Preparation method for CD8 + naive t cells and use thereof in preparation of drugs for delaying senescence

Abstract

A preparation method for CD8+ naive T cells and a use thereof in preparation of drugs for delaying senescence. The CD8+ naive T cells are young CD8+ naive T cells. It is found for the first time that CD8+ naive T cells are related to delaying individual senescence, and the result is successfully verified by means of mouse model experiments. After CD8+ naive T cells from a young mouse body are implanted into an aged mouse body, senescence of the aged mouse is delayed, and the exercise ability is improved. It is proved that CD8+ naive T cells of young individuals really can delay senescence of aged individuals, and a new research and development thought is provided for excavation of drugs for delaying senescence.

Description

CD8 + Method for preparing primitive T cells and application thereof in preparing anti-aging drugs Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a CD8 + A method for preparing primitive T cells and its application in preparing anti-aging drugs. Background Art

[0002] Aging is a global demographic trend, increasing the risk of numerous diseases, including cardiovascular disease, diabetes, neurodegenerative disorders, and cancer. Debate began in the mid-20th century regarding the role of aging in the development of age-related chronic diseases, and understanding the aging process remains a significant challenge for scientists today. During aging, immune system dysfunction occurs, leading to impaired immune responses and accelerating the development and progression of age-related diseases. In recent years, research on aging has shifted from identifying aging phenotypes to uncovering underlying biogenetic pathways, such as DNA damage and stem cell failure.

[0003] Naïve CD8 + T cell subsets (CD8 + Naive T cells) are CD8 + The initial state of T cells before they exert their effects can be rapidly cloned and expanded to form phenotypic and functional heterogeneous effector cells, which then migrate to all corners of the body to exert antiviral and anti-tumor functions. + Naive T cells interact with antigen-presenting cells in the first phase, driving CD8 + Naive T cells differentiate into effector cells, leading to their rapid activation. This interplay continues at the site of infection to modulate the immune response and kill target cells.

[0004] At present, in terms of delaying aging through cells, people mostly use stem cell exosomes to delay aging, and have only verified it at the cellular level but not at the individual level. There is currently no existing technology that has found that immune cells can delay aging. Disclosure of the invention

[0005] The purpose of the present invention is to provide a CD8 + The preparation and application of primitive T cells provide a new intervention strategy for delaying aging.

[0006] In order to achieve the above object, the present invention provides a CD8 + Application of primitive T cells in the preparation of anti-aging drugs, wherein the CD8 + Naive T cells are young CD8+ Naive T cells.

[0007] Preferably, the above-mentioned young CD8 + The original T cells are derived from mammals with the same genetic background. For example, taking mice as an example, "same genetic background" means that the donor mice and recipient mice are of the same mouse strain to avoid cell rejection reactions in mice.

[0008] Preferably, the above-mentioned young CD8 + Primitive T cells can delay the aging of subjects and improve their athletic ability.

[0009] The present invention also provides a method for preparing a cell preparation for delaying aging, the method comprising the following steps:

[0010] Step S1, obtaining tissue cells, adding red blood cell lysis buffer and letting it stand for 4-6 minutes, then adding buffer to dilute and terminate the reaction;

[0011] The above tissues may be one or more combinations of peripheral blood, lymph, spleen or bone marrow.

[0012] The above buffer solution is a PBS solution containing 10% bovine serum albumin.

[0013] Step S2: Centrifuge the cell suspension from step S1 at 300-500 × g for 5 minutes, discard the supernatant, and add cell separation solution to resuspend the cells until the cell concentration reaches 0.5 × 10 5 / μL-2×10 5 / μL;

[0014] Step S3, separation and collection of CD8 + The original T cells are used to obtain the cell preparation for delaying aging. The separation and collection method includes magnetic bead sorting.

[0015] The present invention also provides a cell preparation for delaying aging prepared according to the above preparation method, the cell preparation comprises young CD8 + Naive T cells.

[0016] The cell concentration in the above cell preparation was 1.2×10 4 / μL - 2.4×10 4 / μL.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention discovered CD8 for the first time + The original T cells are associated with delayed individual aging, and this result was successfully verified by mouse model experiments. When CD8 +After the original T cells were transplanted into the old mice, the aging of the old mice was delayed and the motility was enhanced, which confirmed that the CD8 + Primitive T cells can indeed delay aging in elderly individuals, providing a new research and development idea for the discovery of anti-aging drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows the CD8 + Cluster comparison of various cell subsets in primary T cells.

[0020] FIG2 is a schematic diagram of the mouse adoptive transfer model of Example 2 of the present invention.

