Preparation method for and application of metabolic syndrome animal model

Abstract

The present invention relates to the field of biomedicine, and relates to a preparation method for and an application of a metabolic syndrome animal model. Provided are a preparation method for and an application of a metabolic syndrome animal model. The method comprises: modifying a gene in an immune cell in an animal, wherein the modification reduces or eliminates the function or level of Abhd5 in a Treg cell.

Description

A method for preparing an animal model of metabolic syndrome and its application Technical Field

[0001] This disclosure pertains to the field of biomedicine and relates to a method for preparing an animal model of metabolic syndrome and its application. Background Technology

[0002] Metabolic syndrome (MetS) is a complex state of metabolic abnormalities including abdominal obesity, dyslipidemia, hyperglycemia, and hypertension, which can increase the risk of type 2 diabetes, cardiovascular disease, and atherosclerosis. Insulin resistance is a common pathophysiological basis for many metabolic-related diseases and the development of adult MetS. 20%-25% of adults worldwide have MetS, and these individuals have a five times higher risk of developing type 2 diabetes than those without the condition. In 2009, the International Diabetes Federation (IDF) and the American Heart Association / National Heart, Lung, and Blood Institute (AHA / NHLBI) defined MetS as any three of the following five indicators: abdominal obesity, hypertriglyceridemia (TG), hyperlower density lipoprotein cholesterol (HDL-C), hypertension, and fasting hyperglycemia. Establishing MetS-related models is of great significance for in-depth research on the impact of metabolic syndrome on cardiovascular and cerebrovascular diseases and for exploring drug targets for prevention and treatment. Currently, methods for constructing animal models of Metabolic Syndrome (MetS) can be categorized into genetic models, induced models, and genetically modified models. Induced models are further divided into food-induced models, drug-induced models, and food-drug combined-induced models, depending on the inducing substance. Among these, drug-induced models involve injecting specific drugs to construct MetS animal models (Application No. 201710912395.6, China, March 9, 2018). Gene editing methods are also used to induce metabolic syndrome models. While research on metabolic syndrome utilizes methods such as dietary induction and gene editing to construct models, these methods still have drawbacks: 1. They cannot fully simulate all the characteristics of human metabolic syndrome; 2. The key cells and targets driving metabolic syndrome have not been identified. Summary of the Invention

[0003] In some embodiments, this disclosure provides a method for preparing an animal model of metabolic syndrome, including modifying the gene Abhd5 in immune cells of the animal, wherein the modifying substance is capable of reducing or eliminating the function or level of Abhd5.

[0004] In some embodiments, the substance is selected from substances that inhibit Abhd5 activity, substances that degrade Abhd5, and / or gene tools that reduce Abhd5 levels.

[0005] In some implementations, the gene tools for reducing Abhd5 levels include RNA interference, microRNA, gene editing, or gene knockout materials.

[0006] In some implementation schemes, Cre-LoxP technology, TALEN technology, ZFN technology, or CRISPR / Cas technology are used to specifically knock out the Abhd5 gene in immune cells.

[0007] In some implementations, the Abhd5 gene in the immune cells is inactivated and / or deleted.

[0008] In some implementations, the Abhd5 gene in the immune cells includes homozygous destruction.

[0009] In some embodiments, the method for preparing the metabolic syndrome animal model includes the step of: mixing Foxp3-Cre animals and Abhd5... flox / flox Animals were mated and passaged to obtain Treg cells with Abhd5 knockout.

[0010] In some implementations, the Abhd5 flox / flox The loxP site in animals was introduced to both ends of the DNA strand of the Abhd5 gene.

[0011] In some implementations, the Abhd5 flox / flox The Abhd5 gene in animals contains a loxP site at each end of its gene sequence.

[0012] In some implementations, the Cre recombinase in the Foxp3-Cre mouse is placed under the regulation of the Abhd5 gene promoter.

[0013] In some implementations, the animals include rodents.

[0014] In some implementations, the animals include pigs, rats, mice, hamsters, rabbits, pigs, cattle, deer, sheep, goats, chicks, cats, horses, dogs, orangutans, or monkeys.

[0015] In some implementations, the animal model includes one or more of the following phenotypes: obesity, fatty liver, insulin resistance, diabetic nephropathy, osteoporosis, male characteristic degeneration, extensive vacuolation of seminiferous tubules in males, extensive lipid droplet deposition in the interstitial tissue of the testes in males, reduced sperm count in males, and decreased sperm motility in males.

[0016] In some implementations, the metabolic syndrome includes one or more of obesity, atherosclerosis, and diabetes.

[0017] In some implementations, the obesity includes at least one of induced obesity, aging-related obesity, leptin-deficient obesity, and Fas-deficient obesity.

[0018] In some implementations, the leptin-deficient obesity is selected from childhood obesity.

[0019] In some implementations, the diabetes is selected from type 2 diabetes.

[0020] In some implementations, the animals include pigs, rats, mice, hamsters, rabbits, pigs, cattle, deer, sheep, goats, chicks, cats, horses, dogs, orangutans, or monkeys.

[0021] In some implementations, the immune cells include Treg cells.

[0022] In some embodiments, the present invention provides an animal model of metabolic syndrome obtained by the method described. In some embodiments, the present invention provides an animal model of metabolic syndrome in which the in vivo immune cells of the animal model have a modified gene Abhd5, the function or level of the gene Abhd5 in the in vivo immune cells being 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 destruction. In some embodiments, the immune cells comprise Treg cells. In some embodiments, the animal is a Treg cell Abhd5 knockout animal.

