Application of CCL2 in promoting organ or tissue regeneration and delaying senescence

Abstract

The invention discloses application of CCL2 in promoting organ or tissue regeneration and delaying senescence. According to the invention, a plurality of tissue damage regeneration models are utilized, the damage repair characteristics and common regulation and control mechanisms of eight tissues of mammals are systematically analyzed in combination with single cells, and biomarkers and potential intervention targets related to damage repair are deeply excavated by comparing the damage repair differences of young and old tissues, so that the damage repair of the mammals is realized. And a theoretical basis is provided for regeneration function evaluation, early warning and accurate intervention. Meanwhile, the invention also finds that the CCL2 can be used as a key target for improving the regeneration capacity of the senile tissues, which is beneficial to exploring a novel intervention means and developing a new strategy for regulating and controlling regeneration, delaying senescence and preventing and treating senescence-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of CCL2 in promoting organ or tissue regeneration and delaying aging, and particularly relates to the application of CCL2 in promoting regeneration of damaged blood vessels, muscles and skin tissues and delaying aging. Background Art

[0002] "Regeneration" is the process by which an individual responds to damaging stimuli and repairs and remodels tissues. It plays an important role in the body's repair of damage and the maintenance of homeostasis and function. The regenerative capacity of different tissues varies greatly. In addition, during the aging process, the regenerative capacity of tissues also decreases. During the aging process of an individual, stem cell exhaustion, accumulation of chronic inflammation, and imbalance of tissue homeostasis will significantly affect the individual's ability to repair damage, and the weakening of the repair capacity will further cause aging and its related diseases. Therefore, systematic analysis of the cellular and molecular regulatory mechanisms of tissue regeneration and the impact of aging on damage repair has important scientific significance and application value. Summary of the invention

[0003] The technical problem to be solved by the present invention is how to promote organ or tissue regeneration, delay or reverse aging, and how to evaluate or identify the aging degree or regeneration level of the body.

[0004] In order to solve the above technical problems, the present invention first provides a new use of CCL2 protein or a substance that increases the content and / or activity of CCL2 protein.

[0005] The present invention provides the use of CCL2 protein or a substance that increases the content and / or activity of CCL2 protein in any of the following A1)-A8):

[0006] A1) Delay or reverse aging;

[0007] A2) preventing and / or treating aging-related decline in regenerative function;

[0008] A3) Promote organ or tissue regeneration;

[0009] A4) Improve the regenerative capacity of organs or tissues;

[0010] A5) Preparation of products that delay or reverse aging;

[0011] A6) preparing products for preventing and / or treating aging-related decline in regenerative function;

[0012] A7) Preparation of products that promote organ or tissue regeneration;

[0013] A8) Preparation of products that enhance the regenerative capacity of organs or tissues.

[0014] In order to solve the above technical problems, the present invention also provides a CCL2 protein or a substance that increases the content and / or activity of the CCL2 protein and ARG1 + New uses of macrophages or their related preparations.

[0015] The present invention provides a CCL2 protein or a substance that increases the content and / or activity of the CCL2 protein and ARG1 + Use of macrophages or related preparations thereof in any of the following A1)-A8):

[0016] A1) Delay or reverse aging;

[0017] A2) preventing and / or treating aging-related decline in regenerative function;

[0018] A3) Promote organ or tissue regeneration;

[0019] A4) Improve the regenerative capacity of organs or tissues;

[0020] A5) Preparation of products that delay or reverse aging;

[0021] A6) preparing products for preventing and / or treating aging-related decline in regenerative function;

[0022] A7) Preparation of products that promote organ or tissue regeneration;

[0023] A8) Preparation of products that enhance the regenerative capacity of organs or tissues.

[0024] In order to solve the above technical problems, the present invention also provides a product, which includes CCL2 protein or a substance that increases the content and / or activity of CCL2 protein;

[0025] The function of the product is any one of the following applications D1)-D4):

[0026] D1) Delay or reverse aging;

[0027] D2) Preventing and / or treating aging-related decline in regenerative function;

[0028] D3) Promote organ or tissue regeneration;

[0029] D4) Improve the regenerative capacity of organs or tissues.

[0030] Furthermore, the product may also include ARG1 + Macrophages or their related preparations.

[0031] Furthermore, the product may also include pharmaceutically acceptable carriers and / or excipients.

[0032] In any of the above applications or products, the delay or reversal of aging is delay or reversal of cell aging. Further, the delay or reversal of cell aging is embodied in any of the following B1)-B4):

[0033] B1) Promote cell proliferation;

[0034] B2) reduce cellular β-galactosidase content;

[0035] B3) Promote cell migration;

[0036] B4) Promote the ability of cells to form tubes.

[0037] The promoting of organ or tissue regeneration or the improving of organ or tissue regeneration capacity is promoting organ or tissue regeneration after injury or improving organ or tissue regeneration capacity after injury. Further, the promoting of organ or tissue regeneration after injury or the improving of organ or tissue regeneration capacity after injury is embodied in any one of the following C1)-C6):

[0038] C1) Promote blood flow recovery (such as promoting blood flow recovery after hind limb ischemia);

[0039] C2) Promote muscle recovery (e.g. promote muscle recovery after frostbite);

[0040] C3) Promote skin wound healing;

[0041] C4) Promotes the proliferation of skin basal cells;

[0042] C5) reduce the proportion of apoptotic cells;

[0043] C6) Improve the ischemic recovery Tarlov score and ischemic regeneration score (the standards of ischemic recovery Tarlov score and ischemic regeneration score are shown in Table 1 below).

[0044] Furthermore, the cells may be vascular endothelial cells (such as human vascular endothelial cells HAECs), and specifically may be passaged senescent vascular endothelial cells.

[0045] The organ or tissue may be muscle, skin or blood vessel, specifically may be muscle, skin or blood vessel of aged mice (22-23 month old mice).

[0046] Furthermore, the injury may be hind limb ischemia injury or skin wound injury or quadriceps frostbite.

[0047] In order to solve the above technical problems, the present invention also provides a substance for detecting the expression level of CCL2 or a substance for detecting the expression level of ARG1 + New uses for substances at the macrophage level.

[0048] The present invention provides a substance for detecting the expression level of CCL2 or a substance for detecting the expression level of ARG1 + Use of the substance at the macrophage level in any of the following X1)-X4):

[0049] X1) Assessing or assisting in assessing the degree of aging of a subject after injury;

[0050] X2) assessing or assisting in assessing the regeneration level of a subject after injury;

[0051] X3) preparing products for evaluating or assisting in evaluating the degree of aging of a subject after injury;

[0052] X4) Preparation of products for assessing or assisting in assessing a subject's level of regeneration after injury.

[0053] In order to solve the above technical problems, the present invention also provides a product for evaluating or assisting in evaluating the aging degree and / or regeneration level of a subject after injury.

[0054] The product provided by the present invention for evaluating the aging degree and / or regeneration level of a subject after injury includes a substance for detecting the expression level of CCL2 or a substance for detecting ARG1 + Substances at the macrophage level.

[0055] In order to solve the above technical problems, the present invention finally provides any of the following methods Y1)-Y4):

[0056] Y1) A method for delaying or reversing cell senescence, comprising the step of contacting the cell with a CCL2 protein or a substance that increases the content and / or activity of the CCL2 protein;

[0057] Y2) A method for promoting regeneration of an organ or tissue after injury or improving the regenerative capacity of an organ or tissue after injury, comprising the step of contacting a CCL2 protein or a substance that increases the content and / or activity of the CCL2 protein with the organ or tissue;

[0058] Y3) A method for evaluating or assisting in evaluating the degree of aging of a subject after injury, comprising the step of detecting the expression level of CCL2 in a sample from the subject;

[0059] Y4) A method for evaluating or assisting in evaluating a subject's regeneration level after injury, comprising the step of detecting the expression level of CCL2 in a sample from the subject.

