. Background Art

[0002] Aging is a core risk factor for many chronic diseases. It not only accelerates the decline of cognitive function but also significantly increases the risk of neurodegenerative diseases, especially Alzheimer's disease. One of the potential causes of age-related diseases is the widespread accumulation of senescent cells in tissues, which seriously disrupts the integrity and function of tissues. In addition, with the aging process, neuroinflammation intensifies, and the abilities of neurogenesis, synaptic strength, and function gradually decline. Currently, there is no effective means to significantly delay or reverse the aging process.

[0003] To deeply study the pathological mechanism of aging and promote new drug development, constructing an aging animal model is of great significance. Currently, the research on aging-related pathology mainly uses natural aging models and rapid aging models. The natural aging animal model is favored because of its simple modeling, high similarity to the human aging process, and no need for surgery or drug administration. However, the disadvantages of this model are its long modeling cycle, large investment in human and material resources, easy infection with other diseases during breeding, high mortality rate in the old age period, and large individual differences in sample detection.

[0004] The senescence-accelerated mouse (SAM), as an inbred aging model, includes two strains: SAMP (Senescence-Accelerated Mouse Prone, rapidly aging susceptible mouse) and SAMR (Senescence Accelerated Mouse Resistant, anti-rapidly aging mouse). Among them, the SAMP8 sub-strain is mainly characterized by learning and memory impairment and is currently recognized as an ideal natural aging dementia model. Although the genetic information of SAM mice is similar to that of humans and the pathological changes are highly consistent, it has problems such as gene defects, high price, and difficult breeding.

[0005] In addition, there are also D-galactose-induced aging models, β-amyloid injection-induced aging models, γ-ray-induced aging models, thymectomy-induced aging models, aluminum chloride-induced mouse aging models, mouse aging models established by ozone damage method, etc. Although these models have a short modeling time, simple operation, and save manpower, there are still some problems to be solved in the construction process of some models, such as certain danger, low survival rate of mice, and some models do not show memory impairment phenotypes. Therefore, the existing models urgently need to be optimized in terms of phenotypic stability. To deeply study the pathological mechanism of aging, aging-related memory impairment, and its treatment plan, there is an urgent need to establish a more stable and reliable aging model. Summary of the Invention

[0006] Object of the Invention

[0007] In view of the problems or requirements existing in the prior art, the purpose of the present invention is to provide a method for constructing a rapidly aging animal model with good repeatability and a stable phenotype of the constructed model, as well as the application of the rapidly aging animal model constructed according to the construction method in the research on the pathological mechanism of aging or aging-related diseases and drug development.

[0008] Solution

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a method for constructing a rapidly aging animal model, the method comprising: injecting a C5a protein solution into the hippocampal region of an animal by stereotaxic injection of the brain to obtain the model.

[0011] For the above construction method:

[0012] Preferably, the hippocampal region is specifically the dentate gyrus region of the hippocampus;

[0013] Preferably, the C5a protein solution is injected into the bilateral dentate gyrus regions of the hippocampus of the animal; preferably, it is continuously injected for 5-10 days, and more preferably, it is continuously injected for 7 days.

[0014] Preferably, the C5a protein has the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1;

[0015] The amino acid sequence shown in SEQ ID NO:1 is as follows:

[0016] SKQVQLTEKRHQKVMRSLHLGAPRASHLGLARSHLGRPTQVKANLTQSPRRLE NELKQALNQGQEQ.

[0017] Preferably, the C5a protein solution is prepared by the following method: dissolving the C5a protein in DMSO to prepare a C5a protein stock solution (for example, preparing a stock solution with a concentration of 100 nM), and then diluting the C5a protein stock solution to a working concentration with PBS before use (for example, diluting to a working concentration of 10 nM) to obtain the solution;

[0018] Preferably, in the C5a protein solution, the concentration of DMSO is lower than 0.1%;

[0019] Preferably, the working concentration of the C5a protein solution is 5 - 15 nM, preferably 8 - 12 nM, and more preferably 10 nM.

