Preparation of cd73 positive nk cells from different starting sources of umbilical cord blood and application in treatment of alzheimer's disease

Abstract

This invention belongs to the field of medical technology, specifically relating to the preparation of CD73-positive NK cells from umbilical cord blood of different origins and their application in the treatment of Alzheimer's disease. This invention studies CD73-positive NK cells differentiated from different cell types originating from umbilical cord blood (CD34-positive cells, CD3-negative / CD56-positive cells, and CD56-negative cells). + NK cells were found to differentiate into CD73 from all initial cell types derived from umbilical cord blood. + NK cells, and 34 + -73-NK cells showed a higher growth rate, 56 － -73-NK cells exhibited stronger cytotoxicity, and 56 － -73-NK cells showed better overall efficacy than 34- + -73-NK cells. Further research indicates that CD73 + NK cells can inhibit the activation of microglia and astrocytes in the hippocampus of STZ-injected mice, thereby reducing neuroinflammation in the hippocampus and potentially providing a new solution for the treatment of AD.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to the preparation of CD73-positive NK cells from umbilical cord blood of different origins and their application in the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline, accounting for approximately 50%-60% of all dementia cases. The characteristic pathological changes in AD can be roughly summarized as follows: amyloid β-protein (Aβ) accumulates in specific areas of the brain, hyperphosphorylated Tau protein forms neurofibrillary tangles, which in turn triggers a series of inflammatory and oxidative stress responses, leading to a large loss of cholinergic neurons in the cerebral cortex and hippocampus, accompanied by glial cell proliferation, ultimately resulting in slowly progressive cognitive impairment and dementia symptoms.

[0003] Chronic inflammation can lead to a gradual decline in bodily functions and increase the risk of frailty and dementia by mediating neurodegeneration and enhancing central nervous system inflammation. Previous studies have found that elevated and progressive levels of inflammatory factors in the blood of individuals with normal cognitive function are associated with an increased risk of Alzheimer's disease (AD). Furthermore, early-stage AD patients have higher levels of inflammatory markers in their blood and cerebrospinal fluid. Simultaneously, pro-inflammatory signals outside the central nervous system play a significant role in the development and progression of AD. Oxidative stress is the earliest cellular characteristic of AD, and in the early stages of AD, the oxidative stress response in the brain of patients is more pronounced than in individuals with normal cognitive function. Lipid peroxides, isoprostaglandins, and other cellular oxidative parameters are significantly elevated in the brain tissue, serum, plasma, and urine of AD patients, indicating that oxidative stress damage is inextricably linked to the occurrence and transformation of AD. Therefore, an imbalance between inflammatory response and oxidative stress accelerates the progression of neurodegenerative diseases (especially AD) by exacerbating the link between the pathogenesis and specific pathological features of AD. Because the pathogenesis of AD involves multiple factors, disease-modifying therapies targeting a single mechanism or pathway may be less effective than therapies targeting multiple mechanisms. Therefore, exploring the progress in AD treatment from these two aspects is of great significance.

[0004] Immunotherapy (ICT) has emerged as a promising approach for treating cancer and combating pathogens and viruses. Clinical evidence shows that immune cells can respond to disease in multiple ways within the host, representing a significant advancement over traditional cancer treatments such as surgery, chemotherapy, and radiotherapy. Advances in immunology, genetic engineering, gene editing, and synthetic biology have made ICT a potential tool for cancer treatment, as evidenced by the clinical success of chimeric antigen receptor (CAR)-modified T cells. However, CAR-T cells also have limitations, such as antigen escape, off-target effects, associated toxicity, and high production costs. Due to these limitations, natural killer (NK) cells have recently emerged as a promising alternative.

[0005] NK cells are a key component of the innate immune system, capable of eliminating various tumor cells and virus-infected cells through their cytotoxic function. Unlike T cells, NK cells can directly kill target cells without prior sensitization and have fewer side effects (such as cytokine release syndrome and graft-versus-host disease), suggesting that NK cells may be a promising option in ICT (Invasive Chronic and Infectious Disease). NK cells have various receptors on their surface, which localize to target cells by recognizing specific ligands on the target cells. Inhibitory receptors on NK cells include CD94-NKG2A and inhibitory members of the killer Ig-like receptor (KIR) family (such as KIR2DL1, KIR2DL2 / 3), which can bind to major histocompatibility complex class I (MHC I) molecules on the surface of target cells. Activating receptors are categorized into several types: NKp30, NKp44, and NKp46 are innate cytotoxic receptors; NKG2D, NKG2C, and NKp80 are C-type lectin-like receptors; and DNAM1 and CD2 are co-activating receptors. These activating receptors recognize ligands on the surface of target cells and trigger cytotoxic responses to eliminate target cells. NK cells ultimately decide whether to kill target cells by balancing the signals between inhibitory and activating receptors. In addition, NK cells express activating Fcγ receptors, most notably FcγRIIIa (Fcγ receptor IIIa, which mediates antibody-dependent cytotoxicity), also known as CD16a. When antibodies bind to antigens on the surface of target cells, these activating Fcγ receptors induce antibody-dependent cell-mediated cytotoxicity (ADCC), thereby promoting NK cell destruction of target cells. CD73... + NK cells, a subset of NK cells with a unique phenotype discovered in recent years, are also defined as "regulatory NK cells." They play an important role in immune regulation and have emerged as a novel anti-inflammatory treatment modality. Therefore, regarding CD73... +The preparation of NK cells and their application in the treatment of Alzheimer's disease (AD) have significant potential applications. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention differentiates umbilical cord blood mononuclear cells from different sources into CD73. + NK cells were used to compare the differences in their preparation. In a sporadic AD mouse model established by injecting STZ into the lateral ventricle or tail vein, it was found that STZ could inhibit the activation of glial cells in the hippocampus and reduce neuroinflammation, which may provide a new option for the clinical treatment of AD.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides CD73 + Application of NK cells in the preparation of drugs for treating Alzheimer's disease.

