Preparation of CD73 positive NK cells of umbilical cord blood from different starting sources and application of CD73 positive NK cells in treatment of Alzheimer's disease

Abstract

The invention belongs to the technical field of medicines, and particularly relates to preparation of CD73 positive NK cells of umbilical cord blood from different starting sources and application of the CD73 positive NK cells to treatment of Alzheimer's disease. The CD73 + NK cells differentiated from different initial cell types (CD34 positive cells, CD3 negative / CD56 positive cells and CD56 negative cells) of umbilical cord blood are studied, it is found that all the initial cell types of umbilical cord blood sources can be differentiated into the CD73 + NK cells, 34 +-73-NK cells show a higher growth rate, 56-73-NK cells show higher cytotoxicity, and the CD73 + NK cells can be differentiated into the CD73 + NK cells. And the overall effect of the 56-73-NK cell is superior to that of the 34 < + >-73-NK cell. Further research shows that the CD73 + NK can inhibit activation of STZ injection mouse hippocampal microglia and astroglia, so that the neuroinflammation of the hippocampal is reduced, and a new solution is expected to be provided for treatment of AD.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to preparation of CD73 positive NK cells from different starting sources of umbilical cord blood and application of the same in treatment of Alzheimer's disease. BACKGROUND

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

[0003] Chronic inflammation can lead to gradual decline in physical function and increase the risk of frailty and dementia by mediating neurodegeneration and enhancing central nervous system inflammation. Previous studies have found that elevated levels of inflammatory factors in the blood of individuals with normal brain cognitive function are associated with an increased risk of AD, and that early AD patients have higher levels of inflammatory markers in both blood and cerebrospinal fluid. In addition, pro-inflammatory signals outside the central nervous system play an important role in the development and progression of AD. Oxidative stress is the earliest cellular feature 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 brain cognitive function. Lipid peroxides, isoprostanes and other cellular oxidative parameters are significantly elevated in the brain tissue, serum, plasma and urine of AD patients, and oxidative stress damage is closely related to the occurrence and transformation of AD. Therefore, inflammation and oxidative stress imbalance can accelerate the progression of neurodegenerative diseases (especially AD) by exacerbating the association between the pathogenesis and specific pathological features of AD. Since the pathogenesis of AD involves multiple factors, disease-modifying therapies targeting a single mechanism or pathway may not be as effective as therapies targeting multiple mechanisms. Therefore, it is of great significance to explore the progress of AD treatment from these two aspects.

[0004] Immune cell therapy (ICT) has emerged as a promising approach to treat cancer, as well as to combat pathogens and viruses. Clinical evidence has shown that immune cells can react to disease in a variety of ways within the host, which is a significant improvement over traditional cancer treatment methods such as surgery, chemotherapy, and radiotherapy. With advances in immunology, genetic engineering, gene editing, and synthetic biology, immune cell therapy has become 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 certain limitations, such as antigen escape, off-target effects, related toxicity, and high production costs. Influenced by these limiting factors, natural killer cells (NK cells) have recently emerged as a promising alternative.

[0005] NK cells are a key component of the innate immune system, which can eliminate various types of tumor cells and virus-infected cells through its 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, graft-versus-host disease), which suggests that NK cells can be a promising choice for ICT. NK cells have a variety of receptors on their surface, which can locate target cells by recognizing specific ligands on the surface of target cells. Inhibitory receptors of 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 molecules (MHC I) on the surface of target cells. Activating receptors are divided into several categories: NKp30, NKp44, and NKp46 belong to natural cytotoxicity receptors; NKG2D, NKG2C, and NKp80 belong to C-type lectin-like receptors; DNAM1 and CD2 belong to co-activating receptors, which can recognize ligands on the surface of target cells and trigger cytotoxicity to eliminate target cells. NK cells ultimately determine whether to initiate killing of target cells by balancing the signals between inhibitory receptors and activating receptors. In addition, NK cells express activating Fc gamma receptors, among which Fc gamma RIIIa (i.e., Fc gamma receptor IIIa, which mediates antibody-dependent cellular cytotoxicity) is particularly important, which is also known as CD16a. When antibodies bind to antigens on the surface of target cells, these activating Fc gamma receptors induce antibody-dependent cell-mediated cytotoxicity (ADCC), thereby promoting the destruction of target cells by NK cells. Among them, CD73 + NK cells are a unique subset of NK cells discovered in recent years, also defined as "regulatory NK cells", which play an important role in immune regulation and become a new type of anti-inflammatory treatment. Therefore, the role of CD73 +The preparation of NK cells and the exploration of the treatment of AD have important application prospects. SUMMARY

[0006] In order to overcome the deficiencies of the prior art, the present application differentiates umbilical cord mononuclear cells of different sources into CD73 + NK cells, and compared with the preparation differences, the NK cells are injected into STZ-induced sporadic AD mouse models through lateral ventricles or tail veins, so that the NK cells can inhibit the activation of hippocampal glial cells and reduce neuroinflammation, and thus the NK cells are expected to provide a new scheme for the clinical treatment of AD.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is: The present application provides CD73 + The NK cells are used in the preparation of a medicine for treating Alzheimer's disease.

