CN117050157A - Foreign-body symbiotic rejuvenation factor and application thereof in delaying organism aging - Google Patents
Foreign-body symbiotic rejuvenation factor and application thereof in delaying organism aging Download PDFInfo
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Abstract
The invention discloses an application of a foreign-body symbiotic rejuvenation factor in delaying organism aging at the level of multi-tissue cells and molecules, belonging to the technical field of biology. The invention discloses application of a protein CCL3 or a protein YY1 or a protein GILZ or a substance for regulating the expression of the protein CCL3 or the protein YY1 or the protein GILZ coding gene or a substance for regulating the activity or the content of the protein CCL3 or the protein YY1 or the protein GILZ in regulating the aging level of organisms and/or cells and/or tissues. The present invention provides a rich source for studying aging and younger cell and molecule changes at single cell resolution, and reveals key regenerative factors therein (Ccl 3, yy1 and Tsc22d3 (Gilz)).
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of an allosymbiotic rejuvenation factor in delaying organism aging at the level of multi-tissue cells and molecules.
Background
Aging is a systemic degenerative process involving various tissues and organs throughout the body, resulting in diminished regenerative capacity and reduced tissue and organ function. Various means have been reported to delay the senescence-associated phenotype. Among them, allogeneic symbiosis (Heterochronic parabiosis, HP) is a research model system which has received extensive attention in recent years. In the HP mouse model, the circulatory systems of aged and young mice are surgically linked to construct a shared circulatory system in which blood-borne factors produced by both parties are shared with the other individual. Thus, HP provides a unique experimental paradigm for studying how a whole aging organism is revived by the injection of "pro-youth factors". Vice versa, how the young organism is in turn affected by the "pro-aging factors" of the circulatory system. So far, our knowledge of cellular targets of blood-borne factors and how they promote the rejuvenation of aging individuals at a systemic level remains limited.
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 number of adult stem cell populations are able to replenish tissue throughout life to repair age-related injuries and maintain tissue homeostasis. Hematopoietic stem progenitor cells (Hematopoietic stem and progenitor cells, HSPCs) are capable of producing a variety of immune cell types in the hematopoietic and immune system consisting of a number of organs and tissues, including bone marrow, spleen, peripheral blood and hematopoietic stem cells and their immediate progeny. However, with age, hematopoietic stem progenitor cells gradually lose the ability to maintain immune cell composition and exhibit differentiated potential for myeloid migration, resulting in age-related hematopoietic dysfunction and hypoimmunity. Similarly, in other organs and tissues, such as skin, skeletal muscle and brain, there are also resident different types of adult stem cells, namely Hair Follicle Stem Cells (HFSCs) and basal cells responsible for the regeneration of hair follicles and epidermis, fibrogenic/adipogenic progenitor cells (FAPS) and skeletal muscle satellite cells, and brain neural stem cells. These adult stem cells gradually lose regenerative capacity with age, leading to hair loss and skin aging, skeletal muscle and neurodegeneration. Thus, resident adult stem cells in both the hematopoietic system and peripheral organs are compromised by aging, but further research is needed as to whether and to what extent they can be revived.
Disclosure of Invention
The invention aims to solve the technical problem of delaying aging.
To solve the above technical problem, in a first aspect, the present invention provides an application, which may be any one of the following A1) to a 10):
a1 Use of protein CCL3 or protein YY1 or protein GILZ or a substance that modulates the expression of the protein CCL3 or the protein YY1 or the protein GILZ-encoding gene or a substance that modulates the activity or amount of the protein CCL3 or the protein YY1 or the protein GILZ in modulating the level of aging of an organism and/or a cell and/or a tissue;
a2 Protein CCL3 or protein YY1 or protein GILZ or a substance regulating the expression of the protein CCL3 or the protein YY1 or the protein GILZ-encoding gene or a substance regulating the activity or the content of the protein CCL3 or the protein YY1 or the protein GILZ in the preparation of a product for the senescence level of an organism and/or a cell and/or a tissue;
a3 Use of CCL3 or YY1 or GILZ or a substance that determines the content of CCL3 or YY1 or GILZ in the identification or assisted identification of the level of senescence in an organism and/or a cell and/or a tissue;
a4 Use of CCL3 or YY1 or GILZ protein or a substance for determining the content of CCL3 or YY1 or GILZ protein for the preparation of a product for the identification or assisted identification of the level of senescence in an organism and/or a cell and/or a tissue;
A5 Protein CCL3 or protein YY1 or a substance that modulates the expression of a gene encoding said protein CCL3 or said protein YY1 or a substance that modulates the activity or content of said protein CCL3 or said protein YY1 in modulating the reconstitution capacity of hematopoietic stem (progenitor) cells;
a6 Protein CCL3 or protein YY1 or a substance that modulates the expression of a gene encoding said protein CCL3 or said protein YY1 or a substance that modulates the activity or content of said protein CCL3 or said protein YY1 in the preparation of a product that modulates the reconstitution capacity of hematopoietic stem (progenitor) cells;
a7 A protein CCL3 or a protein YY1, or a substance that modulates the expression of a gene encoding the protein CCL3 or the protein YY1, or a substance that modulates the activity or amount of the protein CCL3 or the protein YY1, in modulating lineage differentiation of hematopoietic stem (progenitor) cells;
a8 Protein CCL3 or protein YY1 or a substance that modulates the expression of a gene encoding said protein CCL3 or said protein YY1 or a substance that modulates the activity or content of said protein CCL3 or said protein YY1 in the preparation of a product that modulates lineage differentiation of hematopoietic stem (progenitor) cells;
a9 Protein GILZ or a substance regulating the expression of a gene encoding the protein GILZ or a substance regulating the activity or content of the protein GILZ in regulating the proliferation capacity of skin fibroblasts;
A10 Protein GILZ or a substance regulating the expression of a gene encoding the protein GILZ or a substance regulating the activity or content of the protein GILZ in the preparation of a product regulating the proliferation capacity of skin fibroblasts.
In the above, the protein CCL3 or the protein YY1 or the protein GILZ may be a natural protein such as a protein derived from a mammal, or may be a non-natural protein such as a recombinant protein or the like, as long as the recombinant protein has a function of a natural protein.
The CCL3 protein may be a human CCL3 protein or a mouse CCL3 protein. The amino acid sequence of the human CCL3 protein is GenBank No. NP-002974.1, and the amino acid sequence of the mouse CCL3 protein is GenBank No. NP-035467.1.
The YY1 protein may be a human YY1 protein or a mouse YY1 protein. The amino acid sequence of the human YY1 protein is GenBank No. NP-003394.1, and the amino acid sequence of the mouse YY1 protein is GenBank No. NP-033563.2.
The GILZ protein may be a human GILZ protein or a mouse GILZ protein. The GenBank number of the human GILZ protein is: np_001015881.1, genBank number of the mouse GILZ protein is: np_001070832.1.
Further, in the above application, the substance that regulates the expression of the gene encoding the protein CCL3 or the protein YY1 or the substance that regulates the activity or the content of the protein CCL3 or the protein YY1 may be a biomaterial B1, and the biomaterial B1 may be any one of the following B11) to B17):
B11 A nucleic acid molecule encoding said protein CCL3 or said protein YY 1;
b12 An expression cassette comprising a nucleic acid molecule according to B11);
b13 A recombinant vector comprising the nucleic acid molecule of B11), or a recombinant vector comprising the expression cassette of B12);
b14 A recombinant microorganism comprising the nucleic acid molecule of B11), or a recombinant microorganism comprising the expression cassette of B12), or a recombinant microorganism comprising the recombinant vector of B13);
b15 A transgenic animal cell line comprising the nucleic acid molecule of B11), or a transgenic animal cell line comprising the expression cassette of B12), or a transgenic animal cell line comprising the recombinant vector of B13);
b16 A transgenic animal tissue comprising the nucleic acid molecule of B11), or a transgenic animal tissue comprising the expression cassette of B12), or a transgenic animal tissue comprising the recombinant vector of B13);
b17 A transgenic animal organ containing the nucleic acid molecule of B11), or a transgenic animal organ containing the expression cassette of B12), or a transgenic animal organ containing the recombinant vector of B13).
Further, in the above application, B11) the nucleic acid molecule may be the encoding gene of the protein CCL3 or the protein YY1, the encoding gene of the protein CCL3 is the encoding sequence of the CCL3 gene or the CCL3 gene; the coding gene of the protein YY1 is a Yy1 gene or a coding sequence of the Yy1 gene.
The Ccl3 gene may be a human Ccl3 gene or a mouse Ccl3 gene. The GenBank number of the human CCL3 gene is NM_002983.3, and the coding sequence of the human CCL3 gene is a DNA molecule with the nucleotide sequence shown in 86-364 of NM_ 002983.3; the GenBank number of the mouse Ccl3 gene is NM_011337.2, and the coding sequence is a DNA molecule with the nucleotide sequence shown in 102-380 of NM_ 011337.2.
The Yy1 gene may be a human Yy1 gene or a mouse Yy1 gene. The GenBank number of the human YY1 gene is NM_003403.5, and the coding sequence is a DNA molecule with the nucleotide sequence shown in the 102 th-1346 th positions of NM_ 003403.5. The GenBank number of the mouse Yy1 gene is NM_009537.4, and the coding sequence is a DNA molecule with the nucleotide sequence shown in the 117 th-1361 th positions of NM_ 009537.4.
Further, in the above-mentioned application, the substance that regulates the expression of the gene encoding the protein GILZ or the substance that regulates the activity or content of the protein GILZ may be a biological material B2, and the biological material B2 may be an RNA molecule that inhibits or reduces the expression of the gene encoding the protein GILZ or an RNA molecule that inhibits or reduces the activity or content of the protein GILZ;
further, in the above-mentioned application, the RNA molecule of B21) that suppresses or reduces the expression of the gene encoding the protein GILZ or the RNA molecule that suppresses or reduces the activity or content of the protein GILZ may be i 1) or i 2) or i 3) below:
i1 The target sequence of the RNA molecule is the coding gene of the protein GILZ;
i2 The nucleotide sequence of the target sequence of the RNA molecule is SEQ ID No.1;
i3 The nucleotide sequence of the target sequence of the RNA molecule is SEQ ID No.2.