[0009] This invention has discovered that CD73 + NK cells can inhibit the proliferation and differentiation of pro-inflammatory cells, suppress antibody production, induce related cells to transform into an anti-inflammatory phenotype, enhance their immunomodulatory capacity, and reduce the levels of inflammation-related cytokines such as NO, IL-1β, and IL-6 through paracrine mechanisms, thereby alleviating the inflammatory response. Furthermore, CD73... + NK cells have a strong resistance to oxidation in the body, CD73 + NK cells can further regulate the cellular response to oxidative stress by overexpressing endonuclease 1 / redox factor 1 (APE1 / Ref-1): not only can they inhibit the production of reactive oxygen species (ROS) and regulate the intracellular redox state, but they can also upregulate the gene expression levels of enzymes involved in clearing ROS in vivo.

[0010] Preferably, the CD73 + NK cells are derived from CD34 cells in umbilical cord blood (UCB). + Cells. 34 + NK cells can produce more mature and functional NK cells than those from other sources.

[0011] Preferably, the CD73 + The method for preparing NK cells is as follows: CD34 from umbilical cord blood + Umbilical cord blood mononuclear cells were isolated from the cells, an initial cell suspension was prepared, and then the initial cell suspension was inoculated in complete RPMI 1640 medium and incubated at 37 °C for more than 21 days to obtain the final product.

[0012] More preferably, the RPMI 1640 medium contains 10% (v / v) fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES, 1 mM sodium phosphate, 11 non-essential amino acids, 55 nM 2-ME, 100 U / mL penicillin, 100 mg / mL streptomycin, 1 mM Motolimod, 1 mM Resiquimod, 1 mM Vesatolimod, 1 mM Pidotimod, 1 mM M Laquinimod, 1 mM Tempol, 50 ng / mL IL-2, 10 ng / mL IL-12, 10 ng / mL IL-15, 10 ng / mL IL-18, and 10 ng / mL IL-21.

[0013] Preferably, the CD34 + The cell preparation method is as follows: umbilical cord blood was diluted with DPBS and added to Ficoll. The white blood cell layer was collected by centrifugation, followed by centrifugation to collect the precipitate. Red blood cell lysis buffer was added to lyse any remaining red blood cells. CD34 was then purified by flow cytometry using CD34-PE antibody. + cell.

[0014] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0015] More preferably, the excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, antioxidants, adsorbents, filter aids, and release inhibitors.

[0016] Preferably, the dosage form of the drug includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention studies CD73 derived from different initial cell types of umbilical cord blood (UCB). + NK cells, specifically including CD34 from UCB + Cells (CD34-positive cells), CD3 － CD56 + (CD3-negative / CD56-positive cells) and CD56 － CD73 produced in cells+ NK cells were analyzed, and their function was examined. The results showed that all the starting cell types derived from umbilical cord blood could differentiate into CD73 cells. + NK cells. Among them, 34 + -73-NK cells (CD34) + CD73 (source) + NK cells showed a higher growth rate, 56 － -73-NK cells (CD56) － CD73 (source) + NK cells, on the other hand, exhibited stronger cytotoxicity, and 56 － The overall effect of -73-NK cells was better than that of 34. + -73-NK cells. Further research indicates that CD73 + NK cells can inhibit the activation of microglia and astrocytes in the hippocampus of STZ-injected mice, thereby reducing hippocampal neuroinflammation. This indicates that CD73... + NK cells offer new solutions for the development of innovative cell-based drugs for clinical use and for the treatment of Alzheimer's disease (AD). Attached Figure Description

[0019] Figure 1 CD73 of umbilical cord blood from different origins + Flowchart and results of NK cell preparation; A: Flowchart showing the experimental process from sorting umbilical cord blood mononuclear cells (MNCs) to inducing differentiation into NK cells from different origins; B: Flow cytometry analysis showing the cell sorting process and the proportion of each cell population through staining and gating analysis of cell markers (such as CD34, CD45, CD56, etc.); C: Microscopic observation of cell morphology showing the morphology of NK cells from different origins; D: Chromosome analysis showing the chromosome copy number detection results to confirm cell chromosome ploidy.

[0020] Figure 2 A study on the differentiation and function of NK cells from different sources in umbilical cord blood; A: from "umbilical cord blood → umbilical cord blood mononuclear cells → 34" + 56 + 56 － A: Flowchart of differentiation of NK cells from different origins; B: Cell morphology diagram showing NK cells after 2 weeks of culture and differentiation, 34 + 56 + 56 － Morphology and clonogenesis of three types of NK cells; C, D, E: bar charts, C shows the CD3+ of the three types of NK cells at different culture and differentiation times (0.5, 1, 1.5, 2 weeks). － CD56 + CD56+ CD16 + Cell proportion data, D represents CD56 + CD16 + Differences in cell proportions at different time points; E shows the changes in the number of the three types of NK cells at different differentiation weeks; F: Flow cytometry analysis plot, showing the expression levels of the three types of NK cell surface activating receptors (NKG2D, etc.), inhibitory receptors (KIR2DL1, etc.), and cell lysing granules (perforin, etc.) in the form of overlapping peaks; G: Line graph, with the horizontal axis representing the effector-to-target ratio (1:1, 5:1, 10:1) and the vertical axis representing cytotoxicity (%), showing the killing ability of the three types of NK cells against the three cancer cell lines K562, A2780 cis, and SKOV3.

