Symbiotic young factor and its application in delaying body aging

Abstract

The application discloses application of symbiotic young factor in delaying body aging at multiple tissue cell and molecular levels, and belongs to the technical field of biotechnology.The application discloses application of protein CCL3 or protein YY1 or protein GILZ or a substance for regulating expression of a gene encoding the protein CCL3 or the protein YY1 or the protein GILZ or a substance for regulating activity or content of the protein CCL3 or the protein YY1 or the protein GILZ in regulating aging level of a body and / or cells and / or tissues.The application provides rich resources for studying cell and molecular changes of aging and rejuvenation at single cell resolution, and reveals key regeneration factors (Ccl3, Yy1 and Tsc22d3 (Gilz)).

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of heterologous symbiotic rejuvenation factors in delaying aging at the multi-tissue cellular and molecular levels. Background Technology

[0002] Aging is a systemic degenerative process involving multiple tissues and organs throughout the body, leading to reduced regenerative capacity and decreased tissue and organ function. Several methods have been reported to delay age-related phenotypes. Among them, heterochronic parabiosis (HP) is a research model that has received widespread attention in recent years. In the HP mouse model, the circulatory systems of aged and young mice are surgically connected to create a shared circulatory system, where blood-derived factors produced by one individual are shared with the other. Therefore, HP provides a unique experimental paradigm for studying how a fully aged organism can be revitalized through the injection of "pro-youth factors." Conversely, how young organisms are affected by "pro-aging factors" in the circulatory system. To date, our understanding of the cellular targets of blood-derived factors and how they promote rejuvenation in aged individuals at the systemic level remains limited.

[0003] On the other hand, many features of systemic aging are associated with the damage and depletion of organ-specific adult stem cells. For example, in fully developed organs and tissues, such as bone marrow, skin, brain, and skeletal muscle, a small population of adult stem cells replenishes tissues throughout life to repair age-related damage and maintain tissue homeostasis. In the hematopoietic and immune system, composed of multiple organs and tissues, including bone marrow, spleen, peripheral blood, and hematopoietic stem cells and their progeny, hematopoietic stem and progenitor cells (HSPCs) can produce various types of immune cells. However, with age, HSPCs gradually lose their ability to maintain the composition of immune cells and exhibit myeloid-shifted differentiation potential, leading to age-related hematopoietic dysfunction and weakened immunity. Similarly, other organs and tissues, such as skin, skeletal muscle, and brain, also harbor different types of adult stem cells, namely hair follicle stem cells (HFSCs) and basal cells responsible for hair follicle and epidermal regeneration, fibroblast / adipogenic progenitor cells (FAPS) and satellite cells of skeletal muscle, and neural stem cells of the brain. These adult stem cells gradually lose their regenerative capacity with age, leading to hair loss, skin aging, and degeneration of skeletal muscle and nerves. Therefore, resident adult stem cells in the hematopoietic system and peripheral organs are all affected by aging, but whether and to what extent they can regain their vitality requires further research. Summary of the Invention

[0004] The technical problem this invention aims to solve is how to delay aging.

[0005] To solve the above-mentioned technical problems, in a first aspect, the present invention provides an application, which may be any one of the following A1)-A10):

[0006] A1) The application of protein CCL3 or protein YY1 or protein GILZ or substances that regulate the expression of the gene encoding said protein CCL3 or said protein YY1 or said protein GILZ or substances that regulate the activity or content of said protein CCL3 or said protein YY1 or said protein GILZ in regulating the level of aging in the body and / or cells and / or tissues.

[0007] A2) The application of protein CCL3 or protein YY1 or protein GILZ or substances that regulate the expression of genes encoding said protein CCL3 or said protein YY1 or said protein GILZ or substances that regulate the activity or content of said protein CCL3 or said protein YY1 or said protein GILZ in the preparation of products for the aging level of the body and / or cells and / or tissues.

[0008] A3) The application of protein CCL3 or protein YY1 or protein GILZ or substances for determining the content of protein CCL3 or protein YY1 or protein GILZ in identifying or assisting in the identification of the aging level of the body and / or cells and / or tissues.

[0009] A4) The application of protein CCL3 or protein YY1 or protein GILZ or substances for determining the content of protein CCL3 or protein YY1 or protein GILZ in the preparation of products for identifying or assisting in the identification of the aging level of the body and / or cells and / or tissues.

[0010] A5) The application of protein CCL3 or protein YY1, or substances that regulate the expression of genes encoding said protein CCL3 or said protein YY1, or substances that regulate the activity or content of said protein CCL3 or said protein YY1, in regulating the reconstitution capacity of hematopoietic stem (progenitor) cells.

[0011] A6) The application of protein CCL3 or protein YY1, or substances that regulate the expression of the gene encoding said protein CCL3 or said protein YY1, or substances that regulate the activity or content of said protein CCL3 or said protein YY1, in the preparation of products that regulate the reconstitution ability of hematopoietic stem (progenitor) cells.

[0012] A7) The application of protein CCL3 or protein YY1 or substances that regulate the expression of the gene encoding said protein CCL3 or said protein YY1 or substances that regulate the activity or content of said protein CCL3 or said protein YY1 in regulating the lineage differentiation of hematopoietic stem (progenitor) cells.

[0013] A8) The application of protein CCL3 or protein YY1 or substances that regulate the expression of genes encoding said protein CCL3 or said protein YY1 or substances that regulate the activity or content of said protein CCL3 or said protein YY1 in the preparation of products that regulate the lineage differentiation of hematopoietic stem (progenitor) cells.

[0014] A9) The application of protein GILZ or substances that regulate the expression of the gene encoding said protein GILZ or substances that regulate the activity or content of said protein GILZ in regulating the proliferative capacity of skin fibroblasts.

[0015] A10) The application of protein GILZ or substances that regulate the expression of the gene encoding said protein GILZ or substances that regulate the activity or content of said protein GILZ in the preparation of products that regulate the proliferative capacity of skin fibroblasts.

[0016] In the above text, the protein CCL3, the protein YY1, or the protein GILZ can be a natural protein, such as a protein derived from mammals, or a non-natural protein, such as a recombinant protein. The recombinant protein only needs to have the function of the natural protein.

[0017] The CCL3 protein may be human CCL3 protein or mouse CCL3 protein. The GenBank accession number for the amino acid sequence of the human CCL3 protein is NP_002974.1, and the GenBank accession number for the amino acid sequence of the mouse CCL3 protein is NP_035467.1.

[0018] The YY1 protein may be human YY1 protein or mouse YY1 protein. The GenBank accession number for the amino acid sequence of the human YY1 protein is NP_003394.1, and the GenBank accession number for the amino acid sequence of the mouse YY1 protein is NP_033563.2.

[0019] The GILZ protein may be human GILZ protein or mouse GILZ protein. The GenBank number of the human GILZ protein is NP_001015881.1, and the GenBank number of the mouse GILZ protein is NP_001070832.1.

[0020] Furthermore, in the above applications, the substance regulating the expression of the gene encoding the protein CCL3 or the protein YY1, or the substance regulating the activity or content of the protein CCL3 or the protein YY1, can be biological material B1, which can be any one of the following B11) to B17):

[0021] B11), a nucleic acid molecule encoding the protein CCL3 or the protein YY1;

[0022] B12), an expression cassette containing the nucleic acid molecule described in B11);

[0023] B13), a recombinant vector containing the nucleic acid molecule described in B11), or a recombinant vector containing the expression cassette described in B12;

[0024] B14) recombinant microorganisms containing the nucleic acid molecules described in B11), or recombinant microorganisms containing the expression cassette described in B12), or recombinant microorganisms containing the recombinant vector described in B13);

[0025] B15), a transgenic animal cell line containing the nucleic acid molecule described in B11), or a transgenic animal cell line containing the expression cassette described in B12), or a transgenic animal cell line containing the recombinant vector described in B13;

[0026] B16), transgenic animal tissue containing the nucleic acid molecule described in B11), or transgenic animal tissue containing the expression cassette described in B12), or transgenic animal tissue containing the recombinant vector described in B13;

[0027] B17) Transgenic animal organs containing the nucleic acid molecules described in B11), or transgenic animal organs containing the expression cassette described in B12), or transgenic animal organs containing the recombinant vector described in B13).

[0028] Furthermore, in the above applications, the nucleic acid molecule described in B11) may be the coding gene of the protein CCL3 or the protein YY1, wherein the coding gene of the protein CCL3 is the Ccl3 gene or the coding sequence of the Ccl3 gene; and the coding gene of the protein YY1 is the Yy1 gene or the coding sequence of the Yy1 gene.

[0029] The Ccl3 gene can be the human CCL3 gene or the mouse Ccl3 gene. The GenBank number of the human CCL3 gene is NM_002983.3, and its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 86-364 of NM_002983.3; the GenBank number of the mouse Ccl3 gene is NM_011337.2, and its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 102-380 of NM_011337.2.

[0030] The Yy1 gene can be the human YY1 gene or the mouse Yy1 gene. The GenBank number of the human YY1 gene is NM_003403.5, and its coding sequence is the DNA molecule with nucleotide sequences shown from positions 102 to 1346 of NM_003403.5. The GenBank number of the mouse Yy1 gene is NM_009537.4, and its coding sequence is the DNA molecule with nucleotide sequences shown from positions 117 to 1361 of NM_009537.4.

[0031] Furthermore, in the above applications, the substance that regulates the expression of the GILZ-encoding gene or the substance that regulates the activity or content of the GILZ-encoding gene can be biological material B2. The biological material B2 can be an RNA molecule that inhibits or reduces the expression of the GILZ-encoding gene or an RNA molecule that inhibits or reduces the activity or content of the GILZ-encoding gene.

[0032] Furthermore, in the above applications, the RNA molecule that inhibits or reduces the expression of the gene encoding the protein GILZ, or the RNA molecule that inhibits or reduces the activity or content of the protein GILZ, as described in B21), may be i1), i2), or i3):

[0033] i1) The target sequence of the RNA molecule is the gene encoding the protein GILZ;

[0034] i2) The nucleotide sequence of the target sequence of the RNA molecule is SEQ ID No. 1;

[0035] i3) The nucleotide sequence of the target sequence of the RNA molecule is SEQ ID No. 2.

