CN117771272A - CD8 + Preparation method of primitive T cells and application of primitive T cells in preparation of anti-aging drugs - Google Patents

Abstract

The invention discloses CD8 ⁺ Preparation method of primitive T cells and application of primitive T cells in preparation of anti-aging drugs, wherein CD8 ⁺ Primitive T cells are juvenile CD8 ⁺ Primitive T cells. The invention discovers CD8 for the first time ⁺ Primitive T cells are associated with delaying aging in individuals, and this result was successfully verified by mouse model experiments, when derived from CD8 in young mice ⁺ After the original T cells are planted in the body of the aged mice, the aging of the aged mice is delayed, the exercise capacity is enhanced, and the CD8 of young individuals is proved ⁺ The original T cells can really delay the aging of the aged individuals, and a new research and development idea is provided for the excavation of the anti-aging drugs.

Description

CD8 + Preparation method of primitive T cells and application of primitive T cells in preparation of anti-aging drugs

Technical Field

The invention relates to the technical field of medicines, in particular to CD8 ⁺ A preparation method of primitive T cells and application thereof in preparing anti-aging drugs.

Background

Aging is a trend in the global population, and aging increases the risk of developing a variety of diseases, including cardiovascular disease, diabetes, neurodegenerative disease, cancer, and the like. In the middle of the 20 th century, people began to make a debate about the idea that "aging is the cause of age-related chronic diseases", and understanding the aging process is a serious challenge for scientists today. In the aging process, the immune system is disturbed, so that the immune reaction is disturbed, and the occurrence and development of senile diseases are accelerated. Recent research into senescence has shifted from recognizing the senescent phenotype to finding potential biological pathways such as DNA damage, stem cell failure, etc.

CD8 ⁺ T cell subset (CD 8) ⁺ Primitive T cells) is CD8 ⁺ The T cells, in their initial state prior to their effect, can rapidly clonally expand to form phenotypic and functional heterogeneous effector cells that migrate to the corners of the body to exert antiviral, antitumor functions. In central lymphoid organs, CD8 ⁺ Primary T cells interact with antigen presenting cells in a first phase driving CD8 ⁺ The process of differentiating primitive T cells into effector cells causes them to activate rapidly. This interaction continues at the site of infection to modulate the immune response, killing the target cells.

At present, in the aspect of cell senescence delay, people delay senescence mostly through stem cell exosomes, and only verify at the cell level but not at the individual level, and no related prior art for discovering immune cells capable of delaying senescence exists at present.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and provides a CD8 capable of delaying senescence ⁺ The preparation and application of the original T cells provide a new intervention strategy for deferring senility.

To achieve the above object, the present invention provides CD8 ⁺ Use of primitive T cells for the manufacture of a medicament for delaying senescence, wherein said CD8 ⁺ Primitive T cells are juvenile CD8 ⁺ Primitive T cells.

Preferably, said young age CD8 ⁺ Primitive T cells are derived from the same genetic backgroundIn mammals, e.g., mice, the term "same genetic background" refers to donor mice and recipient mice being of the same species of mouse strain, to avoid cellular rejection in the mice.

Preferably, said young age CD8 ⁺ Primitive T cells can delay aging in a subject and increase locomotor ability in a subject.

The invention also provides a preparation method of the cell preparation for delaying senescence, which comprises the following steps:

s1, obtaining tissue cells, adding erythrocyte lysate, standing for 4-6 minutes, and then adding buffer solution to dilute and terminate the reaction;

the tissue may be one or more of peripheral blood, lymph, spleen or bone marrow.

The buffer was a PBS solution containing 10% bovine serum albumin.

Step S2, centrifuging 300-500 Xg of the cell sap of which the reaction has been stopped in the step S1 for 5 minutes, discarding the supernatant, adding the cell separation solution to resuspend the cells until the cell concentration reaches 0.5X 10 ⁵ /μL-2×10 ⁵ /μL；

Step S3, separating and collecting CD8 ⁺ Primitive T cells, the cell preparation for delaying senescence is obtained. The method for separating and collecting comprises magnetic bead separation.