[0020] Preferably, the injection volume of the C5a protein solution is 0.8 - 5 μL, further preferably 1 - 3 μL, and even more preferably 2 μL;

[0021] Preferably, the injection speed is 0.1 - 0.5 μL / min, preferably 0.1 - 0.3 μL / min, and more preferably 0.2 μL / min;

[0022] Preferably, after the injection, the needle is left in place for 8 - 12 min, more preferably 10 min;

[0023] Preferably, the stereotaxic injection into the brain is performed using a stereotaxic injection system, which includes a stereotaxic apparatus, a microinjection pump, and a digital coordinate display.

[0024] In a feasible embodiment, the animal is a mammal, such as a rodent (e.g., mouse, rabbit), an even - toed ungulate (e.g., pig), a canine (e.g., dog), or a feline (e.g., cat).

[0025] Preferably, the animal is a rodent, further preferably a mouse, and even more preferably a C57BL / 6J mouse.

[0026] In a specific example, the C57BL / 6J mouse is a 24 - week - old mouse.

[0027] In a specific example, the weight of the C57BL / 6J mouse is 28 - 38 g.

[0028] In a specific example, during the model establishment period, the mice are allowed to drink water and eat feed freely, and the sterilized bedding, feed, and sterilized water are changed every 3 days. Specifically, during the model establishment period, the mice are placed in a clean EVC mouse cage, and the sterilized bedding, feed, and sterilized water are changed every 3 days.

[0029] In a second aspect, the present invention provides the application of the rapidly aging animal model constructed by the construction method described in the first aspect above in the following aspects:

[0030] (1) Studying aging or aging - related diseases;

[0031] (2) Application in screening drugs for preventing, inhibiting, or delaying aging;

[0032] (3) Application in screening drugs for preventing, improving, and / or treating aging - related diseases or disorders;

[0033] Preferably, the aging-related disease or disorder is aging-related cognitive impairment.

[0034] In a third aspect, the present invention provides a method for studying aging or aging-related diseases, wherein the study is based on a rapidly aging animal model constructed by the construction method described in the first aspect above.

[0035] In a fourth aspect, the present invention provides a method for screening drugs for preventing, inhibiting or delaying aging, wherein the screening is based on a rapidly aging animal model constructed by the construction method described in the first aspect above.

[0036] In a fifth aspect, the present invention provides a method for screening drugs for preventing, improving and / or treating aging-related diseases, wherein the screening is based on a rapidly aging animal model constructed by the construction method described in the first aspect above.

[0037] Advantageous Effects

[0038] The present invention successfully induced and constructed a rapidly aging animal model by injecting C5a protein into the hippocampal region of animals through stereotaxic technology.

[0039] The method for constructing the rapidly aging animal model provided by the present invention has high repeatability, can efficiently and stably prepare a rapidly aging animal model; its operation process is simple, the modeling period only takes 7 days, the success rate is relatively high, and the mortality rate is low.

[0040] More importantly, the rapidly aging animal model constructed by the method of the present invention exhibits significant and stable learning and memory impairments and aging-related phenotypes, including a decline in learning and memory ability, an increase in the level of aging markers, and damage to synapse-related proteins, etc., and has high representativeness. Therefore, the rapidly aging animal model constructed according to the method of the present invention has broad application potential. It is not only applicable to the study of the pathological mechanisms of aging and its related diseases or disorders, but also provides a powerful tool for the drug development of aging-related diseases or disorders (for example, cognitive function decline caused by aging), showing good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] One or more embodiments are illustrated by way of example in the accompanying drawings, and these exemplary illustrations do not limit the embodiments. The special word "exemplary" here means "serving as an example, an embodiment or an illustration". Any embodiment illustrated as "exemplary" here does not have to be construed as superior to or better than other embodiments.