[0036] The GILZ protein can be human GILZ protein or mouse GILZ protein. The human GILZ protein can be a protein encoded by the human Gilz gene, whose GenBank accession number is XM_005262103.5, and whose coding sequence is the DNA molecule with nucleotide sequences shown at positions 160-762 of XM_005262103.5. The GenBank accession number of the human GILZ protein is NP_001015881.1. The mouse GILZ protein can be a protein encoded by the mouse Gilz gene, whose GenBank accession number is NM_001077364.1, and whose coding sequence is the DNA molecule with nucleotide sequences shown at positions 204-809 of NM_001077364.1. The GenBank accession number of the mouse GILZ protein is NP_001070832.1.

[0037] To address the aforementioned technical problems, in a second aspect, the present invention provides the application of a heterosymbiotic animal in any of the following P1)-P18):

[0038] P1) Reduce the proportion of senescent cells in spleen and / or skin and / or liver and / or brain tissue;

[0039] P2) Reduce the proportion of apoptotic cells in spleen tissue and / or skin tissue and / or liver tissue and / or skeletal muscle tissue;

[0040] P3) Reduce the area of ​​inflammation in liver tissue;

[0041] P4) Reduce the area of ​​fibrosis in liver and / or spleen tissues;

[0042] P5) Increases the diameter of muscle fibers in skeletal muscle tissue;

[0043] P6) Increase the number of hair follicles in skin tissue;

[0044] P7) Increase the proportion of pro-B cells in the bone marrow;

[0045] P8) Increase the expression levels of CCL3 protein and / or YY1 protein in hematopoietic stem and progenitor cells;

[0046] P9) Upregulates the expression levels of the Ccl3 gene and / or the Yy1 gene.

[0047] P10) Delays aging by reducing the proportion of senescent cells in spleen and / or skin and / or liver and / or brain tissues;

[0048] P11) Delays aging by reducing the proportion of apoptotic cells in spleen and / or skin and / or liver and / or skeletal muscle tissues;

[0049] P12) Delays aging by reducing the area of ​​inflammation in liver tissue;

[0050] P13) Delays aging by reducing the area of ​​fibrosis in liver and / or spleen tissues;

[0051] P14) Delays aging by reducing the diameter of muscle fibers in skeletal muscle tissue;

[0052] P15) Delays aging by reducing the number of hair follicles in skin tissue;

[0053] P16) delays aging by increasing the proportion of pro-B cells in the bone marrow;

[0054] P17) Delays aging by increasing the expression levels of CCL3 protein and / or YY1 protein in hematopoietic stem and progenitor cells;

[0055] P18) Delays aging by upregulating the expression levels of the Ccl3 gene and / or the Yy1 gene;

[0056] To address the aforementioned technical problems, in a third aspect, the present invention provides a method for constructing recombinant cells, the method comprising introducing a gene encoding a target protein into recipient cells, promoting or increasing or upregulating the expression of the gene encoding the target protein, or promoting or increasing or upregulating the activity or content of the target protein, thereby obtaining recombinant cells with a senescence level lower than that of the recipient cells;

[0057] The target protein is the CCL3 protein as described in claim 1 or the YY1 protein as described in claim 1.

[0058] To address the aforementioned technical problems, in a fourth aspect, the present invention provides recombinant cells constructed by the above-described method.

[0059] In this invention, the recombinant cells may be recombinant mammalian cells.

[0060] The mammals mentioned can be human or non-human mammals.

[0061] To address the aforementioned technical problems, in a fifth aspect, the present invention provides the application of the recombinant cell line in D1) or D2) below:

[0062] D1) Application in improving the reconstitution capacity of hematopoietic stem cells in the elderly;

[0063] D2) Application in the preparation of products that enhance the reconstitution capacity of aging hematopoietic stem cells.

[0064] The applications or methods provided by this invention can be for the purpose of diagnosing and treating diseases, or for the purpose of diagnosing and treating non-diseases.

[0065] To solve the above-mentioned technical problems, in a sixth aspect, the present invention provides the above-mentioned protein CCL3 or protein YY1 or protein GILZ, or the above-mentioned biological material B1 or / and biological material B2.

[0066] In one embodiment of the invention, the level of aging of the body and / or cells and / or tissues is assessed by evaluating the reconstitution capacity of hematopoietic stem (progenitor) cells.

[0067] In one embodiment of the invention, the reconstitution capacity of hematopoietic stem (progenitor) cells is evaluated through a competitive transplantability experiment.

[0068] In one embodiment of the present invention, the improvement in the reconstructive capacity of hematopoietic stem (progenitor) cells is specifically manifested as follows: in the competitive transplantation experiment, the proportion of T cells from donors of aged mice overexpressing protein CCL3 was significantly higher (P<0.05) than that of the control group; the proportion of whole cells, T cells, and Myeloid cells from donors of aged mice overexpressing YY1 was significantly higher (P<0.05) than that of the control group.

[0069] In one embodiment of the invention, the level of aging of the body and / or cells and / or tissues is assessed by evaluating the proliferative capacity of fibroblasts.

[0070] In one embodiment of the present invention, the proliferative capacity of fibroblasts is assessed by Ki67 staining assay and the percentage of apoptotic cells.

[0071] In one embodiment of the present invention, the reduction in fibroblast proliferation capacity of the transgenic cell line is specifically manifested in the following ways: the number of Ki67-stained cells in human and mouse fibroblasts of the Gilz gene RNA interference group (si-GILZ) is significantly lower than that in the control group (si-NC), indicating that the cell proliferation capacity of the RNA interference group (si-GILZ) is reduced; and the proportion of apoptotic cells in human and mouse fibroblasts of the RNA interference group (si-GILZ) (7.81%, 7.89%) is significantly lower than that in the control group (11.80%, 12.20), indicating that the apoptosis level of the RNA interference group (si-GILZ) is increased.

[0072] In one embodiment of the present invention, the heterosymbiotic animal is a heterosymbiotic mouse model.

[0073] In one embodiment of the invention, the method for preparing the mouse heterologous symbiotic model is as follows: Hair is shaved along a continuous line from the elbow, flank, and knee of the side to be connected in each of the two mice. The skin is prepared by wiping with iodine disinfectant and 70% alcohol for more than three alternating cycles. An autoclave is used while maintaining a sterile area. On each mouse, the skin is incised along the flank from proximal to proximal to the elbow, without disturbing the subcutaneous muscle. The triceps muscles of the animals are connected with two interrupted sutures. The lateral sutures are then made 7-9 times along the body wall. The quadriceps muscles of the animals are connected with two interrupted sutures. The skin of the two individuals is then sutured with interrupted sutures. The mice are then placed supine on a heating pad and allowed to awaken in ambient air. After the parabolic surgery, each pair is individually fed and given a subcutaneous injection of 1 ml of 0.25% bupivacaine in saline solution daily for three days.

[0074] In one embodiment of the invention, the reduction in the proportion of senescent cells in spleen and / or skin and / or liver and / or brain tissue is manifested in reducing SA-β-gal staining positive areas in spleen and / or skin and / or liver and / or brain tissue.

[0075] The reduction of liver tissue inflammation is reflected in the reduction of the area of ​​inflammatory cell infiltration in liver tissue stained by HE.

[0076] The reduction in apoptosis in spleen and / or skin and / or liver and / or skeletal muscle tissues is reflected in the proportion of positive cells in TUNEL staining.

[0077] The reduction in liver and / or spleen fibrosis is reflected in the positive area of ​​Masson staining.

[0078] The increase in skeletal muscle fiber diameter is reflected in the measurement of muscle fiber area during HE staining.

[0079] The increase in the number of hair follicles in skin tissue is reflected in the changes in the number of hair follicles in HE staining.

[0080] In the above applications, the delay in the aging of hematopoietic stem and progenitor cells is reflected in enhancing the reconstructive capacity and / or lineage differentiation capacity of aged hematopoietic stem (progenitor) cells;

[0081] The expression levels of YY1 and / or CCL3 are both protein expression levels.

[0082] The expression levels of Yy1 and / or Ccl3 are both gene expression levels.

[0083] In the above applications, the peripheral blood, bone marrow, spleen tissue, brain tissue, liver tissue, skeletal muscle tissue, and skin tissue are peripheral blood, bone marrow, spleen tissue, brain tissue, liver tissue, skeletal muscle tissue, and skin tissue of mammals, and the mammals include humans.

[0084] This invention systematically analyzed the single-cell transcriptomes of the hematopoietic and immune systems (hematopoietic stem and progenitor cells, bone marrow, peripheral blood, and spleen) and four solid tissues / organs (skin, skeletal muscle, brain, and liver) sampled from isochronic parabiosis (Mus musculus) mice. This approach allows for the construction of a multi-tissue single-cell transcriptome atlas to study the effects of hematopoietic stem cell (HP) on aging and to explore the complex changes and regulatory roles of adult stem cells and their microenvironment. This invention analyzed how a youthful circulatory system environment revitalizes older individuals and how an aged circulatory system environment damages younger individuals, thereby altering cell populations, gene expression characteristics, and cell-cell communication. In our heterosymbiotic atlas, this invention found that hematopoietic stem and progenitor cells are among the most resistant to mutual infiltration and most sensitive to systemic regulation in the hematopoietic and immune systems. Furthermore, this invention also identified regulatory factors with the potential to reverse age-related damage. This study can advance our understanding of age-related systemic changes and help us develop novel age intervention strategies. Attached Figure Description

[0085] Figure 1 SA-β-Gal staining analysis was performed on different tissues of four groups of mice.

[0086] Figure 2 TUNEL staining was used to analyze the proportion of apoptosis in different tissues of four groups of mice.

[0087] Figure 3 HE staining analysis of liver, skin and skeletal muscle of four groups of mice.

[0088] Figure 4 MASSON staining analysis of liver and spleen in four groups of mice.

