Mesenchymal stem cells and adipocytes for preparing mitokine mixtures

Abstract

Provided are: a non-human animal or a part of the same in which the function of Mipep gene is totally or partially lost in adipose tissues or the expression level of Mipep gene in adipose tissues is lowered compared to a wild type; and mesenchymal stem cells or adipocytes in which the function of Mipep gene is totally or partially lost or the expression level of Mipep gene is lowered compared to a wild type. Also provided are: a therapeutic or prophylactic drug that comprises the aforesaid mesenchymal stem cells or adipocytes or a culture supernatant thereof; and a method for preparing a mitokine mixture using the aforesaid non-human cells, mesenchymal stem cells or adipocytes.

Description

Technical Field

[0001] The present invention relates to mesenchymal stem cells and adipose cells for preparing a mitokine mixture. in It relates thereto.

Background Art

[0002] The mitochondrial unfolded protein response (mtUPR) is a stress response triggered to maintain mitochondrial proteostasis against mitochondrial stress. This response involves a group of genes such as chaperones, proteases, and mitokines.

[0003] Typical genes included in the group of mitokine genes are GDF15 (growth / differentiation factor 15) and FGF21 (fibroblast growth factor 21). Among these, GDF15 belongs to the transforming growth factor beta superfamily and is predominantly expressed in the liver, lung, and kidney in healthy animals, but is also expressed in multiple tissues in response to various types of stress. For example, it has been suggested that GDF15 is involved in disease resistance against bacterial and viral infections and sepsis (see Non-Patent Document 1). It has also been clarified that GDF15 exhibits a protective function against ischemia-reperfusion injury by being induced in the heart (see Non-Patent Document 2).

[0004] On the other hand, FGF21 belongs to the endocrine FGF superfamily and was initially identified as a hepatokine, but in recent years it has been reported to be expressed in other tissues such as white adipose tissue, brown adipose tissue, muscle, and pancreas. FGF21 has also been confirmed to be involved in stress, and for example, when FGF21 is administered to diabetic mice or aging mice, it has been shown to inhibit neuronal loss and enhance the production of antioxidant enzymes, thereby increasing the protective effect on neuronal mitochondria (see Non-Patent Literature 3). Furthermore, it has been reported that administration of FGF21 may be a treatment for hypoxia-induced cardiac injury by regulating the expression of galectin-3 (see Non-Patent Literature 4).

[0005] Thus, genes included in the mitokine gene group have been suggested to be related to the treatment or prevention of various diseases. Therefore, there is a desire to efficiently prepare the products of the genes included in the mitokine gene group. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Luan et al.,Cell,2019,178,1231-1244 [Non-Patent Document 2] Kempf et al.,Circ.Res.,2006,98,351-360 [Non-Patent Document 3] Kang et al.,Biomed. Pharmacother.,2020,129,110439 [Non-Patent Document 4] Sun et al.,J.Cell Biochem.,2019,120(12),19529-19540 [Overview of the project] [Problems that the invention aims to solve]

[0007] This invention was proposed in view of the above, and aims to efficiently prepare the products of genes included in the mitokine gene group. [Means for solving the problem]

[0008] As a result of diligent research toward the above objective, the inventors of this invention discovered that deleting the function of the Mipep gene in adipose tissue increases the expression of genes included in the mitokine gene group, and thus completed the present invention. More specifically, the present invention provides the following:

[0009] <1> Non-human animals or parts thereof in which the function of all or part of the Mipep gene is lost in adipose tissue, or in which the expression level of the Mipep gene in adipose tissue is reduced compared to the wild type.

[0010] <2> Mesenchymal stem cells in which all or part of the function of the Mipep gene is lost, or in which the expression level of the Mipep gene is reduced compared to the wild type.

[0011] <3> Adipocytes in which all or part of the function of the Mipep gene is lost, or in which the expression level of the Mipep gene is reduced compared to the wild type.