[0021] Figure 3 CD73 for different MNCs origin + Diagram of NK cell differentiation and function; A: Cell morphology diagram (light microscope image), showing three types of CD73. + NK cells (34) + -73-NK, 56 + -73-NK and 56 － -73-NK) Cell morphology and distribution after 3 weeks of culture and differentiation; B, C, D: bar chart, B shows the CD3 content of the three cell types at different culture and differentiation times (1.5, 2, 2.5, 3 weeks). － CD56 + Expression ratio; C displays CD56 + CD16 + The differences in expression ratios at different time points; D: Comparison of the changes in the number of the three cell types at different differentiation weeks; E: Flow cytometry analysis, showing the expression levels of the three cell surface activation receptors (NKG2D, etc.), inhibitory receptors (KIR2DL1, etc.), and cell lysing granules (Perforin, etc.) in the form of overlapping peaks; F: Line graph, with the horizontal axis representing the effector-to-target ratio (1:1, 5:1, 10:1) and the vertical axis representing cytotoxicity (%), demonstrating the killing ability of the three cell types against the three cancer cell lines K562, A2780 cis, and SKOV3.

[0022] Figure 4 Typical charts and statistical analysis results of IBA1 expression in the mouse hippocampus are presented, showing CD73. + NK cells inhibited the proliferation and activation of microglia in the hippocampus of STZ-injected mice; A: Fluorescence detection of IBA1, a marker of microglia in the hippocampus; B: Quantification of the number of IBA1-positive cells; C: Microglia in different states (left: resting state; right: activated state); D: Ratio of activated microglia to total microglia. Data are expressed as mean ± standard error, n=3 per group (…). , ).

[0023] Figure 5 This section presents typical charts and statistical analysis results of GFAP expression in the mouse hippocampus, and showcases CD73. + NK cells inhibited the activation of astrocytes in the hippocampus of STZ-injected mice; A: Fluorescence detection of GFAP, a marker of activated astrocytes in the hippocampus; B: Quantification of the number of GFAP-positive cells; C: Western blot images of IBA1 and GFAP; D: Quantification of IBA1 and GFAP protein expression levels. Data are expressed as mean ± standard error, n=3 per group ( , ).

[0024] Figure 6 This is an integrated diagram showing the results of immune inflammation and pathological marker detection in AD model mice; A: WB plot, showing different treatment groups (Control, AD, AD+CD73). + Bands of IL-1β, IL-6, TNF-α, and β-actin (internal reference) proteins in the hippocampus of NK and AD+NK mice are used to visually represent protein expression; B and C: bar charts, B is used to quantify the expression levels of IL-1β, IL-6, and TNF-α proteins relative to β-actin in the Western blot results; C presents the relative mRNA expression levels of these inflammatory factors; D: Western blot plot showing Aβ expression in the hippocampus of different groups of mice. 1-42 BACE, p-Tau (Ser396), Tau5, and β-actin (internal control) protein bands show the expression of related pathological proteins; E: bar chart, showing the expression of DAβ. 1-42 Quantitative statistical analysis of the relative β-actin expression levels of proteins such as BACE and p-Tau (Ser396), and analysis of CD73. + Effects of NK on amyloid accumulation and Tau hyperphosphorylation; F: Using immunofluorescence technology, Aβ in the hippocampus was labeled with a specific antibody. Red fluorescence showed the distribution and expression of Aβ, and DAPI (blue) stained the cell nucleus. The "merged" plot showed the co-localization of the two, allowing for a direct observation of the morphological and positional differences of Aβ deposition in the hippocampus of different groups of mice; G: The fluorescence intensity of the Aβ-positive region in F was quantitatively analyzed and compared among different groups (Control, AD, AD+CD73). + The level of Aβ deposition in the hippocampus of NK and AD+NK mice can be used to help determine CD73. + Effects of NK on Aβ accumulation in AD model mice. Detailed Implementation

[0025] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0026] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0027] This invention differentiates umbilical cord blood mononuclear cells from different origins into CD73. + NK cells (a type of regulatory natural killer cell that highly expresses the adenosine metabolizing enzyme CD73 and has enhanced cytotoxicity) were compared, and CD73 from different origins were analyzed. + Differences in NK cell preparation protocols. Simultaneously, a sporadic AD mouse model was established by intraventricular injection of streptozotocin (STZ), and CD73 was administered via intraventricular or tail vein injection after 6 weeks. + NK cells. Immunofluorescence staining (IF) was used to observe hippocampal glial cell activation, neonatal neurons, and Aβ. 1-42 Positive areas showed the presence of CD73. + NK cells inhibited the activation of microglia and astrocytes in the hippocampus of STZ-injected mice. Furthermore, IBA1, GFAP, IL-1β, IL-6, TNF-α, BACE, and Aβ were detected in the hippocampus. 1-42 The protein expression levels of CD73, p-Tau, Tau5, synaptic protein-1 (Syt-1), synaptophysin-1, and brain-derived neurotrophic factor (BDNF) were decreased. Furthermore, real-time quantitative PCR (qPCR) assays showed decreased mRNA expression levels of IL-1β, IL-6, and TNF-α. These results indicate that CD73... + NK cells can reduce neuroinflammation in the hippocampus of AD mice, providing a solution for innovative cell-based drug therapy for AD in clinical practice.

[0028] To fully and clearly present the technical solution and significant advantages of the present invention, the present invention will be described in detail below with reference to specific embodiments.

