Preparation method and application of metabolic syndrome animal model

Abstract

The invention belongs to the field of biological medicine, and relates to a preparation method and application of a metabolic syndrome animal model. The invention provides a preparation method and application of a metabolic syndrome animal model, the preparation method comprises the step of modifying genes in immune cells in an animal body, and the modification reduces or eliminates the function or level of Abhd5 in Treg cells.

Description

TECHNICAL FIELD

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

[0002] Metabolic syndrome (MetS) is a cluster of metabolic abnormalities including abdominal obesity, dyslipidemia, hyperglycemia, and hypertension, which increases the risk of type 2 diabetes, cardiovascular disease, and atherosclerosis. Insulin resistance is the common pathophysiological basis of MetS and many other metabolic-related diseases. About 20-25% of adults worldwide have MetS, and these people have a 5-fold higher risk of developing type 2 diabetes than normal people. In 2009, the International Diabetes Federation (IDF) and the American Heart Association / National Heart, Lung, and Blood Institute (AHA / NHLBI) defined MetS as abdominal obesity, hypertriglyceridemia, low-density lipoprotein cholesterol (HDL-C) hyperlipidemia, hypertension, and fasting hyperglycemia. Any of the five indicators can be diagnosed as MetS if three of them are met. The establishment of a MetS-related model is of great significance for in-depth study of the effects of metabolic syndrome on cardiovascular and cerebrovascular diseases and the exploration of drug prevention and treatment targets. Current methods for constructing MetS animal models can be divided into genetic models, induced models, and genetically modified models. Induced models are further divided into food-induced models, drug-induced models, and food combined with drug-induced models, according to the inducing substances. Among them, the drug-induced model is constructed by injecting special drugs to construct a MetS animal model (Application No. 201710912395.6, China, March 9, 2018). There is also a method of gene editing to induce a metabolic syndrome model. The research on metabolic syndrome is constructed by diet induction, gene editing, etc., but still has disadvantages: 1. It cannot completely simulate all the characteristics of human metabolic syndrome; 2. The key cells and targets driving metabolic syndrome have not been found. SUMMARY

[0003] In some embodiments, the present application provides a method for preparing an animal model of metabolic syndrome, comprising modifying a gene Abhd5 in immune cells in 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 a substance that inhibits the activity of Abhd5, a substance that degrades Abhd5, and / or a genetic tool that reduces the level of Abhd5.

[0005] In some embodiments, the genetic tool that reduces the level of Abhd5 comprises RNA interference, microRNA, gene editing, or gene knockout material.

[0006] In some embodiments, the Abhd5 gene specific knockout in immune cells is performed using Cre-LoxP technology, TALEN technology, ZFN technology, or CRISPR / Cas technology.

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

[0008] In some embodiments, the gene Abhd5 in the immune cells comprises homozygous disruption.

[0009] In some embodiments, the method of preparing the animal model of metabolic syndrome comprises the step of: mating a Foxp3-Cre animal and an Abhd5 flox / flox The Treg cell Abhd5 knockout animal is obtained by mating and passing the animals.

[0010] In some embodiments, the loxP site in the Abhd5 flox / flox The loxP site is introduced into the DNA strand of the Abhd5 gene at both ends.

[0011] In some embodiments, the Abhd5 flox / flox The Abhd5 gene in the animal comprises a gene sequence with a loxP site at both ends.

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

[0013] In some embodiments, the animal comprises a rodent.

[0014] 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.

[0015] In some embodiments, the animal model comprises one or more of the following phenotypes: obesity, fatty liver, insulin resistance, diabetic kidney lesions, osteoporosis, degeneration of male characteristics, a large number of vacuoles in the male animal's spermatogenic tubules, a large number of lipid droplets deposited in the male animal's testicular stroma, a decrease in the number of sperm in the male animal, and a decrease in sperm activity in the male animal.

[0016] In some embodiments, the metabolic syndrome comprises one or more of the following: obesity, atherosclerosis, and diabetes.

[0017] In some embodiments, the obesity comprises at least one of diet-induced obesity, age-induced obesity, and leptin-deficient obesity, Fas-deficient obesity.

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

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

[0020] 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.

[0021] In some embodiments, the immune cell comprises a Treg cell.

[0022] In some embodiments, the animal is a Treg cell Abhd5 knockout animal;

[0023] In some embodiments, the present application provides a cell or a cell line or a primary cell culture or a breakage or an extract thereof from said cell or cell line or primary cell culture, which is from the animal produced by the method or the offspring of said animal.

[0024] In some embodiments, the present application provides a tissue or an organ or a culture of said tissue or organ or a breakage or an extract thereof from said tissue or organ, which is from the animal produced by the method or the offspring of said animal.

[0025] In some embodiments, the present application provides a body fluid of an animal or the offspring of said animal, which is prepared by the method.

[0026] In some embodiments, the present application provides an isolated nucleus from the animal produced by the method or the offspring of said animal.

[0027] In some embodiments, the present application provides the use of the animal model or its offspring derived from 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 drugs for treating lipid metabolism syndrome, or in the research of drug efficacy.

[0028] In some embodiments, the metabolic syndrome comprises one or more of obesity, atherosclerosis, and diabetes.

[0029] In some embodiments, the obesity comprises at least one of diet-induced obesity, aging-induced obesity, and leptin-deficient obesity, or Fas-deficient obesity.

[0030] In some embodiments, the leptin-deficient obesity is selected from childhood obesity.

