A biomarker and its application

Abstract

The present invention discloses a biomarker and an application thereof. The biomarker is a cell expressing CD8+CD16++. The application of the biomarker is to use the biomarker to evaluate the immune aging of an organism. The organism is a human body.

Description

Technical Field

[0001] The invention relates to a biomarker and application thereof, belonging to the technical field of biological cell application. Background Art

[0002] Immunosenescence refers to immune dysfunction mainly caused by aging, and this immune dysfunction leads to high morbidity and mortality in the elderly. The aging of the immune system is a multifactorial cascade event, and different types of immune cells show different sensitivities. Immunosenescence is a typical process of life, and many immune system (cells) inevitably go through this process. It is of great significance to correctly study and master the changes in the immune system during aging. For example, the development of pro-inflammatory cytokine phenotypes and the balance of anti-inflammatory effects can save the elderly from disability; the correct regulation of immune response and apoptosis may help reduce the occurrence of age-related degenerative diseases and inflammatory and neoplastic diseases, so that the body can enter the old age in good health.

[0003] The essence of body aging is the process of continuous accumulation of cellular aging. The aging of the immune system is both an inevitable result of body aging and an important cause of body aging. To date, the specific cause of immune aging has not been thoroughly studied, and it may be related to aging, chronic inflammation and microenvironmental factors. At the same time, chronic inflammation, malignant tumors or other progressive immunodeficiency diseases caused by immune aging seriously affect the length of human life.

[0004] As the research on immunosenescence continues to deepen, many research results have been achieved, but there are still many problems that have not been solved. First, there is no clear and simple biomarker that can be used to quantify and accurately evaluate the degree of immunosenescence. Secondly, most research directions on immunosenescence are the aging of immune cells, which cannot be applied to the body's systemic immunosenescence assessment. In addition, most of the research conclusions on immunosenescence are derived from animal experiments. Whether they can be reasonably extended to humans requires a population research sample system and accurate experimental data. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a biomarker for reflecting the aging of the human immune system.

[0006] The second object of the present invention is to provide an application of the above-mentioned biomarker for evaluating the immune aging of an organism with better efficiency and accuracy.

[0007] The first object of the present invention can be achieved by adopting the following technical solution: a biomarker, which is a cell expressing CD8+CD16++.

[0008] The second object of the present invention can be achieved by adopting the following technical solution: application of a biomarker, where the biomarker is used to evaluate the immune aging of an organism.

[0009] Furthermore, the organism is a human body.

[0010] Furthermore, biomarkers are used to quantitatively assess the immunosenescence of an organism.

[0011] Furthermore, biomarkers are used to assess the immune aging of an organism.

[0012] Furthermore, the expression of biomarkers was detected by flow cytometry to evaluate the immune aging of the organism.

[0013] The formulation design principle of the present invention is as follows:

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The biomarkers of the present invention can directly reflect the state of human immune aging and detect the aging of the body's immune system;

[0016] 2. The application of the biomarkers of the present invention is used to evaluate the immune aging of an organism with better efficiency and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Flow cytometry analysis of peripheral blood CD8+CD16++ cell subsets;

[0018] Figure 2 It is a statistical chart of the proportion of peripheral blood lymphocytes;

[0019] Figure 3-4 This is a statistical chart of the expression ratio of peripheral blood cell senescence markers;

[0020] Figure 5-6 It is a statistical diagram of the expression ratio of peripheral blood cell function exhaustion markers;

[0021] Figure 7-9 This is a statistical diagram of the expression ratio of activating receptors in peripheral blood cells;

[0022] Fig.10 It is a statistical diagram of the expression ratio of inhibitory receptors in peripheral blood cells;

[0023] Fig.11 Flow cytometry analysis of CD8+CD16++ cell subsets in peripheral blood of a 4-year-old boy;

[0024] Fig.12 This is a flow cytometric analysis of CD8+CD16++ cell subsets in the peripheral blood of a 32-year-old male;

[0025] Fig.13 This is a flow cytometer analysis of CD8+CD16++ cell subsets in the peripheral blood of a 68-year-old male. DETAILED DESCRIPTION

[0026] Below, the present invention is further described in conjunction with the accompanying drawings and specific embodiments:

[0027] A biomarker comprising CD8+CD16++ cells.