[0029] 1. Experimental Methods

[0030] 1.1 UCB cell sorting

[0031] Thaw 25 mL of frozen umbilical cord blood in a water bath for 5 min, then dilute with DPBS at a 1:1 volume ratio. Add 10 mL of the diluted blood to 3 mL of Ficoll and centrifuge at 900×g for 30 min. Collect the leukocyte layer using a pipette, and centrifuge the resulting sample again at 700×g for 10 min. Collect the pellet and add erythrocyte lysis buffer, incubating at room temperature for 10 min to lyse any remaining erythrocytes. Stain the treated cells with 7-aminoactinomycin D (7-AAD) viability staining solution and analyze by flow cytometry to distinguish between live and dead cells. For live cells, stain with CD34-PE, CD45-V500, CD3-FITC, and CD56-APC antibodies. After staining, classify the cells by flow cytometry as CD34-positive cells. + Cells, CD3 － CD56 + Cells and CD56 － Cells (i.e., CD56 isolated) + (Remaining cells after cell division). All detection data were analyzed using FlowJo 10.8.1 software.

[0032] 1.2, NK and CD73 + NK training

[0033] (1) Live cells (CD34) obtained by erythrocyte lysis and flow cytometry confirmation during the UCB cell sorting process were collected. + Cells, CD3 － CD56 + Cells and CD56 － Using Gibco mononuclear cell separation medium, umbilical cord blood mononuclear cells were separated by centrifugation at 800g for 15 min to prepare an initial cell suspension.

[0034] (2) Prepare complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 2mM L-glutamine, 25mM HEPES, 1mM sodium phosphate, 11 non-essential amino acids, 55nM 2-ME, 100 U / mL penicillin and 100 mg / mL streptomycin.

[0035] (3) Add 1mM Motolimod + 1mM Resiquimod + 1mM Vesatolimod + 1mM Pidotimod + 1mM Laquinimod + 1mM Tempol to the above culture medium, and add 50ng / mL IL-2 (interleukin-2), 10ng / mL IL-12, 10ng / mL IL-15, 10ng / mL IL-18, and 10ng / mL IL-21. These reagents should be used throughout the culture process.

[0036] (4) The initial cell suspension was seeded in complete RPMI 1640 medium at a cell density of 1×10⁻⁶ cells / year. 6 Cells / mL, and incubated at 37 °C for 21 days.

[0037] Motolimod (VTX-2337) is a selective and potent Toll-like receptor 8 (TLR8, a pattern recognition receptor that promotes immune responses upon activation) agonist, EC50. 50 The selectivity is 100 nM, which is more than 50 times higher than that for TLR7. The following is the preparation method for its 2.75 mg / mL (6.00 mM) working solution (taking 1 mL of working solution as an example): Add 50 μL of 55 mg / mL clear DMSO stock solution to 400 μL of PEG300, mix well until clear; add 50 μL of Tween80 to the above system, mix well until clear; then add 500 μL of ddH2O to bring the volume to 1 mL. The working solution should be prepared fresh before use.

[0038] Resiquimod (R-848, S28463) is an immunomodulator used as a potent TLR7 / TLR8 agonist and can induce the upregulation of cytokines such as TNF-α, IL-6, and IFN-α. Resiquimod can reduce hepatitis C virus (HCV) infection. The following is the preparation method for its 3.15 mg / mL (10.02 mM) working solution (using 1 mL as an example): Add 50 μL of 63 mg / mL clear DMSO stock solution to 400 μL of PEG300, mix thoroughly until clear; add 50 μL of Tween80 to the above system, mix thoroughly until clear; then add 500 μL of ddH2O to bring the volume to 1 mL. The working solution should be prepared fresh before use.

[0039] Vesatolimod (GS-9620) is an oral Toll-Like Receptor-7 (TLR-7) agonist being considered as a potential treatment for HIV infection. The following is the preparation method for its 0.44 mg / mL (1.07 mM) working solution (using 1 mL as an example): Add 50 μL of 8.8 mg / mL clear DMSO stock solution to 400 μL of PEG300, mix thoroughly until clear; add 50 μL of Tween80 to the above system, mix thoroughly until clear; then add 500 μL of ddH2O to bring the volume to 1 mL. The working solution should be prepared fresh before use.

[0040] Pidotimod (Pidotomod) is a synthetic dipeptide molecule with biological immunomodulatory activity against both adaptive and innate immune responses. Its solubility in DMSO is 49 mg / mL (200.59 mM). It should be noted that DMSO is hygroscopic; moisture absorption will reduce the solubility of Pidotimod. Therefore, it is recommended to use freshly opened DMSO.

[0041] Laquinimod (ABR-215062, LAQ) is an effective immunomodulator. The following is the preparation method for its 30.0 mg / mL (84.08 mM) working solution (using 1 mL as an example): Add 300 μL of clear 100 mg / mL propylene glycol stock solution to 50 μL of Tween 80, mix thoroughly until clear; then add 650 μL of D5W to bring the volume to 1 mL. Prepare the working solution immediately before use.

[0042] Tempol (4-Hydroxy-TEMPO) is a superoxide scavenger with neuroprotective, anti-inflammatory, and analgesic effects. The following is the preparation method for its 30.0 mg / mL (174.18 mM) working solution: Taking 1 mL of working solution as an example, add 30 mg of the product to 1 mL of physiological saline (0.9% NaCl solution), mix thoroughly until clear, and use immediately.