[0042] Figure 1 It is a flowchart for constructing a rapidly aging mouse model in Example 1.

[0043] Figure 2 Representative pictures during the process of constructing a rapid aging mouse model in Example 1; among them, Figure A shows the stereotactic injection point coordinates in the DG region, Figure B shows the stereotactic injection device, Figure C shows the injection point after exposing the whole brain after stereotactic injection, and Figure D shows stereotactic injection into the hippocampal DG region.

[0044] Figure 3 Showing the results of the water maze experiment of the mouse model constructed in Example 1; among them, Figures A, B, C, D, and E respectively show the escape latency, average number of times crossing the platform, residence time in the target quadrant, distance from the target platform, and average useful speed of the mouse model in the first five days, as detected in Example 2.

[0045] Figure 4 Showing the detection results of the aging molecular phenotypes of the mouse model constructed in Example 1, as detected in Example 3.

[0046] Figure 5 Showing the detection results of the damage level of GABAergic neurons in the mouse model constructed in Example 1, as detected in Example 4. Detailed implementation manners

[0047] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0048] In addition, to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can be implemented without some specific details. In some embodiments, raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0049] Unless otherwise specified, the implementation of the present invention will adopt conventional techniques in molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, and immunology, which are all within the technical scope of the art.

[0050] In addition, unless otherwise clearly defined in other parts of this article, the scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. To make it easier to understand the present invention, some scientific and technical terms are specifically defined as follows.

[0051] The term "comprising" or variations thereof such as "including" or "having" etc. shall be understood to include the stated element or component without excluding other elements or other components.

[0052] The term "about", when used in conjunction with a numerical value, means a numerical value within a range that has a lower limit 5% less than the specified numerical value and an upper limit 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1% and ±0.1%, as these variations are suitable for the disclosed methods.

[0053] The term "and / or" shall be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0054] As used herein, the term "or" shall be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted inclusively, i.e., including at least one of the items in the list of quantities or elements, but also including more than one, and optionally, additional unlisted items.

[0055] The reagents and animals used in the following examples are all ordinary commercially available products and are all commercially available.

[0056] Example 1: Construction of a mouse model by stereotaxic injection of C5a protein into the DG region of the mouse hippocampus

[0057] Animals and Main Reagents Used

[0058] (1) C57BL / 6J mice

[0059] Healthy SPF-grade C57BL / 6J mice at 24 weeks of age (both male and female), purchased from Beijing Huafukang Biotech Co., Ltd., weighing 28 - 38 g, and properly raised in accordance with laboratory animal ethics.

[0060] (2) C5a protein

[0061] Name: Complement C5 / C5a protein, Mouse;

[0062] Catalog number: HY-P7695;

[0063] Manufacturer: MedChemExpress;

[0064] Purity: >95%;

[0065] Specification: 10 μg;

[0066] Storage conditions: Can be stored at -20°C for two years.

[0067] C5a protein stock solution: Dissolve C5a protein in DMSO to prepare a 100 nM C5a protein stock solution;

[0068] C5a protein solution: Before use, dilute the C5a protein stock solution with sterile filtered phosphate buffered saline (PBS) to prepare a C5a protein solution with a concentration of 10 nM.

[0069] Next, by means of stereotaxic injection, inject 1 nM C5a protein solution and 10 nM C5a protein solution into the bilateral hippocampal DG regions of mice respectively, and continuously inject for one week to construct a mouse model. Each concentration group contains 10 mice.