The GILZ protein may be a human GILZ protein or a mouse GILZ protein. The human GILZ protein can be a protein coded by a human Gilz gene, the GenBank of the human Gilz gene is XM_005262103.5, the coding sequence of the human Gilz gene is a DNA molecule with the nucleotide sequence shown in 160-762 bits of XM_005262103.5, and the GenBank of the human GILZ protein is NP_001015881.1. The mouse GILZ protein can be a protein coded by a mouse Gilz gene, the GenBank number of the mouse Gilz gene is NM_001077364.1, the coding sequence of the mouse GILz gene is a DNA molecule with a nucleotide sequence shown as the 204 th to 809 th positions of NM_001077364.1, and the GenBank number of the mouse GILZ protein is NP_001070832.1.
To solve the above technical problem, in a second aspect, the present invention provides the use of an allogeneic symbiont in any one of the following P1) -P18):
p1), decreasing the proportion of senescent cells in spleen tissue and/or skin tissue and/or liver tissue and/or brain tissue;
p2), decrease the proportion of apoptosis in spleen tissue and/or skin tissue and/or liver tissue and/or skeletal muscle tissue;
P3), reducing the area of liver tissue inflammation;
p4), reducing liver tissue and/or spleen tissue fibrosis areas;
p5), increasing skeletal muscle tissue myofiber diameter;
p6), increasing the number of hair follicles of skin tissue;
p7), increasing pro-B cell ratio in bone marrow;
p8), increasing CCL3 protein or/and YY1 protein expression levels in hematopoietic stem and progenitor cells;
p9), up-regulates the expression level of the Ccl3 gene or/and the Yy1 gene.
P10) delaying senescence by decreasing the proportion of senescent cells in spleen tissue and/or skin tissue and/or liver tissue and/or brain tissue;
p11) delay aging by decreasing the proportion of apoptosis in spleen tissue and/or skin tissue and/or liver tissue and/or skeletal muscle tissue;
p12) delay aging by reducing the area of inflammation of liver tissue;
p13) delay aging by reducing the fibrotic area of liver tissue and/or spleen tissue;
p14) delaying aging by decreasing skeletal muscle tissue myofiber diameter;
p15) delay aging by reducing the number of hair follicles in skin tissue;
p16) delay aging by increasing pro-B cell proportion in bone marrow;
p17) delaying senescence by increasing CCL3 protein or/and YY1 protein expression levels in hematopoietic stem/progenitor cells;
p18) delay senescence by up-regulating the expression level of the Ccl3 gene or/and Yy1 gene;
In order to solve the technical problem, in a third aspect, the present invention provides a method for constructing a recombinant cell, the method comprising introducing a gene encoding a protein of interest into a recipient cell, promoting or increasing or up-regulating the expression of the gene encoding the protein of interest, or promoting or increasing or up-regulating the activity or content of the protein of interest, to obtain a recombinant cell having a senescence level lower than that of the recipient cell;
the target protein is the CCL3 protein of claim 1 or the YY1 protein of claim 1.
In order to solve the above technical problem, in a fourth aspect, the present invention provides a recombinant cell constructed by the above method.
In the present invention, the recombinant cell may be a recombinant mammalian cell.
The mammal may be a human or a non-human mammal.
In order to solve the above technical problem, the present invention provides in a fifth aspect the use of the recombinant cell line described above in D1) or D2) as follows:
d1 Use in improving the reconstitution capacity of aged hematopoietic stem cells;
d2 For the preparation of a product for improving the reconstitution capacity of aged hematopoietic stem cells.
The application or the method provided by the invention can be used for diagnosing and treating diseases or can be used for diagnosing and treating non-diseases.
In order to solve the above technical problems, in a sixth aspect, the present invention provides the protein CCL3 or protein YY1 or protein GILZ, or the biological material B1 or/and the biological material B2.
In one embodiment of the invention, the level of aging of the body and/or cells and/or tissues is assessed by assessing the reconstitution capacity of hematopoietic stem (progenitor) cells.
In one embodiment of the invention, hematopoietic stem (progenitor) cell reconstitution capacity is assessed by competitive transplantation experiments.
In one embodiment of the invention, the enhancing the reconstitution capacity of hematopoietic stem (progenitor) cells is embodied as follows: in competitive transplantation experiments, the aged mouse donor-derived T cell ratio of the over-expressed protein CCL3 was significantly higher (P < 0.05) than in the control group; the ratio of whole cells, T cells, myeloid cells from the donor of the aged mice over-expressing YY1 was significantly higher (P < 0.05) than that of the control group.
In one embodiment of the invention, the level of aging of the body and/or cells and/or tissues is assessed by assessing the proliferative capacity of fibroblasts.
In one embodiment of the invention, the proliferative capacity of fibroblasts is assessed by Ki67 staining experiments and apoptotic cell duty cycle.
In one embodiment of the invention, the transgenic cell line reduces the proliferative capacity of fibroblasts by: the number of cells stained by Ki67 was significantly lower in human fibroblasts and mouse fibroblasts of the RNA interference group (si-Gilz) of Gilz gene than in the control group (si-NC), indicating a decrease in the cell proliferation capacity of the RNA interference group (si-Gilz); and the ratio of apoptotic cells (7.81%, 7.89%) was significantly lower in human and mouse fibroblasts in the RNA interference group (si-GILZ) than in the control group (11.80, 12.20), indicating an elevated level of apoptosis in the RNA interference group (si-GILZ).
In one embodiment of the invention, the allosymbiont is an allosymbiotic mouse model.
In one embodiment of the invention, the method for preparing the mouse allotropic symbiotic model comprises the following steps: each of the 2 mice was shaved along a continuous line from the elbow, flank, and knee to which one side was to be connected. Skin scrub with iodine +70% alcohol for more than 3 alternating cycles in preparation for incision. An autoclave instrument was used and the sterile field was maintained. On each mouse, the skin was cut with scissors along the flank from the proximal knee end to the proximal elbow end without disturbing the subcutaneous muscles. The triceps of the animals were connected with 2 intermittent sutures. The seams were continued 7-9 times along the side panel walls. The quadriceps femoris muscle of the animal was connected with 2 intermittent sutures. The skin of 2 individuals was sutured with intermittent suturing. The mice were then placed on a heated pad in a supine position and allowed to wake up in ambient air. After the parabolic operation, each group received separate feeds, and was subcutaneously injected with 1ml of 0.25% bupivacaine physiological saline daily for 3 days.
In one embodiment of the invention, said reducing the proportion of senescent cells in spleen tissue and/or skin tissue and/or liver tissue and/or brain tissue is reflected in reducing SA- β -gal staining positive areas in spleen tissue and/or skin tissue and/or liver tissue and/or brain tissue.
The reduction of liver tissue inflammation is reflected in a reduction of the area of infiltration of HE-stained inflammatory cells in liver tissue.
The reduction of apoptosis in spleen tissue and/or skin tissue and/or liver tissue and/or skeletal muscle tissue is reflected in a positive proportion of cells in TUNEL staining.
The reduction of liver tissue and/or spleen tissue fibrosis is manifested in positive areas in masson staining.
The increase in skeletal muscle tissue myofiber diameter is manifested by a measurement of myofiber area in HE staining.
The increase in the number of skin tissue follicles is manifested by a change in the number of follicles in HE staining.
In the above applications, the delay of the senescence of hematopoietic stem/progenitor cells is manifested by an enhancement of the reconstitution capacity and/or the lineage differentiation capacity of the aged hematopoietic stem (progenitor) cells;
the YY1 and/or CCL3 expression levels are protein expression levels.
The Yy1 and/or Ccl3 expression levels are gene expression levels.
In the above application, the peripheral blood, the bone marrow, the spleen tissue, the brain tissue, the liver tissue, the skeletal muscle tissue, and the skin tissue are mammalian peripheral blood, bone marrow, spleen tissue, brain tissue, liver tissue, skeletal muscle tissue, and skin tissue, and the mammal includes human.
The single cell transcriptome of four solid tissues/organs (skin, skeletal muscle, brain and liver) affected by hematopoietic and immune organs sampled from allogeneic symbiotic and simultaneous allogeneic symbiotic (Isochronic parabiosis) mice (Mus musculus) was systematically analyzed in the present invention. By the method, a multi-tissue single-cell transcriptome map can be constructed to study the influence of HP on aging, and the complex change and regulation effects of somatic stem cells and microenvironment thereof are explored. The present invention analyzes how a young circulatory system environment rejuvenates an older individual and how an older circulatory system environment compromises a young individual, thereby altering cell populations, gene expression characteristics, and cell-cell communication. In our allotropic symbiotic profile, the present invention found that hematopoietic stem and progenitor cells are one of the most resistant to infiltration by each other in the hematopoietic and immune systems, while being the most susceptible to systemic modulation. In addition, the invention also discovers the regulatory factor with the potential of reversing the aging-related injury. This study may facilitate our understanding of aging-related systemic changes and the development of novel aging intervention strategies.
Drawings
FIG. 1 shows SA-. Beta. -Gal staining analysis in different tissues of four groups of mice.
FIG. 2 shows TUNEL staining analysis of apoptosis ratios in different tissues of four groups of mice.
Fig. 3 is an HE staining analysis of liver, skin and skeletal muscle of four groups of mice.
FIG. 4 is a MASSON staining analysis of liver and spleen of four groups of mice.
FIG. 5 is a pseudo-temporal differentiation trace analysis of hematopoietic system.
FIG. 6 is a set of four groups of mouse hematopoietic stem cell differentiation-related gene scores.
Fig. 7 shows the change in cell proportion of B cell groups in single cell data of four groups of mice.
FIG. 8 shows the flow cytometry to detect the B cell ratio variation of different groups of mice.
FIG. 9 is statistical data on the proportion of bone marrow pro-B cells to total B cells in different groups of mice.
FIG. 10 shows the flow cytometry detection of the changes in the ratio of the pro-B cells of the bone marrow of mice of different groups, and the statistical data.
FIG. 11 is a graph showing differential gene calculation patterns of aged and allogeneic symbiotic mice.