[0023] 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: obesity, fatty liver, insulin resistance, diabetic nephropathy, osteoporosis, male characteristic degeneration, extensive vacuolation of seminiferous tubules in males, extensive lipid droplet deposition in the testicular interstitium in males, reduced sperm count in males, and decreased sperm motility in males. In some embodiments, the metabolic syndrome includes one or more of obesity, atherosclerosis, and diabetes. In some embodiments, the obesity includes at least one of induced obesity, aging-related obesity, leptin-deficient obesity, and Fas-deficient obesity. In some embodiments, the leptin-deficient obesity is selected from childhood obesity. In some embodiments, the diabetes is selected from type 2 diabetes.

[0024] In some implementations, the animal is a Treg cell Abhd5 knockout animal.

[0025] 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 a descendant of said animal.

[0026] 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.

[0027] In some embodiments, this disclosure provides bodily fluids of an animal or its offspring, the animal being prepared by the method.

[0028] 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.

[0029] In some embodiments, this disclosure provides for the use of animal models or their progeny derived from the methods described in the context of pharmacological, immunological, microbiological and medical research, or in the production and use of experimental animal disease models for pathogen research and / or for the development of new diagnostic and / or therapeutic strategies, or in the screening, validation, evaluation or research of drugs for the treatment of lipid metabolism syndromes, and in pharmacodynamic studies.

[0030] In some implementations, the metabolic syndrome includes one or more of obesity, atherosclerosis, and diabetes.

[0031] In some implementations, the obesity includes at least one of induced obesity, aging-related obesity, leptin-deficient obesity, or Fas-deficient obesity.

[0032] In some implementations, the leptin-deficient obesity is selected from childhood obesity.

[0033] In some implementations, the diabetes is selected from type 2 diabetes.

[0034] In some implementation schemes, this disclosure provides a reliable method for preparing an animal model of metabolic syndrome, which provides an important model for the pathogenesis of related metabolic syndrome diseases and is an important tool for future drug development, screening, and exploration of effective treatment methods.

[0035] In some embodiments, the present invention provides a method for screening drugs for treating metabolic syndrome, wherein the method includes:

[0036] a) Administering a drug to an animal model of metabolic syndrome obtained by the method, wherein the animal exhibits symptoms of metabolic syndrome, and,

[0037] b) Determine whether the drug treats the metabolic syndrome symptoms in the animal compared to an animal model of metabolic syndrome obtained by the method without medication.

[0038] In some implementations, this disclosure provides the use of Abhd5 detection reagents in the preparation of diagnostic reagents or kits for lipid metabolism syndrome.

[0039] In some implementations, the detection reagent detects the expression level of the Abhd5 gene.

[0040] In some implementations, the detection reagent detects the mRNA expression level of Abhd5.

[0041] In some implementations, the detection reagent detects the protein expression level of Abhd5.

[0042] In some implementations, the test sample for the detection reagent includes Treg cells.

[0043] In some embodiments, the detection reagent is selected from at least one of the following groups: quantitative PCR dyes, quantitative PCR primers, quantitative PCR probes, antibodies, functional fragments of antibodies, or conjugated antibodies.

[0044] In some implementations, the kit is selected from at least one group consisting of qPCR kits, immunoblotting kits, immunochromatographic kits, flow cytometry kits, immunohistochemistry kits, ELISA kits, or electrochemiluminescence kits. Attached Figure Description

[0045] Figure 1 shows the results of Western blot analysis of ABHD5 expression in Treg cells of mice with Abhd5 knockout Treg cells.

[0046] Figure 2 shows the Abhd5 knockout induction of obesity in Treg cells. Figure 2A is a gross photograph of the mouse. Figure 2B shows the results of quantitative weight measurement of the mouse. Figure 2C shows photographs of visceral fat (row 1), beige fat in the upper limbs (row 2), and beige fat in the lower limbs (row 3). Figure 2D shows the HE staining results of brown adipose tissue (BAT) and beige adipose tissue (BeAT) in mice.

[0047] Figure 3 shows the results of Oil Red O staining of liver tissue.

[0048] Figure 4 shows the results of Masson trichrome staining of liver tissue.

[0049] Figure 5 shows that Abhd5 knockout in Treg cells induces insulin resistance. Figure 5A shows the results of the IPGTT (Insulin Perceptual Glucose Tolerance Test). Figure 5B shows the results of the ITT (Insulin Sensitivity Tolerance Test).

[0050] Figure 6 shows that Abhd5 knockout in Treg cells leads to diabetic-like kidney disease. Figure 6A shows the immunofluorescence staining results of Podocin protein. Figure 6B shows the immunofluorescence staining results of Synaptopodin protein. Figure 6C shows the immunofluorescence staining results of Nephrin protein. Figure 6D shows the immunofluorescence staining results of Desmin protein.

[0051] Figure 7 shows that Abhd5 knockout in Treg cells leads to osteoporosis. Figure 7A is a CT scan of the mouse femur. Figure 7B shows the statistical analysis results of mouse bone parameters (Tb.N and SMI). Figure 7C shows the statistical analysis results of Tb.Th (mean trabecular thickness), Tb.Sp (trabecular separation), and BV / TV% (bone volume fraction).

[0052] Figure 8 shows that Treg lipid metabolism instability leads to the degeneration of male characteristics in mice. Figure 8A is a photograph of the mouse face taken while the mouse is awake. Figure 8B is a photograph of the mouse prepuce gland. Figure 8C shows the HE staining results of the mouse prepuce gland. Figure 8D shows the Masson trichrome staining results.

[0053] Figure 9 shows the HE staining results of mouse testes (left image is observed under a 20x microscope, right image is observed under a 40x microscope).

[0054] Figure 10 shows the results of Oil Red O staining of mouse testes (left image is observed under a 4x microscope, right image is observed under a 10x microscope).