[0089] Figure 5 Analysis of pseudo-temporal differentiation trajectories in the hematopoietic system.

[0090] Figure 6 Gene sets related to hematopoietic stem cell differentiation were scored for four groups of mice.

[0091] Figure 7 The change in the proportion of B cells in the single-cell data of four groups of mice.

[0092] Figure 8To detect changes in the proportion of bone marrow B cells in different groups of mice using flow cytometry.

[0093] Figure 9 Statistical data on the proportion of pro-B cells to total B cells in bone marrow of mice in different groups.

[0094] Figure 10 To detect changes in the proportion of pro-B cells in the bone marrow of mice in different groups and to collect statistical data using flow cytometry.

[0095] Figure 11 A schematic diagram of differential gene computation in aging and xenobiotic mice.

[0096] Figure 12 This is a cell type-specific differential gene regulatory network.

[0097] Figure 13 Analysis of the regulatory network of core transcription factors.

[0098] Figure 14 The core regulatory transcription factor expression levels and target gene scores are used to determine the expression levels of transcription factors.

[0099] Figure 15 This is an intercellular ligand-receptor pair that is restored through heterologous symbiosis.

[0100] Figure 16 To verify the efficiency of YY1 overexpression in mouse LSK cells at the WB level.

[0101] Figure 17 To assess the function of aged hematopoietic stem (progenitor) cells infected with different viruses, schematic diagrams and line graphs are used to demonstrate the ability of overexpression of YY1 to enhance the reconstitution capacity of aged hematopoietic stem (progenitor) cells.

[0102] Figure 18 To verify the efficiency of CCL3 overexpression in mouse LSK cells at the WB level.

[0103] Figure 19 The line graph illustrates the ability of CCL3 overexpression to enhance the remodeling capacity of aged hematopoietic stem (progenitor) cells.

[0104] Figure 20 The bar chart illustrates the effect of CCL3 overexpression on the differentiation of hematopoietic stem (progenitor) cell lineages in older individuals.

[0105] Figure 21 It is a cross-cell type allogeneic symbiotic recovery factor - Tsc22d3 (Gilz).

[0106] Figure 22 To determine the knockdown efficiency of the Gilz gene in human and mouse skin fibroblasts by RT-qPCR.

[0107] Figure 23Immunofluorescence assays of Ki67 were used to demonstrate the proliferation capacity of human and mouse fibroblasts after GILZ / Gilz knockdown.

[0108] Figure 24 Apoptosis detection was used to assess the proliferation capacity of human and mouse fibroblasts after GILZ / Gilz knockdown. Detailed Implementation

[0109] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0110] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from regular biochemical reagent stores. All animal experiments in the following examples were approved by the Animal Protection and Utilization Committee of the Chinese Academy of Sciences. The isolation of human adipose-derived stem cells in the following examples has been reviewed by the Ethics Committee of Peking Union Medical College.

[0111] The experimental data in the following examples were processed using GraghPad Prism 8 statistical software. The experimental results are expressed as mean ± standard deviation. One-tailed t-tests and two-tailed t-tests were used. P < 0.05 (*) indicates statistical significance, P < 0.01 indicates extremely significant difference (**), and P < 0.001 indicates extremely significant difference (***).

[0112] The biological materials used in the following examples were obtained from the following sources: C57BL6 / J mice were purchased from Beijing SPF Biotechnology Co., Ltd. Isoflurane was purchased from Reward Biotech, catalog number R510-22. 4-0 poly(dioxane) suture material and 4-0 polypropylene suture material were purchased from Jinhuan Medical, catalog number F406. The masson kit was purchased from Solarbio, catalog number G1346. The TUNEL kit was purchased from Beyotime Biotech, catalog number C1088.

[0113] Hematopoietic stem cell culture medium: SFEM medium (Stem Cell Technology) with added SCF (final concentration 20 ng / mL, PeproTech), TPO (final concentration 20 ng / mL, PeproTech) and penicillin-streptomycin (final concentration 1%, Gibco).

[0114] HBSS +Solution: Hanks balanced salt solution (HBSS, Solarbio) with 2% FBS and 1% N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES, Solarbio).

[0115] 293T cell culture medium: 89 parts DMEM high glucose medium (Hyclone), 10 parts fetal bovine serum (Gibco), 1 part penicillin / streptomycin (Gibco).

[0116] The biological materials used in the following examples are from the following sources:

[0117] Human embryonic kidney 293T cells: ATCC, CRL-3216;

[0118] Lentiviral packaging vector psPAX2 (plasmid psPAX2 for short): Addgene product, #12260;

[0119] Lentiviral packaging vector pMD2G (pMD2G for short): Addgene product, #12259;

[0120] The SF-LV-EGFP vector is described in the literature “Aging-induced IL27Ra Signaling Impairs Hematopoietic Stem Cells. He H, et al. Blood. 2020 Jul 9;136(2):183-198.” (the name in the literature is SF-LV-cDNA-EGFP vector.). The public can obtain this biological material from the applicant for the purpose of repeating the relevant experiments of this invention only, and it may not be used for other purposes.

[0121] SF-LV-Yy1-EGFP vector: Constructed on the SF-LV-EGFP vector.

[0122] SF-LV-Ccl3-EGFP vector: Constructed on the SF-LV-EGFP vector.

[0123] In the following embodiments, the following are examples: FITC anti-mouse Lineage Cocktail with Isotype Ctrl (clone: ​​145-2C11; RB6-8C5; RA3-6B2; Ter-119; M1 / 70), catalog number 133302; Ki67 (abcam, catalog number: 15580); APC anti-mouse Ly-6A / E (Sca-1) (clone: ​​D7), catalog number 108112; PerCP anti-mouse CD117 (c-Kit) (clone: ​​2B8), catalog number 105822; FITC anti-mouse / human CD45R / B220 (clone: ​​RA3-6B2), catalog number 103206; Brilliant Violet 421. TM Biotin anti-mouse IgM (clone: ​​RMM-1), catalog number 406518; APC anti-mouse CD43 (clone: ​​S11), catalog number 143208; Biotin anti-mouse CD3ε (clone: ​​145-2C11), catalog number 100304; Biotin anti-mouse CD4 (clone: ​​RM4-5), catalog number 100508; Biotin anti-mouse CD8a (clone: ​​53-6.7), catalog number 100704; Biotin anti-mouse / human CD45R / B220 (clone: ​​RA3-6B2), catalog number 103204; Biotin anti-mouse / human CD11b (clone: ​​M1 / 70), catalog number 101204; Biotin anti-mouse Ly-6G / Ly-6C(Gr-1) (clone:RB6-8C5), catalog number 108404; Biotin anti-mouse TER-119 / Erythroid Cells (clone:TER-119), catalog number 116204; PerCP / Cyanine5.5 anti-mouse CD48 (clone:HM48-1), catalog number 103422; APC anti-mouse CD3ε (clone:145-2C11), catalog number 100312; PerCP anti-mouse / human CD11b (clone:M1 / 70), catalog number 101230; Brilliant Violet 605 TMAnti-mouse / human CD45R / B220 (clone: ​​RA3-6B2), catalog number 103244; PE anti-mouse CD45.1 (clone: ​​A20), catalog number 110708, all antibodies were purchased from Biolegend. APC anti-mouse CD117 (c-Kit) (clone: ​​2B8), catalog number 17-1171-83; PE-Cy TM 7Sca-1 anti-mouse (clone:D7), part number: 25-5981-82; Streptavidin Conjugate APC-eFluor TM 780 (catalog number 47-4317-82), DAPI (catalog number 62248), and Propidium iodide (catalog number P3566) were all purchased from Invitrogen. 7-AAD Staining Solution (catalog number 559925) was purchased from BD Biosciences. Mouse monoclonal anti-YY1 (catalog number sc-7341) and Mouse monoclonal anti-CCL3 (catalog number sc-365691) were both purchased from Santa Cruz Biosciences. Anti-APC MicroBeads (catalog number 130-090-855) were purchased from Miltenyi Biotec; mouse and human siRNA were purchased from Ribobio.

[0124] Example 1: The restorative effect of heterologous symbiosis on the aging phenotype in mice.

[0125] I. Experimental Grouping

[0126] The age and sex information of the three groups of C57BL6 / J mice are as follows: in vivo young mice (Iso-Y) (2 months old, n=7, male mice), allogeneic young mice (Het-Y) and allogeneic aged mice (Het-O) (2 months old and 23 months old, n=5, male mice, respectively) and in vivo aged mice (Iso-O) (23 months old, n=9, male mice).

[0127] In addition, to trace and differentiate the donor / host cells, we used the CD45.1 / CD45.2 homologous system to prepare allogeneic young mice (2 months old, n=4, CD45.1 male mice) and allogeneic aged mice (23 months old, n=4, CD45.2 male mice). Each pair of allogeneic mice was kept in the same cage for at least two weeks prior to surgery. The mice's physical condition and overall appearance were checked daily.

[0128] The surgical procedure is as follows:

[0129] Administer analgesia (buprenorphine, SR / sustained release, 0.1 mg / kg subcutaneously) after induction and before surgery. Apply eye lubricant. Use 4-0 poly(dioxane) suture material for all internal sutures. Use 4-0 polypropylene suture material for all external sutures. Shave each of the two mice along a continuous line from the elbow, flank, and knee on the side to be joined. Prepare the incision by wiping the skin with iodine and 70% alcohol for more than three alternating cycles. Use autoclave and maintain a sterile area. On each mouse, make an incision along the flank with scissors, from proximal to the elbow, without disturbing the subcutaneous muscle. Connect the animal's triceps muscle with two interrupted sutures. Suture continuously 7–9 times along the flank connecting body wall. Connect the animal's quadriceps muscle with two interrupted sutures. Suture the skin of the two accessory bodies with interrupted sutures. Then place the mice in a supine position on a heating pad and allow them to wake up in ambient air. Following the parabolic symbiotic procedure, each pair was individually housed and administered a subcutaneous injection of 1 ml of 0.25% bupivacaine saline solution daily for three days. In the xenobiotic group, younger mice were paired on the left side, and older mice on the right side. Tissue samples were collected and histologically examined six weeks post-surgery.