[0031] In some embodiments, the diabetes is selected from type 2 diabetes.

[0032] In some embodiments, the present application 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.

[0033] In some embodiments, the present application provides use of a detection reagent for Abhd5 in the preparation of a diagnostic reagent or kit for lipid metabolism syndrome.

[0034] In some embodiments, the detection reagent detects the expression amount of the Abhd5 gene.

[0035] In some embodiments, the detection reagent detects the mRNA expression amount of the Abhd5.

[0036] In some embodiments, the detection reagent detects the protein expression amount of the Abhd5.

[0037] In some embodiments, the detection sample of the detection reagent comprises Treg cells.

[0038] In some embodiments, the detection reagent is selected from at least one 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.

[0039] In some embodiments, the kit is selected from at least one 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. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Western blot detection of Treg cell ABHD5 expression in Treg cell Abhd5 knockout mice is shown.

[0041] Figure 2 Treg cell Abhd5 knockout induces obesity. Figure 2 A is a photograph of the general appearance of the mice. Figure 2 B shows the results of mouse weight quantification. Figure 2C is the result of photographing visceral fat (row 1), upper limb beige fat (row 2) and lower limb beige fat (row 3). Figure 2 D is the HE staining result of mouse brown adipose tissue (BAT) and beige adipose tissue (BeAT).

[0042] Figure 3 The oil red O staining result of liver tissue is shown.

[0043] Figure 4 The Masson trichrome staining result of liver tissue is shown.

[0044] Figure 5 Treg cell Abhd5 knockout induces insulin resistance. Figure 5 A is the result of IPGTT in the glucose tolerance test. Figure 5 B is the result of ITT in the insulin sensitivity test.

[0045] Figure 6 Treg cell Abhd5 knockout leads to diabetic-like kidney lesions. Figure 6 A is the result of immunofluorescence staining of Podocin protein. Figure 6 B is the result of immunofluorescence staining of Synaptopodin protein. Figure 6 C is the result of immunofluorescence staining of Nephrin protein. Figure 6 D is the result of immunofluorescence staining of Desmin protein.

[0046] Figure 7 Treg cell Abhd5 knockout leads to osteoporosis. Figure 7 A is the CT scan of mouse femur. Figure 7 B is the statistical analysis result of mouse bone parameters (Tb.N and SMI). Figure 7 C is the statistical analysis result of Tb.Th (mean thickness of bone trabecula), Tb.Sp (separation degree of bone trabecula) and BV / TV% (bone volume fraction).

[0047] Figure 8 Treg lipid metabolism instability leads to degeneration of male characteristics in mice. Figure 8 A is the photograph of mouse face in a conscious state. Figure 8 B is the photograph of mouse preputial gland. Figure 8 C is the HE staining result of mouse preputial gland. Figure 8 D is the Masson trichrome staining result.

[0048] Figure 9 The HE staining result of mouse testis is shown (left, 20x magnification; right, 40x magnification).

[0049] Figure 10Oil red O staining results of mouse testis (left panel: 4x magnification, right panel: 10x magnification).

[0050] Figure 11 Sperm number and motility results of mice (NP: all forms of non- forward movement, such as swimming in small circles, tail power cannot drive the head to move, or only tail swing can be observed. PR: sperm actively move in a straight line or along a large circle, regardless of its speed. IM: no movement).

[0051] Figure 12 Cre recombinase is a 38kD protein composed of 343 amino acids encoded by the Cre gene of bacteriophage P1 of Escherichia coli. 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.

[0052] Figure 13 Detection of aging phenotype of Treg cell Abhd5 knockout mice. Figure 13 A shows the general photograph of Treg cell Abhd5 knockout mice and control mice. Figure 13 B shows the immunofluorescence staining results of the classic marker P21 related to aging in Treg cell Abhd5 knockout mice and control mice. Figure 13 C shows the immunofluorescence staining results of the classic marker P16 related to aging in Treg cell Abhd5 knockout mice and control mice. DETAILED DESCRIPTION

[0053] The technical solutions of the present application are further illustrated below by specific examples, which do not represent a limitation on the scope of protection of the present application. Some non-essential modifications and adjustments made by others according to the concept of the present application still fall within the scope of protection of the present application.

[0054] Unless specifically stated, the practice of the present application will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology.

[0055] 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 to which this application belongs. Otherwise, certain terms used herein have the meanings as set forth herein.

[0056] In the event that any definition set forth below contradicts any document incorporated herein by reference, the definition set forth below shall prevail.

[0057] The terms "comprising," "having," "including," and "containing," and other similar forms, as used herein, are intended to be equivalent in meaning to "consisting of" and are open-ended, that is, they mean including, but not limited to, one or more of the elements, components, steps, or the like that follow, and they do not exclude additional, unrecited elements, components, steps, or the like. For example, a composition "comprising" components A, B, and C can consist (i.e., contain only) of components A, B, and C, or can include not only components A, B, and C, but also one or more other components. It is therefore intended that the definition of "comprising" and other similar forms should encompass "consisting of" embodiments as well as "consisting essentially of" embodiments.

[0058] As used herein, and unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents. Unless the context clearly indicates otherwise, the term "comprising" as used herein is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0059] It is to be understood that the present disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments or aspects only and is not intended to limit the scope of the present disclosure.