[0055] Figure 11 shows the results of sperm count and motility testing in mice (NP: all non-forward movements, such as swimming in small circles, where tail movement cannot drive head movement, or only tail wagging can be observed. PR: sperm actively move in a straight line or along a large circle, regardless of their speed. IM: no movement).

[0056] Figure 12 shows that the Cre recombinase (Cyclization Recombination Enzyme), encoded by the Cre gene of E. coli phage P1, is a 38kD protein composed of 343 amino acids. It not only possesses catalytic activity but also, similar to restriction enzymes, can specifically recognize the loxP site, thereby recombinating or deleting genes between LoxP segments. The LoxP (locus of X-over P1) site is 34 bp long, consisting of two 13 bp inverted repeat sequences and an 8 bp spacer region. The inverted repeat sequences are the specific recognition site for the Cre recombinase, while the spacer region determines the orientation of the loxP site.

[0057] Figure 13 shows the detection of the aging phenotype in Treg cell Abhd5 knockout mice. Figure 13A shows gross photographs of Treg cell Abhd5 knockout mice and control mice. Figure 13B shows the immunofluorescence staining results of P21, a classic aging marker, in Treg cell knockout mice and control mice. Figure 13C shows the immunofluorescence staining results of P16, a classic aging marker, in Treg cell knockout mice and control mice. Detailed Implementation

[0058] The following specific embodiments further illustrate the technical solutions of this disclosure. These specific embodiments do not represent a limitation on the scope of protection of this disclosure. Any non-essential modifications and adjustments made by others based on the concept of this disclosure still fall within the scope of protection of this disclosure.

[0059] Unless otherwise specified, the practices disclosed herein will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to this disclosure. Otherwise, certain terms used herein have the meanings set forth in the specification.

[0061] In the event of any conflict between any definition set forth below and any reference incorporated herein by reference, the definition set forth herein shall prevail.

[0062] The terms “comprising,” “having,” “containing,” and “including,” as used herein, and their grammatical equivalents, are synonymous and open-ended, meaning that one or more items following any of these terms do not imply an exhaustive list of those items or items, nor do they imply limitation to the listed items or items. For example, an article “comprising” components A, B, and C may consist of components A, B, and C (i.e., containing only components A, B, and C), or may contain not only components A, B, and C, but may also include one or more other components. Therefore, it is intended and understood that “comprising” and its similar forms and their grammatical equivalents encompass the disclosure of embodiments “consisting substantially of” or “composed of.”

[0063] As used herein, the singular forms “a,” “or,” and “the” included in the appended claims contain plural references unless the context clearly indicates otherwise.

[0064] It should be understood that this disclosure is not limited to the specific methods, schemes, and reagents described herein, and these methods are subject to change. The terminology used herein is for the purpose of describing specific embodiments or aspects only and is not intended to limit the scope of this disclosure.

[0065] The experimental protocol disclosed herein has been reviewed by the Laboratory Animal Management and Use Committee of Jin Feng Laboratory (IACUC-JFLAB) and complies with animal protection, animal welfare and ethical principles, as well as relevant national regulations on laboratory animal welfare and ethics (ethics number: IACUC-JFLAB-2022-001).

[0066] Cre recombinase (Cyclization Recombination Enzyme) is encoded by the Cre gene of E. coli phage P1 and is a 38kD protein composed of 343 amino acids. It not only possesses catalytic activity but also, similar to restriction enzymes, can specifically recognize the loxP site, thereby recombinating or deleting genes between LoxP fragments (Figure 12).

[0067] The LoxP (locus of X-overP1) site is 34 bp long, consisting of two 13 bp inverted repeat sequences and an 8 bp spacer region. The inverted repeat sequences are the specific recognition sites for Cre recombinase, while the spacer region determines the orientation of the LoxP site (Figure 12).

[0068] The Cre / loxP system exhibits several induced recombination mechanisms, based on the interaction between Cre recombinase and the loxP site. When a loxP site and Cre recombinase are present in the gene, Cre recombinase binds to the inverted repeat regions flanking the loxP site, forming a dimer. This dimer binds to dimers at other loxP sites, forming a tetramer. Subsequently, the DNA sequence between the loxP sites is cleaved by Cre recombinase, and the cleavage is reconnected by DNA ligase. The outcome of DNA recombination depends primarily on the orientation and location of the loxP site.

[0069] To achieve the knockout of a specific gene in vivo under specific conditions using the Cre / loxP system, two types of transgenic mice are required:

[0070] (1) The first type of mouse, Foxp3-Cre mouse, was donated by Professor Ye Lilin of the Department of Immunology, Basic Medical College, Army Medical University.

[0071] (2) The second type of mouse, the Flox mouse (i.e., a mouse containing the loxP locus gene sequence), is constructed by in vitro creating a gene sequence with a loxP locus at each end of the ABHD5 gene. This constructed gene sequence is then transferred into mouse embryonic stem cells, allowing homologous recombination to replace the original gene sequence in the cell's genome. These treated mouse embryonic stem cells are then re-implanted into the uterus of pseudopregnant mice, allowing them to develop into a complete embryo, ultimately becoming a transgenic mouse, the Flox mouse (i.e., the Abhd5). flox / flox (Mouse). In this transgenic mouse, the loxP site is introduced at both ends of the ABHD5 gene sequence, which theoretically should not affect the function of the corresponding gene. Therefore, under normal circumstances, the mouse has a normal phenotype.

[0072] Finally, the two mouse types, Foxp3-Cre and Flox, were mated. The offspring mice that produced both of the above genotypes would lack the ABHD5 gene in Treg cells driven by the Foxp3 transcription factor.

[0073] Foxp3-Cre mice were donated by Professor Ye Lilin of the Department of Immunology, Basic Medical College, Army Medical University.