[0130] II. Experimental Methods:

[0131] (1) SA-β-gal staining

[0132] Spleen, skin, liver, and brain tissue from mice in each group were collected for aging-related β-galactosidase staining analysis. Aging-related β-galactosidase staining is a method for detecting aging tissues based on the upregulation of SA-β-gal (senescence-associated β-galactosidase) activity during aging.

[0133] The specific steps are as follows: Frozen sections of mouse tissue were thawed to room temperature, washed with PBS, and fixed with 2% formaldehyde and 0.2% glutaraldehyde for 15 minutes at room temperature. They were then stained with staining solution at 37°C for 48-96 hours. The slides were then fixed with 80% glycerol. Images were obtained using a confocal laser scanning microscope (Leica, CS2) with X-Gal as the substrate, which produces a deep blue product under the catalysis of aging-specific β-galactosidase. The aging of the tissue can be observed under a regular optical microscope, and the percentage or intensity of SA-β-gal positive areas can be quantified using ImageJ.

[0134] The staining solution formula is as follows: 40mM citrate / sodium phosphate buffer, 5mM K4[Fe(CN)6]·6H2O, 5mM K3[Fe(CN)6], 150mM NaCl, 2mM MgCl2, and 1mg / ml X-gal.

[0135] (2) TUNEL staining

[0136] Paraffin-embedded tissues were sectioned to a thickness of 10 μm using a rotary microtome. Sections were dewaxed in xylene, rehydrated in a gradient of ethanol (100%, 100%, 95%, 80%, 70%), and stained with TUNEL using a TUNEL apoptosis detection kit. Images were acquired using a confocal laser scanning microscope (Leica TCS SP5Ⅱ), and the percentage of positive cells was quantified using ImageJ.

[0137] (3) HE staining

[0138] Paraffin-embedded tissues were sectioned to a thickness of 5 μm using a rotary microtome. Sections were dewaxed in xylene, rehydrated in a gradient of ethanol (100%, 100%, 95%, 80%, 70%), incubated in hematoxylin solution, rinsed in tap water to remove excess hematoxylin, differentiated with 1% acidic ethanol for 5 seconds, and then rinsed in tap water for 10 min. Finally, sections were stained with eosin, dehydrated in a gradient of ethanol and xylene, mounted with neutral resin, and images were collected under a microscope. The area of ​​positive regions was quantified using ImageJ.

[0139] (4) Masson staining

[0140] After paraffin embedding, tissue sections were cut into 5 μm thick sections, dewaxed with xylene, and hydrated with 100% ethanol, 95% ethanol, 70% ethanol, and tap water. The sections were then stained overnight with potassium dichromate solution (concentration). After rinsing with tap water for 5–10 minutes, the sections were stained for 10 minutes in iron hematoxylin working solution and 5–10 minutes in carmine and acid fuchsin solution. The slides were then differentiated in phosphomolybdic phosphotungstic acid solution for 10–15 minutes and stained with aniline blue solution for 5–10 minutes. They were then briefly rinsed in distilled water and differentiated in 1% acetic acid solution for 2–5 minutes. The sections were then dehydrated with 70% ethanol, 95% ethanol, and 100% ethanol, removed with xylene, covered with coverslips, and mounted with neutral resin. Images were obtained using a confocal laser scanning microscope (Leica, CS2), and the area of ​​positive regions was quantified using ImageJ.

[0141] III. Experimental Results

[0142] SA-β-gal staining results are as follows Figure 1 As shown, Figure 1Microscopic images of age-related galactosidase-positive cells in the spleen, skin, liver, and brain of mice from the in vivo young group, in the in vivo aged group, in the allogeneic young group, and in the allogeneic aged group are presented, along with bar charts showing the percentage of SA-β-gal positive regions quantified by ImageJ. The vertical axis of the bar chart represents the percentage of SA-β-gal positive regions quantified by ImageJ, and the horizontal axis of the bar chart, from left to right, represents the mice in the in vivo young group, the in vivo aged group, the allogeneic young group, and the allogeneic aged group, respectively.

[0143] Figure 1 The results showed that the proportion of age-related galactosidase-positive areas in the spleen, skin, and liver of the allogeneic young group mice was significantly lower than that in the allogeneic aged group mice (P < 0.001 (skin and liver) or P < 0.01 (spleen) or P < 0.05 (brain)). There was no significant difference in the proportion of age-related galactosidase-positive areas in the spleen, skin, and brain between the allogeneic young group mice and the allogeneic aged group mice (P > 0.05). However, the proportion of age-related galactosidase-positive areas in the liver of the allogeneic young group mice was significantly lower than that in the allogeneic aged group mice (P < 0.05).

[0144] The percentage of age-related galactosidase-positive regions in the spleen, skin, liver, and brain of allogeneic young mice and allogeneic aged mice was between that of the in vivo young group and the in vivo aged group. Compared with the in vivo aged mice, the allogeneic aged mice showed a reduction in age-related galactosidase-positive regions in the spleen, skin, liver, and brain, indicating that age-related phenotypes were alleviated at the cellular level in multiple organs.

[0145] TUNEL staining results are as follows: Figure 2 As shown, Figure 2 Microscopic images of apoptotic cells in the spleen, skin, liver, and skeletal muscle of mice from the in vivo young group, in vivo aged ancestor group, allogeneic young group, and allogeneic aged group are presented, along with bar charts quantified by ImageJ to show the percentage of TUNEL-positive cells. In the bar charts, the vertical axis represents the percentage of apoptotic cells, and the horizontal axis, from left to right, represents the mice from the in vivo young group, in vivo aged ancestor group, allogeneic young group, and allogeneic aged group, respectively.

[0146] Figure 2 The results showed that the proportion of apoptotic cells in the spleen, skin, liver, and skeletal muscle of the allogeneic young group mice was significantly lower than that in the allogeneic aged group mice (P < 0.001 (spleen) or P < 0.01 (liver and skeletal muscle) or P < 0.05 (skin)); there was no significant difference in the proportion of apoptotic cells in the skin, liver, and skeletal muscle between the allogeneic young group mice and the allogeneic aged group mice (P > 0.05), but the proportion of apoptotic cells in the spleen of the allogeneic young group mice was significantly lower than that of the allogeneic aged group mice (P < 0.05).

[0147] The percentage of apoptotic cells in the spleen, skin, liver, and skeletal muscle of allogeneic young mice and allogeneic aged mice was between that of the endologous young group and the endologous aged group. Compared with endologous aged mice, the number of apoptotic cells in the spleen, skin, liver, and skeletal muscle of allogeneic aged mice was reduced, indicating that the aging-related phenotype was alleviated at the cellular level in multiple organs.

[0148] SA-β-gal staining and TUNEL staining analysis showed that, compared with genotype aged mice, allogeneic aged mice had a reduced number of age-related galactosidase-positive cells in the spleen, skin, liver, and brain. Figure 1 A decrease in apoptotic cells in the spleen, skin, liver, and skeletal muscle. Figure 2 This indicates that aging-related phenotypes are alleviated at the cellular level in multiple organs.

[0149] HE staining results are as follows Figure 3 As shown, Figure 3 Microscopic images and corresponding bar charts of the percentage of liver inflammatory infiltration area, the number of hair follicles per unit area of ​​skin, and the diameter of skeletal muscle fibers in mice from the in vivo young group, in vivo aged ancestor group, allogeneic young group, and allogeneic aged group are presented. The horizontal axis of the bar charts represents the mice from the in vivo young group, in vivo aged ancestor group, allogeneic young group, and allogeneic aged group, respectively, from left to right.

[0150] Figure 3 The results showed that the inflammatory infiltration area in the liver of the syngeneic aged group mice was significantly higher than that in the syngeneic young group mice (P < 0.001); there was no significant difference in the inflammatory infiltration area in the liver between the allogeneic young group mice and the allogeneic aged group mice (P > 0.05), and the inflammatory infiltration area in the liver of the allogeneic young group mice and the allogeneic aged group mice was between that of the syngeneic young group mice and the syngeneic aged group mice; the number of hair follicles per unit area in the syngeneic young group mice was significantly higher than that in the syngeneic aged group mice (P < 0.001); the number of hair follicles per unit area in the allogeneic young group mice and the allogeneic aged group mice was significantly higher than that in the syngeneic aged group mice. There was no significant difference in the number of hair follicles among mice (P > 0.05), and the number of hair follicles in the allogeneic young group and the allogeneic aged group was between that in the homologous young group and the homologous aged group. The diameter of muscle fibers in the skeletal muscle of the homologous aged group was significantly higher than that in the homologous young group (P < 0.001), and there was no significant difference in the diameter of muscle fibers in the skeletal muscle between the allogeneic young group and the allogeneic aged group (P > 0.05), and the diameter of skeletal muscle in the allogeneic young group and the allogeneic aged group was between that in the homologous young group and the homologous aged group.

[0151] The results of the masson staining are as follows: Figure 4 As shown, Figure 4Microscopic images of liver and spleen fibrosis in mice from the same young group, same aged group, allogeneic young group, and allogeneic aged group are presented, along with bar charts quantified by ImageJ to show the percentage of fibrosis. The vertical axis of the bar chart represents the fibrosis area in mm². 2 The horizontal axis of the bar chart, from left to right, represents the mice in the same young group, the same old ancestor group, the different young group, and the different old group.

[0152] Figure 4 The results showed that the fibrotic area in the liver and spleen of the aged group mice was higher than that of the young group mice (P < 0.001); there was no significant difference in the fibrotic area in the liver and spleen between the young group mice and the aged group mice (P > 0.05), and the fibrotic area in the liver and spleen of the young group mice and the aged group mice was between that of the young group mice and the aged group mice.

[0153] HE staining and Masson staining analysis showed that, compared with in vivo aged mice, allogeneic aged mice reduced age-related inflammation in the liver, reduced fibrosis in the liver and spleen, and restored the average diameter of skeletal muscle fibers and the number of hair follicles in the skin (which usually decrease with age) in a “rejuvenating” manner.