[0070] The specific construction procedure is as follows:

[0071] Step 1: Select 24-week-old C57BL / 6J mice;

[0072] Step 2: Measure general indicators, including body weight, hair color, and activity status;

[0073] Step 3: Prepare 10 nM C5a protein solution, aliquot it, and store it in a -80 °C refrigerator;

[0074] Step 4: Prepare a stereotaxic apparatus, dental drill, and anesthesia machine; Before transferring the mouse to the stereotaxic frame, first induce anesthesia with an air mixture containing 5% anesthetic gas, and then maintain the anesthetic gas concentration at 2%; As an alternative, 2% isoflurane can be used, or a single injection of pentobarbital sodium (50 mg / kg, intraperitoneal injection) can be used instead of anesthesia, or avertin (400 mg / kg, single injection) can be used for substitution; Then, fix the mouse on the stereotaxic apparatus;

[0075] Step 5: After the mouse is completely anesthetized, perform postoperative analgesia management by intraperitoneal injection of ketoprofen (3 - 5 mg / kg); As an alternative, carprofen (5 - 10 mg / kg, subcutaneous injection) can be used instead of ketoprofen;

[0076] Step 6: Place the anesthetized mouse on a heating pad to ensure stable body temperature;

[0077] Step 7: Moisten the mouse's eyes with erythromycin eye ointment to prevent dryness and infection;

[0078] Step 8: Use depilatory cream to remove the hair on the top of the mouse's head, and disinfect the skin with 75% ethanol, alternating between alcohol and iodophor;

[0079] Step 9: Lock the mouse's front teeth on the front clamp of the stereotaxic apparatus, and then insert the positioning rod into the groove in front of the ear to fix the head; Ensure that the mouse's head is firmly fixed to avoid loosening;

[0080] Step 10: Use a scalpel to make an incision about 1.5 cm long in the front-to-back direction between the two ears, and inject 30 μL of 0.25% bupivacaine locally at the incision to help relieve pain and provide additional analgesia before the postoperative analgesic meloxicam (which belongs to nonsteroidal anti-inflammatory drugs (NSAIDs)) takes effect;

[0081] Step 11: Use a sterile cotton swab to clean the skull surface and ensure that the two anatomical landmarks, Bregma and Lambda, are clearly visible; Bregma is the intersection of the coronal and sagittal sutures, and Lambda is the intersection of the coronal and lambdoid sutures;

[0082] Step 12: Use a microinjector to draw 2 μL of 1 nM C5a protein solution or 10 nM C5a protein solution, and fix the injection needle on the stereotaxic injection instrument; point the injection needle tip to the Bregma point as the zero point of the three axes; ensure that the injection needle moves in the vertical direction to avoid scratching the skull during the operation; then, move the needle to the specified position according to the coordinate system, such as Figure 2 As shown;

[0083] Step 13: According to the Mouse Brain Atlas 4th Edition, the coordinates of DG are: 2.0 mm behind Bregma, ±1.7 mm to the left and right of the midline, and 1.9 mm below the skull surface. Adjust the position of the syringe needle according to the above coordinates;

[0084] Step 14: Lift the syringe on the vertical axis to move the needle tip to a safe position;

[0085] Step 15: Use a fine dental drill (0.6 mm drill bit) to gently drill two shallow holes at the target site; use an oblique angle when drilling, rather than vertically, to avoid the drill bit entering the deep brain tissue; if bleeding occurs, gently absorb the blood with a small sterile cotton ball;

[0086] Step 16: Use the syringe pump controller to inject the C5a protein solution at a rate of 0.2 μL / min;

[0087] Step 17: After the injection, the incision was sutured and the needle was left in place for 10 minutes to prevent fluid leakage; the mouse was then placed on a heating pad until it fully woke up and given the analgesic meloxicam after surgery;

[0088] Step 18: After surgery, place the mice back into clean cages, add sterile feed, and replace sterile water; send the cages to the animal room, replace sterile bedding, feed, and sterile water every 3 days, and observe the vitality and growth status of the mice; stereotaxic injections of 2uL 10nM C5a in bilateral DG regions were given from the first day, and the injections continued for 7 days;

[0089] Step 19: Prepare for the behavioral test 15 days later.