[0028] This biomarker is used to detect the expression of the biomarker by flow cytometry and to quantitatively evaluate the immune aging of the human body.

[0029] Detection can be achieved through flow cytometry, which is a simple process that takes less time and requires less equipment. It reflects the aging of the human immune system and the aging at the non-immune cell level.

[0030] Embodiment 1:

[0031] Detection of the proportion of CD8+CD16++ cell subsets in peripheral blood of people of different age groups

[0032] In order to verify that CD8+CD16++ cell subsets can be used as a marker of immune aging, flow cytometry was used to directly detect the proportions of CD8+CD16+, CD8+CD16++, and CD8++CD16+ cell subsets in the peripheral blood of people of different age groups.

[0033] ) First, the experimental population was divided into three groups: Child (0-12 years old), Young (20-50 years old), and Old (over 60 years old), with 30 cases in each group and 2 mL of venous blood was collected;

[0034] 2) Take a 15 mL centrifuge tube, add 2 mL of lymphocyte separation solution, spread 2 mL of peripheral blood on the cell separation solution, and centrifuge at 800 g for 15 minutes with slow rise and fall;

[0035] 3) After centrifugation, take the second layer of buffy coat cells from the middle to a new centrifuge tube, add 5 mL of PBS solution, mix well, and centrifuge at 300 g for 10 min;

[0036] 4) Aspirate the supernatant, add 5 mL of PBS solution, mix well, and centrifuge at 200 g for 10 min;

[0037] 5) Aspirate the supernatant to obtain PBMC, resuspend the cells with PBS solution, add flow cytometry antibodies CD3-FITC (Biolegend, 300406), CD56-PE (Biolegend, 318306), CD8a-PerCP / Cyanine5.5 (Biolegend, 300924), and CD16-APC (Biolegend, 302012) for staining, incubate at room temperature in the dark for 30 minutes, add 1 mL PBS solution, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant;

[0038] 6) Resuspend the cells in 300uL PBS and detect the proportion of CD8+CD16+, CD8+CD16++, and CD8++CD16+ cell subsets in lymphocytes by flow cytometry.

[0039] Figure 1 This is a flow cytometry plot of peripheral blood CD8+CD16++ cell subset analysis. According to the expression of CD8 and CD16, CD8+CD16+ cells can be divided into two groups: CD8+CD16++ and CD8++CD16+. CD8+CD16++ cells are mainly NK cells, and CD8++CD16+ cells are T cells.

[0040] Figure 2 Statistical chart of the proportion of CD8+CD16+, CD8+CD16++, and CD8++CD16+ cells in peripheral blood lymphocytes of different age groups (* P <0.05;*** P <0.001; **** P <0.0001), the difference was statistically significant. With the increase of age, the proportion of CD8+CD16+ and CD8+CD16++ cells in peripheral blood gradually increased, indicating that cells expressing CD8+CD16++ can be used as an indicator to evaluate immune aging.

[0041] Embodiment 2:

[0042] Detect CD8+, CD16+ cells, and CD8+CD16++ combined expression cell subsets in the peripheral blood of people of different age groups, and the status of expressing other functional or aging markers at the same time

[0043] In order to verify the expression of CD8+CD16++ cell surface markers in the peripheral blood of people of different age groups, flow cytometry was used to detect the expression of functional markers of CD8+CD16+, CD8+CD16++, and CD8++CD16+ cell subsets, such as exhaustion markers (PD-1, Tim-3), cell senescence markers (CD57, KLRG-1), cell activation receptor markers (CD226, CD244, NKG2D), and cell inhibitory receptor markers (NKG2A).

