Application of biomarker in preparation of product for detecting individual aging degree

Abstract

The invention relates to the field of biomedicine, in particular to application of a biomarker in preparation of a product for detecting the aging degree of an individual. And the biomarker is any one or a combination of several of DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4, MT-ND5 and PRDX6 (porcine reproductive and respiratory syndrome). Nine cell markers for aging evaluation and nine molecular markers for aging evaluation are obtained through single cell sequencing, flow cytometry sorting and QPCR identification of animals of different ages, the aging level of individuals can be rapidly evaluated by utilizing the markers, and the blank in the field of aging detection is filled.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a biomarker in preparing a product for detecting the degree of aging of an individual. Background Art

[0002] With the aging of the population and the improvement of people's health awareness, the demand for anti-aging products and services is growing. Accurate aging detection technology can meet people's higher demand for health management and promote the rapid development of the anti-aging industry. During the aging process, the physiological function and metabolic capacity of the human body gradually decline, which is often accompanied by a series of increased health risks. Through aging detection, these potential health problems, such as cardiovascular disease, diabetes, osteoporosis, and cognitive decline, can be discovered early. Aging detection can provide detailed information about the aging status, physiological function and metabolic capacity of individuals, and provide a scientific basis for the formulation of personalized health management plans such as nutrition, exercise, and drug intervention. Although existing aging detection methods, such as telomere length measurement and DNA methylation detection, have revealed the biological mechanism of aging to a certain extent, they still have limitations. The development of new aging detection methods can more comprehensively and accurately reflect the aging status of individuals, including physiological, metabolic, psychological and other aspects. The development of aging detection technology provides an important tool for anti-aging research. By conducting aging detection on individuals of different age groups and different health conditions, researchers can have a deeper understanding of the mechanisms and laws of aging, and provide new ideas for the development of anti-aging drugs and treatments. In addition, aging detection can also be used as an important indicator to evaluate the efficacy of anti-aging drugs, providing strong support for drug research and development. Therefore, the continuous development of new uses of aging-related markers is of great significance to the research of anti-aging products. Summary of the invention

[0003] In view of the above technical problems, the purpose of the present invention is to provide a use of a biomarker in the preparation of a product for detecting the degree of aging of an individual.

[0004] The specific technical solutions provided by the present invention are as follows:

[0005] The present invention provides use of a biomarker in preparing a product for detecting the degree of aging of an individual, wherein the biomarker is any one of DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4, MT-ND5, and PRDX6, or a combination of several of them.

[0006] As a preferred embodiment of the present invention, the biomarker has any of the following uses:

[0007] a1) preparing products for detecting the degree of aging of individuals;

[0008] a2) Preparation of a product that differentiates the degree of senescence of tissues from older individuals from younger individuals.

[0009] As a preferred embodiment of the present invention, the detection sample of the individual aging degree is derived from fresh blood, urine or secretion fluid of a mammal.

[0010] More preferably, the mammal is a human or a dog.

[0011] When the mammal is a dog, old refers to 8 years old and above, and young refers to 3 years old and below.

[0012] As a preferred embodiment of the present invention, the product is a test kit, a chip, a test paper or a detection reagent.

[0013] The present invention provides an application of a substance that downregulates or upregulates the expression level of the biomarker in the preparation of an anti-aging product. The substance that downregulates the expression level of the biomarker is a substance that downregulates the expression level of DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4 or MT-ND5, and the substance that upregulates the expression level of the biomarker is a substance that upregulates the expression level of PRDX6.

[0014] As a preferred embodiment of the present invention, the substance that downregulates the expression level of the biomarker is:

[0015] A small molecule substance that specifically inhibits the expression or activity of the biomarker, or

[0016] Specific antibodies that specifically inhibit the expression or activity of the biomarker.

[0017] As a preferred embodiment of the present invention, the substance that upregulates the expression level of the biomarker is:

[0018] A small molecule substance that specifically activates the expression or activity of the biomarker, or

[0019] Specific antibodies that specifically activate the expression or activity of the biomarker.

[0020] As a preferred embodiment of the present invention, the product is a medicine or a cosmetic.

[0021] As a preferred embodiment of the present invention, the drug includes a pharmaceutically acceptable carrier and / or excipient. The addition of the excipient or carrier is specifically selected according to the specific dosage form of the drug and the characteristics of the substance that up-regulates or down-regulates the expression level of the biomarker, such as a buffer, an emulsifier, a suspending agent, a colorant, a stabilizer, a preservative, an antioxidant, an excipient, a filler, a coagulant and a conditioning agent, a surfactant, a dispersant, a defoaming agent, etc.