[0060] Furthermore, the Y1) method may further include adding ARG1 + The step of contacting macrophages or related preparations with the cells.

[0061] The method Y2) may further include adding ARG1 + The step of contacting macrophages or related preparations with the organ or tissue.

[0062] The Y3) method may also include detecting ARG1 in the sample from the subject + Steps at the macrophage level.

[0063] The Y4) method may also include detecting ARG1 in the sample from the subject + Steps at the macrophage level.

[0064] Furthermore, said Y3) and said Y4) may be method 1 or method 2;

[0065] The method 1 comprises the following steps:

[0066] m1) determining the expression level of CCL2 in a sample from the subject;

[0067] m2) comparing the CCL2 expression level with a reference value;

[0068] m3) assessing or assisting in assessing the degree of aging and / or the level of regeneration of the subject.

[0069] Preferably, the sample is selected from a tissue or blood sample, such as whole blood, serum or plasma.

[0070] Preferably, the reference value is the CCL2 expression level (or average expression level) in the same type of samples from young, healthy individuals (or groups) with normal regenerative function.

[0071] Preferably, the degree of aging includes normal aging, accelerated aging and / or delayed aging.

[0072] Preferably, the regeneration level includes a normal level, a decline in regeneration function and / or an enhancement in regeneration function.

[0073] Preferably, when the CCL2 expression level in the sample from the subject is lower than the reference value, it indicates that the subject is in an aging state and / or the regenerative capacity of the subject may decline.

[0074] The second method comprises the following steps:

[0075] n1) determining ARG1 in a sample from the subject + Macrophage levels and CCL2 expression levels;

[0076] n2) the ARG1 + The macrophage levels and CCL2 expression levels were compared with reference values;

[0077] n3) assessing or assisting in assessing the degree of aging and / or the level of regeneration of the subject.

[0078] Preferably, the sample is selected from a tissue or blood sample, such as whole blood, serum or plasma.

[0079] Preferably, the reference value is ARG1 in the same type of sample from a young, healthy individual (or population) with normal regenerative function. + Macrophage level (or average cell level) and CCL2 expression level (or average expression level).

[0080] Preferably, the degree of aging includes normal aging, accelerated aging and / or delayed aging.

[0081] Preferably, the regeneration level includes a normal level, a decline in regeneration function and / or an enhancement in regeneration function.

[0082] Preferably, when ARG1 in the sample from the subject + If both the macrophage level and the CCL2 expression level are lower than the reference value, it indicates that the subject is in an aging state and / or the regenerative capacity of the subject may decline.

[0083] Any of the above-mentioned substances for increasing the content and / or activity of CCL2 protein may be a nucleic acid molecule encoding CCL2 protein or an expression cassette, vector or microorganism containing the nucleic acid molecule.

[0084] Any of the above-mentioned CCL2 expression levels may be CCL2 protein expression levels or CCL2 mRNA expression levels. The substance for detecting CCL2 expression levels may be a substance for detecting CCL2 expression levels by immunofluorescence tissue staining, plasma ELISA, (single cell) transcriptomics, plasma proteomics or a combination thereof.

[0085] Any of the above ARG1 + Macrophage levels (ARG1-positive macrophage levels, i.e. macrophages expressing ARG1 protein) can be ARG1 + Macrophage number or ARG1 + The detection of ARG1 + Macrophage levels of substances can be detected by immunofluorescence tissue staining ARG1 + Macrophage number or detection of ARG1 by single-cell transcriptome analysis + Macrophages account for the proportion of substances.

[0086] In any of the above-mentioned applications or products, the amino acid sequence of the CCL2 protein is shown in Sequence 1 in the sequence listing, and the protein can be purchased through commercial channels; it can also be prepared by conventional methods in the technical field, such as introducing a nucleic acid molecule encoding the CCL2 protein into an organism or a biological cell and expressing it in the organism or a biological cell to obtain the CCL2 protein, and the nucleic acid molecule encoding the CCL2 protein is shown in Sequence 2 in the sequence listing.

[0087] The amino acid sequence of the ARG1 protein is shown in Sequence 3 in the sequence listing.

[0088] In any of the above-mentioned applications or products, the CCL2 is significantly activated after injury in young individuals, but its activation is blocked after aging, and its expression level is negatively correlated with the body's aging level. It can be used as a marker to evaluate or assist in evaluating the degree of aging and / or regeneration level of a subject after injury. At the same time, the regeneration capacity of blood vessels, muscles and skin of aging individuals can be improved by timely increasing the CCL2 expression level after injury, thereby delaying aging.

[0089] The schematic diagram of the technical route of the present invention is as follows Figure 1 As shown in the figure, the specific method is as follows: First, eight tissues (including liver, skin, small intestine, blood vessels, bone marrow, lung, muscle and heart) of young and old mice exposed to different injuries were studied, and it was found that the regeneration capacity of multiple tissues (except heart) was downregulated with aging, and a single-cell transcriptome map of the regeneration process, cross-tissue and cross-age was constructed. Then, the cellular and molecular mechanisms of the regeneration process were analyzed at the single-cell level, and the differences in stem cell activation after young and old tissue damage were found. At the same time, it was also found that the ARG1-positive macrophage subpopulation would increase significantly during the regeneration of young tissues. This subpopulation highly expressed CCL2, which could interact with vascular cells and promote angiogenesis. In vitro experiments further proved that CCL2 can delay the aging of hemangioendothelioma and improve functions related to vascular regeneration. By intervening with CCL2 in old mice, it was found that CCL2 can promote the regeneration of blood vessels, muscles and skin in old mice. The present invention uses a variety of tissue injury and regeneration models, combined with single cells, to systematically analyze the characteristics and common regulatory mechanisms of 8 types of tissue injury and repair in mammals, and by comparing the differences in injury and repair between young and old, deeply explores the biomarkers and potential intervention targets related to injury and repair, providing a theoretical basis for regeneration function assessment, early warning and precise intervention. At the same time, the present invention found that CCL2 is a key target for improving the regeneration capacity of elderly tissues, which is helpful to explore new intervention methods and develop new strategies for regulating regeneration, delaying aging and preventing and treating aging-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 It is a schematic diagram of the technical route of the present invention.

[0091] Figure 2 It was found that the tissue regeneration ability of old mice was weaker than that of young mice in 7 tissues.

[0092] Figure 3 To construct a multi-tissue single-cell atlas.

[0093] Figure 4 To obtain immune cell subtype clustering.

[0094] Figure 5 ARG1 + Macrophages responded more strongly to regeneration in the younger group.

[0095] Figure 6 The scatter plot of dimensionality reduction results of monocyte / macrophage subset distribution (left) and the scatter plot of tissue remodeling related gene scores in monocytes / macrophages (right).

[0096] Figure 7 ARG1 + Macrophage marker genes were enriched in pathways related to endothelial cell proliferation, vascular endothelial growth factor production, angiogenesis regulation, and wound healing.

[0097] Figure 8 CCL2 in young ARG1 + It is expressed at a higher level in macrophages.

[0098] Fig. 9 Young mice have more ARG1 expressing CCL2 in regenerating skeletal muscle, blood vessels, and skin tissue + Macrophages.

[0099] Fig.10 ARG1 + Macrophages have strong interactions with endothelial cells in multiple tissues.

[0100] Fig.11 The score for young individual endothelial cells is that they have higher expression of CCL family ligand receptors in the early stage of regeneration.

[0101] Fig.12 ARG1 + CCL-related receptor ligand reactions between macrophages and endothelial cells exist in multiple tissues and organs.