[0060] The experimental protocol of the present application was reviewed by the IACUC-JFLAB (Institutional Animal Care and Use Committee) and was in accordance with the principles of animal protection, animal welfare and ethics, and in accordance with the relevant regulations of the national experimental animal welfare ethics (Ethical Number: IACUC-JFLAB-2022-001)

[0061] Cre recombinase (Cyclization Recombination Enzyme) is encoded by the Cre gene of the bacteriophage P1 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 the genes between the Loxp fragments Figure 12 ).

[0062] 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 Figure 12 ).

[0063] There are several ways to induce recombination in the Cre / loxP system, which is based on the interaction of Cre recombinase and loxP sites. When there is a loxP site in the gene and Cre recombinase is present, the Cre recombinase binds 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. Then, 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 direction and position of the loxP site.

[0064] To achieve the knockout of a specific gene under specific conditions in vivo using the Cre / loxP system, two types of transgenic mice are required:

[0065] (1) The first type of mouse, Foxp3-Cre mouse, was donated by Professor Ye Lilin of the Immunology Teaching and Research Section of the Basic Medical College of the Army Medical University;

[0066] (2) The second type of mouse, Flox mouse (i.e., mouse containing loxp site gene sequence), is constructed by constructing a gene sequence containing a loxP site at both ends of the ABHD5 gene in vitro. Then, the in vitro constructed gene sequence is transferred into mouse embryonic stem cells, allowing it to replace the original gene sequence in the cell genome through homologous recombination. The mouse 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, the 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 under normal circumstances, the phenotype of this mouse is normal.

[0067] Finally, the two types of mice, Foxp3-Cre mouse and Flox mouse, are mated to produce offspring mice that contain both genotypes, which will lack the ABHD5 gene in Treg cells driven by the Foxp3 transcription factor.

[0068] The Foxp3-Cre mouse was donated by Professor Ye Lilin of the Immunology Teaching and Research Section of the Basic Medical College of the Army Medical University.

[0069] The Flox mouse was obtained from Saiye (Suzhou) Biotechnology Co., Ltd.

[0070] In the following examples of this paper, each characterization is exhibited in both male and female mice, and there is no gender difference between male and female mice. Moreover, there are relevant characteristics in low and high age groups, and there is no difference between low and high age groups.

[0071] Example 1: Method for constructing a metabolic syndrome animal model (Treg cell Abhd5 knockout mouse)

[0072] 1.1 Method for constructing

[0073] The F1 generation is obtained by mating the Foxp3-Cre mouse with the Flox mouse, and the F2 generation is obtained by the first passage. Each founder mouse (i.e., a mouse with the target gene inserted obtained by microinjection of a fertilized egg) needs to be independently passed.

[0074] The F1 mice born are identified, and their offspring have a 50% probability of carrying the integrated target gene. The F1 positive mice obtained can be used for experiments and continued to be passed (they can also be bred with Foxp3-Cre mice).

[0075] In some embodiments, if a homozygote is needed, the same positive F1 can be used for sibling mating, and the F2 mice born have a 25% probability of being homozygotes. The Treg cell Abhd5 knockout mice are screened, and the line is stably passed, while the passage and lineage are recorded.

[0076] 1.2 Western blot detection of Treg cell ABHD5 expression in Treg cell Abhd5 knockout mice

[0077] The control mice and Treg cell Abhd5 knockout mice are taken, and the Treg cells in the spleen are extracted. The cells are collected by centrifugation, and after adding cell lysis solution and protease inhibitor PMSF (1:100), they are thoroughly blown, lysed on ice for 20 min, and then centrifuged at 4°C at 12000 rpm for 20 min. The supernatant is the total protein lysate after extraction.

[0078] i. BCA method for protein concentration quantification (Bi Yun Tian Company, Catalog No.: P0009):

[0079] In the 96-well plate, the following standard sample is prepared using 0.5 mg / ml protein standard sample solution:

[0080] 0.5 mg / mL protein standard (μL) 0 1 2 4 8 12 16 20 PBS solution (μL) 20 19 18 16 12 8 4 0 Final concentration (mg / mL) 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5

[0081] The samples are diluted as follows:

[0082] Cell group Control Treg cells Knockout Treg cells Total protein lysate 1 μL 1 μL PBS 19 μL 19 μL

[0083] The protein standard and sample are added to each well at 20 μL, and the BCA reaction solution is added at 200 μL (reagent A: reagent B = 50:

[0084] 1), 60℃ without CO2 incubator for 30 min, then use the enzyme marker to measure the absorbance at A562nm. According to the measured absorbance and the known concentration of the above table, draw a standard curve, and calculate the protein concentration of the four samples, take 20 μg of protein for loading for subsequent experiments.

[0085] ii. Electrophoretic separation of proteins:

[0086] The electrophoresis liquid is configured as follows:

[0087] Reagent name Amount Glycine 14.4g Tris 3.03g SDS 1g ddH2O to volume 1L

[0088] Take SDS-PAGE precast gel (Bi Yun Tian Company, P0057A), add the sample to the corresponding hole in turn, and set 2-3 duplicate holes for each sample. Adjust the voltage to 150V for 60 min.

[0089] iii. Transmembrane

[0090] The transmembrane liquid is configured as follows:

[0091] Reagent name Amount Glycine 14.4g Tris 3.03g Methanol 200 mL ddH2O to volume 1L

[0092] After configuration, place it at 4℃ for precooling.

[0093] Use wet transfer method, place sponge pad, filter paper, PVDF membrane, SDS-PAGE gel, filter paper, and sponge pad in turn, and adjust the current to 250mA for 80 min.

