Preparation method for and use of aging animal model

Abstract

The present disclosure relates to the field of biomedicine, and in particular to a preparation method for and a use of an aging animal model. The present disclosure provides a method for preparing an aging animal model, comprising modifying genes in immune cells in an animal body, wherein the modification reduces or eliminates the function or level of Abhd5.

Description

Preparation method of a senescence animal model and application thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biological medicine, and relates to a preparation method of a senescence animal model and application thereof. BACKGROUND

[0002] Senescence-related research has become a current research hotspot. Since natural aging usually takes a long time, researchers construct and accelerate cell and animal aging models by drug induction, gene knockout and other methods. Compared with animal models, cell models can be used for mechanism research, but cannot explore the mechanism of aging from the whole level, therefore, animal aging models have unique research value. In animal experimental research, from invertebrates such as nematodes and fruit flies to vertebrates such as rats and mice, aging models have been developed and widely applied to senescence-related research. Different models have advantages and disadvantages, and have different degrees of application.

[0003] Invertebrates have the advantages of fast modeling, convenient cultivation and observation, and some senescence research can replicate certain human aging characteristics through invertebrate models to quickly explore the mechanism of aging; however, invertebrates lack specific organs and systems (such as blood, bone and adaptive immune system), and these organs and systems are important components of human aging and age-related diseases, so invertebrate aging models have great limitations. In contrast, mice contain 99% of human genes with a high gene homology of 78.5%, and compared with other animals, the mouse genome transformation technology is very mature, and mice are relatively inexpensive, which is an ideal source of experimental animal models. SUMMARY

[0004] In some embodiments, the present disclosure provides a method for preparing a senescence animal model, comprising modifying a gene in an immune cell in the animal, the modification reducing or eliminating the function or level of Abhd5.

[0005] In some embodiments, the modification is selected from a substance that inhibits Abhd5 activity, a substance that degrades Abhd5, and / or a gene tool that reduces the level of Abhd5.

[0006] In some embodiments, the gene tool that reduces the level of Abhd5 comprises RNA interference, microRNA, gene editing or gene knockout material.

[0007] In some embodiments, Cre-LoxP technology, TALEN technology, ZFN technology or CRISPR / Cas technology is used for Abhd5 gene-specific knockout in immune cells.

[0008] In some embodiments, the gene Abhd5 in the immune cells is inactivated and / or deleted.

[0009] In some embodiments, the gene Abhd5 in the immune cells comprises a homozygous disruption.

[0010] In some embodiments, the immune cells comprise CD4+ T cells and CD8+ T cells.

[0011] In some embodiments, the animal is one in which the gene Abhd5 in CD4+ T cells and CD8+ T cells is simultaneously knocked out.

[0012] In some embodiments, the method of preparing the animal model of aging comprises the step of: mating and breeding a CD4 / 8-Cre mouse and an Abhd5flox / flox animal to obtain an animal in which Abhd5 in CD8+ T cells and CD4+ T cells is simultaneously knocked out.

[0013] In some embodiments, the loxP sites in the Abhd5flox / flox animal are introduced into introns of the Abhd5 gene.

[0014] In some embodiments, the Abhd5 gene in the Abhd5flox / flox animal comprises a gene sequence with one loxP site at each end of the gene.

[0015] In some embodiments, the Cre recombinase in the CD4 / 8-Cre mouse is placed under the control of the promoters of the CD4 and CD8 genes.

[0016] In some embodiments, the animal comprises a rodent.

[0017] In some embodiments, the animal model comprises one or more of the following phenotypes: skin lesions, hair loss, skin wrinkling, lymph node enlargement and pigmentation, kyphosis, brown fat hyperemia, and volume reduction, relative to normal group animals, a decrease in lymphocytes, relative to normal group animals, a decrease in grip strength, relative to normal group animals, a significant decrease in the number of mitochondria between muscles, and, a large number of inflammatory and chemotactic factor secretion phenotypes associated with aging.

[0018] In some embodiments, the animal comprises a pig, a rat, a mouse, a hamster, a rabbit, a pig, a cow, a deer, a sheep, a goat, a chicken, a cat, a horse, a dog, a chimpanzee, or a monkey.

[0019] In some embodiments, the immune cells comprise CD8 T cells.

[0020] In some embodiments, the present disclosure provides an animal model of aging obtained by the above method.

[0021] In some embodiments, an aging animal model is provided, wherein the immune cells in the animal model contain a modified gene Abhd5, the function or level of which is reduced or eliminated. In some embodiments, the gene Abhd5 in the immune cells is inactivated and / or deleted. In some embodiments, the gene Abhd5 in the immune cells comprises homozygous deletion. In some embodiments, the immune cells include CD4+. + T cells and CD8 + T cells. In some embodiments, the animal is CD4. + T cells and CD8 + Animals in which the Abhd5 gene in T cells is simultaneously knocked out. In some embodiments, the animals include rodents. In some embodiments, the animals include pigs, rats, mice, hamsters, rabbits, pigs, cattle, deer, sheep, goats, chicks, cats, horses, dogs, orangutans, or monkeys. In some embodiments, the animal model includes one or more of the following phenotypes: skin lesions, hair loss, skin wrinkling, lymphadenopathy and pigmentation, kyphosis, brown adipose tissue congestion and reduced size, reduced lymphocytes compared to the normal group, decreased grip strength compared to the normal group, significantly reduced number of intermuscular mitochondria compared to the normal group, and the presence of a large number of inflammatory factors and chemokines associated with aging secretion.

[0022] In some embodiments, this disclosure provides a cell or cell line or primary cell culture or a fragment of said cell or cell line or primary cell culture or an extract thereof, derived from an animal produced by the method or the offspring of said animal.

[0023] In some embodiments, this disclosure provides a tissue or organ, or a culture of said tissue or organ, or a fragment or extract of said tissue or organ, derived from an animal produced by the method or a descendant of said animal.

[0024] In some embodiments, this disclosure provides bodily fluids of an animal or its offspring, the animal being prepared by the method.

[0025] In some embodiments, this disclosure provides an isolated cell nucleus derived from an animal produced by the method or from the offspring of said animal.

[0026] In some embodiments, the present disclosure provides use of the animal model or offspring thereof obtained by the method in a model system for pharmacological, immunological, microbiological and medical research, or in the production and use of animal experimental disease models, for etiological research and / or for the development of new diagnostic strategies and / or therapeutic strategies, or in the screening, verification, evaluation or research of anti-aging drugs, pharmacodynamic research.

[0027] In some embodiments, the present disclosure provides a reliable preparation method of an aging animal model, which provides an important model for the pathogenesis of related aging diseases, and is an important tool for future drug development, screening, and exploration of effective treatment methods.

[0028] In some embodiments, the present disclosure provides a method for screening anti-aging drugs, wherein the method comprises:

[0029] (1) administering a drug to the aging animal model obtained by the method, wherein the animal has symptoms of aging, and

[0030] (2) determining whether the drug treats the symptoms of aging in the animal compared to the aging animal model obtained by the method without taking the drug.

[0031] In some embodiments, the present disclosure provides use of a detection reagent for Abhd5 in the preparation of an aging diagnostic reagent or kit.

[0032] In some embodiments, the detection reagent detects the expression amount of the gene.

[0033] In some embodiments, the detection reagent detects the mRNA expression amount of the gene.

[0034] In some embodiments, the detection reagent detects the protein expression amount of the gene.

[0035] In some embodiments, the detection sample of the detection reagent for Abhd5 is CD8+ T cells and CD4+ T cells.

[0036] In some embodiments, the detection reagent is at least one selected from the group consisting of a fluorescent quantitative PCR dye, a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody, an antibody functional fragment, or a conjugated antibody.