[0021] A: CD8 in aged mice from Example 2 of the present invention + Flowchart for the construction of the naive T cell adoptive transfer model.

[0022] B: Two months after the initial cell injection in the old mice of Example 2 of the present invention, CD8 + A graph showing the results of naive T cell colonization in aged mice.

[0023] Figure 3 is a mouse adoptive transfer model for evaluating young CD8 + The regulatory role of naive T cells in aging.

[0024] A: Survival rate curve of mice in the control group and treatment group of Example 2 of the present invention, the vertical axis represents the survival rate of mice in each group.

[0025] B: Comparison of the fur status of 30-month-old mice in the control group and the treatment group according to Example 2 of the present invention.

[0026] C: Heat map of the open field test of 10-week-old positive control group mice, 30-month-old control group mice, and 30-month-old treatment group mice in Example 2 of the present invention.

[0027] D: Statistical graph of the total movement distance, total movement time, non-movement time, and frequency of entering the central area of ​​each mouse within 5 minutes for 10-week-old positive control group mice, 30-month-old control group mice, and 30-month-old treatment group mice in the open field test of Example 2 of the present invention.

[0028] E: Graph showing the results of the pole climbing experiment for 10-week-old mice in the positive control group, 30-month-old mice in the control group, and 30-month-old mice in the treatment group in Example 2 of the present invention. Best Mode for Carrying Out the Invention

[0029] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples. However, it will be readily understood by those skilled in the art that the contents described in the examples are intended only to illustrate and explain the present invention and are not intended to limit the present invention described in detail in the claims. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional methods unless otherwise specified, and the test materials used can be obtained from commercial companies unless otherwise specified.

[0030] As mentioned above, in the process of developing biological markers and drugs related to animal aging, the inventors of this application found for the first time through in-depth research that individual CD8 + The original T cells are associated with delayed aging. Furthermore, the inventors of this application successfully verified this result through mouse model experiments. + After the primitive T cells were transplanted into the bodies of aged mice, the aging of the aged mice was delayed and their motor ability was enhanced.

[0031] The following is a detailed description with reference to the embodiments.

[0032] Example 1 CD8 + Naive T cells undergo phenotypic changes during aging, suggesting targeting CD8 + Potential feasibility of naive T cells to delay aging.

[0033] Experimental operation:

[0034] 1. Collect clinical samples

[0035] The inventors previously collaborated with the Department of Orthopedics at Shanghai Sixth People's Hospital to collect bone marrow samples from 8 volunteers undergoing orthopedic surgery. + All immune cells were subjected to single-cell transcriptome sequencing analysis. Inclusion criteria: ① All age groups; ② Healthy individuals with no underlying metabolic disease as determined by physical examination or clinical tests.

[0036] 2. Extract immune cell populations from the above samples

[0037] The 8 volunteers were divided into a young group (3-15 years old) and an elderly group (70-95 years old). Cells were extracted from fresh tissue using a syringe until the tissue block turned from red to white. Red blood cells were further lysed using ACK lysis buffer to prepare a single cell suspension. Each single cell suspension was labeled with anti-human CD45 staining antibody, and CD45 was sorted using a flow cytometer BD FACSAriacell sorter. + The immune cells, including T cells, B cells, NK cells, DC cells, etc., have a viability of over 90%.

[0038] The sorted cells were centrifuged and resuspended in PBS containing 0.04% BSA. Chromium Single Cell 3' v3 (10x Genomics) library preparation was performed by Shanghai Xuran Biotechnology Co., Ltd. according to the manufacturer's instructions. The resulting libraries were sequenced using the Illumina HiSeq 4000 platform. The trimmed data were processed using CellRanger (10x Genomics, version 3.0), and the cells were further screened, processed, and analyzed using the Seurat package (version 3.1.2) in R (version 3.6.3) software. In the preliminary analysis, cells with fewer than 200 genes, more than 5000 genes, or a mitochondrial content of more than 5% were removed. The remaining single cells were clustered according to the graph-based clustering algorithm. As shown in Figure 1, in CD8 + Within the naïve T cell population, the contents of the C6 cell population (highly expressed characteristic gene ANXA1) and the C7 cell population (highly expressed characteristic gene PLAAT4) in the bone marrow samples of the young and elderly groups were significantly different.

[0039] As can be seen from the above, individual CD8 + The phenotype of primitive T cells is associated with aging and changes during the aging process. This can be used as an entry point to intervene in the aging process, which is verified by the following mouse model experiments.