[0008] The present application finds that CD73 + The NK cells can inhibit the proliferation and differentiation of pro-inflammatory cells, inhibit the production of antibodies, can induce the transformation of related cells into an anti-inflammatory phenotype, enhance the immune regulation ability, reduce the levels of inflammation-related cytokines such as NO, IL-1β and IL-6 through a paracrine mechanism, and thus reduce the inflammatory response. + The NK cells have strong resistance to oxidation in vivo, and CD73 + The NK cells can further regulate the response of cells to oxidative stress by overexpressing endonuclease 1 / oxidation-reduction factor 1 (APE1 / Ref-1): not only can the NK cells inhibit the production of reactive oxygen species (ROS) and regulate the intracellular oxidation-reduction state, but also can up-regulate the gene expression level of enzymes involved in the clearance of ROS in vivo.

[0009] Preferably, the CD73 + The NK cells are derived from CD34 + cells of umbilical cord blood (UCB). + The NK cells can produce more mature and functional NK cells than other sources of NK cells.

[0010] Preferably, the CD73 + The preparation method of the NK cells is as follows: umbilical cord mononuclear cells are separated from CD34 + cells of umbilical cord blood, an initial cell suspension is prepared, the initial cell suspension is inoculated in complete RPMI 1640 culture medium, and the initial cell suspension is incubated at 37℃ for more than 21 days to obtain the NK cells.

[0011] More preferably, the RPMI 1640 medium contains 10% (volume percentage) 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 Laquinimod, 1 mM Tempol, 50 ng / mL IL-2, 10 ng / mL IL-12, 10 ng / mL IL-15, 10 ng / mL IL-18, 10 ng / mL IL-21.

[0012] Preferably, the CD34 + The preparation method of the cells is as follows: after diluting the umbilical cord blood with DPBS, adding to Ficoll, collecting the white blood cell layer by centrifugation, collecting the precipitate by centrifugation and adding a red blood cell lysis solution to lyse the residual red blood cells, and then purifying the CD34 + cells by flow cytometry with CD34-PE antibody.

[0013] Preferably, the medicine further comprises a pharmaceutically acceptable excipient.

[0014] More preferably, the excipient comprises at least one of an excipient, a propellant, a solubilizer, a co-solvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a defoaming agent, a thickening agent, a clathrate agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, an antioxidant, an adsorbent, a filter aid, a release retardant.

[0015] Preferably, the dosage form of the medicine comprises tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents or suppositories.

[0016] Compared with the prior art, the beneficial effects of the present application are: The present application studies CD73 + NK cells differentiated from different starting cell types of umbilical cord blood (UCB), specifically including CD73 + cells (CD34 positive cells), CD3 － CD56 + (CD3 negative / CD56 positive cells) and CD56 － cells generated from CD34 +NK cells and their functions were analyzed. The results showed that all the above-mentioned starting cell types derived from umbilical cord blood could differentiate into CD73 + NK cells. Among them, 34 + -73-NK cells (CD34 + derived CD73 + NK cells) showed higher growth rate, 56 － -73-NK cells (CD56 － derived CD73 + NK cells) showed stronger cytotoxicity, and 56 － -73-NK cells were superior to 34 + -73-NK cells in overall effect. Further studies showed that CD73 + NK cells could inhibit the activation of hippocampal microglia and astrocytes in STZ-injected mice, thereby reducing hippocampal neuroinflammation. It can be seen that CD73 + NK cells provide a new solution for the preparation of clinical innovative cell drugs and the treatment of AD. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Flow chart and preparation effect diagram of CD73 + NK cells from different starting sources of umbilical cord blood; A: flow chart, showing the experimental process from the classification of umbilical cord blood mononuclear cells (MNCs) to the induction and differentiation of NK cells from different starting sources; B: flow cytometry analysis chart, showing the cell sorting process and the proportion of each cell population by staining and gating analysis of cell markers (such as CD34, CD45, CD56, etc.); C: microscope observation chart of cell morphology, showing the morphology of NK cells from different sources; D: chromosome analysis chart, showing the results of chromosome copy number detection, used to confirm the chromosome ploidy of cells.