[0037] In some embodiments, the kit is at least one selected from the group consisting of a qPCR kit, an immunoblotting detection kit, an immunochromatography detection kit, a flow cytometry analysis kit, an immunohistochemistry detection kit, an ELISA kit, or an electrochemiluminescence detection kit. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 shows the results of Western blot detection of the genotype of CD4 / CD8 T cell Abhd5 knockout mice. Figure 2 shows that CD4 / CD8 T cell Abhd5 knockout promotes mouse aging phenotype. Figure 2A shows that Abhd5-specific knockout mice develop skin lesions, hair loss, skin wrinkles, lymph node enlargement and pigmentation. Figure 2B shows that knockout mice have obvious kyphosis. Figure 2C shows that the brown fat of knockout mice is congested and the volume is smaller. Figure 2D-2F show that HE staining, Masson staining and a-smooth immunohistochemical staining of brown fat of knockout mice show that lipid droplets are significantly reduced and fibrosis is increased. Figure 2G-2H show that the thymus of knockout mice is significantly smaller than that of control mice, and HE staining shows that the number of lymphocytes in knockout mice is significantly reduced compared with control mice. Figure 2I shows that the grip strength of knockout mice is significantly lower than that of control mice. Figure 2J shows that electron microscopy shows that the number of mitochondria in the muscle of knockout mice is significantly reduced.

[0039] Figure 3 shows that CD4 / CD8 T cell Abhd5 knockout mice develop a large number of pro-aging factors in plasma.

[0040] Figure 4 shows that CD4 / CD8 T cell Abhd5 knockout leads to senile leukemia and suppresses anti-tumor immunity. Figure 4A shows that in vivo imaging and gross specimens, the tumor of knockout mice is significantly larger than that of control mice. Figure 4B is the result of quantitative analysis by weighing the tumor. Figure 4C-D show that the size and weight of the spleen of knockout mice are larger than those of control mice. Figure 4E shows that bone marrow smears show that a large number of mononuclear cells appear in knockout mice, and granulocytes are occasionally seen in control mice.

[0041] Figure 5 shows that CD4 / CD8 T cell Abhd5 knockout leads to abnormal bone marrow differentiation-myeloid differentiation. Figure 5A-5C show the results of cyTOF analysis of mouse spleen. Figure 5D shows the results of single cell sequencing analysis of CD8 + T cells in the spleen. Figure 5E shows the results of single cell sequencing analysis of CD8 + T cells in the bone marrow.

[0042] Figure 6 shows that CD4 / CD8 T cell Abhd5 knockout promotes tissue and organ aging. Figure 6A shows the results of cell aging beta-galactosidase staining of mouse heart, liver and kidney, which shows that the kidney of knockout mice develops obvious aging phenotype. Figure 6B shows that electron microscopy of brain tissue of mice shows that the number of mitochondria in brain tissue of male and female knockout mice is significantly reduced. Figure 6C shows that behavioral experiments such as Y maze and novel object recognition experiment on mice show that knockout mice have anxiety and depression tendency compared with control mice. Figure 6D shows that P-H2AX immunofluorescence staining of mouse liver shows that P-H2AX in the liver of knockout mice is significantly increased, indicating that DNA damage in the liver is significantly aggravated.

[0043] Figure 7 shows that CD4 / CD8 T cell knockout of Abhd5 promotes metabolic imbalance. Figure 7A PET-CT shows that CD8 + T cells and CD4 + Brown adipose tissue and brain metabolic function significantly decreased after ABHD5 was simultaneously deleted in CD8 + T and CD4 + Blood glucose of mice rapidly increased and slowly decreased after ABHD5 was simultaneously deleted in CD8 + T and CD4 + Respiratory entropy of mice decreased, heat production increased and energy consumption decreased after ABHD5 was simultaneously deleted in CD8

[0044] Figure 8 shows that CD4 / CD8 T cell knockout of Abhd5 causes mitochondrial damage. Figure 8A Cell fluorescence staining of control CD8 + T and CD8 + T cells of knockout mice showed a significant decrease in the number of mitochondria and lipid deposition. Figure 8B Electron micrographs show that CD8 + T cells of knockout mice showed a significant decrease in the number of mitochondria and lipid deposition. Figure 8B Electron micrographs show that CD8 + T cells of knockout mice showed a significant decrease in the number of mitochondria and lipid deposition. Figure 8B Electron micrographs show that CD8 + OCR of CD8 + T cells of knockout mice showed a significant decrease in the number of mitochondria and lipid deposition. Figure 8B Electron micrographs show that CD8 13 T cells of knockout mice showed a significant decrease in the number of mitochondria and lipid deposition. Figure 8B Electron micrographs show that CD8 + T cells of knockout mice showed a significant decrease in the number of mitochondria and lipid deposition. Figure 8B Electron micrographs show that CD8 + T cells of knockout mice showed a significant decrease in the number of mitochondria and lipid deposition. Figure 8B Electron micrographs show that CD8

[0045] Figure 9 shows that Cre recombinase (Cyclization Recombination Enzyme) is encoded by the Cre gene of bacteriophage PI of Escherichia coli, and is a 38kD protein composed of 343 amino acids. It not only has catalytic activity, but also, similar to restriction enzymes, can specifically recognize the loxP site, thereby recombining or deleting genes between Loxp fragments. The LoxP (locus of X-over P1) site is 34 bp long, including two 13 bp inverted repeat sequences and an 8 bp spacer region. Among them, the inverted repeat sequence is the specific recognition site of Cre recombinase, and the spacer region determines the direction of the loxP site.

[0046] Figure 10 shows the detection of metabolic syndrome phenotype of CD4 / CD8 T cell knockout Abhd5 mice. Figure 10A shows the general photograph of control mice and knockout group mice. Figure 10B shows the general photograph of liver of control mice and knockout group mice. Figure 10C shows the body weight comparison column chart of control mice and knockout group mice.

[0047] Figure 11 shows the detection of aging phenotype of CD8 + T cell Abhd5 knockout mice. Figure 11A is the HE staining result of brown adipose tissue of knockout mice. Figure 11B is the Sirius red staining result of kidney tissue of knockout mice. Figure 11C is the cell aging beta-galactosidase staining result of brown adipose tissue of knockout mice. Figure 11D is the HE staining result of visceral adipose tissue of knockout mice. Figure 11E is the result of behavioral experiment of Y maze and new object recognition experiment of mice. DETAILED DESCRIPTION

[0048] The technical solutions of the present disclosure are further illustrated by specific examples below, and the specific examples do not represent a limitation on the protection scope of the present disclosure. Some non-essential modifications and adjustments made by others according to the concept of the present disclosure still fall within the protection scope of the present disclosure.

[0049] The experimental scheme of the present disclosure has been reviewed by the IACUC-JFLAB (Institutional Animal Care and Use Committee of Jinfeng Laboratory) and conforms to the principles of animal protection, animal welfare and ethics, and complies with the relevant regulations of national experimental animal welfare ethics (Ethical Number: IACUC-JFLAB-2022-001).

[0050] Cre recombinase (Cyclization Recombination Enzyme) is encoded by the Cre gene of bacteriophage PI of Escherichia coli, and is a 38kD protein composed of 343 amino acids. It not only has catalytic activity, but also, similar to restriction enzymes, can specifically recognize the loxP site, thereby recombining or deleting genes between Loxp fragments (Figure 8).

[0051] LoxP (locus of X-over P1) site is 34 bp long, including two 13 bp inverted repeats and an 8 bp spacer region. The inverted repeats are the specific recognition sites for Cre recombinase, and the spacer region determines the orientation of the loxP site (as shown in Figure 9).

[0052] There are several ways to induce recombination in the Cre / loxP system, which is based on the interaction between Cre recombinase and loxP sites. When loxP sites exist within a gene and Cre recombinase is present, Cre recombinase will bind to the inverted repeat regions at both ends of the loxP site to form a dimer. This dimer binds to other loxP site dimers to form a tetramer. Subsequently, the DNA sequence between the loxP sites is cut off by Cre recombinase, and the cut is reconnected by DNA ligase. The result of DNA recombination mainly depends on the orientation and position of the loxP site.

[0053] Cre recombinase is expressed under the control of the mouse CD4 regulatory elements. The CD4 enhancer, promoter, and silencer together drive CD4 + T / CD8 + expression of specific consecutive stages of T cell development. The integration site of the transgene construct is located at the 60335693-60341285 (qD) site of chromosome 3, and the insert contains at least 15 complete copies of the transgene construct sequence. The Cre / loxP system is used to achieve in vivo knockout of a specific gene under specific conditions, which requires two types of transgenic mice:

[0054] (1) The first type of mouse is generally obtained using embryonic stem cell technology. First, a gene sequence containing a loxP site at both ends of the Abhd5 gene is constructed in vitro, and then the in vitro constructed gene sequence is transferred into embryonic stem cells to replace the original gene sequence in the cell genome through homologous recombination. The embryonic stem cells treated in this way are reimplanted into the uterus of a pseudopregnant mouse to develop into a complete embryo, and ultimately become a transgenic mouse, which is a Flox mouse (i.e., Abhd5 flox / flox Mouse). In this transgenic mouse, loxP sites are introduced at both ends of the Abhd5 gene sequence, which theoretically does not affect the function of the corresponding gene, so the phenotype of this mouse is generally normal.