FIG. 12 is a cell type specific differential gene regulatory network.
FIG. 13 is a core regulatory transcription factor regulatory network analysis.
FIG. 14 shows the expression level of the core regulatory transcription factor and the target gene score.
FIG. 15 is a graph of intercellular ligand receptor interaction pair with symbiotic restoration.
FIG. 16 is a graph showing WB level verification of the efficiency of overexpression of YY1 in mouse LSK cells.
FIG. 17 is a schematic and line graph showing the transplantation of aged hematopoietic stem (progenitor) cell functions evaluated for infection with different viruses, showing that overexpression of YY1 enhances the reconstitution ability of aged hematopoietic stem (progenitor) cells.
Figure 18 is a graph demonstrating the efficiency of WB levels to over-express CCL3 in mouse LSK cells.
Fig. 19 is a line graph showing that overexpression of CCL3 enhances the reconstitution capacity of aged hematopoietic stem (progenitor) cells.
Figure 20 is a bar graph showing the effect of over-expression of CCL3 on the differentiation of aged hematopoietic stem (progenitor) cell lineages.
FIG. 21 is a cross-cell type allotropic recovery factor-Tsc 22d3 (Gilz).
FIG. 22 shows RT-qPCR detection of knockdown efficiency of Gilz gene in human and mouse skin fibroblasts.
FIG. 23 is a graph showing the proliferation potency of human and mouse fibroblasts following GILZ/Gilz knockdown by Ki67 immunofluorescence.
FIG. 24 is an apoptosis assay showing the proliferation capacity of human and mouse fibroblasts following GILZ/Gilz knockdown.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. All animal experiments in the following examples were approved by the national academy of sciences animal protection and utilization committee. The isolation of human adipose stem cells in the following examples has passed the examination by the ethical examination committee of the college of beijing covariate.
Experimental data in the following examples were processed using GraghPad Prism 8 statistical software, experimental results were expressed as mean ± standard deviation, using single-tail t-test, double-tail t-test, P < 0.05 (x) was statistically significant, P <0.01 was extremely significant, and P <0.001 was extremely significant.
The sources of biological material in the following examples are as follows: c57BL6/J mice were purchased from Beijing SPF Biotechnology Co. Isoflurane is available from rawald corporation under the product number R510-22.4-0 polydioxanone suture material and 4-0 polypropylene suture material were purchased from gold ring medical, cat No. F406. The masson kit was purchased from soribao corporation under the product number G1346.TUNEL kit was purchased from bi yun tian company under the product number C1088.
Hematopoietic stem cell culture medium: SFEM medium (Stem Cell Technology) was supplemented with SCF (final concentration of 20ng/mL, peproTech), TPO (final concentration of 20ng/mL, peproTech) and penicillin-streptomycin (final concentration of 1%, gibco).
HBSS + Solution: hanks balanced salt solution (HBSS, solarbio) was added with 2% fbs and 1% N-2 hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES, solarbio).
293T cell medium: 89 parts by volume of DMEM high-glucose medium (Hyclone), 10 parts by volume of fetal bovine serum (Gibco), 1 part by volume of penicillin/streptomycin (Gibco).
The sources of biological material in the following examples are as follows:
human embryonic kidney 293T cells: ATCC, CRL-3216;
lentiviral packaging vector psPAX2 (abbreviated as plasmid psPAX 2): addgene products, #12260;
lentiviral packaging vector pMD2G (abbreviated as plasmid pMD 2G): addgene product, #12259;
SF-LV-EGFP vectors are described in the literature "Aging-reduced IL27Ra Signaling Impairs HematopoieticStem cells.He H, et al, blood.2020Jul 9;136 In 183-198 (literature named SF-LV-cDNA-EGFP vector), the biological material is available to the public from the applicant for only repeated experiments related to the invention and not for other uses.
SF-LV-Yy1-EGFP vector: construction was performed on SF-LV-EGFP vector.
SF-LV-Ccl3-EGFP vector: construction was performed on SF-LV-EGFP vector.
In the examples described below, FITC anti-mouse Lineage Cocktail with Isotype Ctrl (clone: 145-2C11; RB6-8C5; RA3-6B2; ter-119; M1/70), accession number 133302; ki67 (abcam, cat# 15580); APCanti-mouse Ly-6A/E (Sca-1) (clone: D7), accession number 108112; perCP anti-mouse CD117 (c-Kit) (clone: 2B 8), accession number 105822; FIT (FIT)C anti-mouse/human CD45R/B220 (clone: RA3-6B 2), accession number 103206; brilliant Violet 421 TM anti-mouse IgM (clone: RMM-1), accession number 406518; APC anti-mouse CD43 (clone: S11), accession number 143208; biotin anti-mouse CD3 epsilon (clone: 145-2C 11), accession number 100304; biotinanti-mouse CD4 (clone: RM 4-5), cat# 100508; biotin anti-mouse CD8a (clone: 53-6.7), accession number 100704; biotin anti-mouse/human CD45R/B220 (clone: RA3-6B 2), accession number 103204; biotinanti-mouse/human CD11b (clone: M1/70), accession number 101204; biotin anti-mouse Ly-6G/Ly-6C (Gr-1) (clone: RB6-8C 5), cat# 108404; biotin anti-mouse TER-119/Erythroid Cells (clone: TER-119), cat# 116204; perCP/Cyanine5.5 anti-mouse CD48 (clone: HM 48-1), cat# 103422; APCanti-mouse CD3 ε (clone: 145-2C 11), accession number 100312; perCP anti-mouse/human CD11b (clone: M1/70), accession number 101230; brilliant Violet 605 TM anti-mouse/human CD45R/B220 (clone: RA3-6B 2), accession number 103244; PE anti-mouse CD45.1 (clone: A20), accession number: 110708 the above antibodies were all purchased from Biolegend. APC anti-mouse CD117 (c-Kit) (clone: 2B 8), accession number 17-1171-83; PE-Cy TM 7Sca-1anti-mouse (clone: D7), cat#: 25-5981-82; streptavidin ConjugateAPC-eFluor TM 780, the goods number is 47-4317-82; DAPI, cat no: 62248; propidium ion, cat No. P3566, all available from Invitrogen corporation. 7-AAD Staining Solution, product number: 559925 from BD company. Mouse monoclonal anti-YY1, cat# sc-7341; mouse monoclonal anti-CCL3, sc-365691, all available from Santa Cruz. Anti-APC microblades, cat#: 130-090-855 from Miltenyi Biotec company; mouse and human siRNA were purchased from libo.
Example 1 recovery of the senescent phenotype of mice by allosymbiosis
1. Experimental grouping
Age and sex information for three groups of C57BL6/J mice are as follows: group of syngeneic young mice (Iso-Y) (2 months old, n=7, male mice), allogeneic young mice (Het-Y) and allogeneic senior mice (Het-O) (2 months old and 23 months old, n=5, male mice) and syngeneic senior mice (Iso-O) (23 months old, n=9, male mice), respectively.
In addition, to trace back the source and differentiation of donor/host cells, we prepared allogeneic young mice (2 months old, n=4, CD45.1 male mice) and xenogeneic old mice (23 months old, n=4, CD45.2 male mice) using the CD45.1/CD45.2 homology system. Each pair of allogeneic mice needs to be housed in the same cage for at least two weeks prior to surgery. Mice were examined daily for physical condition and overall appearance.
The surgical procedure is as follows:
analgesic (buprenorphine, SR/sustained release, 0.1mg/kg subcutaneous injection) was administered after induction and prior to surgical procedures. Applying eye lubricant. For all in vivo suturing, a 4-0 polydioxanone suture material was used. For all external stitches, a 4-0 polypropylene stitch material was used. Each of the 2 mice was shaved along a continuous line from the elbow, flank, and knee to which one side was to be connected. Skin scrub with iodine +70% alcohol for more than 3 alternating cycles in preparation for incision. An autoclave instrument was used and the sterile field was maintained. On each mouse, the skin was cut with scissors along the flank from the proximal knee end to the proximal elbow end without disturbing the subcutaneous muscles. The triceps of the animals were connected with 2 intermittent sutures. The seams were continued 7-9 times along the side panel walls. The quadriceps femoris muscle of the animal was connected with 2 intermittent sutures. The skin of 2 secondary bodies was sutured with intermittent suturing. The mice were then placed on a heated pad in a supine position and allowed to wake up in ambient air. After the parabolic operation, each group received separate feeds, and was subcutaneously injected with 1ml of 0.25% bupivacaine physiological saline daily for 3 days. In the allosymbiotic group, young mice are connected on the left and old mice are connected on the right. Six weeks after the operation, tissue sampling and histological examination were performed.
2. The experimental method comprises the following steps:
(1) SA-beta-gal staining
The spleen, skin, liver and brain tissues of each group of mice were taken for aging-related beta-galactosidase staining analysis. Senescence-associated beta-galactosidase staining is a method for staining senescent tissue based on up-regulation of SA-beta-gal (senescence-associated-beta-galactose) activity level at the time of senescence.
The method comprises the following specific steps: frozen sections of mouse tissue were returned to room temperature for drying, washed with PBS, and fixed with 2% formaldehyde and 0.2% glutaraldehyde for 15 minutes at room temperature. Then staining with a staining solution at 37 ℃ for 48-96 hours. Slides were then fixed with 80% glycerol. Images were obtained using confocal laser scanning microscopy (Leica, CS 2) with X-Gal as substrate, which produced a dark blue product under the catalysis of senescence-specific β -galactosidase. Aging of the tissue was observed under a common light microscope and the percentage or intensity of the SA- β -gal positive regions was quantified by imageJ.
Wherein, the formula of the staining solution is as follows: citric acid/sodium phosphate buffer 40mM, K 4 [Fe(CN) 6 ]·6H 2 O 5mM、K 3 [Fe(CN) 6 ]5mM、NaCl 150mM、MgCl 2 2mM、X-gal 1mg/ml。
(2) TUNEL staining
Paraffin embedded tissue was sectioned at a thickness of 10 μm using a rotary microtome. Sections were dewaxed in xylene, rehydrated in gradient hexanol (100%, 95%, 80%, 70%) and TUNEL stained using TUNEL apoptosis detection kit. Images were obtained using confocal laser scanning microscopy (Leica TCS SP5 ii) and the percentage of positive cells was quantified using ImageJ.