[0018] Figure 2 Differentiation and function research of NK cells from different sources in umbilical cord blood; A: differentiation flow chart of “umbilical cord blood→umbilical cord blood mononuclear cells→34 + , 56 + , 56 － NK cells from different starting sources”; B: cell morphology chart, showing the morphology and clonal formation of 34 + , 56 + , 56 － three kinds of NK cells after 2 weeks of culture and differentiation; C, D, E: bar charts, C showing the CD3 － CD56 + , CD56 + CD16+ Cell proportion data, D is CD56 + CD16 + Cell proportion difference at each time point; E shows the number change of three kinds of NK cells at different differentiation weeks; F: flow cytometry analysis chart, showing the expression level of three kinds of NK cell surface activating receptors (NKG2D, etc.), inhibitory receptors (KIR2DL1, etc.) and cytolytic granules (perforin, etc.) in the form of overlapping peaks; G: line chart, the abscissa is the effector-target ratio (1:1, 5:1, 10:1), and the ordinate is the cytotoxicity (%), showing the killing ability of three kinds of NK cells to K562, A2780 cis, SKOV3 three cancer cell lines.

[0019] Figure 3 CD73 + Differentiation and function diagram of NK cells; A: cell morphology chart (light microscope chart), showing 3 kinds of CD73 + NK cells (34 + -73-NK, 56 + -73-NK and 56 － -73-NK) cell morphology and distribution after 3 weeks of culture differentiation; B, C, D: column chart, B presents the CD73 － CD56 + expression proportion of three kinds of cells at different culture differentiation times (1.5, 2, 2.5, 3 weeks); C shows the CD56 + CD16 + expression proportion difference at each time point; D compares the number change of three kinds of cells at different differentiation weeks; E: flow cytometry analysis chart, showing the expression level of three kinds of cell surface activating receptors (NKG2D, etc.), inhibitory receptors (KIR2DL1, etc.) and cytolytic granules (Perforin, etc.) in the form of overlapping peaks; F: line chart, the abscissa is the effector-target ratio (1:1, 5:1, 10:1), and the ordinate is the cytotoxicity (%), showing the killing ability of three kinds of cells to K562, A2780 cis, SKOV3 three cancer cell lines.

[0020] Figure 4 Typical chart and statistical analysis results of IBA1 expression in mouse hippocampus are shown, showing that CD73 + NK inhibits the proliferation and activation of hippocampal microglia cells in STZ injected mice; A: fluorescence detection of hippocampal microglia marker IBA1; B: quantification of IBA1 positive cell number; C: microglia cells in different states (left: resting state; right: activated state); D: the proportion of activated microglia cells to total microglia cells. Data are expressed as mean ± standard error, n=3 in each group , ).

[0021] Figure 5 Typical chart of GFAP expression in hippocampus of mice and statistical analysis results are shown. CD73 + NK suppressed the activation of hippocampal astrocytes in STZ-injected mice; A: fluorescence detection of hippocampal activated astrocyte marker GFAP; B: quantification of the number of GFAP positive cells; C: Western Blot photos of IBA1 and GFAP; D: quantification of IBA1 and GFAP protein expression levels. Data are expressed as mean ± standard error, n = 3 in each group , ).

[0022] Figure 6 Integrated chart of immune inflammation and pathological index detection results of AD model mice; A: WB chart showing protein bands of IL-1β, IL-6, TNF-α, β-actin (internal reference) in hippocampus of mice in different treatment groups (Control, AD, AD+CD73 + NK, AD+NK), for intuitive presentation of protein expression; B, C: column chart, B is used to quantify the expression level of IL-1β, IL-6, TNF-α proteins relative to β-actin in WB results; C presents the relative expression amount of these inflammatory factors mRNA; D: WB chart showing Aβ 1-42 , BACE, p-Tau (Ser396), Tau5, β-actin (internal reference) protein bands in hippocampus of mice in different groups, presenting the expression of related pathological proteins; E: column chart, quantifying and statistically analyzing the relative expression level of DAβ 1-42 , BACE, p-Tau (Ser396) and other proteins relative to β-actin, analyzing the effect of CD73 + NK on amyloid accumulation and Tau hyperphosphorylation; F: using immunofluorescence technology, Aβ in hippocampus is labeled by specific antibody, red fluorescence shows the distribution and expression of Aβ, DAPI (blue) stains the cell nucleus, and the "merged" chart presents the co-localization of the two, which can directly observe the morphological and positional differences of Aβ deposition in hippocampus of mice in different groups; G: fluorescence intensity of Aβ positive area in F is quantified and statistically analyzed, comparing the Aβ deposition level in hippocampus of mice in different groups (Control, AD, AD+CD73 + NK, AD+NK), to assist in judging the effect of CD73 + NK on Aβ accumulation in AD model mice. DETAILED DESCRIPTION