[0022] As a preferred embodiment of the present invention, the cosmetics include excipients or carriers acceptable in the cosmetics field. The addition of the excipients or carriers is specifically selected according to the specific dosage form of the cosmetics and the characteristics of the substance that up-regulates or down-regulates the expression level of the biomarker, such as buffers, emulsifiers, suspending agents, colorants, stabilizers, preservatives, antioxidants, excipients, fillers, coagulants and conditioning agents, surfactants, diffusing agents, defoaming agents, etc.

[0023] The present invention collects and analyzes single-cell sequencing data of dogs of different ages, and finds that the number of neutrophils, undifferentiated neutrophils, monocytes, myeloid dendritic cells 2, basophils, CD4+ effector memory T cells, CD8+ effector T cells, CD8+ memory T cells and plasma cells can be used as markers to identify the level of individual aging and distinguish between young and aging individuals. Further comparison of aging biomarkers in corresponding human cell types and QPCR identification found that DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4, MT-ND5, PRDX69 markers in CD8+T cells can evaluate the degree of individual aging.

[0024] Through the above technical scheme, the present invention can distinguish between young and old individuals and detect the degree of aging. The present invention collects blood through minimally invasive methods, performs rapid monocyte separation, and can quickly obtain aging evaluation through flow cytometry sorting and QPCR identification. The present invention does not require the collection of important tissues and organs of the body, and has the significant advantages of being fast, efficient, safe, and simple. The present invention is of great significance. Therefore, the present invention is particularly conducive to breaking the current dilemma in the field of aging detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the study in Example 1. scRNA-seq of peripheral blood mononuclear cells (PBMC) was performed on Beagles dogs aged 3, 6, and 12 years old. The icons used in the present invention are from BioRender.com.

[0026] Figure 2 Preprocessing of ScRNA-seq data. (A) UMAP embedding of raw data (left) and harmonious integrated data (right), where different colors represent cells of different ages. (B) 31 clusters determined by the Leiden clustering algorithm. (C) Expression of PPBP in all cells, where the color bar on the right represents the expression level of PPBP. (D) PPBP expression in each group. (E) Expression profiles of marker genes for nine major cell types.

[0027] Figure 3 Expression of cell type marker genes among cell types.

[0028] Figure 4 Classification of cell subpopulations in ScRNA-seq.

[0029] Figure 5 is the number of each cell type in different samples.

[0030] Figure 6 is the ratio of each cell type in different samples.

[0031] Figure 7 is the Pearson correlation between different cell types in different samples.

[0032] Figure 8 is the Pearson correlation of the same cell type in different samples, and the dashed line indicates the threshold of 0.95.

[0033] Fig. 9 Figure 3 shows the distribution of nine age-related cell types in different samples.

[0034] Fig.10 Identification of aging-related cell types and common aging markers in different species. (A) Dynamic changes of genes during aging were observed in CD8+ effector cells, CD8+ memory cells, CD4+ TEM cells, and CD4- monocytes. The red line indicates that the genes in humans and dogs increased significantly with age, while the blue line indicates that the genes in both species decreased significantly with age. (B) Average expression of cross-species conserved aging markers in CD8+ Effector, CD8+ Memory, CD4+ TEM, and CD4- monocytes, respectively. (C) Dynamic changes of cross-species conserved aging markers during aging observed in CD8+ T cells.

[0035] Fig.11 Expression of reporter genes showing age-related upregulation (red) or downregulation (blue) in CD8+ T cells.

[0036] Fig.12 qPCR validation of age-related upregulated (red), downregulated (blue), and inactive (black) genes in CD8+ T cells. DETAILED DESCRIPTION

[0037] With the aging of the population and the improvement of people's health awareness, the demand for anti-aging products and services is growing. Accurate aging detection technology can meet people's higher demand for health management and promote the rapid development of the anti-aging industry. By conducting aging detection on individuals of different age groups and different health conditions, researchers can have a deeper understanding of the mechanisms and laws of aging, and provide new ideas for the development of anti-aging drugs and treatments. In addition, aging detection can also be used as an important indicator to evaluate the efficacy of anti-aging drugs, providing strong support for drug research and development. Therefore, the continuous development of new uses of aging-related markers is of great significance to the research of anti-aging products.

[0038] The present invention provides use of a biomarker in preparing a product for detecting the degree of aging of an individual, wherein the biomarker is any one of DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4, MT-ND5, and PRDX6, or a combination of several of them.