[0055] (2) The second type of transgenic mouse is generally obtained using oocyte injection or embryonic stem cell technology. In this mouse, Cre recombinase is placed under the control of the CD4 / CD8 gene promoter, which can be expressed under certain conditions, and is a CD4 / 8-Cre mouse.

[0056] Finally, let the two mice Flox mice and CD4 / CD8-Cre mice to mate, the offspring of the same time containing the two genotypes of mice will be in CD8 + T cells and CD4 + T cells Abhd5 gene is deleted.

[0057] CD4 / 8-Cre mice from the Department of Immunology, Basic Medical College, Army Medical University, Professor Li Lin.

[0058] Flox mice from Saiye (Suzhou) Biotechnology Co., Ltd.

[0059] CD8-Cre mice from Saiye (Suzhou) Biotechnology Co., Ltd.: In this mouse, Cre recombinase is placed under the control of the CD8 gene promoter, which can be expressed under certain conditions, CD8-Cre mice.

[0060] CD4 / 8 represents "CD4 and CD8 genes". "CD4 / 8 T cell Abhd5 knockout" means "CD8 + T cells and CD4 + T cells Abhd5" at the same time.

[0061] CD8 + T cells can also be referred to as "CD8 single positive T cells"; CD4 + T cells can also be referred to as "CD4 single positive T cells".

[0062] Example 1 Construction method of a senescent animal model (CD8 + T cells and CD4 + T cells Abhd5 knockout mice) at the same time

[0063] 1.1 Construction method

[0064] F0 generation of mice carrying CD4 / 8-Cre is mated with Flox mice to obtain F1 heterozygotes, and F1 heterozygotes are mated to obtain F2 homozygotes; Each founder mouse (i.e. a mouse carrying the target gene inserted after microinjection of the fertilized egg) needs to be independently passed on;

[0065] Identify the F1 mice born, and their offspring have a 50% chance of carrying the integrated target gene; F1 positive mice obtained can be used for experiments and continued to be passed on (also can be bred with Cre mice).

[0066] In some embodiments, if homozygotes are needed, positive F1 from the same source can be sibling mated, and F2 mice born will have a 25% chance of being homozygous; The screened CD8+ T cells and CD4 + T cells and CD8 + T cells and CD4 + T cells and CD8 + T cells and CD4 + T cells and CD8 + T cells and CD4 + T cells and CD8 + T cells and CD4 + T cells and CD8

[0067] 1.2 Western blot detection of CD4 + T cells and CD4 + T cells and CD8 + T cells and CD4 + T cells and CD8 + T cells and CD4 + T cells and CD8

[0068] T cells and CD4 + T cells and CD8 + T cells and CD4 + T cells and CD8 + T cells and CD8

[0069] i. BCA method for protein concentration quantification (Bi Yun Tian Company, Item No.: P0009):

[0070] In the 96-well plate, the following standard sample solution was prepared using 0.5 mg / ml protein standard sample solution:

[0071] The samples were diluted as follows:

[0072] Corresponding protein standard sample and sample 20 μL were added to each well, BCA reaction solution 200 μL (reagent A: reagent B = 50: 1), 60°C CO2 incubator for 30 min, then measured by enzyme marker at A562 nm absorbance. According to the measured absorbance and the known concentration in the above table, a standard curve was drawn, and the protein concentration of the four samples was calculated. 25 μg of protein was loaded for subsequent experiments.

[0073] ii. Electrophoretic separation of protein:

[0074] The electrophoresis solution was prepared as follows:

[0075] SDS-PAGE precast gel (Bi Yun Tian Company, P0057A) was taken, and the samples were added to the corresponding wells in turn. Each sample can set 2-3 duplicate wells, adjust the voltage to 150V for 60 min.

[0076] iii. Transmembrane

[0077] The transfer membrane solution is configured as follows:

[0078] After configuration, place in 4°C pre-cooling.

[0079] In wet transfer mode, place sponge pad, filter paper, PVDF membrane, SDS-PAGE gel, filter paper, and sponge pad in sequence, and adjust the current to 250 mA for 80 min.

[0080] iv. Blocking

[0081] The electrophoresis washing buffer TBST is configured as follows:

[0082] After the transfer is completed, take out the PVDF membrane and place it in an antibody incubation box, add TBST buffer and wash 3 times, each time for 5 min. After completion, discard the washing liquid, add Western blocking solution (Biyun Tian Company, P0023B), and slowly shake on a shaking table at room temperature for 1 hour.

[0083] v. Primary antibody incubation

[0084] Take Western primary antibody diluent (Bi Yun Tian Company, P0023A) and rabbit anti-ABHD5 antibody (Proteintech, 12201-1-AP), and configure 10 mL of primary antibody according to a ratio of 1:1000 (i.e. 10 μL of ABHD5 antibody + 9.99 mL of Western primary antibody diluent) to add to the antibody endowment box, and slowly incubate at 4°C constant temperature shaking table for 12 hours.

[0085] vi. Secondary antibody incubation

[0086] Recover the primary antibody, wash with TBST buffer 3 times, each time for 5 min, add HRP-goat anti-rabbit secondary antibody (1:5000), and slowly incubate at room temperature on a shaking table for 1 hour.

[0087] vii. ECL development

[0088] Take ECL kit (Affinity, KF8003) 1:1, mix well, and develop on a developing instrument.

[0089] The results are shown in FIG. 1. CD4 + T cells and CD8 + T cells in the group of Abhd5 knockout mice, CD4 + T cells and CD8 + T cells ABHD5 expression is lost, where GAPDH is used as an internal reference gene (indicating that the protein loading amount is consistent), indicating that the ABHD5 gene in CD4 + T cells and CD8 + T cells are knocked out, i.e. CD4 + T cells and CD8+ T cell knockout Abhd5 mice were successfully constructed.

[0090] Example 2 CD8 + T cell and CD4 + T cell knockout Abhd5 mice promote mouse aging phenotype

[0091] 2.1 CD8 + T cell and CD4 + After the T cell knockout Abhd5 mice were anesthetized, the corners of the eyes, the back and the abdomen were photographed, and after the mice were sacrificed, the lymph nodes and the back skin were taken and photographed, and the results are shown in Figure 2A. From the results, it can be seen that the CD8 + T cell and CD4 + T cell knockout Abhd5 mice had skin lesions, hair loss, skin wrinkles, lymph node enlargement and pigmentation.

[0092] 2.2 CD8 + T cell and CD4 + After the T cell knockout Abhd5 mice were anesthetized, they were photographed in a lateral position, and the results are shown in Figure 2B. The knockout mice had obvious kyphosis.

[0093] 2.3 CD8 + T cell and CD4 + After the T cell knockout Abhd5 mice were anesthetized and sacrificed, the brown adipose tissue of the two groups of mice was taken and photographed, and the results are shown in Figure 2C. The brown adipose tissue of the knockout mice was congested and the volume was smaller.

[0094] 2.4 CD8 + T cell and CD4 + After the T cell knockout Abhd5 mice were anesthetized, the brown adipose tissue was taken and subjected to HE staining, Masson staining and α-smooth immunohistochemical staining:

[0095] (1) The paraffin section deparaffinization step is as follows:

[0096] i. The brown adipose tissue was fixed in a 4% paraformaldehyde solution, and before dehydration, it was transferred to a 10% neutral formalin solution, and then dehydrated in 70% ethanol-85% ethanol-95% ethanol I-95% ethanol II-anhydrous ethanol I-anhydrous ethanol II-xylene I-xylene II-xylene III-paraffin I-paraffin II-paraffin III;

[0097] ii. Embedding and sectioning;

[0098] iii. Xylene I-Xylene II-Anhydrous Ethanol I-Anhydrous Ethanol II-95% Ethanol-85% Ethanol-75% Ethanol Rehydration.

[0099] (2) HE staining steps are as follows:

[0100] i. Stain the slices obtained in step (1) above with hematoxylin I for 14s, stain with hematoxylin II for 1min, and wash with water for 5min.