[0094] iv. Blocking

[0095] The electrophoresis washing buffer TBST is configured as follows:

[0096] Reagent name Amount Finished TBS dry powder 1 package Tween-20 2 mL ddH2O to volume 2L

[0097] After transmembrane, take out the PVDF membrane and place it in the antibody incubation box. Add TBST buffer and wash 3 times, each time for 5 min. After completion, discard the washing liquid, add Western blocking liquid (Bi Yun Tian Company, P0023B), and shake slowly on the shaking table at room temperature for 1 hour.

[0098] v. Primary antibody incubation

[0099] Take Western primary antibody diluent (Bi Yun Tian Company, P0023A) and rabbit anti-ABHD5 antibody (Proteintech, 12201-1-AP), and configure 10mL of primary antibody according to the ratio of 1:1000 (i.e. 10 μL of ABHD5 antibody + 9.99mL of Western primary antibody diluent). Add the antibody to the antibody assignment box and incubate slowly on the constant temperature shaking table at 4℃ for 12h.

[0100] vi. Secondary antibody incubation

[0101] The primary antibody was recovered, washed with TBST buffer for 3 times, 5 min each time, and a HRP-goat anti-rabbit secondary antibody (1:5000) was added, and slowly incubated at room temperature for 1 h.

[0102] vii.ECL development

[0103] After mixing the ECL kit (Affinity, KF8003) 1:1, it was developed in the developing instrument.

[0104] The results are shown in Figure 1 ABHD5 expression in Treg cells of Treg cell knockout Abhd5 mice was lost, and Tubulin was used as an internal reference gene (indicating consistent protein loading), indicating that the ABHD5 gene in the Treg cell knockout Abhd5 mouse group was knocked out in the Treg cells, i.e., the Treg cell knockout Abhd5 mouse was successfully constructed.

[0105] Example 2 Treg cell Abhd5 knockout induces obesity

[0106] 1.1, Experimental steps

[0107] After the Treg cell Abhd5 knockout mice (Abhd5 KO group) and normal control group mice (Control) were anesthetized, HE staining was performed:

[0108] (1) Brown fat, beige fat was fixed in 4% paraformaldehyde solution, transferred to 10% neutral formalin solution before dehydration, and 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 in sequence;

[0109] (2) Embedding and sectioning;

[0110] (3) Xylene I-xylene II-anhydrous ethanol I-anhydrous ethanol II-95% ethanol-85% ethanol-75% ethanol rehydration;

[0111] (4) Stained with hematoxylin staining solution I for 14 s, hematoxylin staining solution II for 1 min, and washed with water for 5 min;

[0112] (5) Differentiated for 3-5 s, and washed with water for 1 min;

[0113] (6) Return blue solution treatment for 7 s, and water washing for 20 s;

[0114] (7) 95% ethanol for 1 min, eosin staining for 14 s, anhydrous ethanol I and II for 2 min each, xylene I and xylene II for 2 min each, and neutral gum mounting.

[0115] 1.2、Experimental results

[0116] 1.1.1 After the Treg cell Abhd5 knockout mice and normal control group mice were anesthetized, they were photographed in a prone position, and their body weight was measured. The results are shown in Figure 2 A and Figure 2 B. From the results, it can be seen that the general photograph of the normal control group and the knockout group mice shows that the body shape of the knockout group mice is significantly larger than that of the control group mice.

[0117] 1.1.2 After the Treg cell Abhd5 knockout mice (Abhd5 KO group) and normal control group mice (Control group) were anesthetized, they were sacrificed, and the visceral fat and upper and lower limb beige fat were taken for photographing. The results are shown in Figure 2 C, after the mice were anesthetized, the visceral fat, upper limb beige fat, and lower limb beige fat were taken for photographing, which showed that the knockout group mice were significantly fatter than the control group mice.

[0118] 1.1.3 The HE staining results are shown in Figure 2 D, the results show that the HE staining of brown adipose tissue and beige adipose tissue of the normal control group mice (Control) and the knockout group mice (Abhd5 KO group) found that the fat tissue of the knockout group mice was whitened.

[0119] Among them, the Control group mice refer to normal control mice without knockout treatment.

[0120] Example 3 Treg cell Abhd5 knockout induced fatty liver

[0121] 2.1、Experimental steps

[0122] 2.1.1 Oil red O staining experiment

[0123] After the Treg cell Abhd5 knockout mice were anesthetized and sacrificed, the samples were taken for oil red O staining experiment, and the experimental steps were as follows:

[0124] (1) The liver was prepared as an 8 μm frozen section and stored at -20°C for standby.

[0125] (2) Take out the frozen section and put it in the section holder for 10 minutes.

[0126] (3) Use the Biyun Tian modified oil red O staining kit (C0158S) for staining.

[0127] (4) Add staining washing solution to cover the cells for 20 seconds.

[0128] (5) Suck out the staining washing solution, add oil red O staining working solution, and stain for 20 minutes.

[0129] (6) Remove the oil red O staining working solution, add the staining washing solution, and stand for 30 seconds. Remove the staining washing solution and wash with PBS for 20 seconds.

[0130] (7) Nuclei are counterstained with hematoxylin staining solution.

[0131] (8) PBS washing and microscope photographing.