[0040] Example 2 Mouse model experiment verifies CD8 + Naive T cells can delay aging

[0041] In this example, 4-6 week old mice (equivalent to 5-year-old human) of CD45.1 strain background were used as CD8 + The original T cell donor mice were 16-month-old mice (equivalent to 50 years old in human) with CD45.2 strain background as recipient mice. Both CD45.1 and CD45.2 markers can be detected by flow cytometry. The purpose of choosing different strain backgrounds for donor and recipient mice is to facilitate the differentiation of donor and recipient cells and to subsequently detect CD8 + Whether naive T cells successfully colonize in recipient mice.

[0042] Experimental operation:

[0043] 1. CD8 in young mice + Extraction of naive T cells

[0044] 4-6 week old CD45.1 mice were used as donor mice, and spleen tissue from donor mice was used as CD8 +The tissue source of naive T cells. Mouse spleen tissue was chosen because it is easy to obtain and CD8 + The content of naive T cells is high, and the CD8 + The naive T cells can be used in multiple recipient mice.

[0045] After euthanizing the donor mouse, disinfect the entire body by spraying or soaking it in 75% ethanol. Remove the mouse spleen in a cleanroom and grind it on a 40μm cell sieve until it turns white. Rinse continuously with PBS containing 10% serum during grinding to obtain a mouse spleen cell suspension. A centrifuge tube is attached to the cell sieve, allowing the resulting cell suspension to be directly centrifuged.

[0046] After centrifugation at 400 × g for 5 minutes, discard the supernatant, tap the tube wall with your fingers to disperse the cells, add 3 ml of red blood cell lysis buffer and let it stand for 5 minutes. Add 3 times the volume of the cells in FACS buffer to dilute, centrifuge at 400 × g for 5 minutes, discard the supernatant, and add cell separation buffer (Lymphoprep™, STEMCELL Technologies, Canada) to resuspend the cells to a concentration of 1 × 10 7 / 100μL.

[0047] Using MagniSort TM Mouse CD8 Naïve T cell Enrichment Kit (Invitrogen, Thermo Fisher Scientific, USA) magnetic bead separation of CD8 + Naive T cells, acquired CD8 + Primary T cell suspension.

[0048] 2. Construction of CD8 in Aged Mice + Naive T cell adoptive transfer model

[0049] The CD8 + The original T cells were dissolved in sterile PBS solution and the concentration was 1.2×10 6 / 100μL-2.4×10 6 Resuspend cells at a concentration of 4% PBS / 100 μL.

[0050] Sixteen-month-old mice with CD45.2 strain background were selected as recipient mice and randomly divided into two groups: control group and treatment group. The control group mice were injected with equal volume of normal saline, and the treatment group mice were injected with CD8 + Inject 125 μL of the original T cell suspension into each mouse. Observe the mouse for 1 hour after injection and continue feeding after confirmation. The overall process is shown in Figure 2A.

[0051] Two months after the mice received the initial cell injection, three treatment group mice were randomly selected to verify the CD8 + Whether the original T cells have colonized the recipient mice is determined by the following method:

[0052] First, orbital blood was collected from mice in the treatment group. 100 μL of blood was transferred from each mouse into a 1.5 mL EP tube containing sodium heparin to prevent clotting. In a separate 15 mL centrifuge tube, 8 mL of double-distilled water and 1 mL of fetal bovine serum (FBS) were added and mixed thoroughly. The collected blood was then added to the tube and inverted for 1 minute to lyse red blood cells. The lysis reaction was terminated by adding 1 mL of 10× PBS. The tube was centrifuged at 300 × g for 5 minutes, the supernatant discarded, and the cells resuspended in PBS for flow cytometry staining.

[0053] After orbital bleeding, the mice were euthanized, and after full-body alcohol disinfection, the spleen and lymph nodes were removed and ground, and the femur was taken and the cells in the bone marrow were flushed out using a syringe. The cells from the three parts were prepared into single-cell suspensions and then stained with flow cytometry antibodies together with the cells lysed from the above blood. The fluorescent-labeled antibodies added to the flow cytometry included anti-mouse CD45.1 and anti-mouse CD45.2. The test results are shown in Figure 2B, which shows that the CD45.1-positive cells from the donor mice have been successfully colonized in the recipient mice, and the mouse adoptive transfer model has been completed, and the subsequent steps can be continued.

[0054] 3. Behavioral Observation of the Adoptive Transfer Model in Aged Mice

[0055] The control group (normal aging mice) and the treatment group mice were subjected to lifespan statistics, physiological status observations, open field tests and pole climbing tests to evaluate the mice's autonomous movement ability, free exploration behavior, mental anxiety state and other aging-related aspects.