[0101] ii. Differentiate in the differentiation solution for 3-5 seconds, then wash with water for 1 minute;

[0102] iii. Treat with blueing solution for 7 seconds, then rinse with water for 20 seconds;

[0103] iv. Immerse in 95% ethanol for 1 min, stain with eosin for 14 s, then stain with anhydrous ethanol I and II for 2 min each, and clear with xylene I and xylene II for 2 min.

[0104] v. Neutral resin sealing.

[0105] (3) The steps of α-smooth immunohistochemical staining are as follows:

[0106] i. Dewax the sections obtained in step (1) above into water, microwave on high for 15 min, wash with PBS 3 times, 5 min each time;

[0107] ii. Add blocking solution and block at room temperature for 1 hour; add primary antibody and incubate overnight at 4°C;

[0108] iii. Recover the primary antibody, wash 3 times with PBS, 5 min each time;

[0109] iv. Add HRP-conjugated secondary antibody and incubate at room temperature for one hour;

[0110] v. Wash 3 times with PBS, 5 min each time, add DAB colorimetric solution, and stop when a positive result appears;

[0111] vi. Stain with hematoxylin for 1 min, dehydrate, clear, and mount.

[0112] (4) The steps of Masson staining are as follows:

[0113] i. Dewax the sections obtained in step (1) above into water, immerse them in mordant solution, let them sit at room temperature overnight, and rinse with running water for 10 minutes;

[0114] ii. Stain with azurite blue staining solution for 2-3 minutes, then wash twice with distilled water for 10-15 seconds each time;

[0115] iii. Stain with Mayer's hematoxylin staining solution for 2-3 minutes, then wash twice with distilled water for 10-15 seconds each time;

[0116] iv. Differentiate in acidic solution for a few seconds, wash with water to stop differentiation, and rinse with distilled water for 10 minutes;

[0117] v. Stain with Pomfret and fuchsin solution for 10 min, then add distilled water twice, 10-15 s each time;

[0118] vi. Treat with phosphomolybdic acid solution for 10 min;

[0119] vii. Discard the supernatant and directly add aniline blue for staining for 5 minutes;

[0120] viii. After washing away the aniline blue solution with a weak acid solution, continue to add weak acid solution to cover the section for 2 minutes;

[0121] ix. 95% ethanol-anhydrous ethanol-xylene I-xylene II are sequentially dehydrated to become transparent, and then sealed with neutral resin.

[0122] The results are shown in Figures 2D-2F. HE staining, Masson staining, and α-smooth immunohistochemical staining of brown adipose tissue in knockout mice showed a significant reduction in lipid droplets and an increase in fibrosis.

[0123] CD8 obtained in 2.5 + T cells and CD4 + T cell knockout Abhd5 mice were anesthetized and sacrificed, and thymus tissue was collected for photographic recording and HE staining. The HE staining procedure is as follows:

[0124] i. After mice were anesthetized and euthanized, the thymus was fixed in 4% paraformaldehyde solution and transferred to 10% neutral formalin solution before dehydration;

[0125] ii. Dehydrate sequentially using 70% ethanol, 85% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, anhydrous ethanol II, xylene I, xylene II, xylene III, paraffin I, paraffin II, and paraffin III;

[0126] iii. Embedding and sectioning;

[0127] iv. Xylene I-Xylene II-Anhydrous Ethanol I-Anhydrous Ethanol II-95% Ethanol-85% Ethanol-75% Ethanol Rehydration;

[0128] v. Stain with hematoxylin solution I for 14 seconds, then stain with hematoxylin solution II for 1 minute, and wash with water for 5 minutes.

[0129] vi. Differentiate in differentiation solution for 3-5 seconds, then wash with water for 1 minute;

[0130] vii. Treat with blueing solution for 7 seconds, then rinse with water for 20 seconds;

[0131] viii. Immerse in 95% ethanol for 1 min, then stain with eosin solution for 14 s;

[0132] ix. Apply anhydrous ethanol I and II for 2 min each, xylene I and xylene II for 2 min to clear, and then mount with neutral resin.

[0133] As can be seen from Figures 2G-2H, the thymus of the knockout mice was significantly smaller and atrophied than that of the control mice, and HE staining showed that the number of lymphocytes in the knockout mice was significantly reduced compared with that in the control mice.

[0134] CD8 obtained in 2.6 + T cells and CD4 + T cell knockout Abhd5 mice were placed on a grip strength tester. After they gripped the instrument firmly, they were pulled backward. The readings were recorded after the instrument stabilized. Statistical analysis was performed on the results, with P < 0.05 considered statistically significant. The results are shown in Figure 2I, indicating that the grip strength of the knockout mice was significantly lower than that of the control mice.

[0135] 2.7 Obtain the acquired CD8 + T cells and CD4 + Muscle tissue from T-cell knockout Abhd5 mice was immediately placed in 2.5% glutaraldehyde solution, trimmed to 1 mm size, fixed, dehydrated, and infiltrated, then semi-thinly sectioned and observed and photographed under an electron microscope. The results are shown in Figure 2J; electron microscopy revealed a significant reduction in the number of mitochondria in the muscle tissue of the knockout mice. Example 3 CD8 + T cells and CD4 + A large number of pro-aging factors were observed in the plasma of mice with simultaneous T cell knockout of Abhd5.

[0136] The CD8 obtained by the method in Example 1 + T cells and CD4 + T cells were simultaneously knocked out in Abhd5 mice. After anesthetizing the mice, blood was collected from the retroorbital vein and placed in EDTA anticoagulant tubes. The tubes were centrifuged at 3500 rpm for 5 min, and the supernatant was collected and stored at -80℃. The supernatant was then transported to Wuhan Sewell Pharmaceutical Co., Ltd. on dry ice for the detection of mouse plasma factor 31. The results were statistically analyzed, with P < 0.05 considered statistically significant.

[0137] The results are shown in Figure 3, which displays CD8. + T cells and CD4 + Mice in which Abhd5 was simultaneously knocked out in T cells showed a large number of aging-related secretory phenotypes, such as inflammatory factors and chemokines, in their plasma, promoting the aging phenotype in mice.

[0138] Example 4CD8 + T cells and CD4 +Simultaneous knockout of Abhd5 by T cells leads to spontaneous senile leukemia and suppression of anti-tumor immunity (spontaneous leukemia model).

[0139] The CD8 obtained by the method in Example 1 + T cells and CD4 + Mice with simultaneous T cell knockout of Abhd5 were treated as follows:

[0140] 4.1 Age- and sex-matched control mice and CD8 mice were used. + T cells and CD4 + Mice with simultaneous T cell knockout of Abhd5:

[0141] i. Mouse colon cancer cells MC38 (8*10^5 cells) were injected intraperitoneally.

[0142] ii. On day 10 after inoculation, live imaging was performed on both types of mice. After anesthetizing the mice, 100ul (15mg / mL) of D-fluorescein potassium salt was injected into the peritoneum and the mice were placed in a small animal live imaging device for imaging.

[0143] iii. After euthanizing the mice, photographs were taken to record the size of the abdominal tumors.

[0144] As shown in Figure 4A, in in vivo imaging and gross specimens, the tumors in the knockout group mice were significantly larger than those in the control group mice.

[0145] 4.2 After anesthetizing the mice, the tumor tissues of each group of mice were weighed and quantitatively analyzed; the results are shown in Figure 4B, the tumor weight of the knockout group mice was significantly greater than that of the control group mice.

[0146] 4.3 After anesthetizing the mice, the heart, liver, spleen, lungs and kidneys of each group of mice were weighed and photographed; the results are shown in Figures 4C-4D. The spleen size and weight of the knockout mice were larger than those of the control mice.

[0147] 4.4 After anesthetizing the mice, long bones were taken, both ends were cut off, the bone marrow was flushed out with PB S, centrifuged at low speed, and an appropriate amount of bone marrow suspension was spread on a glass slide and allowed to air dry naturally.

[0148] i. Add 2-3 drops of WG staining solution (the staining kit used is Solarbio's Wright-Giemsa combined staining kit, catalog number G 1021) to cover the entire specimen smear and stain for 3-5 minutes;

[0149] ii. Add an equal volume of WG buffer, gently shake the slide to mix thoroughly with the WG staining solution, and stain for 3-5 minutes;

[0150] iii. Wash with water, dry, and take a photograph under a microscope to record the process.