[0102] Fig.13 Young individuals have higher scores of endothelial proliferation and angiogenesis-related gene sets at the transcriptional level.

[0103] Fig.14 Young individuals have higher levels of endothelial cell regeneration at the tissue protein level.

[0104] Fig.15CCL2 promotes the proliferation of senescent HAECs.

[0105] Fig.16 CCL2 reduces the expression level of senescence markers in HAECs undergoing passage senescence.

[0106] Fig.17 CCL2 promotes the migration of senescent HAECs.

[0107] Fig.18 CCL2 promotes tube formation of senescent HAECs during passage.

[0108] Fig.19 CCL2 promotes the recovery of vascular, skeletal muscle and skin function and angiogenesis in aged mice.

[0109] Fig. 20 CCL2 promotes the proliferation of basal cells in the skin of aged mice.

[0110] Fig.21 CCL2 reduces the proportion of apoptotic cells after hindlimb ischemia in aged mice. DETAILED DESCRIPTION

[0111] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0112] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0113] Example 1. Establishment of a multi-tissue organ injury and regeneration model and establishment of a multi-tissue organ regeneration single cell atlas

[0114] 1. Establishment of a mouse multi-tissue organ regeneration model

[0115] All animal experiments were performed according to approved protocols and in accordance with the animal welfare ethics of the Institute of Zoology, Chinese Academy of Sciences. The mice used were wild-type C57BL / 6J strain (Speffort). Young mice were 2-3 months old and old mice were 22-23 months old. Mice were individually housed in a pathogen-free facility with a controlled temperature (23-24°C) with a 12-h light and 12-h dark cycle, provided with ample food and water, and cages and bedding were changed once a week.

[0116] 1. Construction of multi-tissue and organ injury model

[0117] Young mice and old mice were treated as follows to construct a multi-tissue organ injury model:

[0118] 1) Quadriceps frostbite

[0119] The mice were anesthetized by inhalation of isoflurane (2-3% concentration) and the hair on the legs was shaved. The skin was then disinfected with iodine, and a 1.5 cm skin incision was made on the surface of the quadriceps to expose the quadriceps. If there was adipose tissue above the muscle, the fat was also pushed aside to fully expose the quadriceps. The metal rod was precooled with liquid nitrogen, and immediately attached to the quadriceps for 10 seconds after the metal rod was taken out. The skin was sutured with 5-0 silk thread after the injury, and the wound was disinfected.

[0120] 2) Myocardial ischemia

[0121] Mice were anesthetized by inhalation of isoflurane (2-3% concentration) and the hair on the chest was removed with a depilatory cream. The skin was incised above the left chest and then a prepared purse-string suture was performed. The pectoralis minor and major muscles were incised and a small hole was made in the fourth intercostal space. The heart was gently and quickly pulled out and the left anterior descending coronary artery (LAD) was quickly sutured with 6-0 silk thread. Immediately after suturing, the heart was returned to the chest cavity and the incision was sutured with the previously placed purse-string suture.

[0122] 3) Bleomycin-induced lung injury

[0123] The mice were anesthetized by inhalation of isoflurane (2-3% concentration), and then bleomycin was injected into the lungs of the mice via trachea to induce injury. The bleomycin injection dose was 1.5 mg / kg.

[0124] 4) Skin puncture injury

[0125] The mice were anesthetized by inhalation of isoflurane (2-3% concentration) and their back hair was shaved. Two full-thickness parallel excisional skin wounds with a diameter of 0.4 cm were then made on the back of the mice using a 4 mm biopsy punch.

[0126] 5) Hindlimb ischemia model

[0127] The hair of the mouse hind limb area was removed using a depilatory cream, and after the mouse was anesthetized by inhalation of isoflurane (2-3% concentration), the proximal and distal parts of the right femoral artery were ligated with 6-0 non-absorbable silk sutures, carefully preserving the femoral nerve, and then the skin was sutured with 5-0 silk sutures.

[0128] 6) Partial hepatectomy

[0129] The mice were anesthetized by continuous inhalation of isoflurane (2-3% concentration), and the skin was disinfected with 70% ethanol. A midline abdominal incision (about 3 cm in length) was then made to expose the xiphoid process and liver. The left lobe of the liver was first ligated with 5-0 silk and removed using microdissecting forceps. After this, the right and left parts of the middle lobe were ligated and removed separately. The peritoneum was sutured with 4-0 silk sutures, and the skin was closed with 4mm reflex clips. The postoperative mice were placed on a heating pad for recovery. Finally, the mice were given a subcutaneous injection of bupivacaine hydrochloride to minimize postoperative discomfort.

[0130] 7) Whole body irradiation bone marrow injury model

[0131] Young and old mice were sham-irradiated or irradiated with a sublethal dose (5 Gy) of total body irradiation (TBI) at a dose rate of 0.948 Gy / min using an RS 2000 X-ray biological irradiator (Rad Source Technologies, Inc.). To produce a more uniform dose distribution within the block, a RAD+ reflector block was used.

[0132] 8) Local irradiation small intestine injury model

[0133] To immobilize the mice during irradiation, the mice were administered anesthesia IP with 0.9% sodium chloride (Sigma) containing 10% ketamine and 1% xylazine. The mice were then exposed to 8 Gy of ionizing radiation at a dose rate of 0.948 Gy / min in an RS2000 X-ray biological irradiator (RadSource Technologies, Inc.). To avoid damage to the head, chest, and limbs, irradiation was limited to the ventral surface of the mice using a lead shield.

[0134] 2. Verification of the decline of regenerative function in aged mice

[0135] 1) Calculation of liver organ index

[0136] The mice were weighed on the sham operation group (day 0), 1, 2, 4, and 7 days after injury, and the livers of the mice with partial hepatectomy were dissected and weighed using an analytical balance. The data were obtained by using the ratio of liver mass to the corresponding mouse body weight to obtain the final organ index.

[0137] 2) Tensile force test

[0138] The grip strength of the mouse limbs was tested using a GSM Grip Strength Meter (Cat. No. 47200) from UGO BASILE SRL. The mouse was placed flat on the tension receptor of the grip dynamometer, and the mouse was pulled back horizontally at a constant speed. The dynamometer was designed to automatically record the peak tension. Each mouse was tested 8-10 times, and the final grip strength was determined by the average value after excluding the maximum and minimum values.

[0139] 3) Skin wound area measurement

[0140] The wounds were digitally photographed once a day, and the wound area was analyzed from the images using ImageJ software.

[0141] 4) Doppler blood flow imaging

[0142] Blood flow was measured daily by laser Doppler perfusion imaging (Moor Instruments). The specific steps were as follows: Limb perfusion was assessed by scanning from the abdomen to the toe tips while mice were anesthetized with isoflurane. Two regions of interest covering each hindlimb area were selected and blood flow was analyzed using moorLDI Review software. The blood base recovery ratio between ischemic and control limbs was determined.

[0143] 5) Calculation of small intestinal crypt length

[0144] Sections of paraffin-embedded small intestine samples were deparaffinized with xylene and rehydrated with decreasing concentrations of alcohol (100%, 90%, 80%, 70%, and 50%) and water. Sections were stained in hematoxylin for 3-5 minutes and washed in running tap water. Sections were differentiated with 1% acid alcohol (1% HCl in 70% alcohol) for a few seconds to remove excess dye and rinsed in running water until the sections turned blue. The slides were then counterstained with eosin, dehydrated with increasing concentrations of alcohol (50%, 70%, 80%, 90%, and 100%), and washed with 100% xylene. After covering with a coverslip, images were taken using a microscope. Crypt lengths were measured from the images using ImageJ software.