[0012] <4> A therapeutic or prophylactic agent comprising at least one selected from the group consisting of mesenchymal stem cells or adipocytes in which all or part of the function of the Mipep gene is lost, or the expression level of the Mipep gene is reduced compared to the wild type, and the culture supernatant of the mesenchymal stem cells or adipocytes containing a mitokine.

[0013] <5> The aforementioned therapeutic or preventive medicine is used for the treatment or prevention of at least one disease selected from the group consisting of sepsis, ischemia-reperfusion injury, inflammatory diseases, chronic kidney disease, obesity, atherosclerosis, non-alcoholic fatty liver disease, and other metabolic diseases. <4> A medicine for the treatment or prevention described above.

[0014] <6>A method for preparing a cytokine mixture, comprising recovering a plurality of cytokines from at least one selected from the group consisting of a non-human animal in which all or part of the function of the Mipep gene is lost in adipose tissue, or the expression level of the Mipep gene in adipose tissue is reduced compared to the wild type, and mesenchymal stem cells or adipocytes in which all or part of the function of the Mipep gene is lost, or the expression level of the Mipep gene is reduced compared to the wild type.

Advantages of the Invention

[0015] According to the present invention, products of genes included in a cytokine gene group can be efficiently prepared.

Brief Description of the Drawings

[0016] [Figure 1] It is a diagram showing the Mipep gene expression level. [Figure 2] It is a diagram showing the change of Mipep substrate protein due to Mipep deficiency. [Figure 3A] It is a diagram showing the change of adipose tissue due to Mipep deficiency. [Figure 3B] It is a diagram showing the change of adipose tissue due to Mipep deficiency. [Figure 4A] It is a diagram showing the visceral fat amount due to Mipep deficiency. [Figure 4B] It is a diagram showing the visceral fat amount due to Mipep deficiency. [Figure 5] It is a diagram showing the change of gene expression related to mtUPR. [Figure 6] It is a diagram showing the effect of Mipep deficiency on the amount of plasma GDF15. [Figure 7] It is a diagram showing the effect of Mipep deficiency on resistance to LPS administration.

Modes for Carrying Out the Invention

[0017] The following describes in detail embodiments for carrying out the present invention. However, the present invention is not limited to the following embodiments.

[0018] In this specification, "mitokine" is the English term, and is sometimes referred to as "mytokine." Mitokines are physiologically active substances that transmit stress generated in mitochondria to the extracellular space, and exhibit non-cellular autonomous actions.

[0019] [Non-human animals or parts thereof] In the non-human animals according to this embodiment, all or part of the function of the Mipep gene is lost in adipose tissue, or the expression level of the Mipep gene in adipose tissue is reduced compared to the wild type. As described in the examples below, deletion of the Mipep gene in adipose tissue increases the expression of genes included in the mitokine gene group.

[0020] MIPEP (mitochondrial intermediate peptidase) is a MtSPase responsible for the second processing of substrate proteins processed by mitochondrial processing peptidase (MPP), and is known to be an enzyme that cleaves the octapeptide from the N-terminus of proteins cleaved by MPP. The inventors previously reported, through comprehensive gene expression analysis, that calorie restriction, which has metabolic improvement, anti-aging, and life-extending effects, enhances MIPEP expression, and that SIRT3, a deacetylase localized in mitochondria and involved in the activation of multiple mitochondrial-localized enzymes, is a substrate of MIPEP (Kobayashi et al., FEBS Letters, 2017, 591, 4067-4073).

[0021] The nucleotide and amino acid sequence information for the Mipep gene is available from the GenBank database of the National Center for Biotechnology Information (NCBI). As an example, the nucleotide and amino acid sequences of human Mipep are shown in SEQ ID NOs: 1 and 2.

[0022] Non-human animals in which all or part of the function of the Mipep gene is lost mean animals in which the Mipep gene has been disrupted or recombinant so that it does not perform its original function. The Mipep gene may have one allele in the genome disrupted or mutated so that it does not function, or both alleles may be disrupted or mutated. Non-human animals also include the offspring of such animals.