[0154] Example 2: Effects and Verification of Heterologous Symbiosis on Cell Ratio Changes

[0155] I. Experimental Methods:

[0156] Using the three groups of experimental mice cultured for 6 weeks in Example 1 as experimental samples, the following operations were performed.

[0157] 1. Tissue and cell isolation and sequencing

[0158] (1) Isolation of peripheral blood cells

[0159] Mouse peripheral blood was collected using EDTA-2Na anticoagulant tubes. Red blood cell lysis buffer (BD Biosciences) was added, and cells were lysed at room temperature for 15-20 minutes. Cells were washed twice with pre-chilled PBS and resuspended in PBS containing 2% FBS. Viable cells were sorted using flow cytometry (BDInflux), centrifuged, and resuspended in PBS containing 0.04% BSA for 10x Genomics sequencing.

[0160] (2) Isolation of bone marrow cells

[0161] The femur and tibia of mice were isolated. Bone marrow cells were flushed from the bone marrow cavity using PBS buffer containing 2% FBS and 2mM EDTA. The cell resuspended cells were filtered through a 40μm cell filter to obtain a single-cell suspension. The cells were centrifuged at 1,200 rpm and 4°C for 5 minutes. The cell pellet was resuspended in erythrocyte lysis buffer (BD Biosciences) and lysed at room temperature for 5 minutes. The cells were washed twice with pre-chilled PBS and resuspended in PBS containing 2% FBS. Viable cells were sorted using a flow cytometer (BD Influx), centrifuged, and resuspended in PBS containing 0.04% BSA for 10x Genomics sequencing.

[0162] (3) Lin - c-Kit + Sca-l lo / + Cell separation

[0163] The isolated bone marrow cells were resuspended in PBS with 2% FBS, and then mixed thoroughly with the Lineage mixed antibody, c-Kit, and Sca-1 antibody. The mixture was incubated at 4°C in the dark for 30 minutes. The Lineage mixed antibody included anti-mouse CD3, B220, CD11b, Ly6G / Ly-6C, and Ter-119. The cells were washed twice with PBS and resuspended in PBS with 2% FBS. Sorting was performed using a flow cytometer (BDInflux), and after centrifugation, the cells were resuspended in PBS containing 0.04% BSA for 10x Genomics sequencing.

[0164] (4) Separation of spleen cells

[0165] Mouse spleens were placed in PBS containing 2% FBS, cut into small pieces with scissors, and gently ground using a 5 mL syringe plunger on a 40 μm cell filter. All spleen cells were collected and centrifuged at 1,200 rpm, 4 °C for 5 min. The cell pellet was resuspended in erythrocyte lysis buffer (BD Biosciences) and lysed at room temperature for 5 min. The cells were washed twice with pre-chilled PBS and resuspended in PBS containing 2% FBS. Viable cells were sorted using a flow cytometer (BD Influx), centrifuged, and resuspended in PBS containing 0.04% BSA for 10x Genomics sequencing.

[0166] 2. Cell ratio analysis

[0167] (1) Data preprocessing

[0168] After obtaining the raw high-throughput sequencing data from the NovaSeq platform, the raw BCL format files were first converted to FASTQ format files using bcl2fastq software (version 2.20.0.422). The FASTQ files were then processed using Cell Ranger software (version 3.1.0), aligned to the mm10 mouse reference genome, and a filtered expression matrix was obtained.

[0169] (2) Cell clustering and cell type identification

[0170] Gene expression matrices from single-cell transcriptomes obtained from LSK cells, bone marrow, peripheral blood, spleen, and skin immune cells were analyzed using Scanpy (version 1.4.4) for low-quality cell filtering, sample integration, data standardization, dimensionality reduction, cell clustering, and differential gene expression analysis. First, scrublet software (version 0.2.1) was used to detect and remove potential multi-cell results. The filtered expression matrices were then analyzed according to the following steps: ① Cells meeting any of the following four criteria were first removed: fewer than 500 or more than 6000 genes, fewer than 500 or more than 40000 unique molecular markers (UMIs), mitochondrial gene proportion exceeding 10%, and ribosomal gene proportion exceeding 40%; ② Data from different samples were integrated and standardized using Scanpy's standard workflow, and batch effects between samples were removed using the "sc.external.pp.bbknn" function; ③ Scanpy's "sc.tl.umap", "sc.pp.neighbors", and "sc" functions were used to analyze the data. The "tl.louvain" function performs dimensionality reduction analysis and data clustering based on the first 75 principal components; ④ Differential expression analysis between different cell populations is performed using the "FindAllMarkers" function of the Seurat software package (version 3.2.3) (Wilcoxon rank-sum test), and the fold difference (FC) is calculated. The screening criteria are a corrected P-value less than 0.05 and |logFC| greater than 0.5; ⑤ Cell populations with high expression of Gm42418 or AY036118 genes are further eliminated, and the analysis in steps 2 to 4 is repeated. Then, the cell type is determined based on the classic cell type marker genes of each cell population.

[0171] Following the above process, we obtained a single-cell atlas of the mouse hematopoietic and immune systems containing 74,323 high-quality cells.

[0172] (3) Single-cell pseudo-time trajectory analysis

[0173] PAGA analysis (iter=1000, layout=fa) using the Scanpy package was performed to construct developmental trajectories of single-cell atlases of the mouse hematopoietic and immune systems. Pseudo-temporal analysis was performed on randomly selected cells (3000 cells per group) using the Monocle2 package. The gene selection threshold for ranking was that the cells were expressed in at least 10 cells and the q-value characterizing inter-population expression differences and dispersion was less than 0.01. The DDRTree dimensionality reduction algorithm was used to plot the trajectory structure on a two-dimensional plane, and pseudo-temporal ranking was performed on the randomly selected cells. The Monocle2 "BEAM_rest" function was used to identify 1000 differentially expressed genes (DEGs) clustered into 6 gene clusters (P-value < 0.001), and the Monocle2 "plot_genes_branched_heatmap" function was used to plot the expression changes of these genes on the pseudo-temporal trajectory.

[0174] (4) Gene set scoring analysis

[0175] The expression of the corresponding gene set in each cell was scored using Seurat's "AddModuleScore" function. The changes in gene set scores among the in vivo young, in vivo old, allogeneic young, and allogeneic old groups were calculated using the Wilcoxon test of the ggpubr package (https: / / github.com / kassambara / ggpubr) (version 0.2.4).

[0176] (5) Analysis of changes in cell composition

[0177] Before performing the cell proportion change analysis, we first removed cell types that accounted for less than 2.5% of the total number of cells in each tissue. Data for the same cell type in different groups (Iso-Y, Het-Y, Iso-O, Het-O) were divided by the total number of cells in that group to obtain its corresponding cell proportion, thus further calculating the percentage of a specific cell type in each group. Next, we determined the cell types whose proportion changed due to aging based on the Log2FC of the difference in cell proportions between Iso-O and Iso-Y (|Log2FC|>0.5), and determined the cell types whose proportion changed due to allogeneic symbiosis based on the difference between the Het-Y and Iso-Y groups or between the Het-O and Iso-O groups (|Log2FC|>0.5).

[0178] 3. Conduct experimental verification on cells showing changes indicated by bioinformatics data.

[0179] Flow cytometry analysis was used to identify several cell types with significant changes indicated by bioinformatics data. The specific steps are as follows:

[0180] Frozen bone marrow cells were thawed from cryopreservation medium containing 10% DMSO and 90% FBS (Gibco), and B220, IgM, and CD43 antibodies were added. The cells were incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS (1200 rpm, 5 min, 4°C). Before analysis, 7-AAD staining was performed to exclude dead cells. The proportion of pro-B cells was detected using flow cytometry (BD Fortessa), and data analysis was performed using FlowJo software (Tree Star Inc.).

[0181] II. Experimental Results

[0182] The calculation yielded a set of 6 genes related to the differentiation of hematopoietic stem cells into myeloid and lymphoid lineages. Figure 5 Gene set scoring results for different gene sets among the four groups showed that the potential for hematopoietic stem cells to differentiate into myeloid cells increases during aging (gene set 2), while the potential to differentiate into lymphoid cells decreases (gene sets 5 and 6). Allogeneic symbiosis can restore the potential for hematopoietic stem cells to differentiate into lymphoid cells (gene sets 5 and 6). Figure 6 Cell proportion analysis of single-cell data revealed that the proportion of B cell progenitors (pro-B) decreased with aging, and xenobiotic therapy could effectively restore their reduced numbers. Figure 7 Compared with the iso-synthetic young group (Iso-Y), the proportion of pro-B cells was downregulated in the iso-synthetic aged group; compared with the iso-synthetic young group, the proportion of pro-B cells was downregulated in the allogeneic young group; compared with the iso-synthetic aged group, the proportion of pro-B cells was upregulated in the allogeneic aged group. Figure 8-10 These results are consistent with the bioinformatics analysis data.

[0183] Example 3: Analysis and Validation of Key Differential Genes in Hematopoietic Stem (Progenitor) Cells Due to Aging

[0184] I. Experimental Methods

[0185] 1. Calculation of differentially expressed genes

[0186] (1) Differential expression and cell type-specific differential gene network analysis

[0187] The Wilcoxon rank-sum test from the "FindMarkers" function of the Seurat software package (version 3.2.3) was used as the statistical method to perform differential expression analysis for each cell type among different groups (Iso-O / Iso-Y, Het-Y / Iso-Y, and Het-O / Iso-O). Cell types with fewer than three cells in a particular group were excluded before performing the differential expression analysis. Differentially expressed genes were calculated between the Iso-O and Iso-Y groups to obtain the aging-related differentially expressed gene set (aging DEGs) (|LogFC|>0.25, adjusted P-value<0.05); differentially expressed genes were calculated between the Het-O and Iso-O groups to obtain the HY DEGs associated with the heterologous symbiotic young group (|LogFC|>0.25, adjusted P-value<0.05); differentially expressed genes were calculated between the Het-O and Iso-O groups to obtain the HODEGs associated with the heterologous symbiotic aged group (|LogFC|>0.25, adjusted P-value<0.05) (see Appendix Table 4 for a detailed gene list). In the above differentially expressed gene sets, the Gm42418 and AY036118 genes, which may not be able to characterize biological changes, were deleted. Based on the above gene set, “reversing aging differential genes (aging-R DEGs)” were identified, namely genes that are significantly upregulated or downregulated in HO DEGs and significantly downregulated or upregulated in aging DEGs, respectively; and “promoting aging differential genes (aging-P DEGs)” were identified, namely genes that are significantly upregulated or downregulated in both HY DEGs and aging DEGs.