[0074] Flox mice are derived from Cyagen (Suzhou) Biotechnology Co., Ltd.

[0075] In the following examples, each characteristic was expressed in both male and female mice. No sex difference was observed between male and female mice. Furthermore, both young and old mice showed related characteristics, and no difference was observed between young and old mice.

[0076] Example 1: A method for constructing an animal model of metabolic syndrome (Treg cell Abhd5 knockout mouse)

[0077] 1.1 Construction Method

[0078] Mice expressing Foxp3-Cre were mated with Flox mice to obtain the F1 generation, and the F2 generation was obtained by passing them through the first generation. Each first-generation mouse (i.e., a mouse with the target gene inserted after microinjection of a fertilized egg) needs to be passed on independently.

[0079] The F1 mice born were identified, and their offspring had a 50% chance of carrying the integrated target gene; the obtained F1 positive mice could be used for experiments and continued to be propagated (or bred with Foxp3-Cre mice).

[0080] In some implementation schemes, if homozygotes are required, positive F1 mice from the same source may be mated with siblings, and the resulting F2 generation mice will have a 25% probability of being homozygous. Mice with Treg cell Abhd5 knockout will be screened out, established as stable lineages, and the passage details and pedigree will be recorded.

[0081] 1.2 Western blot detection of ABHD5 expression in Treg cells of Abhd5 knockout mice

[0082] Treg cells were extracted from the spleen of control mice and mice with Abhd5 knockout of Treg cells. The 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 lysate.

[0083] i. BCA method for protein concentration quantification (Beyotime Biotechnology Co., Ltd., catalog number: P0009):

[0084] Prepare the following standards in a 96-well plate using a 0.5 mg / ml protein standard solution:

[0085] The sample was diluted as follows:

[0086] 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 concentration of the four samples. Load 20 μg of protein for subsequent experiments.

[0087] ii. Electrophoretic separation of proteins:

[0088] The electrophoresis buffer is prepared as follows:

[0089] 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.

[0090] iii. Transfer membrane

[0091] The transfer buffer is prepared as follows:

[0092] After preparation, place it at 4℃ for pre-cooling.

[0093] 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.

[0094] iv. Enclosed

[0095] Prepare the electrophoresis wash buffer TBST as follows:

[0096] 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.

[0097] v. Antibiotic incubation

[0098] 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.

[0099] vi. Secondary antibody incubation

[0100] 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.

[0101] vii. ECL development

[0102] Mix ECL kit (Affinity, KF8003) 1:1 and develop in a developer.

[0103] As shown in Figure 1, the Treg cells of the Abhd5 knockout mice lacked ABHD5 expression, and with Tubulin as an internal reference gene (indicating consistent protein loading), it is suggested that the ABHD5 gene was knocked out in the Treg cells of the Abhd5 knockout mouse group, indicating that the Abhd5 knockout mouse was successfully constructed.

[0104] Example 2: Abhd5 knockout in Treg cells induces obesity

[0105] 1.1 Experimental Procedure

[0106] Treg cell Abhd5 knockout mice (Abhd5 KO group) and normal control mice (Control) were anesthetized and tissue samples were collected for HE staining:

[0107] (1) Brown fat and beige fat were fixed in 4% paraformaldehyde solution and transferred to 10% neutral formalin solution before dehydration. They were then dehydrated in sequence by 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.

[0108] (2) Embedding and sectioning;

[0109] (3) Xylene I-Xylene II-Anhydrous Ethanol I-Anhydrous Ethanol II-95% Ethanol-85% Ethanol-75% Ethanol Rehydration;

[0110] (4) Stain with hematoxylin I for 14 seconds, stain with hematoxylin II for 1 minute, and wash with water for 5 minutes.

[0111] (5) Differentiate in the differentiation solution for 3-5 seconds, then wash with water for 1 minute;

[0112] (6) Treat with blueing solution for 7 seconds, then rinse with water for 20 seconds;

[0113] (7) Immerse in 95% ethanol for 1 min, stain with eosin for 14 s, 2 min each of anhydrous ethanol I and II, clear with xylene I and xylene II for 2 min, and mount with neutral resin.

[0114] 1.2 Experimental Results

[0115] 1.1.1 Gross images of Treg cell Abhd5 knockout mice and normal control mice were taken in a prone position after anesthesia, and their body weight was measured. The results are shown in Figures 2A and 2B. The results show that, in gross images, the knockout group mice were significantly larger than the control group mice.

[0116] 1.1.2 Treg cell Abhd5 knockout mice (Abhd5 KO group) and normal control mice (Control group) were sacrificed after anesthesia, and visceral fat and beige fat from the upper and lower limbs were collected for photographic recording. The results are shown in Figure 2C. After anesthetizing the mice, visceral fat, beige fat from the upper and lower limbs were collected and photographed, all of which showed that the knockout group mice were significantly more obese than the control group mice.

[0117] 1.1.3 HE staining results are shown in Figure 2D. The results show that HE staining of brown and beige adipose tissue in normal control mice (Control) and knockout mice (Abhd5 KO group) revealed whitening of adipose tissue in knockout mice.

[0118] The Control group mice refer to normal control mice that were not knocked out.

[0119] Example 3: Abhd5 knockout in Treg cells induces fatty liver

[0120] 2.1 Experimental Procedure

[0121] 2.1.1 Oil Red O Staining Experiment

[0122] Treg cell Abhd5 knockout mice were anesthetized and sacrificed, and samples were collected for Oil Red O staining experiments. The experimental procedures are as follows:

[0123] (1) The liver was prepared into 8μm frozen sections and stored at -20℃ for later use.

[0124] (2) Remove the frozen sections and place them on a slide rack to warm up for 10 minutes.