(3) HE staining
Paraffin embedded tissue was sectioned at a thickness of 5 μm using a rotary microtome. Sections were dewaxed in xylene, rehydrated in gradient hexanol (100%, 95%, 80%, 70%), incubated in hematoxylin solution, rinsed in tap water to remove excess hematoxylin, differentiated for 5s with 1% acid alcohol, and rinsed in tap water for 10min. Finally, sections were stained with eosin, dehydrated in gradient ethanol and xylene, and mounted with neutral gum, images were collected under a microscope, and the area of positive areas was quantified by ImageJ.
(4) Maron dyeing
After paraffin embedding, the tissues were sectioned at 5 μm thickness, deparaffinized with xylene, and hydrated with 100% ethanol, 95% ethanol, 70% ethanol, and tap water. The sections were then stained with potassium dichromate solution (concentration) overnight. After washing with tap water for 5-10 minutes, the sections were stained in the hematoxylin working solution for 10 minutes and in the carmine acid fuchsin solution for 5-10 minutes. Then, the slide glass is differentiated in a phosphomolybdic phosphotungstic acid solution for 10-15 minutes and stained with an aniline blue solution for 5-10 minutes. Then briefly rinsed in distilled water and differentiated in 1% acetic acid solution for 2-5 min. Sections were then dehydrated with 70% ethanol, 95% ethanol and 100% ethanol, then removed with xylene, covered with coverslips and mounted with neutral gum. Images were obtained using confocal laser scanning microscopy (Leica, CS 2) and the area of the positive areas was quantified by ImageJ.
3. Experimental results
The results of SA- β -gal staining are shown in FIG. 1, which shows a micrograph of age-related galactosidase positive cells in the spleen, skin, liver and brain of the syngeneic young, syngeneic aged, allogeneic young and xenogeneic aged mice, respectively, and a bar graph of the ratio of SA- β -gal positive areas quantified by ImageJ. Wherein, the ordinate of the bar graph is the SA-beta-gal positive area ratio quantified by imageJ, and the abscissa of the bar graph represents the syngeneic young group, the syngeneic old group, the allogeneic young group and the allogeneic old group mice from left to right, respectively.
The results in fig. 1 show that: the proportion of the age-related galactosidase positive regions in the spleen, skin and liver of the mice of the syngeneic young group is significantly lower than that of the mice of the syngeneic old group (P < 0.001 (skin and liver) or P < 0.01 (spleen) or P < 0.05 (brain)), the proportion of the age-related galactosidase positive regions in the spleen, skin and brain of the mice of the allogeneic young group and the aged mice of the allogeneic old group is not significantly different (P > 0.05), and the proportion of the age-related galactosidase positive regions in the liver of the mice of the allogeneic young group is significantly lower than that of the mice of the aged-allogeneic old group (P < 0.05).
The proportion of the positive areas of the spleen, skin, liver and brain of the allogeneic young mice and the aged mice is between that of the aged mice in the same young group and the aged mice in the same old group; the decrease in the age-related galactosidase-positive areas in the spleen, skin, liver, and brain of the allogeneic aged mice, as compared to the syngeneic aged mice, suggests that the age-related phenotype is alleviated at the cellular level of multiple organs.
TUNEL staining results are shown in fig. 2, which shows a bar graph of TUNEL positive cell ratios quantified by ImageJ for spleen, skin, liver and skeletal muscle of the syngeneic young group, syngeneic year elder Zu, allogeneic young group and xenogeneic aged group mice, respectively. Wherein, the ordinate of the bar graph is the ratio of apoptotic cells, and the abscissa of the bar graph represents the homozygotic young group, homozygotic year elder Zu, allogeneic young group and xenogeneic aged group mice from left to right, respectively.
The results in fig. 2 show that: the proportion of apoptotic cells in spleen, skin, liver and skeletal muscle of mice in the syngeneic young group is significantly lower than that in mice in the syngeneic old group (P < 0.001 (spleen) or P < 0.01 (liver and skeletal muscle) or P < 0.05 (skin)); the ratio of apoptotic cells in the skin, liver and skeletal muscle of the allogeneic young mice and the xenogeneic aged mice was not significantly different (P > 0.05), and the ratio of apoptotic cells in the spleen of the xenogeneic young mice was significantly lower than that of the xenogeneic aged mice (P < 0.05).
Apoptotic cells in spleen, skin, liver and skeletal muscle of allogeneic young mice and xenogeneic aged mice were in proportion to between the syngeneic young and syngeneic aged groups of mice; apoptotic cells were reduced in spleen, skin, liver and skeletal muscle in the allogeneic aged mice compared to the syngeneic aged mice, indicating that the senescence-associated phenotype was relieved at the cellular level in multiple organs.
Analysis by SA- β -gal staining and TUNEL staining showed that: age-related galactosidase-positive cytopenia in spleen, skin, liver and brain (fig. 1), apoptotic cytopenia in spleen, skin, liver and skeletal muscle (fig. 2) in allogeneic aged mice compared to syngeneic aged mice. Indicating that the senescence-associated phenotype is relieved at the cellular level in multiple organs.
HE staining results are shown in fig. 3, and fig. 3 shows photomicrographs and corresponding bar charts of liver inflammation infiltration area ratio, number of hair follicles per unit area of skin, and myofiber diameter of skeletal muscle of the syngeneic young group, syngeneic year elder Zu, allogenic young group, and xenogic aged group mice, respectively. Wherein the abscissa of the bar graph represents, from left to right, the syngeneic young group, syngeneic year elder Zu, allogeneic young group, and xenogeneic aged group mice, respectively.
The results in fig. 3 show that: the inflammatory infiltration area in the liver of mice in the old group of the same body is obviously higher than that of mice in the young group of the same body (P < 0.001); the areas of inflammatory infiltration in the livers of the allogeneic young mice and the xenogeneic aged mice are not significantly different (P > 0.05), and the areas of inflammatory infiltration in the livers of the xenogeneic young mice and the allogeneic aged mice are between the same-body young mice and the same-body aged mice; the number of hair follicles per unit area of mice in the syngeneic young group is significantly higher than that of mice in the syngeneic old group (P < 0.001); the hair follicle numbers of the allogeneic young mice and the allogeneic old mice are not significantly different (P is more than 0.05), and the hair follicle numbers of the allogeneic young mice and the allogeneic old mice are between that of the homozygote young mice and the homozygote old mice; the skeletal muscle fiber diameter of the mice in the same age group is significantly higher than that of the mice in the same age group (P < 0.001), there is no significant difference in skeletal muscle diameters (P > 0.05) between the mice in the allogeneic age group and the mice in the allogeneic age group, and the skeletal muscle diameters of the mice in the allogeneic age group and the mice in the allogeneic age group are intermediate between those of the mice in the same age group and the mice in the same age group.
The results of the masson staining are shown in fig. 4, and fig. 4 shows photomicrographs of liver and spleen fibrosis and ImageJ quantified histogram of the syngeneic young group, syngeneic year elder Zu, allogeneic young group and xenogeneic aged group mice, respectively. Wherein the ordinate of the bar graph represents the fiberized area in mm 2 The abscissa of the bar graph represents, from left to right, the syngeneic young group, syngeneic year elder Zu, allogeneic young group, and xenogeneic aged group mice, respectively.
The results in fig. 4 show that: the area of fibrosis in liver and spleen of mice in the older group of the same body is higher than that of mice in the younger group of the same body (P < 0.001); there was no significant difference in the areas of fibrosis in the liver and spleen (P > 0.05) for the allogeneic young and xenogeneic aged mice, and the areas of fibrosis in the liver and spleen were between the syngeneic young and syngeneic aged mice.
Analysis by HE staining and masson staining showed that: in allogeneic aged mice, aging-related inflammation in the liver is reduced, fibrosis of the liver and spleen is reduced, and the average diameter of skeletal muscle fibers and the number of hair follicles in the skin (typically decreasing with age) are "rejuvenated" and restored, as compared to syngeneic aged mice.
Example 2 Effect of allograft symbiosis on cell proportion variation and verification
1. The experimental method comprises the following steps:
the following procedure was carried out using three groups of experimental mice cultured for 6 weeks in example 1 as experimental samples.
1. Tissue cell isolation and sequencing
(1) Isolation of peripheral blood cells
Peripheral blood of mice was collected using EDTA-2Na anticoagulation tube. Red blood cell lysate (BD Biosciences) was added and lysed at room temperature for 15-20 minutes. Pre-chilled PBS was washed twice and cells were resuspended in PBS with 2% fbs. Viable cells were sorted by flow cytometry (BD Influx), centrifuged, resuspended in PBS containing 0.04% bsa for 10xGenomics sequencing.
(2) Isolation of bone marrow cells
The femur and tibia of the mice were isolated. Bone marrow cells were flushed from the bone marrow cavity with PBS buffer of 2% fbs and 2mM EDTA. The cell resuspension was passed through a 40 μm cell filter and filtered to give a single cell suspension. 1,200rpm, 4℃and 5 minutes. Cell pellet was resuspended in erythrocyte lysate (BD Biosciences) and lysed at room temperature for 5min. Pre-chilled PBS was washed twice and cells were resuspended in PBS with 2% fbs. Viable cells were sorted by flow cytometry (BD Influx), centrifuged, resuspended in PBS containing 0.04% bsa for 10xGenomics sequencing.
(3)Lin - c-Kit + Sca-l lo/+ Cell separation
Isolated bone marrow cells were resuspended in PBS with 2% FBS, and then linear mixed antibody, c-Kit and Sca-1 antibody were added, thoroughly mixed, and incubated at 4℃for 30 minutes in the absence of light. Wherein the linear mixed antibody comprises: anti-mouse CD3, B220, CD11B, ly6G/Ly-6C and Ter-119. The cells were resuspended in PBS with PBS containing 2% FBS and washed twice with PBS. Sorting was performed using a flow cytometer (BDInflux), after centrifugation, PBS containing 0.04% BSA was added for resuspension for 10xGenomics sequencing.