[0145] 6) Treadmill fatigue test

[0146] The treadmill was set at an incline of 5 degrees and the electrical stimulation was 0.5 mA. The mice were trained on the treadmill twice, with an initial speed of 3 m / min for 2 minutes, 7 m / min for 2 minutes, and then 9 m / min for 1 minute. The mice were tested at an initial speed of 3 m / min for 2 minutes, and then increased by 1.5 m / min per minute until the mice could no longer run on the treadmill. Regardless of the electrical shock and mechanical stimulation, the total number of clicks and the total distance run by the mice were recorded when the mice were exhausted.

[0147] 7) Cardiac ejection fraction detection

[0148] Cardiac function by transthoracic echocardiography was assessed using a Vevo LAZR imaging system (Visual Sonics, Inc.) with a 30 MHz transducer. Mice were anesthetized by inhalation of 2% isoflurane and then examined. Two-dimensional M-mode tracings were obtained at the level of the papillary muscles. Left ventricular ejection fraction (EF) was measured and calculated over three consecutive cardiac cycles.

[0149] 8) Immunostaining of vascular endothelial cells in multiple tissues

[0150] Paraffin sections were dewaxed with xylene and rehydrated with 100% alcohol, 90% alcohol, 80% alcohol, 70% alcohol, 60% alcohol, and 50% alcohol. The sections were rinsed with running tap water and placed in a microwave container containing pH 6.0 sodium citrate buffer (ZSGB-BIO, ZLI-9064). Heat-induced epitope retrieval was performed using microwaves. The slides were washed 3 times with PBS and permeabilized with 0.4% Triton X-100 in PBS for 1 hour at room temperature. The slides were then incubated with 5% donkey serum (Jackson ImmunoResearch) in PBS for 1 hour at room temperature for blocking and incubated with the first antibody (CD31, R&D, AF3628) at 4°C overnight. PBS was washed 3 times and the secondary antibody was incubated at room temperature for 1 hour. The nuclei were stained with Hoechst 33342 (Invitrogen). The slides were washed 3 times and covered with coverslips. The cells were fixed with PFA at room temperature for 15 minutes, and permeabilized with 0.4% Triton X-100 for 30 minutes after washing with PBS. After washing with PBS several times, the cells were blocked with 5% donkey serum at room temperature for 1 hour, and then the primary antibody was replaced and incubated at 4°C overnight. The cells were removed the next day, washed with PBS several times, and then incubated with fluorescent secondary antibodies and nuclear dyes (Hoechst 33342) (room temperature, dark, 1 hour). After completion, the cells were washed with PBS several times and sealed with anti-fade sealing agent. Images were taken by laser scanning confocal microscopy (LSM 900, ZEISS).

[0151] The results are as follows Figure 2 As shown, from Figure 2 It can be seen that the tissue regeneration ability of old mice is weaker than that of young mice in liver, skeletal muscle, skin, blood vessels, small intestine and lung tissues.

[0152] 2. Establishment of a single-cell atlas of multi-tissue and organ regeneration in mice

[0153] 1. Multi-tissue single cell isolation and sequencing

[0154] 1) Single cell isolation and sequencing of muscle or heart tissue

[0155] The injured part of muscle or heart tissue was sampled and stored in liquid nitrogen. When the single nucleus was isolated, the tissue was taken out of the liquid nitrogen and ground into powder in liquid nitrogen using a mortar. The tissue powder was then collected into a 2 mL grinding tube and 1 mL of homogenization buffer was added, the buffer containing 250 mM sucrose, 25 mM KCl, 25 mM MgCl2, 10 mM Tris buffer, 1 uM DTT, 1X protease inhibitor, 0.4 U / uL RNaseIn (Thermo Fisher Scientific), 0.4 U / uL Superasin (Thermo Fisher Scientific), 0.1% Triton X-100 (v / v), 1 uM PI (Thermo Fisher Scientific) and 10 ng / mL Hoechst 33342 (Thermo Fisher). Magnetic bead grinding was then performed using a frozen multi-sample tissue grinding system (TissueLyser-24, Jingxin Industrial Development, China), 60 Hz, 30 s, 3 times, with a 60 s interval between the two times. The cell suspension was collected and filtered twice through a 40um cell filter to remove undigested tissue pieces. The cell suspension was centrifuged at 100g for 2 minutes to precipitate debris. The supernatant was collected in a new tube, centrifuged at 500×g for 5 minutes, the supernatant was removed, and the cell pellet was resuspended in PBS containing 1% BSA (Gemini), 0.4U / uL RNaseIn, and 0.4U / uL Superasin. The nuclei were sorted using FACS (BD Influx) to remove cell debris and collect nuclei that were double positive for propidium iodide and Hoechst. The nuclei were then counted using a dual fluorescence cell counter (Luna-FLTM, LogosBiosystems), and approximately 8,000 nuclei were captured for RNA-seq library construction, followed by sequencing in the NovaSeq 6000 sequencing system (Illumina, 20012866).

[0156] 2) Single cell isolation and sequencing of lung tissue

[0157] The lung tissue was minced into small pieces with micro scissors, and then the tissue was digested with a mixed buffer containing collagenase I (1 mg / mL), collagenase II (1 mg / mL), collagenase IV (1 mg / mL), dispase (1 mg / mL), elastase (0.38 mg / mL), DNase I (1.25 U / mL) and trypsin-EDTA (0.125%) for 20 minutes at 37°C. The digestion was terminated by adding cold DMEM / F-12 (Gibco) containing 10% fetal bovine serum (FBS, Gibco). The mixture was passed through a 70um filter (BD Falcon) and centrifuged at 350×g for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in red blood cell (RBC) lysis buffer (BD Biosciences) for 3 minutes at room temperature to remove red blood cells. The mixture was centrifuged, resuspended in PBS containing 0.3% bovine serum albumin (BSA, Gibco), and passed through a 40um filter (BD Falcon). The isolated cells were sorted by FACS (BD Influx) to remove cell debris and propidium iodide-positive dead cells and generate single-cell suspensions for 10x Genomics sequencing. Single-cell capture was performed using the 10x Genomics single-cell system, and approximately 8,000 cells were captured per sample following the standardized 10x capture and library preparation protocol (10x Genomics Single Cell 3' System), and then sequenced on the NovaSeq 6000 sequencing platform (Illumina, 20012866).

[0158] 3) Single-cell isolation and sequencing of skin tissue

[0159] Skin samples were collected using an 8 mm biopsy punch on the wound and stored in ice-cold phosphate-buffered saline. Skin tissue was minced with microscissors and digested with a mixed buffer of collagenase I (1 mg / mL), collagenase IV (1 mg / mL), dispase (1 mg / mL) and trypsin-EDTA (0.125%) at 37°C for 1 hour and 15 minutes. The mixture was diluted with DMEM / F-12 (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and passed through a 70um filter (BD Falcon). The cell dissociation mixture was centrifuged at 400×g for 5 minutes at 4°C and the supernatant was discarded. The cells were resuspended with red blood cell (RBC) lysis buffer (BD Biosciences) at room temperature for 2-3 minutes and DMEM / F-12 (Gibco) containing 10% fetal bovine serum (FBS, Gibco) was added. The cell suspension was centrifuged and the cells were resuspended in 800uL of 0.3% bovine serum albumin (BSA, Gibco) in PBS. The dissociated cells were passed through a 40um filter (BD Falcon) and then sorted by FACS (BD Influx) to remove cell debris and propidium iodide-positive dead cells to generate single cell suspensions for 10x Genomics sequencing. Single cell capture was performed using the 10x Genomics single cell system, and approximately 8,000 cells were captured per sample according to the standardized 10x capture and library preparation protocol (10x Genomics single cell 3' system), and then sequenced on the NovaSeq 6000 sequencing platform (Illumina, 20012866).