[0039] The present invention collects and analyzes single-cell sequencing data of dogs of different ages, and finds that the number of neutrophils, undifferentiated neutrophils, monocytes, myeloid dendritic cells 2, basophils, CD4+ effector memory T cells, CD8+ effector T cells, CD8+ memory T cells and plasma cells can be used as markers to identify the level of individual aging and distinguish between young and aging individuals. Further comparison of aging biomarkers in corresponding human cell types and QPCR identification found that DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4, MT-ND5, PRDX69 markers in CD8+T cells can evaluate the degree of individual aging.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0041] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0042] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent suppliers.

[0043] The quantitative tests in the following examples were all repeated three times.

[0044] In the following examples, canine monocyte separation solution was purchased from Tianjin Haoyang Huake Biotechnology Co., Ltd., with the catalog number LDS1077; canine CD4 / CD8 / CD3 antibodies were purchased from Bio-Rad, with the catalog number TC014; reverse transcription kit HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) and ChamQ Universal SYBR qPCR MasterMix were purchased from Novagen, with the catalog numbers R312-01 and Q711-02. Chromium NextGEM Single Cell 3'HT Kit v3.1, Chromium NextGEM Chip G Single cell kit, Dual Index Kit TT set A were purchased from 10X GENOMICS, with the catalog numbers PN-1000348, PN-1000120, and PN-3000431. Nuclease-free Water and Low TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) were purchased from Thermo Fisher Scientific, with catalog numbers AM9937 and 12090-015. SPRIselect Reagent Kit was purchased from Beckman Coulter, with catalog number B23318. Phosphate-Buffer Saline (PBS) 1X without calcium and magnesium was purchased from Corning Cellgro, with catalog number 21-040-CV. 10% Tween-20 was purchased from Bio-Rad, with catalog number 1662404. Glycerin (glycerol), 50% (v / v) Aqueous Solution was purchased from Ricca Chemical Company, with catalog number 3290-32. Qiagen Buffer EB was purchased from Qiagen, with catalog number 19086.

[0045] Example 1

[0046] Obtaining and analyzing canine blood single-cell sequencing libraries

[0047] Schematic diagram of canine blood single-cell sequencing construction and analysis is shown in Figure 1 .

[0048] 1. Obtaining single cell suspension from canine blood

[0049] According to the degree of aging of the dogs, one female dog aged 3, 6, and 12 was used. All dogs were in good condition and had no abnormalities.

[0050] Obtain blood mononuclear cell suspension from each dog. The specific steps are as follows:

[0051] (1) Collect 5 ml of fresh blood from the cephalic vein of the forelimb using a blood collection needle and place it in a blood collection tube containing EDTA anticoagulant. After blood collection, place the blood under refrigeration and immediately isolate peripheral blood mononuclear cells (PBMC).

[0052] (2) Transfer the blood to a centrifuge tube and perform density gradient centrifugation using Ficoll reagent. Set the speed to 500g for 25 minutes. Use a pipette to carefully aspirate the PBMC layer and transfer it to a new centrifuge tube to obtain PBMC.

[0053] 2. Construction of canine blood PBMC single cell library

[0054] Then, the 10×Genomics sequencing platform, Chromium Single-Cell 3'Gel Bead and LibraryV2 Kit were used for single-cell oil droplet coating, capture, cell lysis, reverse transcription, pre-amplification, and cDNA library establishment. After quality control, sequencing was performed on the NovaSeq 6000 sequencing platform (Illumina) to obtain single-cell transcriptome data.

[0055] Example 2

[0056] Acquisition and detection of blood molecular markers in aging dogs

[0057] 1. Obtaining the canine blood PBMC senescent cell atlas through bioinformatics analysis

[0058] The original sequencing data files were processed using Cell Ranger (version 8.0, 10×Genomics) and mapped to the CanFam3.1 reference genome. Subsequently, an expression matrix was generated for downstream analysis. For each sample, the count matrix was imported into the Seurat pipeline using the Read10X function. To determine the quality of the data, the percentage of reads mapped to the mitochondrial chromosome in each cell was calculated using the PercentageFeatureSet function, with the 'pattern' parameter set to '^MT-'. Quality control measures were applied to all samples. Cells meeting the following criteria were retained for downstream analysis: (1) 200 < nFeature_RNA < 4000 (the number of genes detected in each cell was between 200 and 4000); (2) percent.mt < 10 (the percentage of mitochondrial gene expression in total gene expression was less than 10%); (3) 500 < nCount_RNA < 30000 (the unique molecular identifier (UMI) count in each cell was between 500 and 30000). Next, all samples were normalized using SCTransform (Hafemeister and Satija 2019). Subsequently, the Harmony (Korsunsky, Millard et al. 2019) algorithm was applied to remove batch effects.