[0044] ) First, the experimental population was divided into three groups: Child (0-12 years old), Young (20-50 years old), and Old (over 60 years old), with 15 cases in each group and 2 mL of venous blood was collected;

[0045] 2) Take a 15 mL centrifuge tube, add 2 mL of lymphocyte separation solution, spread 2 mL of peripheral blood on the cell separation solution, and centrifuge at 800 g for 15 minutes with slow rise and fall;

[0046] 3) After centrifugation, take the second layer of buffy coat cells from the middle to a new centrifuge tube, add 5 mL of PBS solution, mix well, and centrifuge at 300 g for 10 min;

[0047] 4) Aspirate the supernatant, add 5 mL of PBS solution, mix well, and centrifuge at 200 g for 10 min;

[0048] 5) Remove the supernatant, obtain PBMC, resuspend the cells in PBS solution, and divide into two tubes A and B. Add flow cytometry antibodies CD3-FITC (Biolegend, 300406), CD56-PE (Biolegend, 318306), CD8a-PerCP / Cyanine5.5 (Biolegend, 300924), CD16-APC (Biolegend, 302012), PD-1-Brilliant Violet 510™ (Biolegend, 329931), Tim3-APC / Fire™750 (Biolegend, 345043), CD57-Pacific Blue™ (Biolegend, 359608), KLRG1-PE / Dazzle™594 (Biolegend, 367709) were used for staining; flow cytometry antibodies CD3-FITC (Biolegend, 300406), CD56-PE (Biolegend, 318306), CD8a-PerCP / Cyanine5.5 (Biolegend, 300924), CD16-APC (Biolegend, 302012), CD226-BrilliantViolet 421™ (Biolegend, 338331), CD244-PE / Dazzle™594 (Biolegend, 329521), NKG2D-Brilliant Violet 510™ (Biolegend, 320815), and NKG2A-Alexa Fluor®750 (R&D, FAB1059S) were added to tube B for staining. Incubate at room temperature in the dark for 30 min, add 1 mL of PBS solution, centrifuge at 1000 rpm for 5 min, and remove the supernatant;

[0049] 6) Resuspend the cells in 300uL PBS and detect the expression ratio of surface markers of CD8+CD16+, CD8+CD16++, and CD8++CD16+ cell subsets in lymphocytes by flow cytometry.

[0050] Figure 3-4 Statistical chart of the expression ratio of senescence markers in peripheral blood CD8+CD16+, CD8+CD16++, and CD8++CD16+ cells in different age groups (* P <0.05;*** P <0.001; **** P<0.0001), the difference was statistically significant. With the increase of age, the expression of senescence markers CD57 and KLRG1 in CD8+CD16++ cells gradually increased, indicating that cells expressing CD8+CD16++ gradually aged with age.

[0051] Figure 5-6 Statistical chart of the expression ratio of CD8+CD16+, CD8+CD16++, and CD8++CD16+ cell function exhaustion markers in peripheral blood of different age groups (* P <0.05;** P <0.01;*** P <0.001; **** P <0.0001), the difference was statistically significant. With the increase of age, the expression of PD-1 and Tim-3, markers of CD8+CD16++ cell function exhaustion, gradually increased, indicating that the cell function of cells expressing CD8+CD16++ gradually became exhausted with age.

[0052] Figure 7-9 Statistical chart of the expression ratio of activating receptors in peripheral blood CD8+CD16+, CD8+CD16++, and CD8++CD16+ cells in different age groups (* P <0.05;** P <0.01;*** P <0.001), the difference was statistically significant. With the increase of age, the expression of CD8+CD16++ cell activity receptors NKG2D, CD226 and CD244 gradually decreased, indicating that the degree of cell activation of cells expressing CD8+CD16++ gradually decreased with age.

[0053] Fig.10 Statistical chart of inhibitory receptor expression ratio of peripheral blood CD8+CD16+, CD8+CD16++, and CD8++CD16+ cells in different age groups (* P <0.05). With the increase of age, the expression of NKG2A, an inhibitory receptor on CD8+CD16++ cells, did not change significantly, indicating that the inhibitory regulation of cells expressing CD8+CD16++ did not change significantly with the increase of age.

[0054] The number of CD8+CD16+ in conventional T cell subsets decreases with age, and shortened telomere length and decreased telomerase activity lead to cell senescence. CD8+CD16++ is a unique lymphocyte subset that increases with age, making it more meaningful as a new biomarker of immune aging. By directly detecting the proportion of CD8+CD16++ cell subsets in the peripheral blood of people of different age groups by flow cytometry, the degree of immune aging can be evaluated.