[0188] (2) Transcription factor (TF) regulatory network analysis

[0189] Transcription factor regulatory network analysis was performed using the standard analysis workflow of the SCENIC software package (version 1.1.2.2): Using the mm10 database of the RcisTarget software package (version 1.6.0) as a reference database, the GENIE3 software package (version 1.6.0) was called to construct gene expression regulatory networks based on differentially expressed genes in different cell types from bone marrow, peripheral blood, and spleen tissues. Enriched TF-binding motifs, predicted candidate target genes (regulons), and target gene activity information were obtained from RcisTarget, and the transcriptional regulatory network was visualized using the Cytoscape software package (version 3.7.2).

[0190] (3) Intercellular communication analysis

[0191] Intercellular communication analysis based on single-cell sequencing data was performed using the CellPhoneDB software package (version 1.1.0) (www.cellphonedb.org). Only receptors and ligands expressed in at least 10% of cells of a specific cell type were included in subsequent analyses; intercellular communication was discarded if either the ligand or receptor did not meet the criterion of expression in at least 10% of cells. The mean expression levels of each ligand-receptor pair were compared across different cell types, and significant ligand-receptor pairs (P-value < 0.01) were used to predict inter-tissue intercellular communication within the Iso-Y, Iso-O, and Het-O groups.

[0192] 2. Preparation of viral fluid containing recombinant lentiviruses overexpressing the Yy1 or Ccl3 genes

[0193] (1) Construction of recombinant plasmids

[0194] The mouse Yy1 and Ccl3 genes were amplified from the cDNA of bone marrow cells of C57BL6 / J mice by PCR and ligated into the SF-LV-EGFP vector digested with Mlu1 and BamH1 (NEB) enzymes. This yielded the recombinant lentiviral plasmids SF-LV-Yy1-EGFP (a recombinant expression vector obtained by replacing the sequence between the Mlu1 and BamH1 recognition sites of SF-LV-EGFP with the mouse Yy1 gene, while keeping the other nucleotide sequences of SF-LV-EGFP unchanged) and SF-LV-Ccl3-EGFP (a recombinant expression vector obtained by replacing the sequence between the Mlu1 and BamH1 recognition sites of SF-LV-EGFP with the mouse Ccl3 gene, while keeping the other nucleotide sequences of SF-LV-EGFP unchanged).

[0195] The amino acid sequence of the mouse CCL3 protein is represented by the GenBank number NP_035467.1. The nucleic acid molecule encoding the mouse CCL3 protein is the mouse Ccl3 gene, which is represented by the GenBank number NM_011337.2. Its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 102-380 of NM_011337.2.

[0196] The amino acid sequence of the mouse YY1 protein has the GenBank number NP_033563.2, the nucleic acid molecule encoding the mouse YY1 protein is the mouse Yy1 gene, and the GenBank number of the mouse Yy1 gene is NM_009537.4. Its coding sequence is the DNA molecule shown in positions 117-1361 of NM_009537.4.

[0197] The Yy1-F and Yy1-R sequences are as follows:

[0198] Yy1-F:5'-CG ACGCGT GCCACCATGGCCTCGGGCGACACCCTCTACAT-3' (underlined indicates Mlu1 identification site);

[0199] Yy1-R: 5'-CGC GGATCC TCACTGGTTGTTTTTGGCTTTAGCGTGT-3' (underlined indicates BamH1 recognition site).

[0200] The Ccl3-F and Ccl3-R sequences are as follows:

[0201] Ccl3-F:5'-CG ACGCGT GCCACCATGAAGGTCTCCACCACTGCCCTTGCTG-3' (underlined to indicate Mlu1 recognition site);

[0202] Ccl3-R: 5'-CGC GGATCC TCAGGCATTCAGTTCCAGGTCAGTGATG-3' (underlined indicates BamH1 recognition site).

[0203] (2) Preparation of recombinant virus

[0204] 2-1) Using the Lipo3000 transfection kit (Thermo Fisher Scientific), 10 μg of SF-LV-EGFP, SF-LV-Yy1-EGFP, or SF-LV-Ccl3-EGFP lentiviral plasmid, 10 μg of psPAX2 plasmid, and pMD2G plasmid were co-transfected into one 10 cm dish of 293T cells. After culturing for 8 hours, the culture medium was replaced.

[0205] 2-2) Collect the supernatant at 24h, 36h and 72h, filter it with a 0.22μm filter, and store the obtained viral supernatant at 4℃.

[0206] 2-3) Centrifuge at 19400 rpm and 4℃ for 2.5 hours, discard the supernatant, and resuspend in hematopoietic dry culture medium to obtain recombinant lentivirus LV / SF-LV-EGFP (VC), recombinant lentivirus LV / SF-LV-Yy1-EGFP (expressing Yy1 gene) and recombinant lentivirus LV / SF-LV-Ccl3-EGFP (expressing Ccl3 gene), respectively, and store at -80℃.

[0207] 3. LSK cell isolation and lentiviral transfection

[0208] (1) LSK cell isolation

[0209] 1-1) Euthanize 24-month-old mice (C57BL6 / J, CD45.2, 4-6 mice), carefully dissect the femur, tibia, and buttock, place them in a sterile mortar, and add 5 mL of pre-cooled HBSS. + The bone marrow cells were separated by gently crushing the bone with a grinding rod. The bone marrow cells were then filtered through a 40μm cell filter to obtain a single-cell suspension, which was collected by centrifugation at 450g and 4℃ for 5 minutes.

[0210] 1-2) After resuspending the cells, add c-Kit-APC antibody, incubate at 4°C in the dark for 30 minutes, then add HBSS. + After centrifugation and washing of the solution, anti-APC microbeads were added and incubated at 4°C in the dark for 30 minutes.

[0211] 1-3) Enrichment of c-Kit using MACS magnetic columns (Miltenyi Biotec, Germany) + Cells. Magnetic column first using HBSS + Rinse the cells with the solution; add the stained cells to HBSS. + The solution was transferred to a magnetic column, and after c-Kit negative cells had flowed through, HBSS was added. + Rinse once with the solution; remove the magnetic column from the magnetic field and add HBSS. + Pour the solution into the magnetic column, slowly eject the cells using the stopcock, and repeat once. Centrifuge at 450g, 4℃ for 5 minutes, and collect c-Kit. + cell;

[0212] 1-4) Add the Lineage-Biotin mixed antibody to the c-Kit + In cells. Mix thoroughly and incubate at 4°C in the dark for 30 minutes. The Lineage-biotin mixed antibody includes: anti-mouse CD3, CD4, CD8, B220, CD11b, Ly6G / Ly-6C and Ter-119;

[0213] 1-5) Add HBSS + Solution, 450g, centrifuged at 4℃ for 5 minutes, and cells were collected;

[0214] 1-6) Add Strepavidin-APC-eFluor TM 780, Sca-1-PE-Cy TM 7. c-Kit-APC antibody, incubated at 4°C in the dark for 30 minutes, centrifuged at 450g, 4°C for 5 minutes, and cells were collected;

[0215] 1-7) Before sorting, DAPI was added for staining to exclude dead cells, and LSK cells were sorted using flow cytometry (BDFusion).

[0216] (2) Lentiviral transfection

[0217] 2-1)2×10 5 LSK cells were cultured in low-adhesion 96-well plates using HSC medium (Nest Biotech). After 2 hours, concentrated lentivirus (recombinant lentivirus LV / SF-LV-EGFP, recombinant lentivirus LV / SF-LV-Yy1-EGFP, or recombinant lentivirus LV / SF-LV-Ccl3-EGFP) was added for viral transfection efficiency titration. After 72 hours of transfection, cells (recombinant LSK cells transfected with recombinant lentivirus LV / SF-LV-EGFP or recombinant LSK cells transfected with recombinant lentivirus LV / SF-LV-Yy1-EGFP or recombinant LSK cells transfected with recombinant lentivirus LV / SF-LV-Ccl3-EGFP) were collected for Western blotting to verify overexpression efficiency.

[0218] 2-2) 72 h after transfection, collect cells by centrifugation at 450 g, 4 °C for 5 minutes, and add Sca-1-PE-Cy TM 7. CD48-PerCP / Cyanine 5.5 antibody, incubated at 4°C in the dark for 30 minutes, centrifuged at 450g, 4°C for 5 minutes, and the cells were collected;

[0219] 2-3) Before sorting, DAPI was added for staining to exclude dead cells, and GFP was sorted using flow cytometry (BDFusion). + Sca1 + CD48 - Cells to HBSS + In the solution, recombinant GFP cells were obtained separately. + Sca1 + CD48 - / LV / SF-LV-EGFP (recombinant cells obtained by transfection with LV / SF-LV-EGFP), GFP + Sca1 + CD48 - / LV / SF-LV-Yy1-EGFP (recombinant cells obtained by transfection with LV / SF-LV-Yy1-EGFP) and GFP + Sca1 + CD48 - / LV / SF-LV-Ccl3-EGFP (recombinant cells obtained by transfection with LV / SF-LV-Ccl3-EGFP).

[0220] 4. Transplantation and peripheral blood testing

[0221] (1) Mouse transplantation

[0222] 1-1) Mice to be irradiated: 8-week-old CD45.2 recipient mice (C57BL6 / J, 8-10 mice) were irradiated with an X-ray irradiator (RS-2000, Rad Source Technologies) before transplantation. The total dose was 10 Gy (divided into two doses of 5 Gy each, 3 hours apart).