[0090] Example 2: Detection of cognitive function in a mouse model

[0091] In this example, the cognitive function of the mouse model constructed in Example 1 was evaluated through the Morris water maze experiment. Among them, the escape latency in the first five days and the number of times of crossing the platform and the number of times of crossing the target quadrant on the sixth day are commonly used evaluation indicators, which reflect the learning and spatial memory abilities of mice.

[0092] The experiment was divided into two groups of mouse models (i.e., the 1 nM C5a group and the 10 nM C5a group) and a group of wild-type mice (i.e., WT), with 10 mice in each group.

[0093] In the Morris water maze experiment, a circular pool with a diameter of 120 cm and a depth of 50 cm was used. Since C57BL / 6J mice are black, a white pool was used in the experiment to enhance the visual contrast. During the test, it was ensured that the experimental mice could not observe the experimenters to reduce human interference. A circular pool with a diameter of 120 cm and a depth of 50 cm was used. Considering that the C57BL / 6J mice have black hair, a white pool was selected in the experiment to enhance the visual contrast. During the experiment, it was ensured that the mice could not observe the experimenters to minimize human interference.

[0094] In the laboratory, high-contrast fixed spatial reference objects were arranged at positions suspended above the pool or with the inner wall higher than the water level as positioning cues. In the target quadrant of the pool, a platform with a diameter of about 8 - 10 cm was placed. On the first day of the test, the platform was about 1 cm above the water surface, while on the 2nd to 6th days, the platform was adjusted to about 1 cm below the water surface. The water temperature was maintained at 21 - 22 degrees Celsius to simulate a suitable experimental environment.

[0095] To further ensure the effectiveness and accuracy of the experiment, optimize the video tracking system, cover the underwater platform, and reduce the impact on mice. 0.5% - 1% titanium dioxide was used in the pool during the experiment.

[0096] The movement trajectories of mice were recorded using the Smart 3.0 video tracking system, and the cognitive functions of mice were monitored through an automatic video recording system and supporting analysis software. Before the experiment began, the test mice were transferred to the water maze test room to prevent them from observing the water maze equipment and related positioning cues in advance. After the mice were allowed to adapt to the test room environment for approximately 30 minutes, the experiment was initiated. During the experiment, the pool was divided into four quadrants, and five platform sub-regions were established in each quadrant and at the center of the pool for subsequent data analysis. The experimental records included the following parameters: the total distance traveled by the mice, the total time, the average swimming speed; the escape latency (time to reach the platform); the number of platform crossings (target search behavior in the quadrant where the platform is located).

[0097] The experimental results are as Figure 3 shown Figure 3 Figures A, B, C, D, and E in Figure 3 show the escape latency, average number of platform crossings, residence time in the target quadrant, distance from the target platform, and average useful speed of the mice in the first five days, respectively. As can be seen from Figure 3 the results, compared with wild-type mice of the same age, the learning and memory abilities of the mouse model constructed by stereotaxic injection of 10 nM C5a protein solution were significantly decreased, while there was no significant difference in the learning and memory abilities of the mice in the 1 nM C5a injection group and wild-type mice. This result indicates that: the mouse model constructed by stereotaxic injection of 10 nM C5a protein solution into the bilateral hippocampal DG region of mice has obvious aging phenotypes and can thus be used as a rapid aging model.

[0098] Example 3: Detection of aging phenotypes of the mouse model

[0099] P21 and P53 are core regulatory factors of cell senescence and are also marker molecules of senescence. Therefore, in this example, the expression levels of the senescence-related proteins P21 and P53 in the mouse model constructed in Example 1 were detected by Western blot to confirm the senescence state of the constructed mouse model.

[0100] The experiment was divided into the following groups: a group of 24-week-old wild-type mice (i.e., the 6m WT group), a group of 88-week-old wild-type mice (i.e., the 22m WT group), and a model group obtained by stereotaxic injection of 10 nM C5a protein solution into 24-week-old C57BL / 6J mice (i.e., the 6m WT + 10 nM C5a group).