(4) Isolation of spleen cells
The spleens of mice were placed in PBS with 2% FBS, cut into small pieces with scissors, and gently ground on a 40 μm cell filter with a 5 ml syringe plunger. All spleen cells were collected, centrifuged at 1,200rpm and 4℃for 5 minutes. Cell pellet was resuspended in erythrocyte lysate (BD Biosciences) and lysed at room temperature for 5min. Pre-chilled PBS was washed twice and cells were resuspended in PBS with 2% fbs. Viable cells were sorted by flow cytometry (BD Influx), centrifuged, resuspended in PBS containing 0.04% bsa for 10xGenomics sequencing.
2. Cell ratio analysis
(1) Data preprocessing
After obtaining high throughput sequencing raw data from the NovaSeq platform, BCL2FASTQ software (version 2.20.0.422) was first used to convert the raw file in BCL format to a FASTQ format file. The FASTQ file was aligned to the mm10 mouse reference genome by the treatment of Cell range software (version 3.1.0) and then a filtered expression matrix was obtained.
(2) Cell clustering and cell type identification
Gene expression matrices of single cell transcriptomes derived from immune cells of LSK cells, bone marrow, peripheral blood, spleen and skin were analyzed by Scanpy (version 1.4.4) for low quality cell filtration, sample integration, data normalization, data dimension reduction, cell clustering and differential gene expression. The possible multicellular results in the data were first detected using the scrublet software (version 0.2.1) and removed. The filtered expression matrix will be analyzed according to the following steps: (1) cells that meet any of four conditions will be rejected first: less than 500 or more than 6000 genes, less than 500 or more than 40000 specific molecular markers (UMI), more than 10% mitochondrial genes and more than 40% ribosomal genes; (2) integrating and normalizing the data of different samples through Scanpy standard flow, and removing the batch effect of the different samples by using a 'sc.external.pp.bbknn' function; (3) dimensionality reduction analysis and data clustering was performed from the first 75 principal components by the "sc.tl.umap", "sc.pp.neighbors" and "sc.tl.louvain" functions of Scanpy; (4) performing differential expression analysis (Wilcoxon rank sum test) between different cell populations by using the "FindAllMarkers" function of the Seurat software package (version 3.2.3), and calculating a fold difference (FC), wherein the screening standard is that the corrected P value is smaller than 0.05 and the l logFC is larger than 0.5; (5) the cell populations highly expressing Gm42418 or AY036118 genes were further knocked out and the analysis of steps 2 to 4 was repeated, followed by determination of the cell type from the classical cell type marker gene of each cell population.
Through the above procedure we obtained a single cell profile of the hematopoietic and immune system of mice containing 74323 high quality cells.
(3) Single cell pseudo-time trajectory analysis
The development traces of the mouse hematopoietic and immune system single cell profile were constructed by conducting PAGA analysis (iter=1000, layout=fa) in Scanpy software package. Pseudo-time analysis was performed on randomly extracted cells (3000 per group) using the Monocle2 software package. The gene screening threshold for ranking was at least 10 cells and the q value characterizing the difference in expression and dispersion between clusters was less than 0.01. And drawing a track structure on a two-dimensional plane by using a DDRTreee dimension reduction algorithm, and performing pseudo-time sequencing on the randomly extracted cells. Invoking the "BEAM_rest" function of Monocle2 determines 1000 Differentially Expressed Genes (DEGs) (P-value < 0.001) clustered into 6 gene clusters, and plots the change in expression of these genes on pseudo-time trajectories by the "plot_genes_clamped_hetmap" function of Monocle 2.
(4) Gene set scoring analysis
The expression of the corresponding gene set in each cell was scored using the "AddModuleScore" function of seal. The change in gene set scores between the syngeneic young, syngeneic old, allogeneic young and xenogeneic old groups was calculated by the Wilcoxon test of the ggpubr software package (https:// gitub. Com/kassambara/ggpubr) (version 0.2.4).
(5) Analysis of cell composition changes
Before performing the cell ratio change analysis, we first removed cell types whose number was less than 2.5% of the total cell number of each tissue. The data for the same cell type in the different groups (Iso-Y, het-Y, iso-O, het-O) will be divided by the total cell number of the group to obtain their corresponding cell ratios, thereby further calculating the percentage of the particular cell type in each group. Next, we determined the cell type with the ratio change due to aging (|Log2FC| > 0.5) based on the Log2FC of the cell ratio difference between Iso-O and Iso-Y, and the cell type with the ratio change due to intergrowth (|Log2FC| > 0.5) based on the difference between Het-Y and Iso-Y groups or Het-O and Iso-O groups.
3. Experimental verification of cells with changes in the information data cues
Cell types with significant changes were suggested for several biographical data by flow analysis. The method comprises the following specific steps:
cryopreserved bone marrow cells were resuscitated from cell cryopreservation containing 10% dmso and 90% fbs (Gibco), B220, igM and CD43 antibodies were added and incubated at 4 ℃ for 30 min in the absence of light. PBS was washed (1200 rpm,5min,4 ℃ C.) twice. Prior to analysis, 7-AAD was added for staining to exclude dead cells, pro-B cell ratios were detected using a flow cytometer (BD Fortessa) and data analysis was performed using FlowJo software (Tree Star inc.).
2. Experimental results
The 6 gene sets associated with differentiation of hematopoietic stem cells into the myeloid and gonomic lineages were calculated (fig. 5). The results of the genome scoring for the different gene sets between the four groups showed an increase in the potential of hematopoietic stem cells to differentiate into the myeloid lineage during aging (gene set 2), a decrease in the potential to differentiate into the gonomic lineage (gene sets 5 and 6), while the allosymbiosis restored the potential of hematopoietic stem cells to differentiate into the gonomic lineage (gene sets 5 and 6) (fig. 6). Cell proportion analysis of single cell data found that B cell progenitors (pro-B) decreased in proportion to aging, while allograft symbiosis was effective in restoring the decrease in their numbers (fig. 7). Compared with the symbiotic young group (Iso-Y), the pro-B cell proportion of the older group of the same body is down-regulated; compared with the homologous young group, the pro-B cell proportion of the allogeneic young group is down-regulated; the pro-B cell ratios were up-regulated in the aged groups compared to the same aged groups (FIGS. 8-10), and these results were consistent with the analysis data of the letter generation.
EXAMPLE 3 differential Gene analysis and validation of aging Critical to hematopoietic Stem (progenitor) cells
1. Experimental method
1. Differential gene calculation
(1) Differential expression and cell type specific differential gene network analysis
Differential expression analysis was performed for each cell type between the different groups (Iso-O/Iso-Y, het-Y/Iso-Y and Het-O/Iso-O) using Wilcoxon rank sum test in the "FindMarkers" function of the Seurat software package (version 3.2.3) as a method of statistical calculation. Cell types with a number of less than three cells in a group are pre-excluded prior to performing the differential expression analysis. Calculating the difference genes between the Iso-O group and the Iso-Y group to obtain a difference gene set (aging-related) of (I LogFC I >0.25 and adjusted P-value < 0.05); calculating the difference genes between the Het-O group and the Iso-O group to obtain a difference gene set (HY DEGs) related to the allosymbiotic young group (I LogFC I >0.25, adjusted P-value < 0.05); the differential genes between Het-O and Iso-O groups were calculated to give Differential Gene Sets (HODEGs) associated with the intergrowth aged group (|LogFC| >0.25, adjusted P-value < 0.05) (see Table 4 for a detailed list of genes). In the above differential expression gene set, gm42418 and AY036118 genes that may not characterize biological changes were deleted. Based on the above gene set, a "reverse senescence-difference genes (maging-R DEGs)", i.e., genes significantly up-regulated or down-regulated in HO DEGs and significantly down-regulated or up-regulated in maging DEGs, respectively, was determined; a "senescence-promoting differential gene (ing-P DEGs)" was determined, i.e., a gene that was significantly up-or down-regulated in both HY DEGs and ing DEGs.
(2) Transcription Factor (TF) regulatory network analysis
The transcription factor regulation network analysis is realized through a standard analysis flow of an scendic software package (version 1.1.2.2): the mm10 database of the RcisTarget software package (version 1.6.0) is used as a reference database, the GENIE3 software package (version 1.6.0) is called, and a gene expression regulation network is established according to the differential genes of each cell type of bone marrow, peripheral blood and spleen tissue. Information on the enriched TF binding motif, predicted candidate target gene (regulatory) and target gene activity was obtained from RcisTarget and visualized by the Cytoscape software package (version 3.7.2) for transcriptional regulatory networks.
(3) Intercellular communication analysis
Intercellular communication analysis based on single cell sequencing data was performed using CellPhoneDB software package (version 1.1.0) (www.cellphonedb.org). Only receptors and ligands expressed in at least 10% of cells of a particular cell type are included in subsequent assays, and when one of the ligands or receptors does not meet the criteria expressed in at least 10% of cells, the intercellular communication is discarded. Comparing the average expression levels of each ligand-receptor pair between different cell types, a significant ligand-receptor pair (P-value < 0.01) would be used to predict inter-cellular communication across tissues within the three groups Iso-Y, iso-O and Het-O.
2. Preparation of virus liquid containing recombinant lentivirus over-expressing Yy1 or Ccl3 genes
(1) Construction of recombinant plasmids
The mouse Yy1 and Ccl3 genes are amplified from bone marrow cell cDNA of a C57BL6/J mouse by a PCR method, and are connected to an SF-LV-EGFP vector subjected to Mlu1 and BamH1 (NEB) enzyme digestion by an enzyme digestion method, so that a recombinant lentiviral plasmid SF-LV-Yy1-EGFP (a recombinant expression vector in which sequences between Mlu1 and BamH1 recognition sites of SF-LV-EGFP are replaced by the mouse Yy1 gene, and other nucleotide sequences of SF-LV-EGFP are kept unchanged) and SF-LV-Ccl3-EGFP (a recombinant expression vector in which sequences between Mlu1 and BamH1 recognition sites of SF-LV-EGFP are replaced by the mouse Ccl3 gene, and other nucleotide sequences of SF-EGFP are kept unchanged) are obtained.