[0056] Lifespan Statistics and Physiological Observations: The survival rates of mice in the control and treatment groups were tracked and recorded until 30 months of age. At 30 months of age, photographs of the mice were taken. As shown in Figure 3A, the survival rate of mice in the treatment group at 30 months of age was significantly higher than that in the control group. As shown in Figure 3B, the control group mice had darker fur and less hair compared to the treatment group mice.

[0057] Open field test: In this experiment, 10-week-old mice (equivalent to 18-year-old humans) were selected as the positive control group and the open field test was also performed. In this experiment, 10 mice were tested in each group, and each mouse was tested 3 times, and the results were averaged. The specific operation steps are as follows:

[0058] Take a clean 50cm 2A mouse was randomly selected from each group and gently placed in the cage. The top camera and software of Noldus Information Technology were used to record the trajectory of the mouse's free movement within 5 minutes, and a summary heat map was produced as shown in Figure 3C.

[0059] Furthermore, the total distance moved, total moving time, and time spent immobile were calculated for each group of mice. These indicators reflect the differences in the free-movement ability of the mice in each group. The statistical results of each indicator are shown in Figure 3D. As shown in Figure 3D, the normally aged mice (control group, shown in blue in Figure 3D) showed a significant decrease in movement speed and exploratory ability, while the mice in the treatment group (shown in red in Figure 3D) showed no significant behavioral differences from 10-week-old young mice.

[0060] Furthermore, the frequency with which mice in each group entered the center of the square box was counted. This metric reflects the mice's mental state and measures their anxiety through their innate approach-avoidance behavior. As shown in Figure 3D, the data also show that normally aging mice exhibit significant changes, while these changes in the treatment group mice are somewhat delayed.

[0061] Pole climbing test: In this experiment, 10-week-old mice (equivalent to 18-year-old humans) were selected as the positive control group and the pole climbing test was also performed. In this experiment, 6 mice were tested in each group, and each mouse was tested 3 times, and the results were averaged. The specific operation steps are as follows:

[0062] A 55-cm-long, 10-mm-diameter wooden pole was placed in the rat cage. Mice were placed headfirst on top of the pole. The time it took for the mice to fully turn around and turn downward and for their hind paws to completely touch the ground was recorded. The statistical results are shown in Figure 3E. As shown in Figure 3E, the mice in the treatment group had significantly better motor and coordination functions than the normally aged mice.

[0063] In summary, the present invention has identified a unique immune cell subset with anti-aging potential in young individuals and successfully established a corresponding mouse cell therapy model to complete the experimental study on the effect of delaying aging. The present invention first proposed that the key component of the immune system - CD8 + Naïve T cells have the effect of delaying the body's systemic aging, and this has been verified in mouse experiments based on an independently constructed cell therapy model.

[0064] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A CD8 + The use of primitive T cells in the preparation of anti-aging drugs is characterized in that: CD8 + Naive T cells are young CD8 + Naive T cells.

2. The CD8 according to claim 1 + The use of primitive T cells in the preparation of anti-aging drugs is characterized in that: The young CD8 + Naive T cells are derived from mammals with the same genetic background.

3. The CD8 according to claim 1 + The use of primitive T cells in the preparation of anti-aging drugs is characterized in that: The young CD8 + Primitive T cells can delay the aging of subjects and improve their athletic ability.

4. A method for preparing a cell preparation for delaying aging, characterized in that: The following steps are involved: Step S1, obtaining tissue cells, adding red blood cell lysis solution and standing for 4-6 minutes, and then adding buffer to dilute and terminate the reaction; Step S2: Centrifuge the cell solution from step S1 at 300-500×g for 5 minutes, discard the supernatant, and add cell separation solution to resuspend the cells until the cell concentration reaches 0.5×10 5 / μL-2×10 5 / μL; Step S3, separation and collection of CD8 + The original T cells are used to obtain the cell preparation for delaying aging.

5. The CD8 according to claim 4 + A method for preparing primitive T cells, characterized in that: In step S1, the tissue is one or more combinations of peripheral blood, lymph, spleen or bone marrow.

6. The CD8 according to claim 4 + A method for preparing primitive T cells, characterized in that: In step S1, the buffer solution is a PBS solution containing 10% bovine serum albumin.

7. The CD8 according to claim 4 + A method for preparing primitive T cells, characterized in that: In step S3, the separation and collection method includes magnetic bead sorting.

8. A cell preparation for delaying aging prepared by the preparation method according to any one of claims 4 to 7, characterized in that: The cell preparation includes young CD8 + Naive T cells.

9. The cell preparation for delaying aging according to claim 8, characterized in that The cell concentration in the cell preparation is 1.2×10 4 / μL - 2.4×10 4 / μL.

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