[0223] 1-2) Preparation of competitive bone marrow cells: Euthanize 1-2 2-3 month old CD45.1 mice (C57BL6 / J), remove the femur, and separate the bone marrow single-cell suspension (HBSS). + The cells were resuspended in solution and counted using a Vi-Cell XR cell viability analyzer (Beckman Coulter) to obtain CD45.1 competitive bone marrow cells.

[0224] 1-3) 2000 GFPs were obtained through sorting + Sca1 + CD48 - Cell (GFP) + Sca1 + CD48 - / LV / SF-LV-EGFP or GFP + Sca1 + CD48 - / LV / SF-LV-Yy1-EGFP or GFP + Sca1 + CD48 - Add 2×10 to / LV / SF-LV-Ccl3-EGFP) 5 CD45.1 competitive bone marrow cells were transplanted into recipient mice treated with 1-1) via tail vein injection to obtain VC mice (injected with GFP). + Sca1 + CD48 - Mice obtained from competitive bone marrow cells of / LV / SF-LV-EGFP and CD45.1, and YY1 mice (injected with GFP) + Sca1 + CD48 - Mice obtained from competitive bone marrow cells of / LV / SF-LV-Yy1-EGFP and CD45.1, and CCL3 mice (injected with GFP) + Sca1 + CD48 - Mice obtained from competitive bone marrow cells of / LV / SF-LV-Ccl3-EGFP and CD45.1.

[0225] (2) Peripheral blood testing

[0226] 2-1) Four weeks after transplantation, blood was collected from the tail vein of VC mice, YY1 mice and CCL3 mice. CD45.1, CD11b, B220 and CD3 antibodies were added and incubated at 4°C in the dark for 30 minutes. The red blood cells were lysed with red blood cell lysis buffer, and after centrifugation and washing with PBS containing 2% FBS (1200 rpm, 5 min, 4°C), the cells were collected.

[0227] 2-2) Before analysis, DAPI or PI was added for staining to exclude dead cells. Flow cytometry (BDFortessa) was used to detect the chimerism rate of donor-derived cells and to analyze the proportional distribution of Myeloid cells, B cells and T cells.

[0228] 2-3) At 4-week intervals, the tail vein blood of transplanted mice was tested and analyzed using the above method.

[0229] 5. Western blotting to verify the overexpression efficiency of YY1 or CCL3

[0230] (1) Collect LSK cells transfected for 72 h and lyse them at 105 °C for 10 min using 1×SDS lysis buffer (containing 4% SDS and 100 mM Tris-HCl (pH = 6.8));

[0231] (2) Total protein was quantified using the BCA protein kit;

[0232] (3) Protein samples were subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Millipore);

[0233] (4) Add YY1 (Santa Cruz) or CCL3 (Santa Cruz) primary antibody and incubate overnight at 4°C, then incubate for 1 hour at room temperature with HRP-conjugated secondary antibody;

[0234] (5) Exposure and development.

[0235] II. Experimental Results

[0236] The results showed that 171 differentially expressed genes reversing aging (149 upregulated and 22 downregulated) and 540 differentially expressed genes related to aging (131 upregulated and 409 downregulated) were identified in bone marrow hematopoietic stem and progenitor cells, along with 119 differentially expressed genes promoting aging (96 downregulated and 23 upregulated). Figure 11This yields 36 heterologous symbiotic core genes. Among them, Zfp36 (Gene ID: 22695), Cd69 (Gene ID: 12515), Cxcl2 (Gene ID: 20310), Jun (Gene ID: 16476), Junb (Gene ID: 16477), Dusp2 (Gene ID: 13537), Ier2 (Gene ID: 15936), Ier3 (Gene ID: 15937), Nfkbiz (Gene ID: 80859), Ppp1r15a (Gene ID: 17872), Egr1 (Gene ID: 13653), Icam1 (Gene ID: 15894), Gadd45b (Gene ID: 17873), Cxcl10 (Gene ID: 15945), Nfkbia (Gene ID: 18035), Tnfaip3 (Gene ID: 21929), Etv3 (Gene ID: 27049), Ccl3 (Gene Genes Ccrl2 (Gene ID: 54199), Dntt (Gene ID: 21673), Rhob (Gene ID: 11852), Fosb (Gene ID: 14282), Tnf (Gene ID: 21926), Sox4 (Gene ID: 20677), Zfp36l2 (Gene ID: 12193), Gimap6 (Gene ID: 231931), Phlda1 (Gene ID: 21664), and Egr3 (Gene ID: 13655) were downregulated in hematopoietic stem and progenitor cells of senescent individuals in monologous symbiosis and young individuals in xenologous symbiosis, and upregulated in aged individuals in xenologous symbiosis (reversing aging). Furthermore, Prtn3 (Gene ID: 19152), Lars2 (Gene ID: 102436), Hp (Gene ID: 15439), Ms4a3 (Gene ID: 170813), Mpo (Gene ID: 17523), S100a8 (Gene ID: 20201), Elane (Gene ID: 50701), and S100a9 (Gene ID: 20202) were upregulated in hematopoietic stem and progenitor cells of senescent individuals in symbiotic relationships and young individuals in heterologous relationships, and downregulated in elderly individuals in heterologous relationships (reversing aging). Figure 12 Further analysis of core regulatory transcription factors revealed that Atf3, Atf4, and Yy1 are key regulatory factors. Figure 13Both Atf3 and Atf4 were downregulated in hematopoietic stem and progenitor cells of senescent individuals in symbiotic co-existence and young individuals in xenobiotic co-existence, with Atf3 and Atf4 being upregulated in aged individuals in xenobiotic co-existence (reversing aging). Figure 14 Furthermore, analysis of intercellular communication results showed that allogeneic symbiosis could partially restore intercellular ligand-receptor interactions in bone marrow hematopoietic stem and progenitor cells. Further screening revealed that Ccl3-related ligand-receptor pairs were also effectively restored, suggesting they may play a key regulatory role in hematopoietic stem and progenitor cell differentiation. Figure 15 Subsequently, we validated the Yy1 and Ccl3 genes.

[0237] 1. Identification results of YY1 overexpression in LSK cells

[0238] To investigate the role of YY1 in aged hematopoietic stem (progenitor) cells, LSK cells were transfected with lentivirus. Western blot results showed that, at the protein level, YY1 was significantly increased in recombinant LSK cells transfected with recombinant lentivirus LV / SF-LV-Yy1-EGFP. Figure 16 ).

[0239] 2. Overexpression of YY1 enhances the reconstitution ability of aged hematopoietic stem (progenitor) cells.

[0240] The effect of YY1 overexpression on the reconstitution capacity of aged hematopoietic stem (progenitor) cells was evaluated using a competitive transplantation experiment. The experimental results are as follows: Figure 17 As shown. Figure 17 A in the diagram is a flowchart of a competitive transplantation experiment; Figure 17 In section B, the donor source analysis for mouse peripheral blood testing is presented. Figure 17 The vertical axis of B represents the percentage of cells from older mouse donors, expressed as a percentage. The horizontal axis represents the number of weeks after the mouse transplantation experiment, expressed as weeks (7 days). Figure 17 The results from the B-cell analysis showed that, in mouse peripheral blood, the proportion of donors from aged mice overexpressing YY1 (YY1 mice) was significantly higher (P < 0.05) than that from the control group (VC mice). However, in mouse peripheral blood B cells, the proportion of donors from aged mice overexpressing YY1 was not significantly different from that from the control group (P > 0.05). This indicates that overexpression of YY1 can improve the reconstitution capacity of aged hematopoietic stem (progenitor) cells, mainly at the whole cell, T cell, and Myeloid cell levels.

[0241] 3. Identification results of CCL3 overexpression in LSK cells

[0242] To investigate the role of CCL3 in aged hematopoietic stem (progenitor) cells, LSK cells were transfected with lentivirus. Western blot results showed that, at the protein level, CCL3 was significantly increased in recombinant LSK cells transfected with recombinant lentivirus LV / SF-LV-Ccl3-EGFP. Figure 18 ).

[0243] 4. Overexpression of CCL3 enhances the reconstitution capacity of aged hematopoietic stem (progenitor) cells.

[0244] The effect of CCL3 overexpression on the reconstitution capacity of aged hematopoietic stem (progenitor) cells was evaluated using a competitive transplantation experiment. The results are as follows: Figure 19 As shown. Figure 19 The vertical axis represents the percentage of cells from older mouse donors, expressed as a percentage, while the horizontal axis represents the number of weeks after the mouse transplantation experiment, expressed as weeks (7 days). Figure 19 The results showed that in mouse peripheral blood T cells, the proportion of donor-derived cells from aged mice overexpressing CCL3 was significantly higher than that from the control group (VC) (P < 0.05); however, there was no significant difference in the proportion of donor-derived cells from aged mice overexpressing CCL3 between the two groups in whole cells, B cells, and Myeloid cells of mouse peripheral blood (P > 0.05). This indicates that CCL3 overexpression can improve the reconstitution capacity of aged hematopoietic stem (progenitor) cells, mainly at the whole cell, T cell, and Myeloid cell levels.

[0245] 5. Effect of CCL3 overexpression on enhancing lymphoid differentiation of hematopoietic stem (progenitor) cells

[0246] The effect of CCL3 on the differentiation capacity of aged hematopoietic stem (progenitor) cell lineages was further evaluated and analyzed through competitive transplantation experiments. The results are as follows: Figure 20 As shown, each bar chart represents the percentage of myeloid cells, B cells, and T cells from top to bottom. Figure 20 The vertical axis represents the pedigree analysis proportion of aged mice from donor sources, in %, where the sum of myeloid cells, B cells, and T cells is 100%. The horizontal axis represents the number of weeks after mouse transplantation, in weeks (7 days). Figure 20 The results showed that the proportion of T cells in aged mouse cells overexpressing CCL3 was significantly higher than that in the control group (VC) at different time points (P < 0.05). The proportions of myeloid and B cells did not differ significantly from the control group at 12, 16, and 20 weeks after transplantation (P > 0.05). These results indicate that CCL3 overexpression can promote lymphoid differentiation of aged hematopoietic stem (progenitor) cells, mainly at the T cell level.