[0132] 2.1.2 Masson trichrome staining experiment

[0133] After the Treg cell Abhd5 knockout mice and normal control group mice (Control group) were anesthetized and sacrificed, the materials were taken for the Masson trichrome staining experiment, and the experimental steps were as follows:

[0134] (1) Liver section preparation: paraffin section is deparaffinized to water.

[0135] (2) Use the Sevill biological Masson trichrome staining solution (G1006-20ML) to immerse the section into Masson A solution at room temperature overnight.

[0136] (3) The section is immersed in Masson A solution and incubated in a 65° oven for 30 minutes, and then washed with tap water for 30 seconds until the yellow color on the tissue disappears. At the same time, Masson D solution and Masson F solution are preheated in a 65° oven.

[0137] (4) Mix Masson B solution and Masson C solution in equal volumes, immerse the section in the mixed solution for 1 minute, and wash with running water.

[0138] (5) The section is differentiated with 1% hydrochloric acid alcohol for 1 minute until the nucleus is gray-black and the background is almost colorless.

[0139] (6) Wash with tap water, slightly drain the excess water on the section, and immerse the section in Masson D solution for 6 minutes. Wash with running water for 20 seconds.

[0140] (7) Slightly drain the water from the section, and immerse it in Masson E solution for about 1 minute.

[0141] (8) After slightly draining the Masson E solution from the section, directly immerse it in Masson F solution for 30 seconds.

[0142] (9) The section is differentiated with three consecutive cylinders of 1% aqueous acetic acid solution, 7 seconds per cylinder.

[0143] (10) The section is dehydrated with three consecutive cylinders of anhydrous ethanol, transparentized with xylene for 5 minutes, and mounted with neutral balsam.

[0144] 2.2, Experimental results

[0145] The results are as followsFigure 3 As shown, a small amount of oil red O staining was observed in the Control group, indicating that there was almost no lipid accumulation. A large amount of oil red O staining was observed in the ABHD5-KO group, indicating that a large amount of lipid was deposited in the liver tissue.

[0146] Results are shown in Figure 4 As shown, in the Control group, hepatocytes were regularly distributed (black: nucleus; red: cytoplasm), and a small amount of fibrous tissue passed between hepatocytes (fibers: blue). In the ABHD5-KO group, hepatocytes were irregularly distributed, and a large amount of fibrous tissue proliferated.

[0147] The Control group refers to the detection results obtained from normal mice that have not been subjected to knockout treatment.

[0148] Example 4 Treg cell Abhd5 knockout induces insulin resistance

[0149] 3.1, Experimental steps

[0150] 3.1.1 IPGTT experiment

[0151] Treg cell Abhd5 knockout mice (ABHD5 KO group) and normal control mice (Control group) were fasted without water for 14 hours, and 1 g / kg of 20% glucose solution was injected intraperitoneally. Blood glucose levels were measured at 0, 15, 30, 60, and 120 minutes after tail vein blood collection, and a curve was recorded. Two-way Anova test analysis was used, and P<0.05 was statistically significant.

[0152] 3.1.2 ITT experiment

[0153] Treg cell Abhd5 knockout mice (ABHD5 KO group) and normal control mice (Control group) were fasted without water for 4 hours, and 1 U / kg of insulin was injected intraperitoneally. Blood glucose levels were measured at 0, 15, 30, 60, and 120 minutes after tail vein blood collection.

[0154] 3.2, Experimental results

[0155] As shown in Figure 5 A-5B results: The glucose tolerance test IPGTT and insulin sensitivity test ITT showed that the blood glucose of mice after Treg cell Abhd5 knockout increased rapidly and decreased slowly, indicating that Treg cell Abhd5 knockout mice developed insulin resistance.

[0156] Example 5 Treg cell Abhd5 knockout leads to diabetic kidney lesions

[0157] 4.1, Experimental steps

[0158] After the mice were sacrificed after anesthesia, kidney tissue frozen sections were prepared for immunofluorescence staining, and the experimental steps were as follows:

[0159] (1) Take out the kidney tissue frozen section, wash with PBS for 3 times, 5 min each time.

[0160] (2) After 0.3% Triton X-100 permeation for 30 min, wash with PBS for 3 times, 5 min each time.

[0161] (3) Block with goat serum for 30 min.

[0162] (4) Prepare 1:200 primary antibody working solution with PBS solution, completely cover the section, put it into the section incubation box, incubate overnight at 4°C in the dark.

[0163] (5) Take out the section incubation box the next day, warm it at room temperature for 30 min, wash with PBS for 3 times, 5 min each time.

[0164] (6) According to the source of the primary antibody, choose the appropriate color of the secondary antibody, prepare the secondary antibody working solution with PBS solution at 1:100, incubate at room temperature in the dark for 2 h, wash with PBS for 3 times, 5 min each time.

[0165] (7) Drop DAPI nuclear staining solution on the section, incubate in the dark at room temperature for 15 min, then wash with PBS for 3 times, 5 min each time.

[0166] (8) Add anti-fluorescence quenching mounting medium on the section, put it into the section incubation box and store at 4°C.

[0167] (9) Observe the results under laser confocal microscope and collect images.

[0168] 4.2, Experimental results

[0169] 4.2.1 As shown in Figure 6 A, Podocin is a podocyte-associated protein, also known as podocin, which is considered to be a scaffold protein required to maintain or regulate the structural integrity of the slit diaphragm, and is essential for the function of the kidney filtration barrier. Compared with the normal control group of mice (Control group), the expression of Podocin in the knockout group of mice (ABHD5(- / -Foxp3-Cre) group) was significantly reduced, suggesting that the podocyte space was increased and the filtration function was severely impaired.