The invention also provides a cell preparation for deferring senility, which comprises young CD8 ⁺ Primitive T cells.

The concentration of cells in the above cell preparation was 1.2X10 ⁴ /μL-2.4×10 ⁴ /μL。

Compared with the prior art, the invention has the beneficial effects that:

the invention discovers CD8 for the first time ⁺ Primitive T cells are associated with delaying aging in individuals, and this result was successfully verified by mouse model experiments, when derived from CD8 in young mice ⁺ After the original T cells are planted in the body of the aged mice, the aging of the aged mice is delayed, the exercise capacity is enhanced, and the CD8 of young individuals is proved ⁺ Primitive T cells do indeed delay agingAging of the aged individuals provides a new research and development idea for the excavation of the anti-aging drugs.

Drawings

FIG. 1 shows CD8 of the young and old groups according to example 1 of the present invention ⁺ Cluster contrast plots for each cell subpopulation in primitive T cells.

FIG. 2 is a CD8 extracted according to example 2 of the present invention ⁺ Flow assay of primary T cells.

FIG. 3 is a schematic diagram of a mouse adoptive transfer model according to example 2 of the present invention.

A: old mouse CD8 of example 2 of the present invention ⁺ And (3) constructing a flow chart of an original T cell adoptive transfer model.

B: after primary cell injection for 2 months in the aged mice of example 2 of the present invention, young mice were examined for CD8 ⁺ Results of primary T cell engraftment in aged mice.

FIG. 4 evaluation of Young's CD8 by the adoptive transfer model of example 2 mice of the present invention ⁺ Primary T cells regulate aging.

A: example 2 survival curves for mice in control and treatment groups according to the invention, the vertical axis represents survival of mice in each group.

B: example 2 comparative figures of coat status of 30 month old mice in control group and 30 month old mice in treatment group.

C: open field experimental heatmaps of 10 week old positive control mice, control 30 month old mice and treatment 30 month old mice in example 2 of the present invention.

D: in the open field experiment of example 2, statistics of total movement distance, total movement time, non-movement time, and frequency of entering the central region of each mouse were obtained within 5 minutes of the 10-week-old positive control group mice, the control group 30-month-old mice, and the treatment group 30-month-old mice.

E: the results of the pole climbing experiments of the 10-week-old positive control mice, the control mice 30 months of age and the treated mice 30 months of age in the embodiment 2 of the invention are shown in the drawing.

Detailed Description

The technical scheme of the invention will be further described with reference to the accompanying drawings and examples. However, it will be readily understood by those skilled in the art that the description of the embodiments is provided for illustration and explanation of the present invention only and is not intended to limit the invention as described in detail in the claims. Unless otherwise indicated, reagents, methods and equipment employed in the present invention are conventional methods and test materials used, unless otherwise indicated, are available from commercial companies.

As described above, in the development of biological markers and drugs related to animal aging, the inventors of the present application have first found that CD8 is an individual through intensive studies ⁺ Primitive T cells are associated with anti-aging. Further, the inventors of the present application successfully validated this result by mouse model experiments, in vivo CD8 from young mice ⁺ After the original T cells are planted in the body of the aged mice, the aging of the aged mice is delayed, and the exercise capacity is enhanced.

The following is a detailed description of the embodiments.

Example 1CD8 ⁺ Primitive T cells undergo phenotypic changes during aging suggesting targeting CD8 ⁺ Potential feasibility of primitive T cells to delay senescence.

Experimental operation:

1. collecting clinical samples

The inventor performs orthopedic cooperation with a sixth people hospital in Shanghai city in earlier stage, collects bone marrow samples in bone surgery of 8 volunteers in total, and aims at CD45 in bone marrow ⁺ All immune cells were subjected to single cell transcriptome sequencing analysis. Group entry criteria: (1) full age group; (2) healthy people without basal metabolic disease in physical examination or clinical normal examination.