Wherein, the GenBank number of the amino acid sequence of the mouse CCL3 protein is NP-035467.1, the nucleic acid molecule encoding the mouse CCL3 protein is the mouse Ccl3 gene, the GenBank number of the mouse Ccl3 gene is NM-011337.2, and the encoding sequence is DNA molecule with the nucleotide sequence shown in the 102 th-380 th positions of NM-011337.2.
Wherein, the amino acid sequence of the mouse YY1 protein is GenBank number NP-033563.2, the nucleic acid molecule encoding the mouse YY1 protein is the mouse Yy1 gene, the GenBank number of the mouse Yy1 gene is NM-009537.4, and the encoding sequence is DNA molecule with the nucleotide sequence shown in NM-009537.4 positions 117-1361.
The sequences of Yy1-F and Yy1-R are as follows:
Yy1-F：5’-CGACGCGTGCCACCATGGCCTCGGGCGACACCCTCTACAT-3' (Mlu 1 recognition site underlined);
Yy1-R：5’-CGCGGATCCTCACTGGTTGTTTTTGGCTTTAGCGTGT-3' (BamH 1 recognition site underlined).
The sequences of Ccl3-F and Ccl3-R are as follows:
Ccl3-F：5’-CGACGCGTGCCACCATGAAGGTCTCCACCACTGCCCTTGCTG-3' (Mlu 1 recognition site underlined);
Ccl3-R：5’-CGCGGATCCTCAGGCATTCAGTTCCAGGTCAGTGATG-3' (BamH 1 recognition site underlined).
(2) Preparation of recombinant viruses
2-1) 10. Mu.g SF-LV-EGFP or SF-LV-Yy1-EGFP or SF-LV-Ccl3-EGFP lentiviral plasmid, 10. Mu.g psPAX2 plasmid and pMD2G plasmid were co-transfected into 1 10cm dish of 293T cells using Lipo3000 transfection kit (Thermo Fisher Scientific). After 8 hours of incubation, the medium was replaced with fresh one.
2-2) collecting supernatant of 24 hours, 36 hours and 72 hours, filtering with 0.22 μm filter, and storing the filtered virus supernatant at 4 ℃.
2-3) at 19400rpm,4℃for 2.5 hours, discarding the supernatant, re-suspending with hematopoietic stem 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 sub-packaging at-80℃for preservation.
3. LSK cell isolation and lentiviral transfection
(1) LSK cell separation
1-1) euthanized 24 month old mice (C57 BL6/J, CD45.2, 4-6) were carefully dissected out of the femur, tibia and gluteus, placed in a sterile mortar, and 5mL of pre-chilled HBSS was added + The bone is gently crushed by a grinding rod, and bone marrow cells are separated. Filtering the bone marrow cells through a 40 mu m cell filter to obtain single cell suspension, centrifuging at 450g and 4 ℃ for 5 minutes to collect the bone marrow cells;
1-2) after cell resuspension, antibodies to c-Kit-APC were added, incubated at 4℃for 30 minutes in the absence of light, and HBSS was added + After the solution is centrifugally washed, anti-APC micro-beads are added, and the mixture is incubated for 30 minutes at 4 ℃ in a dark place;
1-3) enrichment of c-Kit using MACS magnetic columns (Miltenyi Biotec, germany) + And (3) cells. The magnetic column uses HBSS first + Carrying out rinsing on the solution; adding the stained cells to HBSS + The solution is transferred to a magnetic column, and after c-Kit negative cells flow, HBSS is added + Washing the solution once; evacuating the magnetic column from the magnetic field, adding HBSS + The solution was loaded onto a magnetic column and the cells were slowly pushed out with a piston and repeated once. 450g,4℃and 5 min centrifugation to collect c-Kit + A cell;
1-4) adding Linear-biotin Mixed antibody to c-Kit + In cells. Thoroughly mixed and incubated at 4℃for 30 minutes in the absence of light. Wherein the linear-biotin mixed antibody comprises: anti-mouse CD3, CD4, CD8, B220, CD11B, ly6G/Ly-6C and Ter-119;
1-5) adding HBSS + 450g of the solution, centrifuging at 4 ℃ for 5 minutes, and collecting cells;
1-6) Streptomavidin-APC-eFluor addition TM 780，Sca-1-PE-Cy TM 7, c-Kit-APC antibody, 4 ℃ light shielding incubation for 30 minutes, 450g,4 ℃, centrifugation for 5 minutes, cell collection;
1-7) staining was performed by adding DAPI to exclude dead cells prior to sorting, LSK cells were sorted using a flow cytometer (BD Fusion).
(2) Lentivirus transfection
2-1)2×10 5 Individual LSK cells were cultured in low adhesion 96-well plates (post Biotech) using HSC medium and after 2h concentrated lentivirus (recombinant lentivirus LV/SF-LV-E was addedGFP or recombinant lentivirus LV/SF-LV-Yy1-EGFP or recombinant lentivirus LV/SF-LV-Ccl 3-EGFP) for virus transfection efficiency titration; after 72h of transfection, collecting cells (recombinant LSK cells obtained by transfection of recombinant lentivirus LV/SF-LV-EGFP or recombinant lentivirus LV/SF-LV-Yy1-EGFP or recombinant LSK cells obtained by transfection of recombinant lentivirus LV/SF-LV-Ccl 3-EGFP) WB to verify the overexpression efficiency;
2-2) after 72h of transfection 450g, cells were collected by centrifugation at 4℃for 5 min, sca-1-PE-Cy was added TM 7, CD48-PerCP/cyanine5.5 antibody, 4 ℃ light-shielding incubation for 30 minutes, 450g,4 ℃, centrifugation for 5 minutes, cell collection;
2-3) staining with DAPI to exclude dead cells prior to sorting, sorting GFP with a flow cytometer (BD Fusion) + Sca1 + CD48 - cell-to-HBSS + In the solution, recombinant cell GFP was obtained + Sca1 + CD48 - LV/SF-LV-EGFP (recombinant cells transfected with LV/SF-LV-EGFP), GFP + Sca1 + CD48 - LV/SF-LV-Yy1-EGFP (recombinant cells transfected with LV/SF-LV-Yy 1-EGFP) and GFP + Sca1 + CD48 - LV/SF-LV-Ccl3-EGFP (recombinant cells transfected with LV/SF-LV-Ccl 3-EGFP).
4. Transplantation and peripheral blood detection
(1) Mouse transplantation
1-1) mice ready for irradiation: the 8 week old CD45.2 recipient mice (C57 BL6/J, 8-10) were irradiated with X-ray irradiation apparatus (RS-2000,Rad Source Technologies) at a total dose of 10Gy (in two doses of 5Gy 3 hours apart) prior to implantation;
1-2) preparation of competing bone marrow cells: 1-2 mice (C57 BL 6/J) with 2-3 months of age were euthanized, the femur was removed, and a bone marrow single cell suspension (HBSS) was isolated + Solution resuspension), counting with a Vi-Cell XR Cell viability analytical counting instrument (Beckman Coulter) to obtain CD45.1 competitive bone marrow cells;
1-3) 2000 GFP was obtained in the sorting + Sca1 + CD48 - Cells (GFP) + Sca1 + CD48 - LV/SF-LV-EGFP or GFP + Sca1 + CD48 - LV/SF-LV-Yy1-EGFP or GFP + Sca1 + CD48 - /LV/SF-LV-Ccl 3-EGFP) was added 2X 10 5 CD45.1 competitive bone marrow cells were transplanted into 1-1) treated recipient mice by tail vein injection to obtain VC mice (GFP injection) + Sca1 + CD48 - Mice obtained from competing bone marrow cells with/LV/SF-LV-EGFP and CD 45.1), YY1 mice (GFP injection + Sca1 + CD48 - Mice obtained from competing bone marrow cells with/LV/SF-LV-Yy 1-EGFP and CD 45.1), CCL3 mice (GFP injection) + Sca1 + CD48 - Mice obtained from competing bone marrow cells with LV/SF-LV-Ccl3-EGFP and CD 45.1).
(2) Peripheral blood test
2-1) after mice were transplanted four weeks, tail vein blood was collected from VC mice, YY1 mice and CCL3 mice, CD45.1, CD11B, B220 and CD3 antibodies were added, incubated at 4℃for 30 minutes in the absence of light, red blood cells were lysed with red blood cell lysate, and after centrifugation washing with PBS containing 2% FBS (1200 rpm,5min,4 ℃), cells were collected;
2-2) staining with DAPI or PI to exclude dead cells, detecting donor-derived chimerism by flow cytometry (BDFortessa), and analyzing the ratio distribution of Myeloid cells, B cells and T cells;
2-3) at 4 week intervals, the tail venous blood of the transplanted mice was assayed as described above.
5. WB validation of YY1 or CCL3 overexpression efficiency
(1) LSK cells transfected for 72h were collected and lysed with 1 x SDS lysate (containing 4% SDS and 100mM Tris-HCl (ph=6.8)) at 105 ℃ for 10 min;
(2) Quantifying total protein using BCA protein kit;
(3) Protein samples were run on SDS-PAGE and then transferred to PVDF membrane (Millipore);
(4) Adding a primary antibody of YY1 (Santa Cruz) or CCL3 (Santa Cruz) for incubation at 4 ℃ overnight, and incubating with a secondary antibody coupled with HRP for 1 hour at room temperature;
(5) Exposing and developing.