[0023] "Loss of all Mipep gene function" means that the Mipep gene is completely lost, while "loss of some Mipep gene function" means that the Mipep gene's function is reduced compared to the wild type due to the absence of a portion of the Mipep gene. In such a state, either the Mipep gene is not expressed at all, or if it is expressed, the protein's activity is reduced or lost.

[0024] Non-human animals in which all or part of the function of the Mipep gene is lost can be created by known methods. A standard method, for example, consists of the following steps (a) to (d). (a) Create ES cells in which all or part of the function of the Mipep gene is lost. (b) Create chimeric non-human animals by transplanting ES cells into the embryos of non-human animals and allowing them to give birth. (c) A chimeric non-human animal is crossbred with a wild-type non-human animal of the same species to produce a heterozygous knockout non-human animal. (d) Create homozygous knockout non-human animals by mating heterozygous knockout non-human animals with each other.

[0025] Next, "reduced expression levels of the Mipep gene compared to the wild type" means that the expression level of the Mipep gene is suppressed and lower than that of the wild type. Suppression of Mipep gene expression can be achieved, for example, by reducing the transcription level of the Mipep gene or inhibiting translation.

[0026] There are no particular limitations on the method for suppressing the expression level of the Mipep gene; for example, RNA interference (RNAi) can be used. RNAi can be induced by designing and synthesizing siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (microRNA), etc., for the Mipep gene, incorporating these into a retroviral vector or adenovirus vector, and introducing them into a non-human animal.

[0027] Non-human animals are not particularly limited to animals other than humans, and examples include mice, rats, guinea pigs, hamsters, rabbits, monkeys, cows, miniature pigs, pigs, sheep, goats, dogs, cats, etc. Of these, mice, rats, etc. are preferred as non-human animals because protocols for creating mutant animals have been established and they are easy to breed.

[0028] Parts of a non-human animal are not particularly limited as long as they can be obtained from the non-human animal, and include, for example, tissues, cells, their fragments or extracts; body fluids; etc. derived from the non-human animal.

[0029] [Mesenchymal stem cells] In this embodiment, the mesenchymal stem cells have lost all or part of the function of the Mipep gene, or the expression level of the Mipep gene is reduced compared to the wild type. It has been reported that mesenchymal stem cells express genes included in the mitokine gene group (see, for example, International Publication No. 2017 / 188403), and, similar to adipocytes described later, the loss of function of the Mipep gene enhances mitokine production. Therefore, mesenchymal stem cells can be used, for example, in cell therapy where the cells themselves are transplanted, and more mitokines can be produced in the transplanted body.

[0030] Furthermore, the culture supernatant can be recovered by culturing mesenchymal stem cells. As mentioned above, mesenchymal stem cells produce mitokines, and therefore their culture supernatant contains mitokines. Thus, by using the culture supernatant of mesenchymal stem cells as a therapeutic or preventive medicine, therapeutic or preventive effects can be expected for diseases in which mitokines are effective. Moreover, since the culture supernatant contains a balanced amount of multiple types of mitokines, as well as other beneficial components, even greater effectiveness can be expected compared to administering mitokines themselves.

[0031] The phrases "all functions of the Mipep gene are lost," "some functions of the Mipep gene are lost," and "the expression level of the Mipep gene is reduced compared to the wild type" have the same meaning as described above for non-human animals.

[0032] Mesenchymal stem cells are somatic stem cells that originate from the mesenchyme and possess self-renewal and differentiation capabilities; however, the tissue from which they originate is not particularly limited. Examples of tissues from which they originate include fat, bone marrow, dental pulp, blood (peripheral blood, umbilical cord blood, etc.), placenta, umbilical cord, synovial membrane, periosteum, perichondrium, muscle, ligament, tendon, meniscus, and skin. Of these, fat is preferred as the tissue from which they originate.

[0033] Mesenchymal stem cells may be cells derived from non-human animals as described above, or they may be cells derived from humans.

[0034] Mesenchymal stem cells may be derived from the cells of the individual to be administered the treatment (autologous cells), or from cells of another individual (allogeneic cells).