[0151] All results were statistically analyzed, with P < 0.05 considered statistically significant. The results are shown in Figure 4E. Bone marrow smears revealed a large number of mononuclear cells and occasional granulocytes in the knockout mice, while the control mice were predominantly granulocytes.

[0152] Example 5CD8 + T cells and CD4 + Simultaneous knockout of Ab hd5 by T cells leads to abnormal bone marrow differentiation - lymphoid to myeloid differentiation (immunoresenescence).

[0153] 5.1 The CD8 obtained by the method in Example 1 + T cells and CD4 + T-cell knockout Abhd5 mice were anesthetized and sacrificed. The spleen tissue was completely dissected using surgical instruments, rinsed with physiological saline, and then placed in Miltenyi tissue preservation solution. The specimens were sent to Protin for CyTOF analysis at a constant temperature of 2-8°C. The results are shown in Figures 5A-5D. CyTOF analysis of the mouse spleen showed that the knockout group mice had a moderate effector CD8+. + The proportion of T cells decreased significantly, while the proportion of MDSCs increased significantly.

[0154] 5.2 The CD8 obtained by the method in Example 1 + T cells and CD4 + Mice with Abhd5 knocked out by T cells were anesthetized and sacrificed. The spleen and femur of the mice were completely dissected and flushed out of the bone marrow.

[0155] i. Place the spleen in a 70μm filter and grind it with PBS;

[0156] ii. Transfer the ground cell suspension and bone marrow to centrifuge tubes and centrifuge at 1000 rpm for 3 min;

[0157] iii. Discard the supernatant, add red blood cell lysis buffer and lyse on ice for 2-3 minutes, then add an equal volume of PBS to terminate the lysis;

[0158] iv. Wash twice with PBS, reselect cells with Mojosort Buffer and count them, add Biotin-Antibody Cocktail per 10^7 cells, and incubate on ice for 15 minutes;

[0159] v. After vortexing the Streptavidin Nanobeads, add the same volume of the reagent and let stand on ice for 15 min.

[0160] vi. Add 1*Mojosort buffer to 3mL, place the flow cytometer in a magnet, let it stand at room temperature for 5min, and repeat once;

[0161] vii. The supernatant was poured out as the target cell suspension, centrifuged at 1000 rpm, and then resuspended in pure serum for single-cell sequencing.

[0162] The results are shown in Figures 5E-5F. Further analysis of CD8 in bone marrow and spleen... + Single-cell sequencing analysis of T cells revealed that in bone marrow and spleen, knockout mice exhibited effector CD8... + The number of T cells decreased significantly, and differential gene enrichment analysis revealed that T cells expressed myeloid markers.

[0163] Example 6CD8 + T cells and CD4 + T cell knockout of Abhd5 promotes tissue and organ aging.

[0164] CD8 obtained from the method of Example 1 + T cells and CD4 + Mice with simultaneous T cell knockout of Abhd5 were treated as follows:

[0165] 6.1 Obtain frozen sections of the heart, liver, and kidneys, and perform β-galactosidase staining for cell senescence. The method for β-galactosidase staining for cell senescence is as follows:

[0166] i. Frozen sections are thawed at room temperature for 10 minutes, and tissues are circled with a histochemical pen;

[0167] ii. Add an appropriate amount of β-galactosidase staining fixative to the tissue and fix it at room temperature for 20 min;

[0168] iii. Wash with PBS three times, 5 minutes each time;

[0169] iv. Place the sections in a humidified chamber, add an appropriate amount of β-galactosidase staining working solution, and incubate overnight at 37°C in a CO2-free incubator;

[0170] v. After tissue staining, remove the staining solution, rinse twice with PBS, rinse twice with pure water, add nuclear solid red staining solution for 3 min, and rinse three times with water.

[0171] vi. Dehydrate twice with anhydrous ethanol, 5 min each time, clear with xylene for 5 min, and mount with neutral resin.

[0172] As shown in Figure 6A, the knockout mice exhibited a clear aging phenotype in their kidneys.

[0173] 6.2 Mice were euthanized after anesthesia, and brain tissue was immediately immersed in 2.5% glutaraldehyde solution. The tissue was then trimmed to approximately 1 mm in the glutaraldehyde solution and sent for electron microscopy. The results are shown in Figure 6B. Electron microscopy of mouse brain tissue revealed that the mitochondrial cristae in the brain tissue of both male and female knockout mice were significantly swollen and reduced in number.

[0174] 6.3 Behavioral experiments were conducted on mice, including the Y-maze and novel object recognition tests, among which:

[0175] 6.3.1Y Maze Experiment: This includes two experiments, with an interval of more than 1 hour between them.

[0176] i. The first experiment was a training period. One arm was closed, and the animal was allowed to explore freely in the other two arms for 5 minutes. After the training, the animal was put back into the cage, and the animal odor was eliminated with 75% alcohol.

[0177] ii. One hour later, the second experiment (recall phase) was conducted. All arms were opened, and the animal was allowed to move freely in the three arms for 3 minutes. The time and distance traveled in each arm were recorded.

[0178] 6.3.2 New Object Substitution Recognition Experiment:

[0179] i. Habituation period: Remove the mouse from its cage and place it in the center of the open area (test chamber). Allow the mouse to explore freely for 5 minutes. After 5 minutes, remove the mouse and return it to its cage; thoroughly clean the mouse's equipment with 70% ethanol;

[0180] ii. Training period: Place two identical objects diagonally opposite each other in the test box (i.e., the northeast and southwest corners). After 24 hours of habituation, remove the mouse from its cage and place it in the center of the test box, equidistant from the two identical objects. Allow free exploration for at least 5 minutes. At the end of the experiment, remove the mouse and return it to its cage. Thoroughly clean the equipment and objects the mouse has come into contact with using 70% ethanol.

[0181] iii. Testing Period: Place one familiar object used during T1 and a new object diagonally opposite each other in the testing box. Use the same object positions for each mouse as during T1. During the selection interval from T1 to T2, remove the mouse from the cage and place it in the center of the testing box, equidistant from both objects. Allow free exploration for 5 minutes.

[0182] iv. After the experiment, the mice were removed and placed back into cages. Statistical analysis of the results was performed using the Two-Way Anova test, with P < 0.05 considered statistically significant.

[0183] As shown in Figure 6C, the knockout mice showed a significant decrease in overall movement distance and average speed compared to the control mice. In the new object recognition experiment, there was no statistically significant difference in the recognition of the Novel and Start arms, but the recognition of Other arms was reduced, suggesting that the knockout mice had a tendency to anxiety and depression.

[0184] 6.4 After anesthesia, mice were sacrificed. All organs of the mice were aliquoted with aluminum foil, frozen in liquid nitrogen, and then stored at -80°C. Before use, liver samples from control knockout mice were taken from the -80°C freezer, equilibrated at -20°C for 15 min, embedded in OCT, and then cut into 8μm thick sections at -20°C. The sections were placed on positively charged glass slides, and frozen sections were subjected to P-H2AX immunofluorescence staining.

[0185] The steps for P-H2AX immunofluorescence staining of frozen sections are as follows:

[0186] i. Frozen sections were thawed at room temperature for 10 min, fixed with ice-cold methanol for 5 min, and washed three times with PBS;

[0187] ii. Add 3% hydrogen peroxide solution, incubate for 10 minutes, then wash three times with PBS;

[0188] iii. Add immunostaining blocking solution and incubate at room temperature for 1 hour;

[0189] iv. Discard the staining solution, add P-H2AX and incubate overnight at 4°C;

[0190] v. Recover the primary antibody, wash three times with PBS, add anti-rabbit fluorescent secondary antibody and incubate at room temperature for 1 hour, then wash three times with PBS;

[0191] vi. Add DAPI and stain at room temperature for 3 minutes, then mount the slide;

[0192] vii. Statistical analysis of the positive regions was performed using ImageJ, and the p-value was considered statistically significant at P < 0.05.

[0193] As shown in Figure 6D, the knockout mice showed a significant increase in P-H2AX in their livers, indicating a marked increase in liver DNA damage.

[0194] Example 7CD8 + T cells and CD4 + T cells simultaneously knock out Abhd5, promoting metabolic imbalance.