[0170] 4.2.2 As shown in Figure 6Synaptopodin, as shown in B, is an actin-binding protein in podocytes, also known as synaptopodin. It can induce stress fibers by competitively blocking ubiquitination of RhoA mediated by ubiquitin ligase (Smurf1) through regulating the RhoA signal of the Ras superfamily, maintain the integrity of actin, and regulate podocyte migration. Compared with the normal control group of mice, Figure 6 B shows that the expression of Synaptopodin in the kidney of the knockout group of mice is significantly down-regulated, indicating that the cytoskeleton of podocytes is disordered and the glomerular filtration function is abnormal.

[0171] 4.2.3 As shown in Figure 6 C, Nephrin protein is involved in the integrity of the glomerular filtration barrier and the regulation of kidney function. Abnormal activity and expression are involved in the structural damage of the kidney under the condition of hypertension and the pathogenesis of diabetic nephropathy. Its absence is considered to be one of the specific pathological features of glomerular injury, and is the main component of the slit diaphragm of glomerular epithelial cells. It plays a key role in regulating and stabilizing the integrity of the glomerular filtration barrier and the function of the kidney. In addition, the reduction of Nephrin expression can induce apoptosis, shedding and fusion of podocytes, and ultimately lead to proteinuria and other symptoms of renal insufficiency. Compared with the normal control group of mice, Figure 6 C, the expression of Nephrin in the kidney of the knockout group of mice is significantly down-regulated.

[0172] 4.2.4 As shown in Figure 6 D: Desmin is an intermediate filament protein in the cytoskeleton, and is one of the markers of muscle-derived cells, also known as desmin. Under normal circumstances, podocytes do not express. When podocytes are damaged, the podocyte phenotype is transformed, and Desmin is highly expressed. Studies have found that the expression of desmin is significantly positively correlated with podocyte injury, and desmin can be used as a marker of podocyte injury. Figure 6 The experimental results in D show that compared with the normal control group of mice, the expression of Desmin in the kidney of the knockout group is significantly up-regulated, indicating that the podocyte is severely damaged.

[0173] Figure 6 In D of A-6, the ABHD5(- / -Foxp3-Cre) group refers to Treg cell Abhd5 knockout mice.

[0174] Example 6 Treg cell Abhd5 knockout leads to osteoporosis

[0175] 5.1, experimental steps

[0176] After anesthesia, the mice were sacrificed, and the long bone of the mice was immediately placed in a 4% paraformaldehyde solution for fixation, and then scanned on a Micro CT. The parameters were statistically analyzed, and P<0.05 was considered statistically significant.

[0177] 5.2, Experimental results

[0178] 5.2.1 Results are shown in Figure A-7B. Among them, Figure 7 A-7B. Among them, Figure 7 A CT scan of the femur of control mice and knockout mice showed that the knockout mice had obvious osteoporosis.

[0179] 5.2.2 Figure 7 B shows that statistical analysis of mouse bone parameters found that Tb.N (trabecular number) of knockout mice was significantly reduced, SMI (structure model index) was significantly increased, indicating that the trabeculae were transformed into rods, indicating that the knockout mice had osteoporosis.

[0180] 5.2.3 Figure 7 C shows that statistical analysis of Tb.Th (trabecular mean thickness), Tb.Sp (trabecular separation), and BV / TV% (bone volume fraction) all indicate that knockout mice have a tendency to develop osteoporosis.

[0181] Example 7 Treg lipid metabolism instability leads to degeneration of male characteristics in mice 6.1, Experimental steps

[0182] 6.1.1 After the mice were executed by decapitation, the preputial glands were taken, and the preputial glands were HE stained:

[0183] (1) The preputial glands were fixed in 4% paraformaldehyde solution for 24h, and then transferred to 10% neutral formalin solution before dehydration. Dehydration was performed 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 in sequence;

[0184] (2) Embedding and sectioning;

[0185] (3) Rehydration in xylene I-xylene II-anhydrous ethanol I-anhydrous ethanol II-95% ethanol-85% ethanol-75% ethanol;

[0186] (4) Staining in hematoxylin staining solution I for 14s, hematoxylin staining solution II for 1min, and water washing for 5min;

[0187] (5) Differentiation in differentiation solution for 3-5s, and water washing for 1min;

[0188] (6) Treatment in blue solution for 7s, and water washing for 20s;

[0189] (7) 95% ethanol for 1min, eosin staining solution for 14s, anhydrous ethanol I and II for 2min each, xylene I and II for 2min each, and neutral gum mounting.

[0190] 6.1.2 Masson's trichrome staining experiment of mouse preputial gland (1) Dewax the preputial gland section to water, and immerse it in the medium dyeing solution for dyeing at room temperature for one night, and then wash it with running water for 10 min;

[0191] (2) Drop dyeing with azure blue staining solution for 2-3 min, and wash it with distilled water for 2 times, each for 10-15 s;

[0192] (3) Drop dyeing with Mayer hematoxylin staining solution for 2-3 min, and wash it with distilled water for 2 times, each for 10-15 s;

[0193] (4) Differentiate it with acid differentiation solution for a few seconds, stop the differentiation with water, and then wash it with distilled water for 10 min;

[0194] (5) Drop dyeing with ponceau red staining solution for 10 min, and wash it with distilled water for 2 times, each for 10-15 s;

[0195] (6) Treat it with phosphomolybdic acid solution for 10 min; pour away the solution, and then directly drop aniline blue dyeing solution for 5 min;

[0196] (7) If the aniline blue solution is washed away with an acid solution, continue to drop the weak acid solution to cover the section for 2 min;

[0197] (8) Dehydrate and transparentize it with 95% ethanol-anhydrous ethanol-xylene I-xylene II in sequence, and then seal it with neutral balsam.