2. Experimental results
The result shows that: 171 senescence-reversing differential genes (149 upregulation and 22 downregulation), and 540 senescence-associated differential genes (131 upregulation and 409 downregulation), 119 senescence-promoting differential genes (96 downregulation and 23 upregulation) were calculated in bone marrow hematopoietic stem progenitor cells (fig. 11). In turn, 36 allosymbiotic core genes can be obtained. Wherein 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 4639), 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), ccClca 3 (Gene ID 21929), etv (Gene ID 27049), ccl3 (Gene ID 20302), ccl 2 (Gene ID 54199), dntt 89 (Gene ID 62), ppp1 (in the intergrown, pzm, and the intergrown of individuals (in the intergrowth), the intergrowth of the individuals, the intergrowth of the individuals, and the individuals, the intergrowth, and the intergrowth are expressed in the intergrowth, and the intergrowth. In addition, prtn3 (GeneID: 19152), lars2 (GeneID: 102436), hp (GeneID: 15439), ms4a3 (GeneID: 170813), mpo (GeneID: 17523), S100a8 (GeneID: 20201), elane (GeneID: 50701), S100a9 (GeneID: 20202) up-regulate expression in hematopoietic stem progenitors of syngeneic aged individuals and allogeneic young individuals, and down-regulate expression in allogeneic aged individuals (reverse aging) (FIG. 12). Further analysis of core regulatory transcription factors found that Atf3, atf4 and Yy1 are key regulatory factors therein (fig. 13), and were all down-regulated in hematopoietic stem progenitors of syngeneic aged individuals and syngeneic young individuals, with Atf3 and Atf4 up-regulated in syngeneic aged individuals (reverse senescence) (fig. 14). In addition, analysis of the results of intercellular communication showed that allogeneic symbiosis could partially restore intercellular ligand receptor interactions of bone marrow hematopoietic stem progenitor cells, wherein further screening found that the cc l 3-related ligand receptor pairs were also effectively restored therein, presumably playing a key regulatory role in hematopoietic stem progenitor cell differentiation (fig. 15). Subsequently, we validated the Yy1 gene and the Ccl3 gene.
1. Identification of over-expressed YY1 in LSK cells
To investigate the role of YY1 in aged hematopoietic stem (progenitor) cells, lentiviral transfection was performed on LSK cells, and WB results showed that, at the protein level, YY1 was significantly increased in recombinant LSK cells obtained by recombinant lentiviral LV/SF-LV-YY1-EGFP transfection (fig. 16).
2. Overexpression of YY1 enhances the reconstitution capacity of aged hematopoietic stem (progenitor) cells
The effect of over-expression YY1 on the ability of aged hematopoietic stem (progenitor) cells to reconstitute was assessed by competitive transplantation experiments, the results of which are shown in fig. 17. FIG. 17A is a flow chart of a competitive transplantation experiment; in fig. 17, B is a donor-derived analysis of the peripheral blood test of mice, and the ordinate of B in fig. 17 represents the cell fraction of the donor source of the aged mice in% and the abscissa represents the number of weeks after the mouse transplantation experiment in weeks (7 days). Fig. 17B shows that: in whole cells, T cells and Myeloid cells of the peripheral blood of the mice, the donor source ratio of the aged mice (YY 1 mice) which over-express YY1 is obviously higher (P < 0.05) than that of the control group (VC mice); the donor-derived ratio of aged mice overexpressing YY1 was not significantly different from that of the control group (P > 0.05) in B cells of the peripheral blood of the mice. It is shown that overexpression of YY1 can enhance the reconstitution capacity of aged hematopoietic stem (progenitor) cells, mainly in global levels, T cells and Myeloid cells.
3. Identification result of LSK cell over-expression CCL3
To investigate the role of CCL3 in aged hematopoietic stem (progenitor) cells, lentiviral transfection of LSK cells, WB results showed a significant increase in the protein level of CCL3 in recombinant LSK cells transfected with recombinant lentivirus LV/SF-LV-CCL3-EGFP (fig. 18).
4. Overexpression of CCL3 enhances the reconstitution capacity of aged hematopoietic stem (progenitor) cells
The effect of over-expression CCL3 on the ability of aged hematopoietic stem (progenitor) cells to reconstitute was assessed by competitive transplantation experiments, the results of which are shown in fig. 19. The ordinate of fig. 19 represents the fraction of cells derived from the donor of the aged mice in units of weeks after the mice transplantation experiment, and the abscissa represents the number of weeks (7 days). Fig. 19 shows: in T cells of the mouse peripheral blood, the donor source ratio of the aged mice over-expressing CCL3 was significantly (P < 0.05) higher than that of the control group (VC); the donor-derived ratio of aged mice overexpressing CCL3 was not significantly different from the control group (P > 0.05) in whole cells, B cells and Myeloid cells of the mouse peripheral blood. It was shown that overexpression of CCL3 can enhance the reconstitution capacity of aged hematopoietic stem (progenitor) cells, predominantly at the global level, T cells and Myeloid cells. The results show that over-expression of CCL3 can increase the reconstitution capacity of aged hematopoietic stem (progenitor) cells, predominantly at the T cell level.
5. Effect of overexpression of CCL3 on enhanced differentiation of hematopoietic stem (progenitor) cells into the lineage
The effect of CCL3 on the lineage differentiation ability of aged hematopoietic stem (progenitor) cells was further assessed by competitive transplantation experiments and the results are shown in fig. 20, where each bar graph represents the ratio of myeloid lineage, B cell and T cell, respectively, from top to bottom. The ordinate of fig. 20 represents the proportion of the lineage analysis of the test donor source of the aged mice in% in which the sum of the myeloid cells, B cells, T cells is 100%, and the abscissa represents the number of weeks after the mouse transplantation experiment in weeks (7 days). The results in FIG. 20 show that the proportion of T cells detected at various time points in aged mouse cells overexpressing CCL3 was significantly higher (P < 0.05) than in the control group (VC). The proportion of myeloid and B cells was not significantly different from the control group at 12, 16 and 20 weeks of transplantation (P > 0.05). The results show that overexpression of CCL3 can increase the direction of differentiation of aged hematopoietic stem (progenitor) cells towards the lineage, predominantly at the T cell level.
Example 4 differential Gene validation of aging Critical to skin fibroblasts
1. Experimental method
1. Mouse or human fibroblast culture
The human skin tissue was approved by the ethics committee of the Beijing co-ordinates hospital and the donor signed informed consent. The upper eyelid tissue of a 27 year old healthy person was minced with scissors, attached to the bottom of a 6-well plate, and 0.5ml of fibroblast growth medium (90% dmem,10% fetal bovine serum, 1% neaa,1% glutamax,0.10% plasmocin) was added. Cells were grown to 80% and passaged with 0.25% trybasin. The cells obtained were used for subsequent experimental analysis.
Mouse fibroblasts were obtained from 7 week male mouse back skin tissue, sheared with scissors, 8mg/mL Collagenase 4 was filtered through a 40 micron filter membrane in a 37C water bath for 1 hour 30 minutes, 10mL of fibroblast growth medium was added, inoculated in 10cm dishes, the cells grown to 80%,0.25% Trypsin was used for digestive passages. The cells obtained were used for subsequent experimental analysis.
2. Tsc22d3 (Gilz) gene knockdown
The human GILZ protein can be protein coded by human Gilz gene, the GenBank number of the human Gilz gene is XM_005262103.5, the coding sequence is DNA molecule with the nucleotide sequence shown in 160-762 of XM_005262103.5, and the GenBank number of the human GILZ protein is: np_001015881.1. The mouse GILZ protein may be a protein encoded by a mouse GILZ gene, the mouse GILZ gene having GenBank No. nm_001077364.1, the nucleotide sequence encoded by the mouse GILZ gene being a DNA molecule having nucleotide sequences shown at positions 204 to 809 of nm_001077364.1, the mouse GILZ protein having GenBank No.: np_001070832.1.
The inflammatory environment in aged tissues was simulated using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific), transfecting fibroblasts with negative control and siRNA (Ruibo) against the target gene for 48 hours, and then treating the fibroblasts with 50ng/mL IL-6 for 24 hours. The GILZ gene siRNA is shown in table 1:
Table 1: gilz gene siRNA
Cells were collected for RT-qPCR, ki67 immunostaining and apoptosis analysis.
2、RT-qPCR
After 48 hours of transfection of cells of RNAi sequences with human or mouse, total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific) and 2. Mu.g of total RNA was reverse transcribed into cDNA with master mix (Promega). RT-qPCR was performed with iTaq Universal SYBR GreenSuper Mix (Bio-Rad). The expression of the mouse or human Gilz gene was examined using GAPDH as an internal control, and the primer sequences for Gilz gene are shown in table 2.
Table 2: primer sequence of Gilz gene
The detection results are shown in FIG. 23. The results showed that the siRNA was effective in reducing the expression of the GILZ gene in mouse and human fibroblasts.
3. Ki67 immunofluorescence staining
To measure changes in cell proliferation, mouse or human fibroblasts were seeded on 24-well plate glass plates and tested 72 hours after siRNA treatment according to step 2, each group consisting of three experimental replicates and two independent experiments. Not less than 300 cells per sample were counted for data analysis.
Fibroblasts were fixed with 4% PFA for 25min at 25min,0.4%Triton X-100 permeabilities, incubated with blocking buffer (10% donkey serum) for 1h at room temperature, and stained overnight with Ki67 antibody (abcam 15580) at 4 ℃. Cells were then incubated with secondary antibody and Hoechst33342 (Thermo Fisher Scientific) for 1 hour and imaged by scanning with a confocal laser scanning microscope (Zeiss 900 confocal system). And counting the proportion of Ki67 positive cells.
The results showed that the cell proliferation ability was significantly reduced by treating human or mouse fibroblast cells with IL6 to knock down GILZ.
4. Apoptotic cell detection
To measure changes in apoptosis, mouse or human fibroblasts were isolated at 1×10 e Cells were seeded into 12-well plates per well and tested 72 hours after siRNA treatment according to step 2, each group contained three experimental replicates and two independent experiments.
Freshly collected fibroblasts were stained according to the instructions of Annexin V-EGFP apoptosis detection kit (Vazyme Biotechnology, A211-02), then flow analyzed using a BD LSRFortesa flow cytometer, and the data analyzed using FlowJo software (TreeStar).
The results showed that the number of apoptosis was significantly increased by treating human or mouse fibroblasts with IL6 to knock down GILZ.