[0160] 4) Single-cell isolation and sequencing of vascular tissue

[0161] For vascular tissue, the muscle tissue around the ligated femoral artery was separated and minced by micro scissors. The tissue was collected in a 15 mL test tube and first digested with collagenase II (10 mg / mL) at 37 ° C for 10 minutes. The mixture was passed through a 70 um filter (BD Falcon) and neutralized on ice with 10 mL DMEM / F-12 (Gibco) containing 10% fetal bovine serum (FBS, Gibco). The undigested tissue was then collected in a new tube and a mixed buffer solution of collagenase I (2 mg / mL), dispase (2 mg / mL), elastase (1 mg / mL) and DNase I (2.5 U / mL) was added and reacted for 1 hour at 37 ° C. The mixture was passed through a 70 um filter (BD Falcon) and mixed with the cell suspension on ice before. The mixed cell suspension was centrifuged and the cells were resuspended for 5 minutes with a combination of Accumax (Thermo, 00-4666-56) and Triple to dissociate cell aggregates. The cells were incubated with 1× red blood cell (RBC) lysis buffer (BD Biosciences) at room temperature for 2 minutes and then resuspended in PBS containing 0.4% BSA. Live single cells were sorted by FACS (BD Influx) to remove cell debris and propidium iodide-positive cells to generate single cell suspensions for 10x Genomics sequencing. Single cell capture was performed using the 10xGenomics single cell system, and approximately 8,000 cells were captured per sample according to the standardized 10x capture and library preparation protocol (10xGenomics single cell 3' system), and then sequenced on the NovaSeq 6000 sequencing platform (Illumina, 20012866).

[0162] 5) Single cell isolation and sequencing of liver tissue

[0163] For liver tissue, mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg / kg) and blood was collected by removing the eyeballs. The mice were then placed on the edge of a dissecting tray and the limbs were fixed with pins. The fur was moistened with 70% ethanol, and then a 5 cm long incision was made in the abdomen. The intestines in the abdominal cavity were moved to expose the portal vein and the inferior vena cava. The inferior vena cava was cannulated, and the liver tissue was perfused with preheated perfusion buffer (PB: 0.15M NaCl; 5mM KCl; 25mM NaHCO3; 5mM glucose; 20mM Hepes) to wash the blood in the liver vessels. The liver was then perfused with preheated PBE (perfusion buffer containing 1mM EDTA) to eliminate calcium. Preheated digestion buffer (perfusion buffer plus 1mg / mL type II collagenase, 1mg / mL type IV collagenase, and 5mM CaCl2) was perfused into the liver to digest the hepatocytes. The liver was gently dissected, and the cell suspension was collected and filtered through a 70um cell strainer to remove undigested tissue pieces. The cell suspension was mixed with the culture medium, centrifuged at 350×g for 5 minutes to remove debris, and then resuspended with 5 mL of red blood cell (RBC) lysis buffer (BD Biosciences) at room temperature for 2-3 minutes. The cell culture medium was centrifuged and resuspended in hepatocyte culture medium (Lonza, CC-3198). The culture medium was passed through a 40um filter (BD Falcon) and then sorted by FACS (BD Influx) to remove cell debris and propidium iodide-positive dead cells to generate a single cell suspension for 10xGenomics sequencing. Single cell capture was performed using the 10x Genomics single cell system, and approximately 8,000 cells were captured per sample according to the standardized 10x capture and library preparation protocol (10x Genomics single cell 3' system), and then sequenced on the NovaSeq 6000 sequencing platform (Illumina, 20012866).

[0164] 6) Isolation and sequencing of hematopoietic stem and progenitor cells

[0165] The femur and tibia were separated together and any remaining muscle and connective tissue were carefully removed. The tibia was washed with PBS (without Mg) supplemented with 2% FBS (Gibco) using a 5 mL syringe on ice. 2+ and Ca 2+Bone marrow cells were isolated from the femur and tibia of mice by crushing limb bones with BD Biosciences (Gibco). Bone marrow cells were collected and passed through a 40 μm filter (BD Falcon). Single cell suspensions were centrifuged at 1200 rpm for 5 minutes at 4°C, and then resuspended in 5 mL 1× red blood cell (RBC) lysis buffer (BD Biosciences) for 5 minutes at room temperature. After centrifugation at 1200 rpm for 5 minutes at 4°C, cells were resuspended in PBS containing 2% FBS, and then live single cells were sorted by FACS (BD Influx) to remove propidium iodide-positive dead cells. To isolate hematopoietic stem and progenitor cells (LSK, Lin-Sca-1+c-Kit+), bone marrow cells after erythrocyte lysis were stained with a combination of antibodies, including FITC anti-mouse lineage cocktail and Isotype Ctrl (Biolegend, 133302), APC anti-mouse Ly-6A / E (Sca-1) antibody (Biolegend, 108112) and PerCP anti-mouse CD117 (c-kit) antibody (Biolegend, 105822) on ice for 30 minutes. The cell suspension was washed twice with cold PBS containing 2% FBS, and Lin-Sca-1+c-Kit+ cells were sorted by FACS (BD Influx). The 10x Genomics single-cell system was used to capture single cells of bone marrow cells (all cells) and bone marrow stem cells (Lin-Sca-1+c-Kit+ cells). According to the standardized 10x capture and library preparation protocol (10xGenomics single-cell 3' system), approximately 5,000 to 8,000 cells were captured for each sample and then sequenced on the NovaSeq 6000 sequencing platform (Illumina, 20012866).

[0166] 7) Single-cell isolation and sequencing of small intestinal tissue

[0167] For small intestinal tissue, mice were anesthetized by intraperitoneal administration of pentobarbital. Blood was collected and the body was perfused with saline from the heart. The gastrointestinal tract was then carefully collected and the small intestine was cleaned with saline sold. The mixture of small intestinal fragments was cut into small pieces and reacted with collagenase I (1.8 mg / mL), collagenase IV (1.8 mg / mL), dispase (1.8 mg / mL) and DNase I (2.75 U / mL) at 37°C for 20 minutes. The mixture was blown in with a 5 mL syringe and DF12 containing 10% FBS was added to terminate digestion. The suspension was filtered twice through a 40 um cell strainer and then centrifuged at 1200 rpm for 5 minutes. The cells were resuspended with 2 mL PBS containing 0.4% BSA and sorted with FACS (BD Influx) to remove dead cells positive for propidium iodide to generate a single cell suspension for 10x Genomics sequencing. Single-cell capture was performed using the 10x Genomics Single Cell System. Approximately 8,000 cells were captured per sample following the standardized 10x capture and library preparation protocol (10x Genomics Single Cell 3' System) and then sequenced on the NovaSeq 6000 sequencing platform (Illumina, 20012866).

[0168] 2. Data processing

[0169] 1) Preliminary processing of scRNA-seq and snRNA-seq raw data

[0170] The present invention uses Cell ranger software (version 4.0.0) to perform pre-processing and quality control on single-cell sequencing data, such as processing multiple PCR sequencing data (de-multiplexing), cell label (barcode) identification, and the construction of single-cell gene expression matrix. Cell ranger software is used to align the FASTQ sequencing file of each sample with the mouse reference genome (mm10), and the default parameters are used to perform quality assessment and generate a digital gene expression matrix, and the filtered gene expression matrix is ​​used for downstream analysis. For snRNA-seq data, CellBender software (version 0.2.0) is used to remove background signal contamination caused by technical errors.

[0171] 2) Cell type identification and construction of multi-tissue cell atlas

[0172] Seurat software (version 4.0.2) was used to perform quality control, cell type identification, and subsequent differential gene analysis on the gene-cell expression matrix obtained above. Specifically, the following steps were included: 1) The cell expression matrix after integration of each tissue was separated according to the sample using the SplitObject function of Seurat software (version 4.0.2). Then, all single-sample expression matrices were integrated using Scanpy's standard integration process and batch effects caused by technical reasons were removed using "sc.external.pp.bbknn". 2) The first 100 principal components (PCA) were used to reduce the dimensionality of the integrated data using the "sc.tl.tsne", "sc.pp.neighbors", and "sc.tl.leiden" functions. Finally, the cell types were marked in the atlas using the previously annotated cell types.