[0198] 6.2, Experimental results

[0199] 6.2.1 Photograph and record the face of the mouse in a conscious state, and the results are shown in Figure 8 A, compared with the normal control group of mice, the Treg cell Abhd5 specific knockout male mice (Abhd5 KO group) have sparse mustaches.

[0200] 6.2.2 Photograph and record the preputial gland of the mouse.

[0201] After the mouse is executed by decapitation, the preputial gland is taken and photographed, and the results are shown in Figure 8 B, compared with the normal control group of mice, the knockout group of mice has smaller and atrophied preputial glands.

[0202] 6.2.3 The results of HE staining are shown in Figure 8 C, the knockout group of mice has smaller and atrophied preputial glands.

[0203] 6.2.4 The results of Masson's trichrome staining are shown in Figure 8 D, the knockout mice have smaller and atrophied preputial glands, and the Masson staining shows that the collagen fibers are obviously increased (blue), indicating that the fibrosis is obviously increased.

[0204] Example 8: Treg lipid metabolism instability leads to the formation of numerous vacuoles in the seminiferous tubules.

[0205] 7.1 Experimental Procedure

[0206] (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.

[0207] (2) Embedding and sectioning;

[0208] (3) Xylene I-Xylene II-Anhydrous Ethanol I-Anhydrous Ethanol II-95% Ethanol-85% Ethanol-75% Ethanol Rehydration;

[0209] (4) Stain with hematoxylin I for 14 seconds, stain with hematoxylin II for 1 minute, and wash with water for 5 minutes.

[0210] (5) Differentiate in the differentiation solution for 3-5 seconds, then wash with water for 1 minute;

[0211] (6) Treat with blueing solution for 7 seconds, then rinse with water for 20 seconds;

[0212] (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.

[0213] 7.2 Experimental Results

[0214] Experimental results are as follows Figure 9 As shown, HE staining results revealed that Treg cells specifically knocked out Abhd5 in male mice, resulting in the formation of numerous vacuoles and abnormal morphology of the seminiferous tubules in the testes.

[0215] Example 9: Treg lipid metabolism instability promotes the deposition of large amounts of lipid droplets in the testicular interstitium.

[0216] 8.1 Experimental Procedure

[0217] (1) Frozen sections of the testis were thawed at room temperature for 10 minutes;

[0218] (2) Add an appropriate amount of staining and washing solution to cover the cells for 20 seconds, then remove the staining and washing solution.

[0219] (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;

[0220] (4) Add an appropriate amount of dyeing washing solution, stand for 30 seconds, then remove the dyeing washing solution, wash with PBS for 20 seconds;

[0221] (5) Add an appropriate amount of hematoxylin staining solution, dye for 6-8 minutes, wash with PBS for 1 min, seal with water-based sealing solution, seal with anti-fluorescence quenching sealing solution, and then observe and take pictures under a microscope.

[0222] 8.2, Experimental results

[0223] As shown in Figure 10 compared with the normal control group, the Treg cell Abhd5 specific knockout male mice had a large amount of lipid deposition in the testicular interstitium.

[0224] Example 10 Treg lipid metabolism instability significantly inhibits sperm number and activity in mice

[0225] 9.1, Experimental steps

[0226] (1) After the mice were executed by decapitation, the epididymal tail was peeled off;

[0227] (2) Put into a 1.5 mL EP tube preheated at 37°C with 400 μL PBS, cut the epididymis into a sperm suspension, and incubate in a 37°C water bath for 15 min;

[0228] (3) Mix the upper suspension and take 10 μL to drop into a Makler sperm counting plate to detect the total number of sperm and sperm motility. Repeat three times, and count about 200 sperm per repeat sample.

[0229] 9.2, Experimental results

[0230] The experimental results are shown in Figure 11 compared with the normal control group, the Treg cell Abhd5 specific knockout male mice had a significant decrease in sperm number, a significant decrease in the number of forward-moving sperm, and a significant increase in the number of immotile sperm, indicating a significant decrease in sperm motility.

[0231] Test Example 1 Detection of Treg cell Abhd5 knockout mouse aging phenotype

[0232] The Treg cell Abhd5 knockout mice and control mice were photographed after anesthesia. As shown in Figure 13 A, the knockout mice did not show obvious signs of aging such as skin lesions, hair loss, and gray hair, but showed obvious obesity.

[0233] After the mice were anesthetized and executed, kidney tissue frozen sections were prepared and obtained, and immunofluorescence staining was performed, and the experimental steps were as follows:

[0234] (1) Take out the kidney tissue frozen section, wash with PBS for 3 times, 5 min each time.

[0235] (2) 0.3% Triton X-100 room temperature permeability 30 min, PBS wash 3 times, 5 min each time.

[0236] (3) Goat serum blocking 30 min.

[0237] (4) PBS solution 1:200 with primary antibody working solution, completely cover the slice, put into the slice incubation box, avoid light 4℃ incubation overnight.