2. Immune cell population from which the above sample was taken

The 8 volunteers were divided into young groups (3-15 years) and old groups (70-95 years), and cells were extracted in fresh tissues using a syringe until the tissue mass was changed from red to white. The red blood cells were further lysed using ACK lysate to prepare a single cell suspension. Labeling each single cell suspension with anti-human CD45 staining antibody and sorting CD45 by flow cytometer BD FACSAriacell sorter ⁺ Comprises T cells, B cells,NK cells, DC cells and the like have the activity rate of more than 90%.

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 Xudi Biotech company according to the manufacturer's instructions. The resulting library was sequenced using Illumina HiSeq 4000 platform. The trimmed data was processed using cellrange (10 x Genomics, version 3.0) and the cells were further screened, processed and analyzed using the setup software package (version 3.1.2) in the R (version 3.6.3) software. In the preliminary analysis, less than 200, more than 5000 or more than 5% of the cells with mitochondrial content of the genes were removed. And carrying out cluster analysis on the residual single cells according to a graph-based clustering algorithm. As shown in figure 1 a, at CD8 ⁺ Within the T cell population, the C6 cell population (high expression characteristic gene ANXA 1) and the C7 cell population (high expression characteristic gene PLAAT 4) had significant differences in the levels in the bone marrow samples of the young and old groups.

From the above, it can be seen that CD8 of the individual ⁺ The primary T cell phenotype is associated with aging, changes occur during aging, and the primary T cell phenotype is taken as an incision point to interfere with the aging process, and is verified by a mouse model experiment.

Example 2 mouse model test to verify CD8 in young individuals ⁺ Primitive T cells can delay senescence

In this example, 4-6 week old mice (equivalent to 5 years of human) against the CD45.1 strain background are CD8 ⁺ Primitive T cell donor mice were recipient mice that were 16 months old mice (equivalent to 50 years of human) in the CD45.2 strain background. Both CD45.1 and CD45.2 markers can be detected using a flow cytometer. The purpose of selecting different strain backgrounds for donor mice and recipient mice is: CD8 capable of conveniently distinguishing donor cell and acceptor cell and subsequently detecting young mice ⁺ Whether the naive T cells successfully colonize the recipient mice.

Experimental operation:

1. young mice CD8 ⁺ Extraction of primitive T cells

At 4-6 weeks of ageCD45.1 strain mice were used as donor mice, and spleen tissue of donor mice was used as CD8 ⁺ Tissue origin of primitive T cells. The reason for selecting mouse spleen tissue is that mouse spleen is readily available and CD8 ⁺ Higher primary T cell content and CD8 in single donor mouse ⁺ Primitive T cells can be used in multiple recipient mice.

After euthanizing donor mice, the whole body was sterilized by spraying with 75% ethanol or soaking in 75% ethanol. The spleen of the mice was removed in an ultra clean bench, and the spleen of the mice was ground on a 40 μm cell sieve back until blushing, and continuously rinsed with a PBS solution containing 10% serum during the grinding process to obtain a spleen cell suspension of the mice. Wherein, the cell screen is connected with a centrifuge tube, and the obtained cell suspension can be directly centrifuged.

After centrifugation at 400 Xg for 5min, the supernatant was discarded, the cells were scattered by knocking the walls of the pellet centrifuge with a finger, and 3ml of the erythrocyte lysate was added and allowed to stand for 5min. Diluting with FACS buffer 3 times the volume of the cells, centrifuging at 400 Xg for 5min, discarding the supernatant, adding cell separation solution (Lymphoprep ^TM STEMCELL Technologies, canada) to achieve a cell concentration of 1X 10 ⁷ /100μL。

Using magnisont ^TM Mouse CD8T cell Enrichment Kit (Invitrogen Siemens, USA) magnetic bead sorting CD8 ⁺ Primitive T cells, obtaining CD8 ⁺ Primitive T cell suspensions. As shown in FIG. 2, the CD8 was detected using flow cytometry ⁺ The fluorescence-labeled antibody added by the original T cell suspension and the flow cytometry comprises anti-mouse CD45.1, anti-mouse CD45.2, anti-mouse CD62L, anti-mouse CD44, and the result shows that the extracted CD8 ⁺ The original T cell suspension has good purity.