[0043] 1.3 Copy Number Variation (CNV) Analysis

[0044] NK or CD73 assays were performed using the VeriSeq PGS kit and the MiSeq kit V3 (Illumina). + CNV analysis of NK samples. Total CNVs for all chromosomes were plotted using BlueFuse Multi software version 4.5 to visualize NK or CD73. + NK ploidy.

[0045] 1.4 Flow cytometry analysis

[0046] The human cancer cell lines used in this flow cytometry analysis included K562, A2780cis (ECACC), and SKOV3, all generously provided by Shenzhen Second People's Hospital. The culture conditions for each cell line were as follows:

[0047] K562 and A2780cis cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% docetaxel; SKOV3 cells were cultured in McCoys 5A medium supplemented with 10% fetal bovine serum and 1% docetaxel. The medium was changed every two days, and subcultures were performed weekly. Note that for A2780cis cells, only 1 µM docetaxel was added to the medium on the day of subculturing. All cell lines were cultured at 37°C and 5% CO2.

[0048] The cell staining and subsequent procedures for flow cytometry analysis are as follows:

[0049] For surface marker staining, suspension cells were first collected into 1.5 mL centrifuge tubes and washed with 2% FACS buffer (PBS + 2% FBS). After washing, diluted antibody (Table 1) was added, and staining was performed at 4°C in the dark for 30 min. For intracellular staining, cells were first fixed and permeabilized: treated with 4% paraformaldehyde and 0.2% PBST (PBS + 0.2% Tween 20) at room temperature for 30 min. After 30 min, cells were washed with 2% FACS buffer, then diluted antibody (Table 1) was added for staining, and finally washed again with 2% FACS buffer.

[0050] Cells treated with the above staining were analyzed using a CytoFLEX flow cytometer, and the data were analyzed using FlowJo 10.6 software.

[0051] Table 1 Antibodies used in flow cytometry analysis

[0052]

[0053] 1.5 Cytotoxicity test

[0054] The cytotoxicity of NK cells against tumor cells was detected using the CellTrace Erythrocyte Proliferation Kit (Invitrogen, Waltham, MA, USA, #C34572) / 7-AAD flow cytometry. The specific steps were as follows: NK cells or CD73... +NK cells were mixed with far-infrared stained target cells at different E:T ratios in V-bottom 96-well plates, with each E:T ratio sample mixed in three independent wells and then incubated for 4 hours. After incubation, the incubated cells from each sample in the three independent wells were collected into 1.5 mL centrifuge tubes and resuspended in FACS buffer (PBS + 2% FBS) containing 7-AAD. The percentage of target cell lysis was analyzed using CytoFLEX flow cytometry and FlowJo software, where the percentage of specific lysis was calculated based on fluorescence measurements of the fluorescent dye relative to individual cancer cells.

[0055] 1.6. Karyotype Classification

[0056] Using the standard G-band method for NK or CD73 + NK cells were subjected to karyotype analysis.

[0057] 1.7 Laboratory Animals

[0058] Thirty four-week-old male C57BL / 6 mice were purchased from the Guangdong Provincial Animal Center. These mice were housed in a 12-hour light-dark environment at a temperature of 20±2℃ and humidity of 50±5%, with free access to food and water. When the mice reached 12 weeks of age, they were randomly divided into a normal control group (6 mice) and an STZ group (18 mice), with 6 mice per cage. All animal handling procedures were approved by the Laboratory Animal Ethics Committee of Shenzhen Second People's Hospital and followed the "Guideline for the Care and Use of Laboratory Animals" from the National Institutes of Health (NIH Publication No. 85-23, revised in 1985).

[0059] The animal model and experimental design were as follows: All mice were anesthetized by intraperitoneal injection of 5% hydroxychloroquine (0.1 mL / 10 g) and fixed on a stereotaxic apparatus. After surface disinfection, the scalp was incised. The Bregma point was located, and the coordinates of the bilateral ventricles were drilled using an electric cranial drill (coordinates: 1.0 mm to the left and right of the Bregma point, 0.5 mm posteriorly, and 2.5 mm deep). Then, in the STZ group, streptozotocin (STZ) was injected into the lateral ventricles via an autosampler (injection dose: 0.3 mg / kg, injection rate: 0.5 μL / min, injection volume: 1 μL per side; STZ dissolved in ACSF for later use). After injection, the needle was left in place for 5 minutes, then withdrawn. The mouse scalp was sutured, and an appropriate amount of physiological saline was injected into the peritoneal cavity. The mice were then placed on an electric blanket until awakened, and then placed back into their cages.

[0060] After a one-month recovery period, all mice were divided into four groups: a normal control group, an AD group, an AD+NK group, and an AD+CD73 group. +NK group, 6 mice per group. Stereotypers and drug delivery catheters (62003, 62102, RWD) were used to cannulate the right ventricle of mice. The normal control group received no treatment. Mice in the AD group received STZ injection into the lateral ventricle, and mice in the AD+NK group received 20 μL of NK cells (10 μL / day) via tail vein injection daily. 6 NK cells were dissolved in 100 μL PBS, and AD+CD73 was used. + Mice in the NK group were injected daily via tail vein with 20 μL of CD73. + NK cells (10 6 CD73 + NK cells were lysed in 100 μL PBS. All mice received the drug for 5 days. After drug administration, all mice underwent a series of behavioral tests and related experiments.