[0195] CD8 obtained from the method of Example 1 + T cells and CD4 + Mice with simultaneous T cell knockout of Abhd5 were treated as follows:

[0196] 7.1 After anesthetizing mice, F-18DG was injected via the tail vein, followed by PET-CT scanning. The results are shown in Figure 7A. PET-CT revealed CD8... + T cells and CD4 + Mice lacking ABHD5 in both T cells showed a significant decrease in brown adipose tissue and brain metabolic function.

[0197] 7.2 Insulin sensitivity test (ITT) and glucose tolerance test (IPGTT) were performed. The ITT procedure was as follows: mice were fasted for 4 hours but allowed free water, and then injected intraperitoneally with 1 U / kg insulin. Blood glucose levels were measured by tail vein sampling at 0, 15, 30, 60, and 120 minutes. The IPGTT procedure was as follows: mice were fasted for 14 hours but allowed free water, and then injected intraperitoneally with 1 g / kg of 20% glucose solution. Blood glucose levels were measured by tail vein sampling at 0, 15, 30, 60, and 120 minutes. The results were recorded as curves. Two-way ANOVA was used for analysis, and P < 0.05 was considered statistically significant. The results are shown in Figure 7B. CD8 + T and CD4 + Mice with simultaneous loss of ABHD5 in T cells showed a rapid increase in blood glucose and a slow decrease.

[0198] 7.3 Control group mice and CD8 mice + T cells and CD4 + Mice in the T cell knockout Abhd5 group were starved for 6 hours and placed in metabolic cages. The levels of inhaled oxygen and exhaled CO2 were measured and plotted. A p-value < 0.05 was considered statistically significant. The results are shown in Figure 7C. Metabolic cage analysis showed that CD8+... + T and CD4 + Mice lacking both T and ABHD5 showed decreased respiratory entropy, increased heat production, and decreased energy consumption, with the body's metabolism tending to consume fat for energy.

[0199] Example 8CD8 + T cells and CD4 + Simultaneous knockout of Abhd5 by T cells leads to mitochondrial damage.

[0200] 8.1 From control mice and CD8 + T cells and CD4 + CD8+ cells were extracted from the spleens of mice in the T-cell simultaneous knockout Abhd5 group. + T cells were washed twice with PBS, and then co-stained with prepared Mitotracker (1 mM), Bodipy (5 mM), and Hochest (10 mg / mL) dye (10 mL of Mitotracker stock solution, 6 μL of Bodipy, and 5 μL of Hochest) at 37°C for 1 hour. The cells were then transferred to a glass slide using a slide shaker and photographed under a laser confocal microscope. The results are shown in Figure 8A, with the control CD8... + T and CD8 with Abhd5 knocked out + T cell cytofluorescence staining results showed that knockout mice had CD8 + T cells showed a significant reduction in the number of mitochondria and lipid deposition.

[0201] 8.2 Compare with CD8 + T cells and CD8 cells with Abhd5 knocked out + After centrifuging T cells at 100 rpm for 3 min, the supernatant was discarded, and 1 mL of 2.5% glutaraldehyde was added directly for fixation. The results are shown in Figure 8B. Electron microscopy images show CD8+ in mice with Abhd5 knockout. + T cells showed significant mitochondrial swelling.

[0202] 8.3 Take the control CD8 + T cells and CD8 cells with Abhd5 knocked out + T cell count:

[0203] i. Resuspend the cells in preheated detection solution (100 mL RPMI + 1 mL 1.0 mmol glucose solution + 1 mL 100 mmol / L sodium pyruvate solution + 1 mL 200 mmol / L glutamine solution, preheated at 37°C), seed 2*10^5 cells per well into a culture plate, and centrifuge slowly at 200g for 1 min.

[0204] ii. Before detection, place the cells in a CO2-free incubator and incubate at 37°C for 45-60 min;

[0205] iii. Add the detection drug (oligomycin A + 4 mL detection solution to a final concentration of 13.5 μmol / L; Bam15 + 4 mL detection solution to a final concentration of 25 μmol / L; rotenone / antimycin A + 4 mL detection solution to a final concentration of 5.5 μmol / L) to the corresponding drug addition wells A, B, and C respectively, and then use the culture plate to perform the detection.

[0206] iv. Statistical analysis of the results showed that P < 0.05 was statistically significant.

[0207] The results are shown in Figure 8C. Through the Seahorse experiment, CD8 knockout of Abhd5 under CD3 / 28 stimulation was achieved. + T cell OCR was significantly reduced, and ECAR value was significantly increased; without CD3 / 28 stimulation, CD8 knockout of Abhd5 cells resulted in a significant decrease in CD8+. + The T cell glycolytic capacity was enhanced, and there were no significant statistical differences between the two groups in the other results.

[0208] 8.4 Pass 13 C-labeled glucose for metabolic flux experiments:

[0209] i. Extract CD8 +T cells were seeded in 1 μg / ml CD3-coated plates, and 0.5 μg / ml CD28 antibody was added. The culture medium was RPMI 1640 (glucose-free) + 10% FBS + 1% penicillin antibody + 1% sodium pyruvate + 1% non-essential amino acids + 1% L-glutamine + 50 μM β-mercaptoethanol + 1% HEPES + 11 mM 13 C-labeled glucose +75μM UK5099;

[0210] ii. After culturing for 66 hours, collect the cells, centrifuge and discard the supernatant, transfer to a 1.5 mL EP tube, add 1 mL of ice-cold methanol-water mixture (methanol:water = 4:1), and store at -80℃.

[0211] The results are shown in Figures 8D-8E. 13 Metabolic flux experiments using C-labeled glucose showed that knockout mice exhibited significant differences in metabolites in the glycolysis and TCA cycle pathways.

[0212] 8.5 Extraction of control CD8 + T cells and CD8 cells with Abhd5 knocked out + T cells were treated with 1 mL Trozil, incubated for 5 min, then mixed with 1 / 5 volume of chloroform by vortexing. The mixture was centrifuged at 12000 g at 4°C for 15 min. The supernatant was transferred to a high-quality EP tube, and 1 volume of isopropanol (Trozil volume) was added. The mixture was incubated at room temperature for 10 min, then centrifuged at 12000 g at 4°C for 10 min. The precipitate was washed with an equal volume of 75% ethanol (Trozil volume), the supernatant was discarded, and the precipitate was retained. 30 μL of RNase-free water was added to dissolve the RNA. The RNA was then reverse transcribed into cDNA (in two steps).

[0213] Step (1): Genomic DNA removal reaction. Prepare the reaction solution in each reaction tube according to the following components:

[0214] Reaction Procedure

[0215] Step (2), perform reverse transcription:

[0216] Prepare the reaction mixture according to the following components.

[0217] Reaction Procedure

[0218] Quantitative analysis of genes related to thermogenesis and mitochondrial biogenesis was performed using quantitative real-time PCR.

[0219] Prepare the reaction mixture according to the following components.

[0220] Each sample was replicated in 3 separate wells.

[0221] Reaction procedure:

[0222] Statistical analysis of the results showed that P < 0.05 was statistically significant.

[0223] The results are shown in Figure 8F for the two CD8 groups. + RT-qPCR quantification of T cells revealed that the knockout group of CD8 + Most genes related to T-cell thermogenesis and mitochondrial biosynthesis were significantly downregulated. All results were statistically analyzed, with P < 0.05 considered statistically significant.

[0224] Experimental Example 1CD8 + T cells and CD4 + Detection of metabolic syndrome phenotype in T cell-simultaneous Abhd5 knockout mice

[0225] CD8 + T cells and CD4 + Gross images were taken of Abhd5 knockout mice and control mice after anesthesia, as shown in Figure 10A. Figure 10A shows gross images of the control and knockout mice. It can be seen that there is no significant difference in body size between the two groups of mice, and the knockout mice did not show signs of metabolic syndrome with obesity.

[0226] Mice were euthanized after anesthesia, and CD8 were collected. + T cells and CD4 + The livers of both Abhd5 mice and control mice were simultaneously knocked out using T cells and photographed. Figure 10B shows gross images of the livers of the control and knockout mice. It can be seen that the livers of both groups of mice are of uniform color, and the knockout mice did not show fatty liver symptoms of metabolic syndrome.