[0035] Mesenchymal stem cells may be cells differentiated from ES cells, cells differentiated from induced pluripotent stem cells (such as iPS cells), established cell lines, Muse cells (Multi-lineage differentiating Stress Euduring Cells), etc.

[0036] Typically, mesenchymal stem cells used are those that maintain an undifferentiated state and are negative for differentiation markers (such as CD24).

[0037] The method for preparing mesenchymal stem cells is not particularly limited, and any known method for preparing mesenchymal stem cells can be used. For example, one method involves seeding cells containing mesenchymal stem cells obtained from the aforementioned source tissue into a culture dish, allowing them to adhere to the culture dish and proliferate, and then proliferating a portion of the resulting cells again on the culture dish.

[0038] The method for deleting all or part of the function of the Mipep gene in mesenchymal stem cells is not limited; for example, genome editing methods using CRISPR / Cas nucleases can be used. Similarly, the method for suppressing the expression level of the Mipep gene in mesenchymal stem cells is not particularly limited; for example, the RNAi method described above can be used in non-human animals.

[0039] [Fat cells] In this embodiment, the adipocytes have lost all or part of the function of the Mipep gene, or the expression level of the Mipep gene is reduced compared to the wild type. As shown in the examples described later, such adipocytes produce a large amount of mitokines. Therefore, adipocytes can be used, for example, in cell therapy in which the cells themselves are transplanted, and more mitokines can be produced in the transplanted body.

[0040] Furthermore, the culture supernatant can be recovered by culturing adipocytes. As mentioned above, adipocytes produce a large amount of mitokines, so their culture supernatant contains a large amount of mitokines. Therefore, by using the culture supernatant of adipocytes as a therapeutic or preventive medicine, therapeutic or preventive effects can be expected for diseases in which mitokines are effective. Moreover, since the culture supernatant contains a balanced amount of multiple types of mitokines, as well as other useful components, even greater effectiveness can be expected compared to administering mitokines themselves.

[0041] The phrases "all functions of the Mipep gene are lost," "some functions of the Mipep gene are lost," and "the expression level of the Mipep gene is reduced compared to the wild type" have the same meaning as described above for non-human animals.

[0042] Adipocytes may be cells isolated from the non-human animals mentioned above, or they may be cells differentiated from the mesenchymal stem cells mentioned above.

[0043] There are no particular limitations on the method for differentiating mesenchymal stem cells into adipocytes. For example, methods involving contacting mesenchymal stem cells with dexamethasone, insulin, 3-isobutyl-1-methylxanthine, rosiglitazone, glucocorticoids, phosphodiesterase inhibitors, etc., are known. Alternatively, commercially available "differentiation inducers" can also be used.

[0044] Adipocytes may be cells derived from non-human animals as described above, or they may be cells derived from humans.

[0045] The adipocytes may be derived from the cells of the individual to be administered the drug (autologous cells), or from cells of another individual (allogeneic cells).

[0046] The method for deleting all or part of the function of the Mipep gene in adipocytes is not limited; for example, genome editing methods using CRISPR / Cas nucleases can be used. Similarly, the method for suppressing the expression level of the Mipep gene in adipocytes is not particularly limited; for example, the RNAi method described above can be used in non-human animals.

[0047] [Medicine for treatment or prevention] A therapeutic or prophylactic medicine contains at least one selected from the group consisting of mesenchymal stem cells or adipocytes in which all or part of the function of the Mipep gene is lost, or the expression level of the Mipep gene is reduced compared to the wild type, and the culture supernatant of said mesenchymal stem cells or said adipocytes containing a mitokine.

[0048] As mesenchymal stem cells or adipocytes, the same cells as those described above for mesenchymal stem cells and adipocytes can be used.

[0049] The culture supernatant of mesenchymal stem cells or adipocytes containing mitokines is the culture supernatant obtained during the process of culturing mesenchymal stem cells or adipocytes in which all or part of the function of the Mipep gene is lost, or the expression level of the Mipep gene is reduced compared to the wild type. The method for obtaining the culture supernatant is not particularly limited, but an example is described below.