[0023] The specific embodiments of the present application are further described below. It is to be understood that the description of these embodiments is intended to help understand the present application and is not intended to limit the present application. In addition, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0025] The present application differentiates umbilical cord blood mononuclear cells from different starting sources into CD73 + NK cells (a kind of regulatory natural killer cells with enhanced cytotoxicity function, which highly express adenosine metabolic enzyme CD73), and compares CD73 + The differences of NK cell preparation schemes. At the same time, a sporadic AD mouse model is established by intracerebroventricular injection of streptozotocin (STZ), and after 6 weeks, CD73 + NK cells are given by intracerebroventricular injection or tail vein injection, respectively. The activation of glial cells, newborn neurons and Aβ 1-42 positive area in the hippocampal region is observed by immunofluorescence staining (IF), and it is found that CD73 + NK cells can inhibit the activation of hippocampal microglia and astrocytes in STZ-injected mice. Moreover, the protein expression levels of IBA1, GFAP, IL-1β, IL-6, TNF-α, BACE, Aβ 1-42 , p-Tau, Tau5, synaptotagmin-1 (Syt-1), synaptophysin-1 and brain-derived neurotrophic factor (BDNF) in hippocampal tissue are reduced. In addition, real-time quantitative PCR (qPCR) determination shows that the mRNA expression levels of IL-1β, IL-6 and TNF-α are reduced. These results show that CD73 + NK cells can reduce the neuroinflammation in the hippocampus of AD mice, and provide a solution for the treatment of AD by innovative cell drugs in the clinic.

[0026] In order to comprehensively and clearly present the technical solutions of the present application and its significant advantages, the present application will be described in detail below combined with specific embodiments.

[0027] 1. Experimental method 1.1, UCB cell sorting Freeze 25 mL of cord blood in a cryogenic tube at -80°C. Thaw the frozen cord blood in a water bath for 5 min and dilute with DPBS at a 1 : 1 volume ratio. Then, add 10 mL of the diluted blood to 3 mL of Ficoll and centrifuge at 900 x g for 30 min. Collect the white blood cell layer with a pipette and centrifuge the resulting sample at 700 x g for 10 min. Collect the pellet and add red blood cell lysis solution and incubate at room temperature for 10 min to lyse the remaining red blood cells. Stain the treated cells with 7-Aminoactinomycin D (7-AAD) viability stain and analyze by flow cytometry to distinguish between live and dead cells. For live cells, add CD34-PE, CD45-V500, CD3-FITC, and CD56-APC antibodies for staining. After staining, analyze by flow cytometry and classify the cells as CD34 + cells, CD3 － CD56 + cells, and CD56 － cells (i.e., the remaining cells after isolating CD56 + cells). Analyze all data using FlowJo 10.8.1 software.

[0028] 1.2, NK and CD73 + Culture of NK cells (1) Take the live cells (CD34 + cells, CD3 － CD56 + cells, and CD56 － cells) from the UCB cell sorting process that were lysed by red blood cells and confirmed by flow cytometry, and isolate the cord blood mononuclear cells using a mononuclear cell isolation solution (Gibco) at 800 g for 15 min to prepare an initial cell suspension.

[0029] (2) Prepare complete RPMI 1640 medium supplemented with 10% 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, and 100 mg / mL streptomycin.

[0030] (3) Add 1 mM Motolimod + 1 mM Resiquimod + 1 mM Vesatolimod + 1 mM Pidotimod + 1 mM Laquinimod + 1 mM Tempol to the above medium, along with 50 ng / mL IL-2 (interleukin-2), 10 ng / mL IL-12, 10 ng / mL IL-15, 10 ng / mL IL-18, and 10 ng / mL IL-21, and maintain the use of these reagents throughout the entire culture.

[0031] (4) The initial cell suspension was inoculated in complete RPMI 1640 medium at a cell density of 1 x 10 6 cells / mL and incubated at 37 °C for 21 days.

[0032] Among them, Motolimod (VTX-2337) is a selective and effective Toll-like receptor 8 (TLR8, a pattern recognition receptor, which promotes immune response after activation) agonist, EC 50 100 nM, more than 50 times more selective than TLR7. The following is the preparation method of its 2.75 mg / mL (6.00 mM) working solution (take 1 mL working solution as an example): take 50 μL of 55 mg / mL clear DMSO stock solution and add it to 400 μL of PEG300, mix well to make it clear; add 50 μL of Tween80 to the above system, mix well to make it clear; then continue to add 500 μL of ddH2O to make up to 1 mL. The working solution is prepared and used immediately.