[0061] 1.8 Immunofluorescence staining (IF)

[0062] Mice in each group were randomly selected for cardiac perfusion. Perfusion was initially with PBS until no blood flowed out, then switched to 4% paraformaldehyde. Reperfusion was performed for 3 minutes after a generalized convulsion. The entire brain of each mouse was completely removed, fixed overnight in 4% paraformaldehyde solution at room temperature, and then dehydrated in 30% sucrose solution until it sank. The entire brain was placed in a disposable embedding cassette, filled with OCT embedding solution, and frozen overnight at -80°C. 30 μm thick sections were prepared using a microtome, attached to glass slides, and dried in a fume hood for 10 minutes after reaching room temperature. The tissue sections were labeled with an immunohistochemical pen and blocked with goat serum at room temperature for 30 minutes. After liquid removal, the sections were incubated overnight at 4°C in the dark with diluted rabbit anti-GFAP (1:100), rabbit anti-DCX (1:100), mouse anti-Aβ (1:100), rabbit anti-Tau (1:100), or rabbit anti-Iba-1 (1:100). After washing with PBST, the sections were incubated with goat anti-rabbit FITC or goat anti-rabbit Cy3 at 37°C for 1 hour. After washing again with PBST, anti-fluorescence quenching mounting medium was added for mounting. The plates containing DAPI were then imaged using a laser scanning confocal fluorescence microscope.

[0063] The Nissl staining procedure is as follows:

[0064] Ventricular sections were defatted with xylene, stained deep blue with Nissl solution, washed with double-distilled water, and then differentiated dropwise with Nissl Differentiation Buffer (MCE) for 4–8 seconds until most of the staining was removed. The sections were then dehydrated with anhydrous ethanol, defatted again with xylene, and finally mounted with neutral resin. The sections were dried in a fume hood for 2 hours, and then observed and images were collected under a microscope.

[0065] The procedure for thiamine S staining is as follows:

[0066] The sections were fixed with 95% ethanol, then covered with 1% thiamine S for 1 hour. After differentiation with 50% ethanol, images were collected under a fluorescence microscope.

[0067] 1.9 Western Blot

[0068] When detecting exosome marker proteins, the exosome suspension was mixed with SDS protein loading buffer for Western blot experiments to detect the expression of surface marker proteins, including anti-CD63 (1:1000), anti-TSG101 (1:1000), and anti-HSP70 (1:1000), with cell culture supernatant as a control.

[0069] To detect hippocampal protein expression in mice, three mice in each group were immediately sacrificed after cervical dislocation. Brain tissue was then dissected, and the hippocampal tissue was isolated and stored in liquid nitrogen. Western blot was performed using the method described in previous studies. Tissues were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors, and protein concentrations in each sample were determined using a BCA protein quantification kit. Protein loading buffer and protein samples were mixed in boiling water and heated for 10 minutes. After cooling, samples were added to a 12.5% ​​SDS-PAGE gel. After electrophoresis, membrane transfer was performed, and proteins were rapidly blocked. Primary antibodies were incubated overnight at 4°C. The major antibody dilution ratios were as follows: anti-Aβ... 1-42 Anti-Tau5 (1:1000), anti-p-Tau (1:1000), anti-BACE (1:500), anti-GFAP (1:1000), anti-synaptophysin-1 (1:1000), anti-BDNF (1:1000), anti-IL-1β (1:500), anti-IL-6 (1:1000), anti-TNF-α (1:1000), and anti-β-actin (1:1000). Cell membranes were then treated with the corresponding HRP-labeled secondary antibody according to the protein of interest. Protein levels were analyzed using ImageJ and normalized relative to the internal control β-actin.

[0070] 1.10 Quantitative Real-Time PCR (qPCR)

[0071] Quantitative real-time polymerase chain reaction (qPCR) was used to determine the levels of IL-1β mRNA, IL-6 mRNA, and TNF-α mRNA in all experimental groups. RNA was extracted from partial hippocampal tissue samples from three mice in each group. The extraction procedure was as follows: tissue was lysed using Trizol, chloroform was added, and the samples were incubated and centrifuged. The supernatant was thoroughly mixed with isopropanol and incubated overnight at -20°C. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in 75% ethanol. After centrifugation again, the supernatant was discarded, and the precipitate was resuspended in enzyme-free water for RNA quantification. After quantification, reverse transcription was performed using Evo M-MLV RT Premix, followed by real-time fluorescence quantification using cDNA, upstream primer, downstream primer, enzyme-free water, and SYBR Green. PCR was performed in a thermal cycler as follows: 95°C pre-denaturation for 3 minutes, followed by 40 cycles, each cycle consisting of 95°C denaturation for 10 seconds and 55°C annealing for 30 seconds.

[0072] The primer sequences for IL-1β-F are 5′-ctttgaagttgacggaccc-3′, IL-1β-R are 5′-tgagtgatactgcctgcctg-3′, IL-6-F are 5′-agtccggagaggagacttca-3′, IL-6-R are 5′-atttccacgatttcccagag-3′, TNF-α-F are 5′-caccaccatcaaggactc aa-3′, TNF-α-R are 5′-aggcaacctgaccactctcc-3′, β-actin-F are 5′-agtgtgacgttgacatccgt-3′, and β-actin-R are 5′-tgctaggagccagagcagta-3′. Experimental results were obtained using... The method is used to calculate and perform normalization analysis.

[0073] 1.11 Statistical Analysis

[0074] Data are expressed as mean ± standard deviation. Statistical significance was determined by t-test or Fisher's exact test between two groups and by one-way ANOVA between three groups. , , and It was considered statistically significant. Statistical analysis was performed using GraphPad Prism version 6.01.