2. Construction of aged mice CD8 ⁺ Adoptive transfer model of primitive T cells

Extracting the obtained CD8 ⁺ The primary T cells were dissolved in sterile PBS solution at 1.2X10 ⁶ /100μL-2.4×10 ⁶ Cells were resuspended at a concentration of 100. Mu.L.

16 month old mice on the CD45.2 strain background were selected as recipient mice, which were randomly divided into two groups, a control group and a treatment group. Control mice were injected with an equivalent amount of physiological saline and the treated mice were injected with CD8 from the donor mice via the monthly tail vein ⁺ The original T cell suspensions were each injected at 125 μl. The mice were observed for 1 hour after injection, and after confirmation, the mice were kept on feeding, and the overall flow chart was shown in fig. 3 a.

Three treatment groups of mice were randomly selected 2 months after mice received primary cell injection treatment to verify CD8 from donor mice ⁺ Whether the naive T cells have been engrafted in the recipient mice is as follows:

first orbital bleeds were performed on the treated mice, each of which was bled 100 μl to 1.5ml EP tube containing anticoagulated heparin sodium to prevent clotting. Another 15ml centrifuge tube was added with 8ml double distilled water and 1ml Fetal Bovine Serum (FBS) and mixed well, then the above-mentioned taken mouse blood was added and mixed well upside down for 1 minute to lyse red blood cells, and then 1ml 10 XPBS was added to terminate the lysis reaction. Centrifugation was performed at 300 Xg for 5min, the supernatant was discarded, and the cells were washed with PBS for flow cytometry staining.

After the orbit blood collection is completed, the mouse is euthanized, the spleen and the lymph nodes are taken out for grinding after the whole body is sterilized by alcohol, and the femur is taken out to flush out cells in the bone marrow by using a syringe. The 3-part cells were prepared as single cell suspensions and stained with flow cytometry antibodies together with the lysed cells. Flow cytometry added fluorescent-labeled antibodies included anti-mouse CD45.1 and anti-mouse CD45.2. The results of the test are shown in FIG. 3B, which shows that CD45.1 positive cells from donor mice have successfully colonized in recipient mice, and that the construction of the adoptive transfer model of the mice is completed and the subsequent steps can be continued.

3. Behavior observation of adoptive transfer model of aged mice

The mice in the control group (normal aging mice) and the treatment group are subjected to life statistics, physiological state observation, open field experiments and pole climbing experiments to evaluate the aging-related aspects such as the autonomous movement ability, free exploration behaviors, mental anxiety states and the like of the mice.

Life statistics and physiological state observations: the survival rates of control and treatment mice were recorded by tracking until both mice were 30 months of age, with their outsides photographed at 30 months of age. As shown in a of fig. 4, the survival rate of the mice of the treatment group was significantly better than that of the control group at 30 months of age. As shown in fig. 4B, the control mice were dull in hair color and less in hair amount than the treated mice.

Open field experiments: in this experiment, 10-week-old mice (equivalent to 18 years of human age) were taken as a positive control group and subjected to open field experiments together. In this experiment, 10 were tested in each group, 3 times each, and the results averaged. The operation steps are as follows:

clean 50cm ² One mouse was randomly drawn from each group and gently placed in the cage, and the trace of the free movement of the mouse was recorded for 5 minutes using a Noldus Information Technology top camera and software, and a summary heat map was made as shown in fig. 4C.