[0247] Example 4: Verification of key differentially expressed genes in skin fibroblasts during aging

[0248] I. Experimental Methods

[0249] 1. Mouse or human fibroblast culture

[0250] Human skin tissue was obtained after review by the ethics committee of Peking Union Medical College Hospital, and all donors signed informed consent forms. Upper eyelid tissue from healthy 27-year-old individuals was minced and placed at the bottom of 6-well plates, with 0.5 ml of fibroblast growth medium (90% DMEM, 10% fetal bovine serum, 1% NEAA, 1% GlutaMax, 0.10% plasmocin) added. Cells were passaged after digestion with 0.25% Tryposin when they reached 80% cellularity. The obtained cells were used for subsequent experimental analysis.

[0251] Mouse fibroblasts were obtained from the dorsal skin tissue of 7-week-old male mice. The tissue was minced with scissors, incubated in a water bath at 37°C for 1 hour and 30 minutes with 8 mg / mL Collagenase 4, filtered through a 40-micron filter, and then seeded in 10 mL of fibroblast growth medium in 10 cm culture dishes. Cells were cultured until 80% full growth was achieved, and then passaged using 0.25% Trypsin. The obtained cells were used for subsequent experimental analysis.

[0252] 2. Tsc22d3(Gilz) gene knockdown

[0253] Human GILZ protein is encoded by the human Gilz gene, whose GenBank accession number is XM_005262103.5. Its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 160-762 of XM_005262103.5. The GenBank accession number for human GILZ protein is NP_001015881.1. Mouse GILZ protein is encoded by the mouse Gilz gene, whose GenBank accession number is NM_001077364.1. Its coding sequence is the DNA molecule whose nucleotide sequence is shown at positions 204-809 of NM_001077364.1. The GenBank accession number for mouse GILZ protein is NP_001070832.1.

[0254] Fibroblasts were transfected with Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) for 48 hours using a negative control targeting the target gene and siRNA (Ripex). The fibroblasts were then treated with 50 ng / mL IL-6 for 24 hours to simulate the inflammatory environment of aging tissue. GILZ gene siRNA is shown in Table 1.

[0255] Table 1: Gilz gene siRNA

[0256]

[0257] Cells were collected for RT-qPCR, Ki67 immunostaining, and apoptosis analysis.

[0258] 2. RT-qPCR

[0259] Forty-eight hours after transfecting human or mouse cells with RNAi sequences, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific), and 2 μg of total RNA was reverse transcribed into cDNA using master mix (Promega). RT-qPCR was performed using iTaq Universal SYBR GreenSuper Mix (Bio-Rad). GAPDH was used as an internal control to detect the expression of the Gilz gene in mice or humans. The primer sequences for the Gilz gene are shown in Table 2.

[0260] Table 2: Primer sequences for the Gilz gene

[0261]

[0262] Test results are shown Figure 23 The results showed that siRNA effectively reduced the expression of the GILZ gene in mouse and human fibroblasts.

[0263] 3. Ki67 immunofluorescence staining

[0264] To measure changes in cell proliferation, mouse or human fibroblasts were seeded on 24-well glass slides and treated with siRNA as per step 2. The samples were then analyzed 72 hours later. Each group consisted of three replicates and two independent experiments. At least 300 cells were counted from each sample for data analysis.

[0265] Fibroblasts were fixed with 4% PFA for 25 min, permeabilized with 0.4% Triton X-100 for 25 min, incubated with blocking buffer (10% donkey serum) at room temperature for 1 h, and stained overnight with Ki67 antibody (abcam 15580) at 4°C. Then, cells were incubated with secondary antibody and Hoechst 33342 (Thermo Fisher Scientific) for 1 h, and scanned using a confocal laser scanning microscope (Zeiss 900 confocal system). The proportion of Ki67-positive cells was calculated.

[0266] The results showed that knocking down GILZ in human or mouse fibroblasts and then treating them with IL6 significantly reduced cell proliferation.

[0267] 4. Apoptotic cell detection

[0268] To measure changes in apoptosis, mouse or human fibroblasts were used at a concentration of 1 x 10⁻⁶. e Cells were seeded in each well of a 12-well plate and were analyzed 72 hours after siRNA treatment as per step 2. Each group contained three experimental replicates and two independent experiments.

[0269] Freshly collected fibroblasts were stained according to the instructions of the Annexin V-EGFP apoptosis detection kit (Vazyme Biotechnology, A211-02), and then analyzed by flow cytometry using a BD LSRFortesa flow cytometer. The data were analyzed using FlowJo software (TreeStar).

[0270] The results showed that knocking down GILZ in human or mouse fibroblasts and then treating them with IL6 significantly increased the number of apoptotic cells.

[0271] II. Experimental Results

[0272] The results of bioinformatics calculations are as follows Figure 21 As shown. Figure 21 The results showed that, compared with younger mice, the expression of the Tsc22d3 (Gilz) gene was downregulated in myeloid progenitor cells (CMP), pluripotent progenitor cells (MPP), and lymphoid progenitor cells (CLP) of hematopoietic stem and progenitor cells in older mice; the expression of the Tsc22d3 (Gilz) gene was downregulated in basal stem cells (Basal-1), spinous cells (Spi), reticular fibroblasts (RetiFib), and hair follicle stem cells (HFSC) in the skin; the expression of the Tsc22d3 (Gilz) gene was relatively similar in type IIX myofibroblasts (Fast_IIX) of skeletal muscle; and the expression of the Tsc22d3 (Gilz) gene was relatively consistent in Kupffer cells, Zone 1 hepatocytes (Hepatocyte_Z1), cholangiocytes, endothelial cells (EC), Zone 3 hepatocytes (Hepatocyte_Z3), and Zone 4 hepatocytes (Z1). Tsc22d3 (Gilz) gene expression was downregulated in Zone 2 hepatocytes (Hepatocyte_Z2). Compared to autologous aged mice, Tsc22d3 (Gilz) gene expression was upregulated in myeloid progenitor cells (CMP), pluripotent progenitor cells (MPP), and lymphoid progenitor cells (CLP) of hematopoietic stem and progenitor cells from allogeneic aged mice; Tsc22d3 (Gilz) gene expression was upregulated in basal stem cells (Basal-1), spinous cells (Spi), and reticular fibroblasts (RetiFib) of the skin; and Tsc22d3 (Gilz) gene expression was upregulated in Zone 2 hepatocytes (Hepatocyte_Z2) of the liver. Figure 21The results suggest that Tsc22d3 (Gilz) expression is downregulated with aging in various peripheral tissue cell types, and that xenobiotics can partially restore this change.

[0273] The results of RT-qPCR detection of the relative expression level of the target gene are as follows: Figure 22 As shown. Figure 22 The results showed that the number of human and mouse fibroblasts in the RNA interference group (si-GILZ) was significantly lower than that in the control group (si-NC), indicating that RNAi successfully constructed human and mouse fibroblasts with silenced Tsc22d3 (Gilz) gene.

[0274] The results of Ki67 immunofluorescence staining are as follows: Figure 23 As shown. Figure 23 The results showed that the number of Ki67-stained human and mouse fibroblasts in the RNA interference group (si-GILZ) was significantly lower than that in the control group (si-NC), indicating that the cell proliferation capacity of the RNA interference group (si-GILZ) was reduced.

[0275] The results of the apoptotic cell ratio detection are as follows Figure 24 As shown. Figure 24 The results showed that the proportion of apoptotic cells in human and mouse fibroblasts in the RNA interference group (si-GILZ) (7.81% and 7.89%) was significantly lower than that in the control group (11.80 and 12.20), indicating that the apoptosis level in the RNA interference group (si-GILZ) was increased.

[0276] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Institute of Zoology, Chinese Academy of Sciences <120> Heterologous symbiotic rejuvenation factors and their application in delaying the aging process <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Mouse (Mus musculus) <400> 1 ccatggacct cgtgaagaa 19 <210> 2 <211> 19 <212> DNA <213> Homo sapiens <400> 2 gtgagaacac cctgttgaa 19

Claims

1. Application, characterized in that, The application is any one of the following 1) to 4): 1) The application of protein CCL3 or biomaterials related to said protein CCL3 in the preparation of products that reduce the aging level of hematopoietic stem cells; 2) The application of protein CCL3 or substances for determining the content of said protein CCL3 in the preparation of products for identifying or assisting in the identification of the aging level of hematopoietic stem cells; 3) The application of protein CCL3 or biomaterials related to said protein CCL3 in the preparation of products that enhance the reconstitution capacity of hematopoietic stem cells; 4) The use of protein CCL3 or biomaterials related to said protein CCL3 in the preparation of products that promote T cell differentiation of hematopoietic stem cells; The biomaterial is any one of the following B11) to B17): B11), encoding the CCL3 protein nucleic acid molecule; B12), an expression cassette containing the nucleic acid molecule described in B11); B13), a recombinant vector containing the nucleic acid molecule described in B11), or a recombinant vector containing the expression cassette described in B12; B14) recombinant microorganisms containing the nucleic acid molecules described in B11), or recombinant microorganisms containing the expression cassette described in B12), or recombinant microorganisms containing the recombinant vector described in B13); B15), a transgenic animal cell line containing the nucleic acid molecule described in B11), or a transgenic animal cell line containing the expression cassette described in B12), or a transgenic animal cell line containing the recombinant vector described in B13; B16), transgenic animal tissue containing the nucleic acid molecule described in B11), or transgenic animal tissue containing the expression cassette described in B12), or transgenic animal tissue containing the recombinant vector described in B13; B17), a transgenic animal organ containing the nucleic acid molecule described in B11), or a transgenic animal organ containing the expression cassette described in B12), or a transgenic animal organ containing the recombinant vector described in B13).

2. The application according to claim 1, characterized in that: B11) The nucleic acid molecule is the encoding gene of the protein CCL3, and the encoding gene of the protein CCL3 is... Ccl3 Gene or Ccl3 The coding sequence of a gene.

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