[0050] First, adipose tissue from a non-human animal in which the function of all or part of the Mipep gene is lost in the adipose tissue, or in which the expression level of the Mipep gene in the adipose tissue is reduced compared to the wild type, is collected subcutaneously, and after performing collegase treatment as necessary, etc., it is cultured in the presence of an appropriate culture medium. From the start of culture, the culture supernatant is collected, for example, once every few days. The collected culture supernatant is preferably sterilized appropriately (sterilization under non-heating conditions such as filter filtration and UV sterilization is preferable), and it is preferable to check for residual virus.

[0051] The culture supernatant of mesenchymal stem cells or adipocytes produced in this manner contains many mitokines, but the mitokines included are not particularly limited and include, for example, GDF15, FGF21, ANGPTL6, etc.

[0052] Therapeutic or prophylactic medicines are preferably used for diseases in which the action of genes included in the mitokine gene group is effective. Examples of such diseases include sepsis, ischemia-reperfusion injury, inflammatory diseases, chronic kidney disease, obesity, atherosclerosis, non-alcoholic fatty liver disease, and other metabolic diseases.

[0053] A therapeutic or prophylactic medicine may further contain, in addition to mesenchymal stem cells, adipocytes, or culture supernatant, pharmaceutically acceptable excipients (pharmaceutical excipients) as long as they do not impair their function. Such excipients include, but are not limited to, stabilizers, preservatives, buffers, pH adjusters, suspending agents, flavoring agents, colorants, and viscosity modifiers. A therapeutic or prophylactic medicine may also contain components derived from the culture medium.

[0054] A therapeutic or preventive medicine may contain mesenchymal stem cells, adipocytes, or culture supernatant directly in any solvent, or it may contain them in any form such as a sheet, tube, layer, or solid-phase conjugate.

[0055] One example of a method of administering a therapeutic or preventive drug to humans, in the form of adipocytes, is to place adipocytes in physiological saline or culture medium, for example, 1 x 10⁶ cells. 3 ~1 × 10 7 The dosage may be adjusted to a concentration of 1 / mL and administered locally by injection or catheter into the abdominal cavity or subcutaneous tissue. Other forms of administration for therapeutic or prophylactic medicines include intravenous administration, intra-arterial administration, intramuscular administration, intranasal administration, intra-spinal canal transplantation, intra-articular transplantation, and intragingival injection.

[0056] The target population for therapeutic or preventive medicines is not particularly limited, but mammals are preferably mentioned. Mammals may include both humans and non-human animals.

[0057] The dosage of therapeutic or preventive medicines is determined appropriately according to the recipient, route of administration, target disease, symptoms, etc.

[0058] Furthermore, therapeutic or preventive medicines may be administered in combination with other drugs, depending on the purpose of administration. The type and amount of drugs used in combination with therapeutic or preventive medicines should be appropriately selected based on the desired effect, and they may be administered together with the therapeutic or preventive medicine or separately.

[0059] [Method for preparing a mitokine mixture] As shown in the examples described below, by deleting the function of the Mipep gene in adipose tissue, the expression of multiple genes included in the cytokine gene group increases. Therefore, a non-human animal in which all or part of the function of the Mipep gene is lost in adipose tissue, or the expression level of the Mipep gene in adipose tissue is decreased compared to the wild type, and a mesenchymal stem cell or an adipose cell in which all or part of the function of the Mipep gene is lost, or the expression level of the Mipep gene is decreased compared to the wild type. It is possible to recover multiple types of cytokines from at least one selected from the group consisting of and prepare a cytokine mixture.

[0060] Examples of the cytokines contained in the prepared cytokine mixture include the same cytokines as those described in the above-mentioned therapeutic or prophylactic pharmaceuticals.

Examples

[0061] Hereinafter, examples will be shown to specifically explain the present invention, but the present invention is not limited to these examples.

[0062] [Production of knockout mice] Knockout mice of the Mipep gene were produced by the following method.