[0075] 2. Experimental Results

[0076] 2.1 Generating NK from different origins of UCB

[0077] In order to study CD73 + Whether the differentiation capacity and function of NK cells are related to the origin of umbilical cord blood (UCB) mononuclear cells (MNCs) can be investigated by classifying MNCs into three categories of NK cells (CD34) from different origins. + (34) + CD3 － CD56 + (56) + ) and CD56 － (56) － ), and induce its differentiation to generate NK (34) + NK, 56 + NK and 56 － NK), and then further induce NK to differentiate into CD73. + NK cells, and comparisons ( Figure 1 (A) After MNCs were isolated from UCBs, cryopreserved UCB cells were stained with 7-aminoactinomycin D (7-AAD) live / dead dye and various hematologic markers before classification. The total UCB cell population, excluding debris, accounted for 73.9% (A). Figure 1 R1 in B), the proportion of single cells was 72.03% of the total cells ( Figure 1 (R2 in B). In single cells, live cells were gated using only 7-AAD dye. The live cell population comprised 56.65% of the total cells. Figure 1 (R3 population in B). Live cells were isolated into CD34. + CD34 was then isolated using CD34 antibody. + Cells and CD34 － Cells, including CD34 + Cells accounted for 0.2% of the total UCB ( Figure 1 (R4 group in B), while CD34 － Cells accounted for 56.43% of the total UCB ( Figure 1 (R5 population in B). Cells expressing CD45 versus CD34. － Cells have been isolated, representing 52.83% of the total UCB. Figure 1 (R6 in B). In CD34 － CD45 + Within the gating range, CD3 is further sorted out. － CD56 + The group, and the remaining CD56 + The cells have also been isolated. Sorted CD56 + Cells (CD56) － Cells accounted for 51.76% of the total UCB. Figure 1 (R7+9 group in B), while CD3－ CD56 + Cells accounted for 0.87% of the total UCB ( Figure 1 (R10 group in B). CD34 + The population consists of HSCs (UCB-HSCs), CD3 － CD56 + The population is composed of NK cells (UCB-NK cells), while CD56... － The population consists of the remaining non-NK cells (UCB-nNK cells) after sorting. These three sorted populations were used as the starting source of NK cells. Figure 1 (B in the text). Based on these three groups, 34 strains were induced and cultured respectively. + 56 + and 56 － Three types of NK cells exhibit the typical cell morphology of human natural killer cells. Figure 1 (C) . Chromosome copy number analysis confirmed that all NK lineages were normal diploids ( Figure 1 (D in the middle).

[0078] 2.2 Comparison of NK cells from different sources in umbilical cord blood

[0079] To investigate the three origins of MNCs (i.e., NK-34) + NK, 56 + NK and 56 － The potential differences in NK cell differentiation rates among MNCs (McNs) can be investigated using established protocols to differentiate MNCs into NK cells (34). + NK, 56 + NK and 56 － NK) Figure 2 (A) Two weeks after NK cell differentiation, all three NK cell types exhibited similar cell morphology and produced cell clones ( Figure 2 (B) in the text. 56 + NK's CD3 － CD56 + The expression rate increased faster than other cell lines, followed by 34. + NK and 56 － NK ( Figure 2 In the C, p < 0.05). However, all three types of CD73 + NK cells have similar purity, differentiating from their original source MNSc into CD73. + Two weeks after NK cells, CD3 － CD56 + The ratios all exceeded 90% ( Figure 2 (C) in the text. 34 + NK and 56 +CD56 in NK + CD16 + Their levels of expression were comparable, but at 56 － In NK cells, the expression levels were significantly lower at 1.5 and 2 weeks of differentiation. Figure 2 D in ). 34 + NK cells showed significantly better growth than the other two cell lines. Figure 2 E, p < 0.05. Further analysis was conducted on cell surface markers from three types of NK cell expression, including activating receptors (NKG2D, DNAM-1, NKp30, NKp44, and NKp46) and inhibitory receptors (KIR2DL1, KIR2DL2 / 3, and NKG2A), as well as cytolytic granules (perforin and granzyme B). Figure 2 The expression of all three types of NK was similar across all surface markers (F). Figure 2 (F in the middle).

[0080] In cytotoxicity assays using leukemia cell lines (K562) and ovarian cancer cell lines (A2780 cis and SKOV3), no significant differences were observed among the three NK cell types at each ratio of interaction with K562 and A2780 cis cells. However, 34 + NK cells against SKOV3 cells at a ratio of 5:1 (34) + NK: 28.2%, 56 + NK: 15.4%, 56 － NK: 8.3%) and 10:1 (34) + NK: 37.4%, 56 + NK: 25.6%, 56 － The cytotoxicity at the NK (17.2%) ratio was significantly higher than that at 56%. + NK and 56 － NK ( Figure 2 G in ).

[0081] In summary, the above data indicate that all three types of MNCs can differentiate into NK cells, and in the absence of feeder cells, they not only exhibited significant cell growth but also presented the phenotype of mature NK cells. Compared with NK cells from other originating MNCs, 34 + NK cells exhibited higher cell numbers and stronger cytotoxicity in the SKOV3 cell line. Therefore, 34 + NK cells can produce more mature and functional NK cells than those from other sources.