[0055] The results of the detection of CD8+CD16++ cell subset ratios in the peripheral blood of people of different age groups showed that this method can provide a biomarker for assessing the degree of human immune aging;

[0056] The results of the surface marker expression test of CD8+CD16++ cells in the peripheral blood of people of different age groups showed that with the increase of age, the cell aging marker CD57 increased, the cell function exhaustion marker PD-1 increased, and the cell activation receptor NKG2D decreased. This further shows that the CD8+CD16++ cell subset proposed in the patent method can be used as a marker of the degree of human immune aging.

[0057] Embodiment 3:

[0058] Proportion of CD8+CD16++ cell subsets in peripheral blood of individuals of different ages

[0059] Flow cytometry was used to directly detect the proportions of CD8+CD16+, CD8+CD16++, and CD8++CD16+ cell subsets in the peripheral blood of three individuals of different age groups.

[0060] ) Three healthy subjects were randomly selected, including a 4-year-old boy, a 32-year-old man, and a 68-year-old man, and 2 mL of venous blood was collected from each of them;

[0061] 2) Take a 15 mL centrifuge tube, add 2 mL of lymphocyte separation solution, layer 2 mL of peripheral blood collected from three individuals on the cell separation solution, and centrifuge at 800 g for 15 min.

[0062] 3) After centrifugation, take the second layer of buffy coat cells from the middle to a new centrifuge tube, add 5 mL of PBS solution, mix well, and centrifuge at 300 g for 10 min;

[0063] 4) Aspirate the supernatant, add 5 mL of PBS solution, mix well, and centrifuge at 200 g for 10 min;

[0064] 5) Aspirate the supernatant to obtain PBMC, resuspend the cells with PBS solution, add flow cytometry antibodies CD3-FITC (Biolegend, 300406), CD56-PE (Biolegend, 318306), CD8a-PerCP / Cyanine5.5 (Biolegend, 300924), and CD16-APC (Biolegend, 302012) for staining, incubate at room temperature in the dark for 30 minutes, add 1 mL PBS solution, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant;

[0065] 6) Resuspend the cells in 300uL PBS and detect the expression ratio of surface markers of CD8+CD16+, CD8+CD16++, and CD8++CD16+ cell subsets in lymphocytes by flow cytometry.

[0066] Fig.11 This is a flow cytometric analysis of CD8+CD16++ cell subsets in the peripheral blood of a 4-year-old boy. Based on the expression of CD8CD16, CD8+CD16+ cells can be divided into two subpopulations: CD8+CD16++ and CD8++CD16+. Among them, CD8+CD16++ cells are mainly NK cells, accounting for only about more than 1%, and CD8++CD16+ cells are T cells.

[0067] Fig.12 This is a flow cytometric analysis of CD8+CD16++ cell subsets in the peripheral blood of a 32-year-old male. Based on the expression of CD8 and CD16, CD8+CD16+ cells can be divided into two subpopulations: CD8+CD16++ and CD8++CD16+. Among them, CD8+CD16++ cells are mainly NK cells, and CD8++CD16+ cells are T cells. It can be observed that CD8+CD16++NK cells have increased compared with 4-year-old boys, reaching a level of nearly 6%.

[0068] Fig.13 This is a flow cytometer analysis of CD8+CD16++ cell subsets in the peripheral blood of a 68-year-old male. According to the expression of CD8 and CD16, CD8+CD16+ cells can be divided into two subpopulations: CD8+CD16++ and CD8++CD16+. Among them, CD8+CD16++ cells are mainly NK cells, and CD8++CD16+ cells are T cells. It can be observed that CD8+CD16++NK cells have increased significantly, reaching more than 20%, which can be used as an indicator of immune system aging. The proportion of CD8++CD16+ has not continued to increase with age.

[0069] For those skilled in the art, various other corresponding changes and modifications can be made according to the technical solutions and concepts described above, and all of these changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. An application of a biomarker, characterized in that: The biomarker is used to evaluate human immune aging; the biomarker is cells expressing CD8+CD16++ in peripheral blood.

2. The use of the biomarker according to claim 1, characterized in that: The biomarkers are used to quantitatively evaluate human immune aging.

3. The use of the biomarker according to claim 1, characterized in that: The expression of biomarkers was detected by flow cytometry to evaluate human immune aging.

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