[0063] [Production of Targeting vector and introduction into ES cells] The targeting vector was constructed by inserting the targeting region of the mouse genome into a DT-A / conditional KO FW vector. The inserts (5' arm, targeting arm, 3' arm) were templated using clone RP23-142O16 (Advanced Geno Techs) from a mouse BAC (Bacterial Artificial Chromosome) library and amplified using KOD FX Neo (TOYOBO) with the primers shown in Table 1. The 5' arm was then treated with AscI and NotI, the targeting arm with PmeI and SacII, and the 3' arm with SwaI and XhoI. These were then ligated into a DT-A / conditional KO FW vector treated with similar restriction enzymes, and the targeting vector was purified. After linearization of the targeting vector by XhoI treatment, it was introduced into C57BL / 6N-derived ES cells using electroporation. The primers used are as follows:

[0064] [Table 1]

[0065] Southern Blotting Using RP23-142O16 as a template, the 3' probe was amplified by KOD FX Neo using the primers shown in Table 1. Subsequently, genomic DNA was extracted from the above ES cells into which the targeting vector had been introduced by the phenol-chloroform method and treated with ApaLI. Then, 20 μg of the ApaLI-treated genome DNA was applied to a 0.80% agarose gel, and after electrophoresis, the gel was placed on ethidium bromide and imaged. The gel was then treated in the following order: acid treatment (0.25 M HCl infiltration at room temperature for 15 min), base treatment (0.50 M NaOH, 1.5 M NaCl infiltration at room temperature for 15 min), and neutralization treatment (0.50 M Tris-HCl (pH 8.0), 1.5 M NaCl infiltration at room temperature for 20 min). After processing, the DNA was transferred to a nylon membrane (Gene Screen Plus) using the osmotic pressure of 10×SSC (1.5M NaCl, 150mM sodium citrate), and the DNA was cross-linked with 150mJ UV. After cross-linking, the DNA was immersed in a buffer containing 5×SSCP (0.75M NaCl, 75mM sodium citrate, 50mM NaH2PO4, 5.0mM EDTA), 50% Formamide, 2×Denhardt's solution, 1.0% SDS, 100μg / mL salmon testis DNA, and pre-hybridization was performed overnight at 42°C. 32 The 3' probe labeled with phosphorus (P) was mixed in buffer and hybridized overnight at 42°C. Afterward, the buffer was removed, and the membrane was washed in a 2×SSC, 0.10% SDS solution at 42°C for 15 minutes, followed by two washes in a 0.1×SSC, 0.10% SDS solution at 65°C for 15 minutes each. This membrane was placed on a photosensitive plate and left overnight before being photographed with a Fujifilm FLA-7000.

[0066] <Creating a mouse> ES cells in which the insertion of the target allele at the desired site was confirmed by Southern blotting were mixed with 8-cell stage fertilized eggs of ICR mice, cultured overnight, and then transplanted into the uterus of pseudopregnant mice to create chimeric mice (aggregation method). The chimeric mice were crossed with C57BL / 6JJcl to create C57BL / 6J mice possessing a genome derived from the introduced ES cells. CAG-FLPe mice (Kanki et al., Exp. Anim., 2006, 55, 137-141) were crossed with the above mice, and neomycin resistance sequences remaining in the ES cell-derived genome were deleted using the flippase-FRT system, resulting in Mipep flox / flox We created mice. Then, we crossed them with Adiponectin-Cre mice to generate adipose tissue-specific Mipep knockout mice.

[0067] [Test Example 1] In Test Example 1, the expression level of the Mipep gene in Mipep knockout mice (hereinafter also referred to as "Mipep KO mice") was confirmed by real-time PCR. Real-time PCR was performed using the following method.

[0068] mRNA was extracted from adipose tissue using ISOGENII (Nippon Gene), and its concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Total RNA was reverse transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO) to obtain cDNA. Subsequently, Real-time RT-PCR using THUNDERBIRD SYBR qPCR Mix (TOYOBO) was performed on a CFX connect real-time PCR system (Bio-rad). The reaction conditions for each reaction followed the recommended protocols of the reagent manufacturers. The primer sequences used are as follows.