[0238] (5) The next day, take out the slice incubation box, room temperature 30 min, PBS wash 3 times, 5 min each time.

[0239] (6) According to the source of the primary antibody, choose the appropriate color of the secondary antibody, with PBS solution 1:100 preparation of secondary antibody working solution, room temperature incubation 2h, PBS wash 3 times, 5 min each time.

[0240] (7) DAPI nuclear staining solution drop on the slice, avoid light room temperature incubation 15 min, PBS wash 3 times, 5 min each time.

[0241] (8) On the slice, drop anti-fluorescence quenching mounting medium for mounting, put into the slice incubation box 4℃ preservation.

[0242] (9) Laser confocal microscope observation results and image acquisition.

[0243] Results as Figure 13 B and Figure 13 C show that there is no significant difference between the two groups in the mouse kidney with Abhd5 knockout in Treg cells and the classic marker P16, P21 related to aging.

[0244] The above results show that the mouse with Abhd5 knockout in Treg cells does not show obvious aging phenotype, but shows classic metabolic syndrome phenotype.

Claims

1. A method for preparing an animal model of metabolic syndrome, characterized in that, This includes modifying the Abhd5 gene in the immune cells of the animal, wherein the modifying substance can reduce or eliminate the function or level of Abhd5.

2. The method as described in claim 1, characterized in that, The substance is selected from substances that inhibit Abhd5 activity, substances that degrade Abhd5, and / or gene tools that reduce Abhd5 levels. Preferably, the gene tools for reducing Abhd5 levels include RNA interference, microRNA, gene editing, or gene knockout materials; Preferably, the Abhd5 gene in immune cells is specifically knocked out using Cre-LoxP technology, TALEN technology, ZFN technology, or CRISPR / Cas technology. Preferably, the Abhd5 gene in the immune cells is inactivated and / or deleted; Preferably, the Abhd5 gene in the immune cells comprises homozygous deletion; Preferably, the immune cells include Treg cells; Preferably, the animal is an animal with Treg cells knocked out of Abhd5; Preferably, the method for preparing the metabolic syndrome animal model includes the steps of: mixing Foxp3-Cre animals and Abhd5... flox / flox Animals were mated and passaged to obtain animals with Abhd5 knockout Treg cells; Preferably, the Abhd5 flox / flox The loxP site in animals was introduced to both ends of the DNA strand of the Abhd5 gene. Preferably, the Abhd5 flox / flox The Abhd5 gene in animals contains a loxP site at each end of its gene sequence. Preferably, the Cre recombinase in the Foxp3-Cre mouse is regulated by the Abhd5 gene promoter; Preferably, the animal includes rodents; Preferably, the animals include pigs, rats, mice, hamsters, rabbits, pigs, cattle, deer, sheep, goats, chicks, cats, horses, dogs, orangutans, or monkeys; Preferably, 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 male animals, extensive lipid droplet deposition in the interstitial tissue of the testes in male animals, reduced sperm count in male animals, and decreased sperm motility in male animals. Preferably, the metabolic syndrome includes one or more of obesity, atherosclerosis, and diabetes. Preferably, the obesity includes at least one of induced obesity, aging-related obesity, leptin-deficient obesity, and Fas-deficient obesity; Preferably, the leptin-deficient obesity is selected from childhood obesity; Preferably, the diabetes mellitus is selected from type 2 diabetes mellitus.

3. 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, characterized in that, It comes from the animal produced by the method of any one of claims 1-2 or the offspring of said animal.

4. A tissue or organ, or a culture of said tissue or organ, or a fragment or extract of said tissue or organ, characterized in that, It comes from the animal produced by the method of any one of claims 1-2 or the offspring of said animal.

5. The bodily fluid of an animal or its offspring, characterized in that, The animal is prepared by the method according to any one of claims 1-2.

6. An isolated cell nucleus, characterized in that, It comes from the animal produced by the method of any one of claims 1-2 or the offspring of said animal.

7. The use of animal models or their offspring obtained by the method of any one of claims 1-2 as model systems 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 and / or therapeutic strategies, or in the screening, validation, evaluation or research of drugs for the treatment of lipid metabolism syndrome, and in pharmacodynamic studies. Preferably, the metabolic syndrome includes: One or more of the following: obesity, atherosclerosis, and diabetes; Preferably, the obesity includes at least one of induced obesity, aging-related obesity, leptin-deficient obesity, or Fas-deficient obesity; Preferably, the leptin-deficient obesity is selected from childhood obesity; Preferably, the diabetes mellitus is selected from type 2 diabetes mellitus.

8. Application of Abhd5 detection reagents in the preparation of diagnostic reagents or kits for lipid metabolism syndrome.

9. The application as described in claim 8, characterized in that, The detection reagent is used to detect the expression level of the Abhd5 gene; Preferably, the detection reagent detects the mRNA expression level of Abhd5; Preferably, the detection reagent detects the protein expression level of Abhd5; Preferably, the test sample for the test reagent includes Treg cells.

10. The application as described in claim 8, characterized in that, The detection reagent is selected from at least one of the following groups: quantitative PCR dye, quantitative PCR primer, quantitative PCR probe, antibody, antibody functional fragment, or conjugated antibody. Preferably, the kit is selected from at least one of the following groups: qPCR kit, immunoblotting kit, immunochromatographic kit, flow cytometry kit, immunohistochemistry kit, ELISA kit, or electrochemiluminescence kit.

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