[0125] (3) Staining was performed using the Beyotime Modified Oil Red O Staining Kit (C0158S).

[0126] (4) Add staining washing solution to cover the cells for 20 seconds.

[0127] (5) Remove the staining washing solution, add Oil Red O staining working solution, and stain for 20 minutes.

[0128] (6) Remove the Oil Red O staining working solution, add staining washing buffer, and let stand for 30 seconds. Remove the staining washing buffer and wash with PBS for 20 seconds.

[0129] (7) Hematoxylin staining solution for cell nucleus counterstaining.

[0130] (8) Wash with PBS and take pictures under a microscope.

[0131] 2.1.2 Masson's Trichrome Staining Experiment

[0132] Treg cell Abhd5 knockout mice and normal control mice (Control group) were sacrificed after anesthesia, and tissue samples were collected for Masson's trichrome staining experiment. The experimental procedure is as follows:

[0133] (1) Preparation of liver sections: Dewaxing paraffin sections to water.

[0134] (2) Use Seville Biomass Trichrome Staining Solution (G1006-20ML) to soak the sections in Masson A solution at room temperature overnight.

[0135] (3) Soak the sections in Masson A solution and incubate in a 65°C oven for 30 minutes. Rinse with tap water for 30 seconds until the yellow color on the tissue fades. At the same time, preheat Masson D solution and Masson F solution in a 65°C oven.

[0136] (4) Mix equal volumes of Masson B solution and Masson C solution, immerse the slices in the mixture for 1 minute, and rinse briefly with running water.

[0137] (5) The slices were differentiated with 1% hydrochloric acid alcohol for 1 minute until the cell nuclei turned gray-black and the background was almost colorless.

[0138] (6) Rinse briefly with tap water, drain excess water from the slices, and immerse the slices in Masson D solution for 6 minutes. Rinse with running tap water for 20 seconds.

[0139] (7) Slice the slices and drain off the water slightly. Soak them in Masson E solution for about 1 minute.

[0140] (8) After slightly draining the Masson E solution from the section, directly immerse it in Masson F solution for staining for 30 seconds.

[0141] (9) The slices were rinsed and differentiated in three consecutive tanks of 1% glacial acetic acid aqueous solution for 7 seconds each.

[0142] (10) The sections were dehydrated in three consecutive cylinders of anhydrous ethanol. After clearing with xylene for 5 minutes, they were mounted with neutral resin.

[0143] 2.2 Experimental Results

[0144] The results are shown in Figure 3. The Control group showed only a small amount of sporadic Oil Red O staining, indicating almost no lipid accumulation. The ABHD5-KO group showed abundant Oil Red O staining, indicating significant lipid deposition in the liver tissue.

[0145] The results are shown in Figure 4. In the Control group, hepatocytes were regularly distributed (black: nuclei; red: cytoplasm), with a small amount of fibrous tissue passing between the hepatocytes (fiber: blue). In the ABHD5-KO group, hepatocytes were randomly distributed, with a large amount of fibrous tissue proliferation.

[0146] The Control group refers to the test results obtained from normal mice that have never been knocked out.

[0147] Example 4: Abhd5 knockout in Treg cells induces insulin resistance

[0148] 3.1 Experimental Procedure

[0149] 3.1.1 IPGTT Experiment

[0150] Treg cell Abhd5 knockout mice (Abhd5 KO group) and normal control mice (Control group) were fasted for 14 hours but allowed free access to water. They were then injected intraperitoneally with 1 g / kg 20% ​​glucose solution. Blood glucose levels were measured by tail vein sampling at 0, 15, 30, 60, and 120 minutes and recorded as curves. Two-way Anova test was used for analysis, and P < 0.05 was considered statistically significant.

[0151] 3.1.2 ITT Experiment

[0152] Treg cell Abhd5 knockout mice (Abhd5 KO group) and normal control mice (Control group) were fasted for 4 hours but allowed free access to water. They were injected intraperitoneally with 1 U / kg of insulin, and blood glucose levels were measured by tail vein sampling at 0, 15, 30, 60 and 120 minutes.

[0153] 3.2 Experimental Results

[0154] As shown in Figures 5A-5B, the glucose tolerance test (IPGTT) and insulin sensitivity test (ITT) showed that the blood glucose level in mice with Abhd5 knockout in Treg cells rose rapidly and fell slowly, indicating that mice with Abhd5 knockout in Treg cells developed insulin resistance.

[0155] Example 5: Abhd5 knockout in Treg cells leads to diabetic nephropathy.

[0156] 4.1 Experimental Procedure

[0157] Mice were anesthetized and sacrificed. Frozen sections of kidney tissue were prepared and subjected to immunofluorescence staining. The experimental steps are as follows:

[0158] (1) Take out frozen sections of kidney tissue, wash with PBS 3 times, 5 min each time.

[0159] (2) After permeation with 0.3% Triton X-100 at room temperature for 30 min, wash with PBS 3 times for 5 min each time.

[0160] (3) Block with goat serum for 30 minutes.

[0161] (4) Prepare a primary antibody working solution with PBS solution at a ratio of 1:200, completely cover the slide, place it in a slide incubation box, and incubate overnight at 4°C in the dark.

[0162] (5) The next day, take out the slide incubation box, warm it to room temperature for 30 minutes, and wash it with PBS 3 times for 5 minutes each time.

[0163] (6) Select a secondary antibody of appropriate color according to the source of the primary antibody, prepare the working solution of the secondary antibody with PBS solution at 1:100, incubate at room temperature in the dark for 2 hours, and wash with PBS 3 times, 5 minutes each time.

[0164] (7) DAPI nuclear staining solution was dropped onto the slide and incubated at room temperature in the dark for 15 min. Then, the slide was washed with PBS 3 times for 5 min each time.