[0069] [Table 2]

[0070] As shown in Figure 1, the expression level of the Mipep gene was found to be reduced in the adipose tissue of Mipep KO mice.

[0071] [Test Example 2] In Experimental Example 2, changes in the MIPEP substrate in adipose tissue due to MIPEP knockout were confirmed by Western blotting. Western blotting was performed using the following method.

[0072] The tissue was homogenized with an appropriate amount of SDS sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 3M urea, 6% glycerol), and then sonicated. The resulting lysate was centrifuged (12000 × G, 4°C, 30 min), the supernatant was collected, and incubated at 95°C for 5 minutes. The amount of protein in the supernatant was measured using the BCA Protein Assay Kit (Thermo Fisher Scientific), and the concentration was adjusted to 1 mg / mL using SDS sample buffer. A 1 / 10 volume of 0.25% BPB / 5% 2-mercaptoethanol = 1:1 mixture was added, and the mixture was reduced at 95°C for 5 minutes. The prepared sample was electrophoresed by SDS-PAGE and transferred to a nitrocellulose membrane. After transfer, the membrane was shaken at room temperature for 1 hour in a blocking solution (2.5% skim milk (WAKO) / 0.25% BSA in TTBS (25mM Tris-HCl (pH7.4), 140mM NaCl, 2.5mM KCl, 0.1% Tween 20) = 1:1). Then, the primary antibody was added to the reaction mixture (Immuno Shot Reagent I (Cosmo Bio, IS-001) / blocking solution = 2:1) and reacted with the membrane at 4°C overnight or for two nights. After the primary antibody reaction, the membrane was washed with TTBS (5 minutes x 2 times and 10 minutes x 2 times), and the secondary antibody was added to the reaction mixture (ImmunoShot Reagent II (Cosmo Bio, IS-002) / blocking solution = 2:1) and reacted with the membrane at room temperature for 1 hour. After the secondary antibody reaction, the membrane was washed with TTBS (5 minutes x 2 times and 10 minutes x 2 times), chemiluminescence was performed using ImmunoStar LD (WAKO), and images were taken with a LAS-3000 lumino-image analyzer. The results were quantified using Multi Gauge 3.1.The primary antibodies used were anti-SIRT3 antibody (Cell Signaling, #5490), anti-COX4 antibody (Cell Signaling, #4844), anti-MDH2 antibody (Cell Signaling, #8610), anti-Clpx antibody (abcam, ab168338), anti-SPG7 antibody (Thermo Fisher Scientific, PA5-87106), and anti-MRPL32 antibody (Thermo Fisher Scientific, PA5-109980). The secondary antibody used was HRP-labeled anti-rabbit IgG antibody (West Grove).

[0073] As shown in Figure 2, in Mipep KO mice, the protein bands for SIRT3 and COX4, substrates of MIPEP, shifted upward, and their expression levels decreased. On the other hand, MDH2, also a substrate of MIPEP, similarly shifted upward in the Mipep KO mice, but its protein expression level increased. These upward shifts in the bands are thought to be due to maturation failure. Furthermore, as shown in Figure 2, in Mipep KO mice, the expression levels of Clpx, a subunit of the protease ClpXP, and SPG7, a subunit of the protease m-AAA, increased, while the expression level of MRPL32, a substrate of m-AAA, decreased. From these results, it is thought that MIPEP also uses proteases as substrates and plays an important role in mitochondrial proteostasis.

[0074] [Test Example 3] In Experimental Example 3, changes in adipose tissue due to Mipep knockout were examined. First, the condition of the adipose tissue was checked, then the adipose tissue was fixed in 10% neutral buffered formalin solution (10% formaldehyde in PBS), embedded in paraffin, sectioned to a thickness of 5 μm, and stained with hematoxylin-Eosin (HE).

[0075] First, as shown in Figure 3A, a significant reduction in adipose tissue was observed in Mipep KO mice.