[0227] For CD8 + T cells and CD4 + The body weights of mice in both the T cell knockout Abhd5 group and the control group were recorded and statistically plotted. Figure 10C shows a bar chart comparing the body weights of the control and knockout groups, indicating no significant statistical difference in body weight between the two groups.

[0228] The above results indicate that CD8 + T and CD4 + Mouse models with simultaneous Abhd5 knockout of T cells did not exhibit a clear metabolic syndrome phenotype, but rather a senescent phenotype.

[0229] Experimental Example 2CD8 + Detection of aging phenotype in T cell Abhd5 knockout mice

[0230] Crossbreeding CD8-Cre mice with Flox mice produces offspring mice that carry both of these genotypes, resulting in CD8-Cre mice. + The Abhd5 gene is missing in T cells.

[0231] CD8 obtained in 10.1 + After T-cell Abhd5 knockout mice were anesthetized and sacrificed, brown adipose tissue and visceral adipose tissue were collected for HE staining.

[0232] The steps for HE staining are as follows:

[0233] i. After mice were anesthetized and euthanized, brown fat and visceral fat were fixed in 4% paraformaldehyde solution and transferred to 10% neutral formalin solution before dehydration.

[0234] ii. Dehydrate sequentially using 70% ethanol, 85% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, anhydrous ethanol II, xylene I, xylene II, xylene III, paraffin I, paraffin II, and paraffin III;

[0235] iii. Embedding and sectioning;

[0236] iv. Xylene I-Xylene II-Anhydrous Ethanol I-Anhydrous Ethanol II-95% Ethanol-85% Ethanol-75% Ethanol Rehydration;

[0237] v. Stain with hematoxylin solution I for 14 seconds, then stain with hematoxylin solution II for 1 minute, and wash with water for 5 minutes.

[0238] vi. Differentiate in differentiation solution for 3-5 seconds, then wash with water for 1 minute;

[0239] vii. Treat with blueing solution for 7 seconds, then rinse with water for 20 seconds;

[0240] viii. Immerse in 95% ethanol for 1 min, then stain with eosin solution for 14 s;

[0241] ix. Apply anhydrous ethanol I and II for 2 min each, xylene I and xylene II for 2 min to clear, and then mount with neutral resin.

[0242] As can be seen from Figures 11A and 11D, there was no significant difference in brown adipose tissue and visceral fat between the knockout mice and the control mice.

[0243] CD8 obtained in 10.2 + T-cell Abhd5 knockout mice were anesthetized and sacrificed, and kidney tissue was collected for Sirius red staining. The steps for Sirius red staining are as follows:

[0244] i. After mice were anesthetized and euthanized, their kidneys were fixed in 4% paraformaldehyde solution and transferred to 10% neutral formalin solution before dehydration;

[0245] ii. Dehydrate sequentially using 70% ethanol, 85% ethanol, 95% ethanol I, 95% ethanol II, anhydrous ethanol I, anhydrous ethanol II, xylene I, xylene II, xylene III, paraffin I, paraffin II, and paraffin III;

[0246] iii. Embedding and sectioning;

[0247] iv. Xylene I-Xylene II-Anhydrous Ethanol I-Anhydrous Ethanol II-95% Ethanol-85% Ethanol-75% Ethanol Rehydration;

[0248] v. Add iron hematoxylin staining solution, stain for 8 minutes, and wash with distilled water for 20 seconds to remove excess staining solution;

[0249] vi. Stain with Sirius red staining solution for 20 minutes;

[0250] vii. Rinse with running water to remove surface staining solution;

[0251] viii. 75% ethanol - 85% ethanol - 95% ethanol - anhydrous ethanol I - anhydrous ethanol II - xylene I - xylene II are sequentially dehydrated and cleared, then sealed with neutral resin.

[0252] As shown in Figure 11B, the knockout mice showed a reduction in collagen fibers (red) in their kidneys compared to the control mice, indicating that the knockout mice did not exhibit significant changes such as aging in their kidneys.

[0253] 10.3 Frozen sections of brown adipose tissue from control and knockout mice were obtained and stained for cellular senescence β-galactosidase staining. The method for cellular senescence β-galactosidase staining is as follows:

[0254] i. Frozen sections are thawed at room temperature for 10 minutes, and tissues are circled with a histochemical pen;

[0255] ii. Add an appropriate amount of β-galactosidase staining fixative to the tissue and fix it at room temperature for 20 min;

[0256] iii. Wash with PBS three times, 5 minutes each time;

[0257] iv. Place the sections in a humidified chamber, add an appropriate amount of β-galactosidase staining working solution, and incubate overnight at 37°C in a CO2-free incubator;

[0258] v. After tissue staining, remove the staining solution, rinse twice with PBS, rinse twice with pure water, add nuclear solid red staining solution for 3 min, and rinse three times with water.

[0259] vi. Dehydrate twice with anhydrous ethanol, 5 min each time, clear with xylene for 5 min, and mount with neutral resin.

[0260] The results are shown in Figure 11C, CD8 + No aging phenotype was observed in brown adipose tissue of mice after Abhd5 knockout of T cells.

[0261] 10.4 Behavioral experiments were conducted on mice, including the Y-maze and novel object recognition tests.

[0262] 10.4.1Y Maze Experiment: Includes two experiments, with an interval of more than 1 hour.

[0263] i. The first experiment was a training period. One arm was closed, and the animal was allowed to explore freely in the other two arms for 5 minutes. After the training, the animal was put back into the cage, and the animal odor was eliminated with 75% alcohol.

[0264] ii. One hour later, the second experiment (recall phase) was conducted. All arms were opened, and the animal was allowed to move freely in the three arms for 3 minutes. The time and distance traveled in each arm were recorded.

[0265] 10.4.2 New Object Substitution Recognition Experiment:

[0266] i. Habituation period: Remove the mouse from its cage and place it in the center of the open area (test chamber). Allow the mouse to explore freely for 5 minutes. After 5 minutes, remove the mouse and return it to its cage; thoroughly clean the mouse's equipment with 70% ethanol;

[0267] ii. Training period: Place two identical objects diagonally opposite each other in the test box (i.e., the northeast and southwest corners). After 24 hours of habituation, remove the mouse from its cage and place it in the center of the test box, equidistant from the two identical objects. Allow free exploration for at least 5 minutes. At the end of the experiment, remove the mouse and return it to its cage. Thoroughly clean the equipment and objects the mouse has come into contact with using 70% ethanol.

[0268] iii. Testing Period: Place one familiar object used during T1 and a new object diagonally opposite each other in the testing box. Use the same object positions for each mouse as during T1. During the selection interval from T1 to T2, remove the mouse from the cage and place it in the center of the testing box, equidistant from both objects. Allow free exploration for 5 minutes.

[0269] iv. After the experiment, the mice were removed and placed back into cages. Statistical analysis of the results was performed using the Two-Way Anova test, with P < 0.05 considered statistically significant.

[0270] The results are shown in Figure 11E, CD8 + Compared to control mice, T-cell Abhd5 knockout mice showed no statistically significant differences in the number and time of new arm entry and the number and time of new object exploration, suggesting that CD8+ knockout mice... +The brains of T-cell Abhd5 knockout mice did not exhibit obvious aging phenotypes.

[0271] 10.5 Western blot detection of CD8 + CD86 in Abhd5 knockout T-cell mice + T cells express ABHD5

[0272] Take control mice and CD8 + CD8+ cells were extracted from the spleen of mice with T-cell knockout Abhd5. + T cells were collected by centrifugation, and cell lysis buffer and protease inhibitor PMSF (1:100) were added. The cells were thoroughly pipetted and lysed on ice for 20 min. After centrifugation at 12000 rpm at 4℃ for 20 min, the supernatant was collected as the total protein lysis buffer.

[0273] ii. BCA method for protein concentration quantification (Beyotime Biotechnology Co., Ltd., catalog number: P0009):

[0274] Prepare the following standards in a 96-well plate using a 0.5 mg / ml protein standard solution:

[0275] The sample was diluted as follows:

[0276] Add 20 μL of protein standard and sample to each well, along with 200 μL of BCA reaction solution (reagent A: reagent B = 50:1). Incubate at 60℃ without CO2 for 30 min, then measure the absorbance at 562 nm using a microplate reader. Plot a standard curve based on the measured absorbance and the corresponding concentrations shown in the table above, and calculate the protein concentrations of the four samples. Load 25 μg of protein for subsequent experiments.