[0101] The specific experimental procedure is as follows:

[0102] Total proteins were extracted from the hippocampi of mice, and the protein quantification was performed using the BCA method. 100 μg of the total protein sample was loaded onto a 10% SDS-PAGE gel for electrophoresis separation. Then, the protein bands separated by electrophoresis were electrotransferred onto a nitrocellulose (NC) membrane. Subsequently, the NC membrane was blocked in 5% non-fat milk for 1-2 hours to reduce non-specific binding. After blocking, the membrane was washed with TBST buffer, and then primary antibodies were added, including anti-p21 (1:1000, sc-6246, Santa Cruz), anti-p53 (1:1000, 2524S, CST), and GAPDH (1:10000, 60004-1-Ig, Proteintech) monoclonal antibodies, and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST buffer for 10 minutes each time to remove unbound antibodies. Subsequently, HRP-labeled goat anti-rabbit secondary antibody (1:10000, ZB5301, Zhongshan Golden Bridge) or goat anti-mouse secondary antibody (1:10000, ZB2305, Zhongshan Golden Bridge) was added and incubated at room temperature for 1 hour. Then, the membrane was washed again by shaking three times with TBST buffer for 10 minutes each time. Finally, ECL chromogenic reagent (WBKLS0500, Millipore) was added for chemiluminescence reaction, and the bands were scanned and observed using a gel imaging system to quantitatively analyze the expression levels of the target proteins.

[0103] The results of Western blot were as Figure 4 shown Figure 4 and demonstrated that: compared with wild-type mice of the same age, the protein levels of P21 and P53 in the mouse model constructed by stereotactic injection of 10 nM C5a were significantly increased, and there was no obvious difference from the corresponding protein levels in 22-month-old senescent mice.

[0104] These results indicated that the mouse model constructed by stereotactic injection of 10 nM C5a had exactly the same expression levels of senescence-related proteins as senescent mice, that is, it had a significant molecular phenotype of senescence.

[0105] Example 4: Detection of GABAergic neuron injury in the mouse model

[0106] In this example, the expression of proteins related to GABAergic neuron injury in the mouse model constructed in Example 1 was detected by Western blot, including the expression levels of SST and GAD65, as well as the ratios of NMDAR2B / pNMDAR2B1303 and KCC2 / NKCC1.

[0107] The experiment was divided into the following groups: wild-type mice at 24 weeks of age (i.e., 6m WT group), wild-type mice at 88 weeks of age (i.e., 22m WT group), and a model group obtained by stereotactic injection of 10 nM C5a protein solution into C57BL / 6J mice at 24 weeks of age (i.e., 6m WT + 10 nM C5a group).

[0108] The specific experimental procedure is as follows:

[0109] Total protein of the mouse hippocampus was extracted, and the protein was quantified by the BCA method. 100 μg of the total protein sample was loaded onto a 10% SDS-PAGE gel for electrophoresis separation. Then, the protein bands separated by electrophoresis were electrotransferred onto a nitrocellulose (NC) membrane. Subsequently, the NC membrane was blocked in 5% skim milk for 1 - 2 hours to reduce non-specific binding. After blocking, the membrane was washed with TBST buffer, and then primary antibodies were added, including anti-SST (PA597200, 1:1000, Thermo), anti-GAD65 (1:1000, A5290, selleck), anti-KCC2 (1:1000, 94725S, CST), anti-NKCC1 (1:1000, 214581S, CST), anti-NMDAR2B (1:1000, ab93610, abcam), anti-NMDAR2B phospho Ser 1303 (1:1000, ab81271, abcam), and β-actin (1:10000, ab8227, proteintech) monoclonal antibodies, and incubated overnight at 4°C. The next day, the membrane was washed three times with TBST buffer for 10 minutes each time to remove unbound antibodies. Subsequently, HRP-labeled goat anti-rabbit secondary antibody (1:10000, ZB5301, Zhongshan Golden Bridge) or goat anti-mouse secondary antibody (1:10000, ZB2305, Zhongshan Golden Bridge) was added and incubated at room temperature for 1 hour. Then, the membrane was washed again by shaking three times with TBST buffer for 10 minutes each time. Finally, ECL chromogenic reagent (WBKLS0500, Millipore) was added for chemiluminescence reaction, and the bands were scanned by a gel imaging system to quantitatively analyze the expression levels of the target proteins.