[0165] (8) Add anti-fluorescence quenching mounting medium to the slides for mounting, and store in a slide incubator at 4°C.

[0166] (9) Observe the results and acquire images using laser confocal microscopy.

[0167] 4.2 Experimental Results

[0168] 4.2.1 As shown in Figure 6A, Podocin is a podocyte-associated protein, also known as podocyte protein, and is considered a scaffold protein required to maintain or regulate the integrity of the slit membrane structure, which is crucial for the function of the renal filtration barrier. Compared with normal control mice (Control group), the expression of Podocin in the kidneys of knockout mice (ABHD5(- / -Foxp3-Cre) group was significantly reduced, indicating that the intercellular space of podocytes was enlarged and the filtration function was severely impaired.

[0169] 4.2.2 As shown in Figure 6B, Synaptopodin, an actin-binding protein also known as synaptopodin, is present in podocytes. It can induce stress fibers, maintain actin integrity, and regulate podocyte migration by competitively blocking RhoA ubiquitination mediated by the Ras superfamily RhoA signaling enzyme (Smurf1). Compared with normal control mice, Figure 6B shows that Synaptopodin expression in the kidneys of knockout mice was significantly downregulated, suggesting a disruption of the podocyte cytoskeleton and abnormal glomerular filtration function.

[0170] 4.2.3 As shown in Figure 6C, Nephrin protein is involved in the integrity of the glomerular filtration barrier and the regulation of kidney function. Abnormal activity and expression of Nephrin are associated with renal structural damage in hypertensive states and the pathogenesis of diabetic nephropathy. Its absence is considered one of the specific pathological features of glomerular injury. Nephrin is a major component of the glomerular epithelial cell slit septum and plays a crucial role in regulating and stabilizing the integrity of the glomerular filtration barrier and kidney function. Furthermore, reduced Nephrin expression induces podocyte apoptosis, shedding, and fusion, ultimately leading to symptoms of renal insufficiency such as proteinuria. Compared with normal control mice, the Nephrin expression in the kidneys of the knockout group mice in Figure 6C was significantly downregulated.

[0171] 4.2.4 As shown in Figure 6D: Desmin is an intermediate filament protein in the cytoskeleton and one of the markers of myogenic cells, also known as desmin. Normally, podocytes do not express it. When podocytes are damaged, the podocyte phenotype changes, and Desmin is expressed in large quantities. Studies have found a significant positive correlation between desmin expression and podocyte injury, and desmin can serve as a marker of podocyte damage. The experimental results in Figure 6D show that, compared with the normal control group mice, the knockout group showed a significant upregulation of Desmin expression in the kidneys, indicating severe podocyte damage.

[0172] In Figures 6A-6D, the ABHD5(- / -Foxp3-Cre) group refers to Treg cell Abhd5 knockout mice.

[0173] Example 6: Treg cell Abhd5 knockout leads to osteoporosis

[0174] 5.1 Experimental Procedure

[0175] Mice were euthanized after anesthesia, and the long bones were immediately fixed in 4% paraformaldehyde solution. They were then scanned using MicroCT, and the parameters were statistically analyzed. P < 0.05 was considered statistically significant.

[0176] 5.2 Experimental Results

[0177] 5.2.1 The results are shown in Figures 7A-7B. Figure 7A shows CT scans of the femurs of the control group and knockout group mice, indicating that the knockout group mice had significant osteoporosis.

[0178] 5.2.2 Figure 7B shows that statistical analysis of mouse bone parameters revealed that the knockout group mice had significantly reduced Tb.N (number of trabeculae) and significantly increased SMI (structural pattern index), indicating that the trabeculae were transforming into rod-like structures, suggesting that the knockout mice developed osteoporosis.

[0179] 5.2.3 Figure 7C shows that statistical analysis of Tb.Th (mean trabecular thickness), Tb.Sp (trabecular separation), and BV / TV% (bone volume fraction) all indicate that knockout mice have a tendency to osteoporosis.

[0180] Example 7: Treg lipid metabolism instability leads to the degradation of male characteristics in mice. 6.1 Experimental procedures

[0181] 6.1.1 Samples were collected from mice after euthanasia by cervical dislocation, and the prepuce glands of mice were stained with hematoxylin and eosin (HE) for staining.

[0182] (1) The prepuce glands were fixed in 4% paraformaldehyde solution for 24 hours. Before dehydration, they were transferred to 10% neutral formalin solution and dehydrated in sequence: 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.

[0183] (2) Embedding and sectioning;

[0184] (3) Xylene I-Xylene II-Anhydrous Ethanol I-Anhydrous Ethanol II-95% Ethanol-85% Ethanol-75% Ethanol Rehydration;

[0185] (4) Stain with hematoxylin I for 14 seconds, stain with hematoxylin II for 1 minute, and wash with water for 5 minutes.

[0186] (5) Differentiate in the differentiation solution for 3-5 seconds, then wash with water for 1 minute;

[0187] (6) Treat with blueing solution for 7 seconds, then rinse with water for 20 seconds;

[0188] (7) Immerse in 95% ethanol for 1 min, stain with eosin for 14 s, 2 min each of anhydrous ethanol I and II, clear with xylene I and xylene II for 2 min, and mount with neutral resin.

[0189] 6.1.2 Masson trichrome staining experiment of mouse prepuce gland (1) Dewax the prepuce gland sections to water, immerse them in mordant solution, let them sit at room temperature overnight, and rinse with running water for 10 min;

[0190] (2) Stain with azurite blue staining solution for 2-3 minutes, then wash twice with distilled water for 10-15 seconds each time;

[0191] (3) Mayer hematoxylin staining solution was applied for 2-3 min, and then washed twice with distilled water for 10-15 s each time.