[0173] The analysis method of the above step 1) is as follows: For the single-cell expression matrix of each sample obtained from each tissue, first, cells with a gene number between 500 and 6000 and a mitochondrial gene ratio of less than 20%, as well as genes expressed in at least 3 cells are retained. Then, DoubletFinder software (version 2.0.3) is used to predict and remove "multiple cells" (multiple cells enter the same droplet for sequencing) due to technical reasons. In order to better eliminate the heterogeneity caused by technical factors such as sequencing depth in single-nuclear RNA sequencing data, SCTransform software (version 0.3.2) is used to normalize and scale the matrix. Next, the "PrepSCTIntegration" and "FindIntegrationAnchors" functions are used to select data integration anchors for the post-quality control data of each sample, and these anchors are used to integrate the data sets of all samples of the same tissue using the "IntegrateData" function.

[0174] The analysis method of step 2) above is as follows: the integrated data is used to identify the cell type. First, the data is scaled using “ScaleData”, and the principal component analysis (PCA) dimension is calculated using the “RunPCA” function. Then, the data is unsupervisedly clustered using the “FindNeighbors” and “FindClusters” functions, and the data is divided into multiple cell clusters. The “RunUmap” function is used to reduce the dimensionality of the data. In order to analyze the gene expression specificity of each cell family, the “FindAllMarkers” function (|avg_log2FC| is greater than 0.5, p_val_adj is less than 0.05) is used to identify the highly expressed genes (marker genes) specific to each cell cluster, and then the cell type is identified based on the expression of the marker genes of each cell cluster. Then, the gene expression matrix containing the cell labels obtained for each tissue is integrated using Scanpy software (version 1.6.1), and a multi-tissue cell atlas is constructed ( Figure 3 ).

[0175] Example 2: Analysis of the regenerative capacity of multiple tissues and organs and identification of regeneration-related cells and markers

[0176] Identification of regeneration-related ARG1+ macrophages

[0177] 1. Immune cell subset analysis

[0178] Immune cells in solid tissues and immune cells in bone marrow tissues were extracted from the entire dataset, and re-clustering and dimensionality reduction were performed respectively. The specific steps include the following: First, the five major immune cell types identified in the dataset were annotated according to well-known marker genes, including Cd68 + Monocytes / macrophages, Batf3 + Dendritic cells, S100a8 + Neutrophils, Cd3e + ILC / T cells, and Cd79a + B cells. Further, based on the marker genes of each subgroup, 23 subgroups were identified in solid tissue immune cells ( Figure 4 The changing trend of each subpopulation during the regeneration process was described using the cell ratio of each subpopulation in the data of young and old time points ( Figure 5 ), we can see from the figure: ARG1 + Macrophages responded more strongly to regeneration in the younger group.

[0179] 2. Quasi-trajectory inference

[0180] Three different algorithms, including DDRTree, DiffusionMap, and partition-based graph abstraction (PAGA), were used to infer the differentiation trajectory of monocytes / macrophages. The specific steps include the following: 1) Pseudo-time was calculated using the first 400 feature genes of the "differentialGeneTest" function of the R package monocle (version 2.22.0), and the differentiation trajectory of monocytes / macrophages was inferred after dimensionality reduction and sorting with mononuclear precursor cells as the starting cell type. 2) DiffusionMap was used to reduce the dimensionality of monocytes / macrophages using the R package destiny (version 3.10.0) to generate a diffusion map. Mononuclear precursor cells were manually selected as root cells, and pseudo-time values ​​were extracted along the generated trajectory. 3) The partition-based graph abstraction method (PAGA) was used for monocyte / macrophage subsets using the Python package Scanpy, and pseudo-time trajectory inference was performed using default parameters. Based on the calculations of these three algorithms, the evolutionary relationship between monocyte / macrophage subsets was inferred.

[0181] 3. Genomic scoring analysis

[0182] Gene sets were downloaded from MSigDB (https: / / www.gsea-msigdb.org / gsea). Gene set scores were calculated using the “AddModuleScore” function of Seurat. Statistical analysis of gene set scores between old and young cells was performed using the ggpubr package (version 0.4.0) by the Wilcoxon test ( Figure 6 ). It was also found that ARG1+ macrophages have the characteristics of promoting angiogenesis, wound healing, endothelial growth, etc. ( Figure 7 ), and found that ARG1+ macrophages in young mice can express higher levels of CCL2 ( Figure 8 ).

[0183] 4. Tissue Immunofluorescence Staining

[0184] Paraffin sections of skeletal muscle, blood vessels, and skin were dewaxed with xylene and rehydrated with 100% alcohol, 90% alcohol, 80% alcohol, 70% alcohol, 60% alcohol, and 50% alcohol. The sections were rinsed with running tap water and placed in a microwave container containing pH 6.0 sodium citrate buffer (ZSGB-BIO, ZLI-9064). Heat-induced epitope retrieval was performed using microwaves. The slides were washed 3 times with PBS and permeabilized with 0.4% Triton X-100 in PBS for 1 hour at room temperature. The slides were then incubated with 5% donkey serum (Jackson ImmunoResearch) in PBS for 1 hour at room temperature for blocking and incubated with the primary antibody (ARG1, Santa Cruz, sc-271430; CCL2, ABclonal, A21991) at 4°C overnight. Wash 3 times with PBS and incubate with the secondary antibody at room temperature for 1 hour. Cell nuclei were stained with Hoechst 33342 (Invitrogen). The slides were washed three times and covered with coverslips. The cells were fixed with PFA for 15 minutes at room temperature and permeabilized with 0.4% Triton X-100 for 30 minutes after washing with PBS. After washing several times with PBS, the cells were blocked with 5% donkey serum for 1 hour at room temperature, and then the primary antibody was replaced and incubated overnight at 4°C. The cells were removed the next day, washed with PBS several times, and then incubated with fluorescent secondary antibodies and nuclear dyes (Hoechst 33342) (room temperature in the dark, 1 hour). After completion, the cells were washed several times with PBS and sealed with anti-fade mounting medium. Images were taken with a laser scanning confocal microscope (LSM 900, ZEISS). Through immunofluorescence staining of young and old tissues after injury, it was found that more ARG1 can be activated in young individuals after injury. + and CCL2 double positive macrophages ( Fig. 9 ).

[0185] 2. ARG1 + Analysis of macrophage-endothelial cell interaction

[0186] The interaction between cells of each sample was studied using the R package CellChat (version v.2.1.1). To evaluate the interaction between cells, CellChat (version 1.5.0) was used to evaluate the expression of ligand and receptor pairs in cell populations based on the database CellChatDB. The "computeCommunProb" function was used to calculate the communication probability and infer the cellular communication network. The communication probability of a signaling pathway is calculated by summarizing the probabilities of its related ligand-receptor pairs. The communication probability here only represents the interaction strength, not entirely the probability. The function "computeCommunProbPathway" is used to infer intercellular communication at the signaling pathway level. The generated intercellular communication matrix at the signaling pathway level was hierarchically clustered, and the clustering results were further divided into 4 groups. The R packages pheatmap and ggplot2 were used to visualize the time-related cell interaction patterns. Through cell and cell interaction analysis, ARG1 was found + Macrophages primarily interact with endothelial cells ( Fig.10 ). It was also found that different CCL receptors are expressed in endothelial cells of different tissues, and the CCL2 receptor is rapidly activated after the loss of young tissues compared to old tissues, and is highly expressed in the middle stage of injury, but is significantly downregulated in the late stage of injury ( Fig.11 ). Further studies found that ARG1 + The interaction of CCL signaling pathway between macrophages and endothelial cells was higher in the young group ( Fig.12 ), while endothelial cells in young tissues have stronger proliferation, angiogenesis and generation capabilities ( Fig.13 To further explore the changes in endothelial generation capacity with aging, immunofluorescence staining of young and old tissues after injury revealed that more CD31-positive cells can be activated in young individuals after injury ( Fig.14 ), which also indicates that the angiogenesis capacity of tissues decreases with aging.