[0076] Next, as shown in Figure 3B, in the WAT of Mipep KO mice, cells exhibiting lipoblast-like morphology, representing undifferentiated adipocytes with abnormal maturation, were observed in some areas. Furthermore, an increase in lipid droplets was confirmed in the BAT of Mipep KO mice.

[0077] [Test Example 4] In Experimental Example 4, changes in visceral fat volume in the adipose tissue around the genitals of mice due to Mipep knockout were examined. This was confirmed using 19-20 week old mice and computed tomography (CT) imaging as described below. A third-generation CT scanner, the Latheta LCT-200 (Hitachi-Alola), was used, with a constant tube voltage of 50kV and current of 0.5mA. The mice were placed in a 48mm diameter holder, the scanner was rotated 360°, and data was collected. Images were acquired with a resolution of 96μm per pixel, a width of 192μm per image, and a spacing of 600μm. The density range of -550 to -140HU was evaluated and analyzed as WAT (Wet Absorption). The estimated X-ray exposure of the mice was maintained at less than 40mSv during imaging.

[0078] As shown in Figures 4A and 4B, visceral fat (vWAT) increased in wild-type mice when fed a high-fat diet (HFD), whereas no such increase was observed in Mipep knockout mice.

[0079] [Test Example 5] In Experimental Example 5, changes in mtUPR-related gene expression in adipose tissue due to Mipep knockout were confirmed. Confirmation was performed by real-time PCR using the same method as in Experimental Example 1. The primer sequences used are as follows.

[0080] [Table 3]

[0081] As shown in Figure 5, Mipep KO mice showed increased expression levels of genes belonging to the mitokine gene group compared to wild-type mice. On the other hand, no increase in expression levels was observed in genes belonging to the chaperone or protease gene group. Furthermore, significantly lower expression levels were confirmed in Mipep KO mice for genes related to adipocyte differentiation.

[0082] Although the data is not shown, a comprehensive analysis of gene expression using the Illumina NextSeq500 RNA-seq analyzer revealed that Mipep KO mice showed significantly suppressed expression of mitochondrial-related genes compared to wild-type mice, confirming that mitochondrial biosynthesis was inhibited.

[0083] [Test Example 6] In Study Example 6, changes in plasma GDF15 protein levels due to Mipep knockout were confirmed. Confirmation was performed using the Mouse / Rat GDF-15 Quantitive ELISA Kit (R&D Systems). The reaction was carried out according to the manufacturer's protocol.

[0084] As shown in Figure 6, the amount of GDF15 in plasma was also higher in Mipep KO mice compared to wild-type mice.

[0085] [Test Example 7] In Study Example 7, the effect of Mipep knockout on survival after LPS administration was examined. Six male Mipep knockout mice and six wild-type mice, aged 40-45 weeks, were administered 15 mg of LPS (Sigma-Aldrich) per kg of body weight intraperitoneally. The number of surviving mice was counted daily after administration, and the survival rate was calculated with 6 mice set as 100%, and this was represented on a survival curve.

[0086] As shown in Figure 7, Mipep KO mice exhibited significantly higher resistance to LPS compared to wild-type mice.

Claims

1. Non-human animals in which the Mipep gene is knocked out in adipose tissue, or in which the expression level of the Mipep gene in adipose tissue is reduced compared to the wild type.

2. Mesenchymal stem cells in which the Mipep gene is knocked out, or in which the expression level of the Mipep gene is reduced compared to the wild type.

3. Adipocytes in which the Mipep gene is knocked out, or in which the expression level of the Mipep gene is reduced compared to the wild type.

4. A method for preparing a mitokine mixture, comprising recovering multiple mitokines from at least one selected from a group consisting of a non-human animal in which the Mipep gene is knocked out in adipose tissue or in which the expression level of the Mipep gene in adipose tissue is reduced compared to the wild type, and mesenchymal stem cells or adipocytes in which the Mipep gene is knocked out or in which the expression level of the Mipep gene is reduced compared to the wild type.

Images