[0192] (4) Differentiate in acidic solution for a few seconds, wash with water to stop differentiation, and rinse with distilled water for 10 minutes;

[0193] (5) Stain with Pomfret and fuchsin solution for 10 min, then add distilled water twice for 10-15 s each time;

[0194] (6) Treat with phosphomolybdic acid solution for 10 min; discard the supernatant and directly add aniline blue for staining for 5 min;

[0195] (7) If the aniline blue solution is washed away with acid solution, continue to add weak acid solution to cover the slice for 2 minutes;

[0196] (8) 95% ethanol-anhydrous ethanol-xylene I-xylene II are sequentially dehydrated and transparent, and then sealed with neutral resin.

[0197] 6.2 Experimental Results

[0198] 6.2.1 The faces of mice were photographed and recorded while the mice were awake. The results are shown in Figure 8A. Compared with the normal control group, male mice with Abhd5-specific knockout of Treg cells (Abhd5 KO group) had sparse beards.

[0199] 6.2.2 Photographing and recording of mouse prepuce glands.

[0200] Mice were euthanized by cervical dislocation, and the prepuce glands were photographed and recorded. The results are shown in Figure 8B. Compared with the normal control group, the knockout group mice showed that the prepuce glands were smaller and atrophied.

[0201] 6.2.3 HE staining results are shown in Figure 8C. The knockout group mice showed reduced prepuce gland volume and atrophy.

[0202] 6.2.4 The results of Masson trichrome staining are shown in Figure 8D. Knockout mice showed a decrease in the volume and atrophy of the prepuce glands. Masson staining showed a significant increase in collagen fibers (blue), indicating a significant increase in fibrosis.

[0203] Example 8: Treg lipid metabolism instability leads to the formation of numerous vacuoles in the seminiferous tubules.

[0204] 7.1 Experimental Procedure

[0205] (1) After mice were euthanized by cervical dislocation, testes were fixed in Davidson's fixative and transferred to 10% neutral formalin solution before dehydration. The solution was then dehydrated in sequence as follows: 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.

[0206] (2) Embedding and sectioning;

[0207] (3) Xylene I-Xylene II-Anhydrous Ethanol I-Anhydrous Ethanol II-95% Ethanol-85% Ethanol-75% Ethanol Rehydration;

[0208] (4) Stain with hematoxylin I for 14 seconds, stain with hematoxylin II for 1 minute, and wash with water for 5 minutes.

[0209] (5) Differentiate in the differentiation solution for 3-5 seconds, then wash with water for 1 minute;

[0210] (6) Treat with blueing solution for 7 seconds, then rinse with water for 20 seconds;

[0211] (7) Immerse in 95% ethanol for 1 min, stain with eosin for 14 s, 2 min each of anhydrous ethanol I and II, clear with xylene I and xylene II for 2 min, and mount with neutral resin.

[0212] 7.2 Experimental Results

[0213] The experimental results are shown in Figure 9. HE staining results showed that Treg cells with Abhd5 specific knockout in male mice resulted in the formation of a large number of vacuoles in the seminiferous tubules and abnormal seminiferous tubule morphology.

[0214] Example 9: Treg lipid metabolism instability promotes the deposition of large amounts of lipid droplets in the testicular interstitium.

[0215] 8.1 Experimental Procedure

[0216] (1) Frozen sections of the testis were thawed at room temperature for 10 minutes;

[0217] (2) Add an appropriate amount of staining and washing solution to cover the cells for 20 seconds, then remove the staining and washing solution.

[0218] (3) Add an appropriate amount of Oil Red O staining working solution, stain for 10-20 minutes, and remove the Oil Red O staining working solution;

[0219] (4) Add an appropriate amount of staining and washing solution, let stand for 30 seconds, then remove the staining and washing solution and wash with PBS for 20 seconds.

[0220] (5) Add an appropriate amount of hematoxylin staining solution, stain for 6-8 minutes, wash with PBS for 1 minute, mount with aqueous mounting solution, mount with anti-fluorescence quenching mounting solution, and then observe and photograph under a microscope.

[0221] 8.2 Experimental Results

[0222] As shown in Figure 10, compared with the normal control group, Treg cells specifically knocked out a large amount of lipid deposition in the testicular interstitium of male mice.

[0223] Example 10: Treg lipid metabolism homeostasis significantly inhibited sperm count and activity in mice.

[0224] 9.1 Experimental Procedure

[0225] (1) After the mice were euthanized by dislocation of their necks, the epididymal tail was removed;

[0226] (2) Place the sperm suspension in a 1.5 mL EP tube containing 400 μL PBS preheated at 37 °C, cut the epididymis into small pieces, and incubate in a water bath at 37 °C for 15 min.

[0227] (3) After mixing the upper suspension, take 10 μL and drop it into a Makler sperm counting chamber to detect the total number of sperm and sperm motility. Repeat three times, with each replicate sample counting approximately 200 sperm.

[0228] 9.2 Experimental Results

[0229] The experimental results are shown in Figure 11. Compared with the normal control group, male mice with Abhd5-specific knockout of Treg cells showed a significant decrease in sperm count, a significant reduction in the number of progressively motile sperm, and a significant increase in the number of stationary sperm, indicating a significant decrease in sperm motility.

[0230] Example 1: Detection of aging phenotype in Treg cell Abhd5 knockout mice

[0231] Gross photographs were taken of Treg cell Abhd5 knockout mice and control mice after anesthesia. The results are shown in Figure 13A. The knockout mice did not show obvious signs of aging such as skin lesions, hair loss, and graying hair, but they did show obvious obesity.