[0082] 2.3 CD73 differentiated from NK cells of different origins in umbilical cord blood + NK cell comparison

[0083] The study investigated the redifferentiation of three MNCs into CD73 cells two weeks after differentiation into NK cells. + NK cells (34) + -73-NK, 56 + -73-NK and 56 － -73-NK) 2.5 cell differentiation rate difference between cycles ( Figure 3 A in the text). 34 + -73-NK's ​​CD3 value increased within 2.5 weeks after differentiation. － CD56 + Expression significantly higher than 56 + -73-NK and 56 － -73-NK, but no significant difference was observed at week 3 ( Figure 3 B in In week 3, 34 + -73-NK CD56 + CD16 + Expression significantly lower than 56 + -73-NK and 56 － -73-NK ( Figure 3 C in At 3 weeks, 56 － -73-NK cells showed significantly higher growth than 34-NK cells. + -73-NK and 56 + -73-NK ( Figure 3 D in However, no differences were found in the expression of activating receptors, inhibitory receptors, and cytolytic granules. Figure 3 (E in the text). Next, we performed cytotoxicity assays to determine the CD73 content after differentiation for each cell line. + Do NK cells exhibit functional differences? When 56 － -73-NK and K562 (34) + -73-NK: 44.12%, 56 + -73-NK: 64.22%, 56 － -73-NK: 80.42%) or with A2780 cis (34) + -73-NK: 21.2%, 56 + -73-NK: 26.9%, 56 － When co-cultured with -73-NK (59.6%), its cytotoxicity was significantly higher than the other two cell lines; when co-cultured with SKOV3 at a 1:1 ratio (34... +-73-NK: 14.4%, 56 + -73-NK: 20.9%, 56 － At -73-NK: 36.8%), its cytotoxicity was also significantly higher than the other two cell lines ( Figure 3 F in Compared to the other two cell lines, 34 + -73-NK showed cytotoxicity against A2780 cis cells at a ratio of 5:1 (34... + -73-NK: 51.7%, 56 + -73-NK: 66.9%, 56 － -73-NK: 84.8%) and 10:1 (34) + -73-NK: 61.8%, 56 + -73-NK: 78.3%, 56 － The percentage of -73-NK was significantly reduced at 88.5% ( Figure 3 F in In summary, each CD73 + NK cell lines share similar phenotypes but differ in differentiation patterns. However, some CD73... + NK may have better functionality, such as starting source 56 － -73-NK. Therefore, the subsequent value is 56. － Research was conducted on -73-NK.

[0084] 2.4, CD73 + NK cells inhibited the activation of microglia in the hippocampus of STZ-injected mice.

[0085] Figure 4 The results showed that, compared with the control group mice, the AD group mice had a significantly increased number of IBA1-positive cells and protein expression in the hippocampus, while AD+CD73... + The NK group showed the opposite change, while the AD+NK group did not show this change.

[0086] 2.5, CD73 + NK cells inhibited the activation of astrocytes in the hippocampus of STZ-injected mice.

[0087] Figure 5 The results showed that, compared with the control group mice, the AD group mice had a significantly increased number of GFAP-positive cells and protein expression in the hippocampus, while AD+CD73... + The NK group showed the opposite change, while the AD+NK group did not show this change.

[0088] 2.6, CD73 + NK cells reduced neuroinflammation in the hippocampus of AD mice.

[0089] like Figure 6 As shown, the protein levels of inflammatory factors IL-1β, IL-6, and TNF-α in the hippocampus of model mice ( Figure 6 (A, B) and mRNA ( Figure 6 The expression levels of CD73 in mice were significantly higher than those in the control group. + This condition was reversed after NK cell therapy, while NK cell injections failed to achieve the same effect. Meanwhile, CD73... + NK reduced amyloid accumulation and Tau hyperphosphorylation in the hippocampus of STZ-injected AD mouse models. Compared with control mice, the AD group showed lower levels of BACE and Aβ in the hippocampus. 1-42 The expression levels of p-Tau (Ser396) were significantly increased. Figure 6 (D, E in the text). There was no significant difference between the AD group and the AD+NK group, but AD+CD73... + The expression of these proteins was significantly reduced in the NK group. Figure 6 (D, E in the text). IF experiment results showed that Aβ in the hippocampus of the model group mice... 1-42 The fluorescence intensity was significantly higher in AD mice than in the control group. Compared with the AD group, AD+CD73 + The fluorescence intensity of Aβ-positive and p-Tau-positive cells in the hippocampus was significantly reduced in the NK group, while no significant changes were observed in the AD+NK group. Figure 6 (F, G in the text).

[0090] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. CD73 + Use of NK cells for the preparation of a medicament for the treatment of Alzheimer's disease, characterized in that, The CD73 + NK cells derived from CD34 + cells of umbilical cord blood; The CD73 + The method for preparing the NK cells is: isolating umbilical cord blood mononuclear cells from CD34 + cells in umbilical cord blood, preparing an initial cell suspension, and then inoculating the initial cell suspension in complete RPMI 1640 medium and incubating at 37°C for more than 21 days to obtain; the RPMI 1640 medium contains 10% fetal bovine serum, 2mM L-glutamine, 25mM HEPES, 1mM sodium phosphate, 11 kinds of non-essential amino acids, 55nM 2-ME, 100 U / mL penicillin, 100 mg / mL streptomycin, 1mM Motolimod, 1mM Resiquimod, 1mM Vesatolimod, 1mM Pidotimod, 1mM Laquinimod, 1mM Tempol, 50ng / mL IL-2, 10ng / mL IL-12, 10ng / mL IL-15, 10ng / mL IL-18, 10ng / mL IL-21.

2. Use according to claim 1, characterized in that, The CD34 + The method for preparing the cells is as follows: dilute the cord blood with DPBS, add to Ficoll, collect the white blood cell layer by centrifugation, collect the precipitate by centrifugation, add red blood cell lysing solution to lyse the residual red blood cells, and then purify the CD34 + cells by flow cytometry using CD34-PE antibody.

3. Use according to claim 1, characterized in that, The medicament also includes a pharmaceutically acceptable adjuvant.

4. Use according to claim 3, characterized in that, The dosage form of the medicament includes a powder, a solution, a suspension, or an emulsion.

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