[0059] The results are shown in Figure 2 As shown, based on single-cell transcriptome sequencing, 31 cell populations of canine blood PBMC were identified according to the marker genes of each cell type. Further using known cell type markers, the 31 clusters were further grouped and annotated into nine major cell types ( Figure 3 ). In the present invention, plot1cell was used to visualize these annotated cells, and this tool shows the UMAP embedding of the cells and the distribution of each cell type at different ages ( Figure 4 ). The abundance of each cell type showed obvious changes during aging ( Figure 5 ). In canines, the number of CD4+ T cells gradually decreased with age, especially in the middle-aged and old-aged stages ( Figure 6 ). The total number of eosinophils / basophils decreased linearly with age, while myeloid-derived suppressor cells increased linearly ( Figure 6 ).

[0060] 2. Bioinformatics analysis to obtain differentially expressed genes in senescent cells of canine blood PBMC

[0061] To study the changes in different types of cells during canine aging, the present invention calculated the Pearson correlation coefficient (PCC) between all ages and all cell types ( Figure 7Paired PCC comparisons were performed, and cell types with a PCC below a threshold of 0.95 in at least one pair of comparisons were identified as cell types that changed with age ( Figure 8 In total, nine cell types that change with age were identified, including neutrophils, immature neutrophils, CD4- monocytes, myeloid dendritic cells type 2 (myeloid cDC2), basophils, CD4+ effector memory T cells (CD4+TEM), CD8+ effector T cells, CD8+ memory T cells, and plasma cells. UMAP embedding of these cell types also showed heterogeneity between different ages ( Fig. 9 ).

[0062] The present invention further defines a monitoring system for aging assessment and evaluates its potential for cross-species application. The present invention tests the consistency between human aging and canine aging. By comparing aging biomarkers in corresponding human cell types, four cell types were identified in which both humans and dogs showed significant and consistent directional changes in these biomarkers ( Fig.10 a, b). In particular, the present invention tested the above consistent markers in CD8+ T cells, which can represent the aging process across species ( Fig.10 c, Fig.11 ), specifically DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4, MT-ND5, and PRDX6. With the increase of age, the expression levels of DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4, and MT-ND5 are gradually increased, while the expression level of PRDX6 is gradually decreased.

[0063] Example 3

[0064] Application of Molecular Markers of Senescence in Canine Blood PBMC

[0065] The present invention collects PBMC from clinical young and old dogs, uses CD3, CD4, and CD8 antibodies to perform flow cytometry sorting to obtain CD8+T cells. The total RNA of CD8+T cells is collected, reverse transcribed into cDNA, and Q-PCR identification is performed on the 10 CD8+T cell molecules screened above, and the primers used are shown in Table 1.

[0066] Table 1 Primer sequences

[0067]

[0068]

[0069] The results showed that nine markers, including DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4, MT-ND5, and PRDX6, were consistent with the predicted trends ( Fig.12 ), indicating that the 9 markers provided by the present invention can be used to assess the degree of aging of an individual.

[0070] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Use of a biomarker in preparing a product for detecting the degree of aging of an individual, characterized in that: The biomarker is any one of DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4, MT-ND5, and PRDX6, or a combination of several of them.

2. The use according to claim 1, characterized in that The biomarker has any of the following uses: a1) preparing products for detecting the degree of aging of individuals; a2) Preparation of a product that differentiates the degree of senescence of tissue from older individuals from younger individuals.

3. The use according to claim 1, characterized in that The samples for testing the degree of individual aging are derived from fresh blood, urine or secretions of mammals.

4. The use according to claim 3, characterized in that The mammal is a human or a dog.

5. The use according to claim 1, characterized in that: The product is a test kit, a chip, a test paper or a detection reagent.

6. Use of a substance that downregulates or upregulates the expression level of the biomarker in the preparation of an anti-aging product, characterized in that: The substance that downregulates the expression level of the biomarker is a substance that downregulates the expression level of DDX24, EML4, IQGAP2, MT-ATP6, MT-CYB, MT-ND1, MT-ND4 or MT-ND5, and the substance that upregulates the expression level of the biomarker is a substance that upregulates the expression level of PRDX6.

7. The use according to claim 6, characterized in that The substance that downregulates the expression level of the biomarker is: A small molecule substance that specifically inhibits the expression or activity of the biomarker, or Specific antibodies that specifically inhibit the expression or activity of the biomarker.

8. The use according to claim 6, characterized in that The substance that upregulates the expression level of the biomarker is: A small molecule substance that specifically activates the expression or activity of the biomarker, or Specific antibodies that specifically activate the expression or activity of the biomarker.

9. The use according to claim 6, characterized in that: The product is a medicine or a cosmetic.

10. The use according to claim 9, characterized in that The medicine or cosmetic comprises an acceptable carrier and / or auxiliary material.

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