[0110] The results of Western blot were as Figure 5 shown Figure 5 and showed that compared with wild-type mice of the same age, the SST, GAD65, and KCC2 / NKCC1 ratios in the mouse model constructed by stereotactic injection of 10 nM C5a were significantly decreased, and the NMDAR2B Ser1303 / NMDAR2B ratio was significantly increased, and there was no obvious difference in the corresponding protein levels or ratios compared with those of 22-month-old aging mice.

[0111] These results indicate that the mouse model established by stereotaxic injection of 10 nM C5a has the same level of GABAergic neuron damage as that of aged mice, that is, it has a significant phenotype of age-related nerve damage.

[0112] In summary, according to the method of the present invention, a rapidly aging mouse model can be successfully established, and the model mice exhibit cognitive impairment and early characteristic phenotypes of aging in terms of cognitive function, brain tissue pathology, and molecular level. Therefore, it can be used as a rapidly aging animal model to study the occurrence and development mechanisms of aging and cognitive impairment or to screen anti-aging therapeutic drugs.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments or equivalently replace some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for constructing a rapid aging animal model, characterized in that, The construction method includes: injecting a C5a protein solution into the hippocampal region of an animal by stereotaxic injection of the brain to obtain the same.

2. The construction method according to claim 1, wherein The hippocampal region is the dentate gyrus region of the hippocampus.

3. The construction method according to claim 2, wherein Inject the C5a protein solution into the bilateral hippocampal dentate gyrus regions of the animal; preferably, inject continuously for 5 - 10 days, more preferably, inject continuously for 7 days.

4. The construction method according to any one of claims 1-3, characterized in that, The C5a protein has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

5. The construction method according to any one of claims 1-4, characterized in that, The C5a protein solution is prepared by the following method: dissolve the C5a protein in DMSO to prepare a mother solution of the C5a protein, and then dilute the mother solution of the C5a protein with PBS to the working concentration before use to obtain the same. Preferably, in the C5a protein solution, the concentration of DMSO is less than 0.1%. Preferably, the working concentration of the C5a protein solution is 5 - 15 nM, preferably 8 - 12 nM, more preferably 10 nM.

6. The construction method according to any one of claims 1-5, characterized in that The injection volume of the C5a protein solution is 0.8 - 5 μL, preferably 1 - 3 μL, more preferably 2 μL.

7. The construction method according to any one of claims 1-6, characterized in that, The injection speed is 0.1 - 0.5 μL / min, preferably 0.1 - 0.3 μL / min, more preferably 0.2 μL / min. And / or, after the injection is completed, keep the needle in place for 8 - 12 min, preferably 10 min.

8. The construction method according to any one of claims 1-7, characterized in that, The stereotaxic injection of the brain is performed using a stereotaxic injection system for the brain, and the stereotaxic injection system for the brain includes: a stereotaxic apparatus for the brain, a microinjection pump, and a digital coordinate display.

9. The construction method according to any one of claims 1-8, characterized in that, The animal is a mammal, preferably a rodent, more preferably a mouse, and even more preferably a C57BL / 6J mouse.

10. Application of the rapidly aging animal model constructed by the construction method according to any one of claims 1 - 9 in the following aspects: (1) Studying aging or aging - related diseases; (2) Application in screening drugs for preventing, inhibiting or delaying aging; (3) Application in screening drugs for preventing, improving and / or treating aging - related diseases or disorders; Preferably, the aging - related disease or disorder is aging - related cognitive impairment.

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