2. Experimental results
The result of the letter generation calculation is shown in fig. 21. Fig. 21 shows that: myeloid Progenitor (CMP), pluripotent group (MPP) and gonomic progenitor (CLP) in hematopoietic stem progenitor cells of mice of the same age group down-regulate Tsc22d3 (Gilz) gene expression compared to mice of the same age group; down-regulation of Tsc22d3 (Gilz) gene expression in Basal stem cells (Basal-1), acanthocellular cells (Spi), reticulocytes (RetiFib) and follicular stem cells (HFSC) in skin; the difference in Tsc22d3 (Gilz) gene expression in type IIX myofibroblasts of skeletal muscle (fast_iix) is small; tsc22d3 (Gilz) gene expression was down-regulated in kupffer_cell, zone 1 hepatic parenchymal cell (hepatocyte_z1), cholangiocyte (Cholangiocyte), endothelial Cell (EC), zone3 hepatic parenchymal cell (hepatocyte_z3) and Zone 2 hepatic parenchymal cell (hepatocyte_z2) in the liver. Myeloid Progenitor (CMP), pluripotent group (MPP) and gonomic progenitor (CLP) in hematopoietic stem progenitor cells of allogeneic aged mice are upregulated by Tsc22d3 (Gilz) gene expression, as compared to autologous aged mice; upregulation of Tsc22d3 (Gilz) gene expression in Basal stem cells (Basal-1), acanthosis cells (Spi) and reticulocytes (retififb) in skin; tsc22d3 (Gilz) gene expression was up-regulated in Zone 2 liver parenchymal cells (hepatocyte_z2) in the liver. From the results of fig. 21, it can be inferred that: tsc22d3 (Gilz) down regulates expression with aging in a variety of peripheral tissue cell types, while allograft symbiosis can partially restore its changes.
The results of RT-qPCR detection of the relative expression level of the target gene are shown in FIG. 22. Fig. 22 shows that: both human and mouse fibroblasts of the RNA interference group (si-GILZ) were significantly lower than the control group (si-NC), indicating that RNAi approach successfully constructed human and mouse fibroblasts with Tsc22d3 (Gilz) gene silencing
The results of Ki67 immunofluorescence staining are shown in figure 23. The results in fig. 23 show that: the number of cells stained with Ki67 was significantly lower in human fibroblasts and mouse fibroblasts in the RNA interference group (si-GILZ) than in the control group (si-NC), indicating a decrease in the cell proliferation capacity of the RNA interference group (si-GILZ).
The results of the ratio detection of apoptotic cells are shown in FIG. 24. The results in fig. 24 show that: the proportion of apoptotic cells (7.81%, 7.89%) was significantly lower in human and mouse fibroblasts in the RNA interference group (si-GILZ) than in the control group (11.80, 12.20), indicating an elevated level of apoptosis in the RNA interference group (si-GILZ).
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
<110> animal institute of China academy of sciences
<120> allosymbiotic rejuvenation factor and its use in delaying aging
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> mice (Mus musculus)
<400> 1
ccatggacct cgtgaagaa 19
<210> 2
<211> 19
<212> DNA
<213> Homo sapiens (Homo sapiens)
<400> 2
gtgagaacac cctgttgaa 19
Claims (10)
1. An application, which is any one of the following A1) -a 10):
a1 Use of protein CCL3 or protein YY1 or protein GILZ or a substance that modulates the expression of the protein CCL3 or the protein YY1 or the protein GILZ-encoding gene or a substance that modulates the activity or amount of the protein CCL3 or the protein YY1 or the protein GILZ in modulating the level of aging of an organism and/or a cell and/or a tissue;
a2 Protein CCL3 or protein YY1 or protein GILZ or a substance regulating the expression of the protein CCL3 or the protein YY1 or the protein GILZ-encoding gene or a substance regulating the activity or the content of the protein CCL3 or the protein YY1 or the protein GILZ in the preparation of a product for the senescence level of an organism and/or a cell and/or a tissue;
a3 Use of CCL3 or YY1 or GILZ or a substance that determines the content of CCL3 or YY1 or GILZ in the identification or assisted identification of the level of senescence in an organism and/or a cell and/or a tissue;
a4 Use of CCL3 or YY1 or GILZ protein or a substance for determining the content of CCL3 or YY1 or GILZ protein for the preparation of a product for the identification or assisted identification of the level of senescence in an organism and/or a cell and/or a tissue;
A5 Protein CCL3 or protein YY1 or a substance that modulates the expression of a gene encoding said protein CCL3 or said protein YY1 or a substance that modulates the activity or content of said protein CCL3 or said protein YY1 in modulating the reconstitution capacity of hematopoietic stem cells;
a6 Protein CCL3 or protein YY1 or a substance that modulates the expression of a gene encoding said protein CCL3 or said protein YY1 or a substance that modulates the activity or content of said protein CCL3 or said protein YY1 in the preparation of a product that modulates the reconstitution capacity of hematopoietic stem cells;
a7 A protein CCL3 or a protein YY1, or a substance that modulates the expression of a gene encoding the protein CCL3 or the protein YY1, or a substance that modulates the activity or content of the protein CCL3 or the protein YY1, in modulating lineage differentiation of hematopoietic stem cells;
a8 Protein CCL3 or protein YY1 or a substance that modulates the expression of a gene encoding said protein CCL3 or said protein YY1 or a substance that modulates the activity or content of said protein CCL3 or said protein YY1 in the manufacture of a product that modulates lineage differentiation of hematopoietic stem cells;
a9 Protein GILZ or a substance regulating the expression of a gene encoding the protein GILZ or a substance regulating the activity or content of the protein GILZ in regulating the proliferation capacity of skin fibroblasts;
A10 Protein GILZ or a substance regulating the expression of a gene encoding the protein GILZ or a substance regulating the activity or content of the protein GILZ in the preparation of a product regulating the proliferation capacity of skin fibroblasts.
2. The use according to claim 1, characterized in that: the substance regulating the expression of the gene encoding the protein CCL3 or the protein YY1 or the substance regulating the activity or the content of the protein CCL3 or the protein YY1 is a biological material B1, and the biological material B1 is any one of the following B11) to B17):
b11 A nucleic acid molecule encoding said protein CCL3 or said protein YY 1;
b12 An expression cassette comprising a nucleic acid molecule according to B11);
b13 A recombinant vector comprising the nucleic acid molecule of B11), or a recombinant vector comprising the expression cassette of B12);
b14 A recombinant microorganism comprising the nucleic acid molecule of B11), or a recombinant microorganism comprising the expression cassette of B12), or a recombinant microorganism comprising the recombinant vector of B13);
b15 A transgenic animal cell line comprising the nucleic acid molecule of B11), or a transgenic animal cell line comprising the expression cassette of B12), or a transgenic animal cell line comprising the recombinant vector of B13);
B16 A transgenic animal tissue comprising the nucleic acid molecule of B11), or a transgenic animal tissue comprising the expression cassette of B12), or a transgenic animal tissue comprising the recombinant vector of B13);
b17 A transgenic animal organ containing the nucleic acid molecule of B11), or a transgenic animal organ containing the expression cassette of B12), or a transgenic animal organ containing the recombinant vector of B13).
3. The use according to claim 2, characterized in that: b11 The nucleic acid molecule is the coding gene of the protein CCL3 or the protein YY1, and the coding gene of the protein CCL3 is a Ccl3 gene or a coding sequence of the Ccl3 gene; the coding gene of the protein YY1 is a Yy1 gene or a coding sequence of the Yy1 gene.
4. The use according to claim 1, characterized in that: the substance regulating the expression of the gene encoding the protein GILZ or the substance regulating the activity or the content of the protein GILZ is a biological material B2, and the biological material B2 is an RNA molecule inhibiting or reducing the expression of the gene encoding the protein GILZ or an RNA molecule inhibiting or reducing the activity or the content of the protein GILZ.
5. The use according to claim 1, characterized in that: b21 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 is i 1) or i 2) or i 3) below:
i1 The target sequence of the RNA molecule is the coding gene of the protein GILZ;
i2 The nucleotide sequence of the target sequence of the RNA molecule is SEQ ID No.1;
i3 The nucleotide sequence of the target sequence of the RNA molecule is SEQ ID No.2.
6. Use of a heterosymbiont in any one of the following P1) -P18):
p1), decreasing the proportion of senescent cells in spleen tissue and/or skin tissue and/or liver tissue and/or brain tissue;
p2), decrease the proportion of apoptosis in spleen tissue and/or skin tissue and/or liver tissue and/or skeletal muscle tissue;
p3), reducing the area of liver tissue inflammation;
p4), reducing liver tissue and/or spleen tissue fibrosis areas;
p5), increasing skeletal muscle tissue myofiber diameter;
p6), increasing the number of hair follicles of skin tissue;
p7), increasing pro-B cell ratio in bone marrow;
p8), increase CCL3 protein or/and YY1 protein expression levels in hematopoietic stem cells;
p9), up-regulates the expression level of the Ccl3 gene or/and the Yy1 gene.
P10) delaying senescence by decreasing the proportion of senescent cells in spleen tissue and/or skin tissue and/or liver tissue and/or brain tissue;
p11) delay aging by decreasing the proportion of apoptosis in spleen tissue and/or skin tissue and/or liver tissue and/or skeletal muscle tissue;
P12) delay aging by reducing the area of inflammation of liver tissue;
p13) delay aging by reducing the fibrotic area of liver tissue and/or spleen tissue;
p14) delaying aging by decreasing skeletal muscle tissue myofiber diameter;
p15) delay aging by reducing the number of hair follicles in skin tissue;
p16) delay aging by increasing pro-B cell proportion in bone marrow;
p17) delaying senescence by increasing CCL3 protein or/and YY1 protein expression levels in hematopoietic stem cells;
p18) delays senescence by up-regulating the expression level of the Ccl3 gene or/and the Yy1 gene.
7. A method of constructing a recombinant cell, characterized by: the method comprises the steps of introducing a target protein coding gene into a receptor cell, promoting or improving or up-regulating the expression of the target protein coding gene, or promoting or improving or up-regulating the activity or content of the target protein, so as to obtain a recombinant cell with the aging level lower than that of the receptor cell;
the target protein is the CCL3 protein of claim 1 or the YY1 protein of claim 1.
8. Recombinant cells constructed by the method of claim 7.
9. Use of the recombinant cell line of claim 8 in the following D1) or D2):
d1 Use in improving the reconstitution capacity of aged hematopoietic stem cells;
D2 For the preparation of a product for improving the reconstitution capacity of aged hematopoietic stem cells.
10. The protein CCL3 or protein YY1 or protein GILZ of claim 1, or the biological material B1 or/and the biological material B2 of any one of claims 2-5.
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