[0277] ii. Electrophoretic separation of proteins:

[0278] The electrophoresis buffer is prepared as follows:

[0279] Take the SDS-PAGE pre-cast adhesive (Beyotime Corporation, P0057A), and add the sample into the corresponding well in sequence. Each sample can be set with 2-3 duplicate wells. Adjust the voltage to 150V and finish after 60 minutes.

[0280] iii. Transfer

[0281] The transfer buffer is prepared as follows:

[0282] After preparation, place it at 4℃ for pre-cooling.

[0283] Using a wet rotation method, place the sponge pad, filter paper, PVDF membrane, SDS-PAGE adhesive, filter paper, and sponge pad in sequence, then adjust the current to 250mA and run for 80 minutes to finish.

[0284] iv. Enclosed

[0285] Prepare the electrophoresis wash buffer TBST as follows:

[0286] After transfer, remove the PVDF membrane and place it in an antibody incubation box. Wash three times with TBST buffer, 5 min each time. After washing, discard the washing buffer and add Western blocking buffer (Beyotime Biotech, P0023B). Block on a shaker at room temperature for 1 hour with gentle shaking.

[0287] v. Antibiotic incubation

[0288] Take Western primary antibody dilution buffer (Beyotime Biotech, P0023A) and rabbit anti-ABHD5 antibody (Proteintech, 12201-1-AP), and prepare 10 mL of primary antibody (i.e., 10 μL of ABHD5 antibody + 9.99 mL of Western primary antibody dilution buffer) at a ratio of 1:1000. Add the solution to the antibody loading cassette and incubate slowly on a shaker at 4°C for 12 h.

[0289] vi. Secondary antibody incubation

[0290] The primary antibody was recovered, and the cells were washed three times with TBST buffer for 5 min each time. HRP-goat anti-rabbit secondary antibody (1:5000) was added, and the cells were slowly incubated on a shaker at room temperature for 1 h.

[0291] vii. ECL development

[0292] Mix ECL kit (Affinity, KF8003) 1:1 and develop in a developer.

[0293] The results are shown in Figure 11F, CD8 + CD8+ T-cell knockout Abhd5 mice + T cells showed a lack of ABHD5 expression, and GAPDH was used as an internal reference gene (indicating consistent protein loading), suggesting that the ABHD5 gene is absent in CD8 cells. + T cells are knocked out, i.e., CD8 + The Abhd5 T-cell knockout mouse model was successfully constructed. These results suggest that CD8... + T-cell Abhd5 knockout mice did not exhibit obvious aging phenotypes, while CD8 + T cells and CD4 + Mice with simultaneous T cell knockout of Abhd5 exhibit a significant aging phenotype.

Claims

1. A method of preparing a model of an aging animal, wherein, comprises a modification in an immune cell in the animal that reduces or eliminates the function or level of Abhd5.

2. The method of claim 1, wherein, the modification is selected from the group consisting of an agent that inhibits Abhd5 activity, an agent that degrades Abhd5, and / or a genetic tool that reduces Abhd5 levels; preferably, the genetic tool that reduces Abhd5 levels comprises RNA interference, microRNA, gene editing, or gene knockout material; preferably, the Abhd5 gene specific knockout in the immune cell is performed using Cre-LoxP technology, TALEN technology, ZFN technology, or CRISPR / Cas technology; preferably, the gene Abhd5 in the immune cell is inactivated and / or deleted; preferably, the gene Abhd5 in the immune cell comprises a homozygous disruption; Preferably, the immune cells comprise CD4 + T cells and CD8 + T cells; Preferably, the animal is CD4 + T cells and CD8 + Animals in which the gene Abhd5 in T cells is simultaneously knocked out; Preferably, the method for preparing the animal model of aging comprises the steps of: crossing CD4 / CD8-Cre mice and Abhd5 flox / flox Animals are mated and passaged to obtain CD4 + T cells and CD8 + Abhd5 simultaneously knockout animals in T cells; Preferably, the Abhd5 flox / flox The loxP site in the animal is introduced into an intron of the Abhd5 gene; Preferably, the Abhd5 flox / flox A gene sequence having a loxP site at each end of the Abhd5 gene in an animal; preferably, the Cre recombinase in the CD4 / 8-Cre mouse is placed under the control of the CD4 and CD8 gene promoters; preferably, the animal comprises a rodent; preferably, the animal comprises a pig, a rat, a mouse, a hamster, a rabbit, a pig, a cow, a deer, a sheep, a goat, a chicken, a cat, a horse, a dog, a chimpanzee, or a monkey; preferably, the animal model comprises one or more of the following phenotypes: skin lesions, hair loss, skin folds, enlarged lymph nodes and pigmentation, kyphosis, brown fat hyperemia, and reduced volume, reduced lymphocytes relative to normal group animals, reduced grip strength relative to normal group animals, significantly reduced intermyofiber mitochondria relative to normal group animals, and a high level of inflammatory and chemotactic factor secretion associated with aging.

3. An animal model of aging obtained by the method of claim 1 or 2.

4. A model of aging in an animal, wherein, the animal model has a modified gene Abhd5 in an immune cell in the animal model; preferably, the gene Abhd5 in the immune cell is inactivated and / or deleted; preferably, the gene Abhd5 in the immune cell comprises a homozygous disruption; Preferably, the immune cells comprise CD4 + T cells and CD8 + T cells; Preferably, the animal is CD4 + T cells and CD8 + Animals in which the gene Abhd5 in T cells is simultaneously knocked out; preferably, the animal comprises a rodent; preferably, the animal comprises a pig, a rat, a mouse, a hamster, a rabbit, a pig, a cow, a deer, a sheep, a goat, a chicken, a cat, a horse, a dog, a chimpanzee, or a monkey; 5. Preferably, the animal model comprises one or more of the following phenotypes: skin lesions, hair loss, skin folds, enlarged lymph nodes and pigmentation, kyphosis, brown fat hyperemia, and reduced volume, reduced lymphocytes relative to normal group animals, reduced grip strength relative to normal group animals, significantly reduced intermyofiber mitochondria relative to normal group animals, and a high level of inflammatory and chemotactic factor secretion associated with aging. A cell or cell line or primary cell culture or a disruption of said cell or cell line or primary cell culture or an extract thereof, wherein, from an animal produced by the method of any one of claims 1-2 or a progeny of the animal.

6. A tissue or organ or a culture of said tissue or organ or a fragmentation of said tissue or organ or an extract, wherein, from an animal produced by the method of any one of claims 1-2 or a progeny of the animal.

7. A bodily fluid of an animal or progeny of said animal, wherein, the animal is produced by the method of any one of claims 1-2.

8. An isolated cell nucleus, wherein, from an animal produced by the method of any one of claims 1-2 or a progeny of the animal.

9. Use of the animal model or its offspring obtained by the method of any one of claims 1-2 as a model system for pharmacological, immunological, microbiological and medical research, or for the production and use of animal experimental disease models for etiological research and / or for the development of new diagnostic strategies and / or therapeutic strategies, or for the screening, validation, evaluation or investigation of anti-aging drugs, or for the study of drug efficacy.

10. A method of screening for an anti-aging drug, wherein, The method comprises: (1) administering a drug to the senescent animal model obtained by the method of claim 1 or 2, wherein the animal has symptoms of aging, and (2) determining whether the drug treats the symptoms of aging in the animal compared to the senescent animal model obtained by the method of claim 1 or 2 without the drug.

11. Use of a detection reagent for Abhd5 in the preparation of a senescence diagnostic reagent or kit. Preferably, the detection reagent detects the expression amount of the gene; Preferably, the detection reagent detects the mRNA expression amount of the gene; Preferably, the detection reagent detects the protein expression amount of the gene; Preferably, the detection sample for the Abhd5 detection reagent is CD8 + T cells and CD4 + T cells; Preferably, the detection reagent is selected from at least one of the group consisting of a fluorescent quantitative PCR dye, a fluorescent quantitative PCR primer, a fluorescent quantitative PCR probe, an antibody, an antibody functional fragment, or a conjugated antibody; Preferably, the kit is selected from at least one of the group consisting of a qPCR kit, an immunoblotting detection kit, an immunochromatographic detection kit, a flow cytometry analysis kit, an immunohistochemical detection kit, an ELISA kit, or an electrochemiluminescence detection kit.

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