[0187] Example 3: CCL2 can delay the senescence of hemangioendothelial cells and improve the functions related to angiogenesis. 1. CCL2 delays endothelial cell senescence and functional decline in vitro

[0188] 1. Endothelial cell culture and treatment

[0189] Human vascular endothelial cells HAECs (Lonza, CC-2535) were cultured in an incubator at 37°C with 5% CO2 using EGM-2 endothelial culture medium (Lonza, CC-3202). The culture medium required the addition of an anti-antibody (added at a volume ratio of 1:5000 to the culture medium) and penicillin-streptomycin (added at a volume ratio of 1:100 to the culture medium). The culture dish was covered with collagen matrix. The primary cell line was serially passaged (cells were digested in an incubator for 5 minutes using accumax cell digestion solution and digested with culture medium; then collected into a centrifuge tube, centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded; and passed to a new culture medium covered with collagen in proportion to complete the passage), until about 12-13 generations (passage senescence).

[0190] Depending on whether the passaged senescent cells were treated with CCL2 recombinant protein (PeproTech, Cat#250-10), they were divided into experimental group and control group:

[0191] Experimental group (CCL2): CCL2 recombinant protein was added to the HAECs cell culture system (about 5E4 cells / 12-well plate) to a final concentration of 10 ng / ml in the HAECs cell culture system, and then cultured in a 37°C constant temperature incubator with 5% CO2 for about one week (the cells grew until they just covered the culture dish).

[0192] Control group: The cells were not treated with CCL2 recombinant protein and were cultured in a 37°C constant temperature incubator with a 5% CO concentration for the same period of time.

[0193] 2. Cell Immunofluorescence Staining

[0194] The experimental and control group cells obtained in step 1 were subjected to immunofluorescence staining. The specific steps are as follows: sterile glass slides were added to the 24-well plate, UV irradiated for more than 30 minutes, coated with appropriate matrix (hESC-matrigel, hNSC-matrigel, hMSC-gelatin) according to the cell type, placed in a 37°C incubator for more than 3 hours or overnight, and the cells were plated on the glass slides at an appropriate density and cultured. The next day or when the cell density reached the staining density, the samples were washed twice with PBS, 500 μL of 4% paraformaldehyde was added to fix the samples, incubated at room temperature for 20 minutes, and washed three times with PBS. 0.5 mL of 0.4% TritonX-100 was added, placed at room temperature for 10 minutes, and washed three times with PBS. 10% donkey serum was added at a volume of 350 μL / well and placed at room temperature for 1 hour. After the primary antibody was diluted with 10% donkey serum, 100 μL / sample was incubated in a wet box, overnight at 4°C, and washed three times with PBS. Dilute the corresponding secondary antibody with PBS (usually 1:500), and add the nuclear dye Hoechst at a ratio of 1:1000. Incubate at room temperature for 60 minutes, and wash three times with PBS. After sealing with mounting medium, observe and take pictures with a LEICA laser confocal microscope.

[0195] The results are as follows Fig.15 As shown in the figure, it can be seen that CCL2 can promote the proliferation of senescent HAECs.

[0196] 3. Cell SA-β-gal staining

[0197] The experimental and control cells obtained in step 1 were stained with SA-β-gal. The specific steps are as follows: the cells were fixed with 2% formaldehyde and 0.2% glutaraldehyde for 5 minutes and washed with PBS several times. Then, the cells were stained with a staining solution containing 1 mg / ml X-gal at 37°C in the dark for 12 hours. After removal, the cells were washed with PBS and photographed under an optical microscope. The proportion of cells positive for SA-β-gal was counted using imageJ software.

[0198] The results are as follows Fig.16 As shown in the figure, it can be seen that CCL2 can reduce the levels of senescence markers in passaged senescent HAECs cells.

[0199] 4. Scratch test

[0200] The cells in the experimental group and the control group obtained in step 1 were subjected to a scratch test. The specific steps are as follows: The scratch test is used to measure the cell migration ability. The cells were cultured in a 12-well plate until they were confluent, and two parallel scratches were made in each well using a 2μL pipette tip. After taking pictures using a microscope, the cells were returned to the 37°C incubator for culture, and pictures were taken again after 8 hours. The scratch width was analyzed using imageJ, and the migration rate of endothelial cells after scratching was calculated.

[0201] The results are as follows Fig.17 As shown in the figure, it can be seen that CCL2 can promote the migration of senescent HAECs.

[0202] 5. Tube Formation Experiment

[0203] The cells in the experimental group and the control group obtained in step 1 were subjected to a tube formation experiment. The specific steps are as follows: cells were cultured at 5×10 4 Cells were seeded in 24-well plates covered with matrigel and cultured at 37°C. Photos were taken using an incucyte. Parameters such as the number of tubes, number of nodes, and branch length were analyzed using ImageJ-Angiogenesis analyze software.

[0204] The results are as follows Fig.18 As shown in the figure, it can be seen that CCL2 can promote the tube formation of aging HAECs.

[0205] 2. CCL2 supplementation promotes multi-organ regeneration in aged mice

[0206] 1. CCL2 intervention in hindlimb ischemia model

[0207] The hindlimb ischemia model was constructed using old mice as experimental materials. Hindlimb ischemia surgery (Hindlimb ischemiaand laser doppler perfusion imaging) was performed under a sterile environment. The hindlimb ischemia model specifically refers to the method in Example 1. Immediately after surgery, the blood flow of mice was measured by laser Doppler perfusion imaging (Moor instrument). Starting from the first day after surgery, CCL2 recombinant protein (PeproTech, Cat#250-10) (dissolved in PBS, 1ng / μL) was injected into the injury site intramuscularly for three consecutive days, 50ng / mouse / each injection. The control group was injected with the same dose of PBS (solvent). Blood flow was measured every other day and analyzed using moorLDI Review software. Tensile testing and weight measurement (weight is used to correct tension) were completed on the ninth day after surgery, and samples from the injury site were collected for immunofluorescence staining. Tarlov score and modified version of ischemic regeneration score were performed on the ninth day after surgery. The specific scoring criteria are shown in Table 1, and double-blind scoring was performed by two experimenters.

[0208] Table 1. Tarlov score and ischemia regeneration score

[0209] Tarlov scoring function (Tarlov scoring) 0Cannot move 1. Almost no movement, no weight bearing 2Frequent and vigorous exercise without weight bearing 3 support weights, can walk 1, 2 steps 4 walks with only minor impairments 5. Normal but slow walking 6. All-around fast walking Modified ischemic score tissue grading 0Autoamputation of lower leg 1 Calf necrosis 2 Foot necrosis 3Discoloration of two or more toes 4 Single toe color change 5. Two or more nails are discolored 6 Single nail discoloration 7 No necrosis

[0210] The results are as follows Fig.19 and 21As shown, the results showed that in vivo injection of CCL2 accelerated vascular regeneration after ischemic injury in aged mice, which was specifically manifested in that compared with the control group, it promoted the speed of blood flow recovery (higher percentage of blood flow recovery), accelerated the functional recovery of the hind limbs of mice after injury (increased Tarlov score and ischemic regeneration score), showed higher vascular regeneration ability in immunofluorescence staining, and had a lower proportion of cell apoptosis.