[0232] Mice were anesthetized and sacrificed. Frozen sections of kidney tissue were prepared and subjected to immunofluorescence staining. The experimental steps are as follows:

[0233] (1) Take out frozen sections of kidney tissue, wash with PBS 3 times, 5 min each time.

[0234] (2) After permeation with 0.3% Triton X-100 at room temperature for 30 min, wash with PBS 3 times for 5 min each time.

[0235] (3) Block with goat serum for 30 minutes.

[0236] (4) Prepare a primary antibody working solution with PBS solution at a ratio of 1:200, completely cover the slide, place it in a slide incubation box, and incubate overnight at 4°C in the dark.

[0237] (5) The next day, take out the slide incubation box, warm it to room temperature for 30 minutes, and wash it with PBS 3 times for 5 minutes each time.

[0238] (6) Select a secondary antibody of appropriate color according to the source of the primary antibody, prepare the working solution of the secondary antibody with PBS solution at 1:100, incubate at room temperature in the dark for 2 hours, and wash with PBS 3 times, 5 minutes each time.

[0239] (7) DAPI nuclear staining solution was dropped onto the slide and incubated at room temperature in the dark for 15 min. Then, the slide was washed with PBS 3 times for 5 min each time.

[0240] (8) Add anti-fluorescence quenching mounting medium to the slides for mounting, and store in a slide incubator at 4°C.

[0241] (9) Observe the results and acquire images using laser confocal microscopy.

[0242] The results, as shown in Figures 13B and 13C, indicate that there were no significant differences in the classic aging markers P16 and P21 in the kidneys of mice with Abhd5 knocked out in Treg cells between the two groups.

[0243] The results show that mice with Abhd5 knocked out in Treg cells did not exhibit obvious aging phenotypes, but rather showed classic metabolic syndrome phenotypes.

Claims

1. A method of preparing an animal model of metabolic syndrome, wherein, comprises a modification in an immune cell in the animal of a gene Abhd5, the modification being capable of reducing or eliminating the function or level of Abhd5.

2. The method of claim 1, wherein, the agent 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 level; preferably, the genetic tool that reduces Abhd5 level 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 cell comprises a Treg cell; preferably, the animal is a Treg cell Abhd5 knockout animal; Preferably, the method for preparing the animal model of metabolic syndrome comprises the steps of: crossing Foxp3-Cre animals and Abhd5 flox / flox Animals are mated and passaged to obtain Treg cell Abhd5 knockout animals; Preferably, the Abhd5 flox / flox The loxP sites in the animal are introduced into the DNA strands flanking 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 Foxp3-Cre mouse is placed under the control of the Abhd5 gene promoter; 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: obesity, fatty liver, insulin resistance, diabetic-like kidney lesions, osteoporosis, regression of male characteristics, extensive vacuolation of the seminiferous tubules in male animals, extensive lipid droplet deposition in the testicular interstitium in male animals, reduced number of sperm in male animals, reduced sperm motility in male animals; preferably, the metabolic syndrome comprises one or more of the following: obesity, atherosclerosis, diabetes; preferably, the obesity comprises at least one of diet-induced obesity, age-induced obesity and leptin-deficient obesity, Fas-deficient obesity; preferably, the leptin-deficient obesity is selected from childhood obesity; preferably, the diabetes is selected from type 2 diabetes.

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

4. An animal model of metabolic syndrome, wherein, the animal model has a modified gene Abhd5 in an immune cell in vivo, the function or level of the gene Abhd5 in the immune cell in vivo being reduced or eliminated; 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 cell comprises a Treg cell; preferably, the animal is a Treg cell Abhd5 knockout animal; 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: obesity, fatty liver, insulin resistance, diabetic-like kidney lesions, osteoporosis, regression of male characteristics, extensive vacuolization of the seminiferous tubules in male animals, extensive lipid droplet deposition in the testicular interstitium in male animals, reduced sperm number in male animals, reduced sperm motility in male animals; Preferably, the metabolic syndrome comprises one or more of the following: obesity, atherosclerosis, diabetes; Preferably, the obesity comprises at least one of induced obesity, aging obesity and leptin-deficient obesity, Fas-deficient obesity; Preferably, the leptin-deficient obesity is selected from childhood obesity; Preferably, the diabetes is selected from type 2 diabetes.

5. 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 the 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 the 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 the 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 progeny 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 drugs for the treatment of lipid metabolism syndrome, or for the investigation of drug efficacy; Preferably, the metabolic syndrome comprises: one or more of the following: obesity, atherosclerosis, diabetes; Preferably, the obesity comprises at least one of induced obesity, aging obesity and leptin-deficient obesity, or Fas-deficient obesity; Preferably, the leptin-deficient obesity is selected from childhood obesity; Preferably, the diabetes is selected from type 2 diabetes.

10. A method of screening for a drug for treating metabolic syndrome, wherein, The method comprises: a) administering a drug to the animal model of metabolic syndrome obtained by the method of claim 1 or 2, wherein the animal has symptoms of metabolic syndrome, and, b) determining whether the drug treats the symptoms of metabolic syndrome in the animal as compared to the animal model of metabolic syndrome 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 diagnostic reagent or kit for lipid metabolism syndrome. Preferably, the detection reagent detects the expression amount of the Abhd5 gene; Preferably, the detection reagent detects the mRNA expression amount of the Abhd5; Preferably, the detection reagent detects the protein expression amount of the Abhd5; Preferably, the detection sample of the detection reagent comprises Treg cells; Preferably, the detection reagent is selected from at least one of the following: 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 detection reagent is selected from at least one of the following: 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, said 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 kit, an immunohistochemistry detection kit, an ELISA kit or an electrochemiluminescence detection kit. Preferably, said 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 kit, an immunohistochemistry detection kit, an ELISA kit or an electrochemiluminescence detection kit.

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