Further, the total movement distance, total movement time, and non-movement time of each group of mice were counted, these indicators represent the difference in free movement ability of each group of mice, and the statistical results of each indicator are shown in D of fig. 4. As shown in D of fig. 4, normal aged rats (control group, blue display in D of fig. 4) were significantly reduced in both locomotor speed and exploratory ability, whereas treated mice (red display in D of fig. 4) were behavioural not significantly different from 10-week old young and adult rats.

Further, the frequency of each group of mice entering the central area of the square box is counted, the index shows the mental state of the mice, and whether the mice have anxiety is measured through the innate evasion of the mice. As shown in D of fig. 4, the data results also show that normal aging aged rats have undergone a dramatic change, while the mental change in the treated mice is delayed.

Pole climbing experiment: in this experiment, a 10-week-old mouse (equivalent to 18 years of human age) was used as a positive control group, and a pole climbing experiment was performed together. In this experiment, 6 tests were performed in each group, 3 times each, and the results were averaged. The operation steps are as follows:

a wooden pole with a length of 55cm and a diameter of about 10mm was placed in the rat cage, the head of the mouse was placed upward on the top of the pole, and the time for the complete turning down of the mouse and the time for the complete landing of the hind paw were recorded, and the statistical results are shown in fig. 4E. As shown in E of fig. 4, the treated mice were significantly better than normal aged rats in terms of locomotion and coordination function.

In conclusion, the invention completes the experimental effect research of deferring aging after successfully constructing the corresponding mouse cell treatment model by identifying the unique immune cell subgroup with anti-aging potential in the young individuals. The invention provides a key component in the immune system for the first time, namely CD8 from young individuals ⁺ T cells have a delay effect on the whole body aging of the organism, and verification is completed in a mouse experiment according to an autonomously constructed cell therapy model.

While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims (9)

1.CD8 ⁺ The application of primitive T cells in preparing anti-aging drugs is characterized in that the CD8 ⁺ Primitive T cells are juvenile CD8 ⁺ Primitive T cells.

2. A CD8 according to claim 1 ⁺ The application of primitive T cells in preparing anti-aging drugs is characterized in that the young CD8 ⁺ Primitive T cells are derived from mammals with the same genetic background.

3. A CD8 according to claim 1 ⁺ The application of primitive T cells in preparing anti-aging drugs is characterized in that the young CD8 ⁺ Original, originalT cells can delay aging in a subject and increase exercise capacity in a subject.

4. A method of preparing a cell preparation for use in delaying senescence comprising the steps of:

s1, obtaining tissue cells, adding erythrocyte lysate, standing for 4-6 minutes, and then adding buffer solution to dilute and terminate the reaction;

step S2, centrifuging 300-500 Xg of the cell sap of which the reaction has been stopped in the step S1 for 5 minutes, discarding the supernatant, adding the cell separation solution to resuspend the cells until the cell concentration reaches 0.5X 10 ⁵ /μL-2×10 ⁵ /μL；

Step S3, separating and collecting CD8 ⁺ Primitive T cells, the cell preparation for delaying senescence is obtained.

5. A CD8 as claimed in claim 4 ⁺ The preparation method of the primitive T cells is characterized in that in the step S1, the tissue is one or a combination of more than one of peripheral blood, lymph, spleen or bone marrow.

6. A CD8 as claimed in claim 4 ⁺ The preparation method of the primitive T cells is characterized in that in the step S1, the buffer solution is PBS solution containing 10% bovine serum albumin.

7. A CD8 as claimed in claim 4 ⁺ A method for preparing primitive T cells, wherein in step S3, the method for separation and collection comprises magnetic bead sorting.

8. A cell preparation for delaying senescence prepared according to the preparation method of any one of claims 4 to 7, characterized in that the cell preparation comprises juvenile CD8 ⁺ Primitive T cells.

9. The cell preparation for delaying senescence according to claim 8, wherein the concentration of cells in the cell preparation is 1.2 x 10 ⁴ /μL-2.4×10 ⁴ /μL。