[0211] 2. CCL2 intervenes in skin wound healing

[0212] A skin wound injury model was constructed using old mice as experimental materials. The skin wound healing model was carried out under a sterile environment. The skin wound injury model specifically refers to the method in Example 1. From the day of completion of the operation to the third day, CCL2 recombinant protein (PeproTech, Cat#250-10) (dissolved in PBS, 1ng / μL) was injected in situ into the wound for four consecutive days, 50μL / mouse / each injection, and the control group was injected with the same dose of PBS (solvent). For wound healing records, the wound site was photographed at the end of the operation, on the second, fourth, and seventh days after surgery, and the wound area was analyzed by imageJ software. The experimental mice were euthanized on the seventh day after surgery, and samples of the injured site were collected for immunofluorescence staining.

[0213] The results are as follows Fig.19 and 20 As shown, the results showed that in vivo injection of CCL2 accelerated skin regeneration after skin injury in aged mice, which was specifically manifested in that compared with the control group, the skin injury area was smaller at the same time, the positive area of ​​vascular fluorescence staining at the tissue level was higher, and the basal cell proliferation ratio was higher.

[0214] 3. CCL2 intervention in muscle frostbite

[0215] A quadriceps frostbite model was constructed using old mice as experimental materials. The quadriceps frostbite surgery was performed under a sterile environment. The quadriceps frostbite model was specifically referred to the method in Example 1. CCL2 recombinant protein (PeproTech, Cat#250-10) (dissolved in PBS, 1 ng / μL) was injected intramuscularly at the site of injury on the day of the end of the operation, the next day, and the third day, 50 μL / mouse / each injection, and the control group was injected with the same dose of PBS (solvent). For tension measurement, tension measurements were performed before surgery (baseline), on the second day, fifth day, and seventh day after surgery. The experimental mice were euthanized on the eighth day after surgery, and samples of the injury site were collected for immunofluorescence staining.

[0216] The results are as follows Fig.19As shown, the results showed that in vivo injection of CCL2 accelerated muscle regeneration after frostbite in aged mice, which was specifically reflected in that the recovery rate of relative tension after muscle frostbite was higher and the positive area of ​​vascular fluorescence staining at the tissue level was higher than that of the control group.

[0217] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of CCL2 protein or a substance that increases the content and / or activity of CCL2 protein in any of the following A1)-A8): A1) Delay or reverse aging; A2) preventing and / or treating aging-related decline in regenerative function; A3) Promote organ or tissue regeneration; A4) Improve the regenerative capacity of organs or tissues; A5) Preparation of products that delay or reverse aging; A6) preparing products for preventing and / or treating aging-related decline in regenerative function; A7) Preparation of products that promote organ or tissue regeneration; A8) Preparation of products that enhance the regenerative capacity of organs or tissues.

2. CCL2 protein or a substance that increases the content and / or activity of CCL2 protein and ARG1 + Use of macrophages or related preparations thereof in any of the following A1)-A8): A1) Delay or reverse aging; A2) preventing and / or treating aging-related decline in regenerative function; A3) Promote organ or tissue regeneration; A4) Improve the regenerative capacity of organs or tissues; A5) Preparation of products that delay or reverse aging; A6) preparing products for preventing and / or treating aging-related decline in regenerative function; A7) Preparation of products that promote organ or tissue regeneration; A8) Preparation of products that enhance the regenerative capacity of organs or tissues.

3. The use according to claim 1 or 2, characterized in that: The delaying or reversing of aging is delaying or reversing cellular aging; Or, the delaying or reversing of cell senescence is embodied in any one of the following B1)-B4): B1) Promote cell proliferation; B2) reduce cellular β-galactosidase content; B3) Promote cell migration; B4) Promote the ability of cells to form tubes; Or, the promoting organ or tissue regeneration or improving the regenerative capacity of an organ or tissue is promoting the regeneration of an organ or tissue after damage or improving the regenerative capacity of an organ or tissue after damage; Or, the promoting regeneration of an organ or tissue after damage or the improving the regeneration capacity of an organ or tissue after damage is embodied in any one of the following C1)-C6): C1) Promote blood flow restoration; C2) Promote muscle tension recovery; C3) Promote skin wound healing; C4) Promotes the proliferation of skin basal cells; C5) reduce the proportion of apoptotic cells; C6) Improve ischemic recovery Tarlov score and ischemic regeneration score.

4. A product comprising CCL2 protein or a substance that increases the content and / or activity of CCL2 protein; The function of the product is any one of the following applications D1)-D4): D1) Delay or reverse aging; D2) Preventing and / or treating aging-related decline in regenerative function; D3) Promote organ or tissue regeneration; D4) Improve the regenerative capacity of organs or tissues.

5. The product according to claim 4, characterized in that: The product also includes ARG1 + Macrophages or their related preparations.

6. The product according to claim 4 or 5, characterized in that: The delaying or reversing of aging is delaying or reversing cellular aging; Or, the delaying or reversing of cell senescence is embodied in any one of the following B1)-B4): B1) Promote cell proliferation; B2) reduce cellular β-galactosidase content; B3) Promote cell migration; B4) Promote the ability of cells to form tubes; Or, the promoting organ or tissue regeneration or improving the regenerative capacity of an organ or tissue is promoting the regeneration of an organ or tissue after damage or improving the regenerative capacity of an organ or tissue after damage; Or, the promoting regeneration of an organ or tissue after damage or the improving the regeneration capacity of an organ or tissue after damage is embodied in any one of the following C1)-C6): C1) Promote blood flow restoration; C2) Promote muscle tension recovery; C3) Promote skin wound healing; C4) Promotes the proliferation of skin basal cells; C5) reduce the proportion of apoptotic cells; C6) Improve ischemic recovery Tarlov score and ischemic regeneration score.

7. Substances for detecting CCL2 expression level or substances for detecting ARG1 + Use of the substance at the macrophage level in any of the following X1)-X4): X1) Assessing or assisting in assessing the degree of aging of a subject after injury; X2) assessing or assisting in assessing the regeneration level of a subject after injury; X3) preparing products for evaluating or assisting in evaluating the degree of aging of a subject after injury; X4) Preparation of products for assessing or assisting in assessing a subject's level of regeneration after injury.

8. A product for evaluating or assisting in evaluating the aging degree and / or regeneration level of a subject after injury, comprising a substance for detecting the expression level of CCL2 or a substance for detecting ARG1 + Substances at the macrophage level.

9. Any of the methods described in Y1) to Y4) below: Y1) A method for delaying or reversing cell senescence, comprising the step of contacting the cell with a CCL2 protein or a substance that increases the content and / or activity of the CCL2 protein; Y2) A method for promoting regeneration of an organ or tissue after injury or improving the regenerative capacity of an organ or tissue after injury, comprising the step of contacting a CCL2 protein or a substance that increases the content and / or activity of the CCL2 protein with the organ or tissue; Y3) A method for evaluating or assisting in evaluating the degree of aging of a subject after injury, comprising the step of detecting the expression level of CCL2 in a sample from the subject; Y4) A method for evaluating or assisting in evaluating a subject's regeneration level after injury, comprising the step of detecting the expression level of CCL2 in a sample from the subject.

10. The method according to claim 9, characterized in that: The method Y1) further comprises the step of: + A step of contacting macrophages or related preparations with the cells; The method Y2) further comprises the step of: + A step of contacting macrophages or related preparations thereof with the organ or tissue; The Y3) method further comprises detecting ARG1 in the sample from the subject + Steps at the macrophage level; The Y4) method further comprises detecting ARG1 in the sample from the subject + Steps at the macrophage level.

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