[0033] Resiquimod (R-848, S28463) is an immune response modulator used as an effective TLR7 / TLR8 agonist and can induce upregulation of cytokines such as TNF-alpha, IL-6 and IFN-alpha. Resiquimod can reduce hepatitis C virus (HCV) infection. The following is the preparation method of its 3.15 mg / mL (10.02 mM) working solution (take 1 mL working solution as an example): take 50 μL of 63 mg / mL clear DMSO stock solution and add it to 400 μL of PEG300, mix well to make it clear; add 50 μL of Tween80 to the above system, mix well to make it clear; then continue to add 500 μL of ddH2O to make up to 1 mL. The working solution is prepared and used immediately.

[0034] Vesatolimod (GS-9620) is an oral Toll-Like Receptor-7 (TLR-7) agonist used as a potential treatment for HIV infection. The following is the preparation method of its 0.44 mg / mL (1.07 mM) working solution (take 1 mL working solution as an example): take 50 μL of 8.8 mg / mL clear DMSO stock solution and add it to 400 μL of PEG300, mix well to make it clear; add 50 μL of Tween80 to the above system, mix well to make it clear; then continue to add 500 μL of ddH2O to make up to 1 mL. The working solution is prepared and used immediately.

[0035] Pidotimod (Pidotomod) is a synthetic dipeptide molecule with biological immunological activity on both the adaptive immune response and the innate immune response. The solubility of this substance in DMSO is 49 mg / mL (200.59 mM). It should be noted that DMSO is hygroscopic, and after hygroscopicity, the solubility of Pidotimod will decrease, so it is recommended to use freshly opened DMSO.

[0036] Laquinimod (ABR-215062, LAQ) is an effective immunomodulator. The following is the preparation method of its 30.0 mg / mL (84.08 mM) working solution (take 1 mL of working solution as an example): take 300 μL of 100 mg / mL clear propylene glycol stock solution and add it to 50 μL of Tween 80, mix well to make it clear; then continue to add 650 μL of D5W to make up to 1 mL. The working solution is prepared and used immediately.

[0037] Tempol (4-Hydroxy-TEMPO) is a superoxide scavenger with neuroprotective, anti-inflammatory and analgesic effects. The following is the preparation method of its 30.0 mg / mL (174.18 mM) working solution: take 1 mL of working solution as an example, take 30 mg of the product and add it to 1 mL of normal saline (0.9% NaCl solution), mix well to make it clear, and prepare and use it immediately.

[0038] 1.3, Copy number variation (CNV) analysis NK or CD73 + CNV analysis of NK samples. Using BlueFuse Multi software version 4.5 to plot the total CNV of all chromosomes to visualize NK or CD73 + Ploidy of NK.

[0039] 1.4, Flow cytometry analysis The human cancer cell lines involved in this flow cytometry analysis include K562, A2780cis (ECACC) and SKOV3, all of which were donated by Shenzhen Second People's Hospital. The culture conditions of each cell line are as follows: K562 and A2780cis cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% doxycycline; SKOV3 cells were cultured in McCoys 5A medium containing 10% fetal bovine serum and 1% doxycycline. The medium was changed every two days and the cells were passaged once a week. It should be noted that for A2780cis cells, only 1 mM doxycycline was added to the medium on the day of passaging. All cell lines were placed in a culture environment of 37°C, 5% CO2.

[0040] When performing flow cytometry analysis, the staining and subsequent operation of the cells were as follows: For surface marker staining, the suspended cells were first collected into 1.5 mL centrifuge tubes, washed with 2% FACS buffer (PBS + 2% FBS), and after washing, the diluted antibody (Table 1) was added for staining at 4°C in the dark for 30 min. For intracellular staining, the cells were first fixed and permeabilized: 4% paraformaldehyde and 0.2% PBST (PBS + 0.2% Tween 20) were used at room temperature for 30 min. After 30 min, the cells were washed with 2% FACS buffer, and then the diluted antibody (Table 1) was added for staining, and finally washed again with 2% FACS buffer.

[0041] The cells treated by the above staining were analyzed using a CytoFLEX flow cytometer, and the data obtained were analyzed using FlowJo 10.6 software.

[0042] Table 1 Antibodies used for flow cytometry analysis 1.5 Cytotoxicity test CellTrace Far Red Cell Proliferation Kit (Invitrogen, Waltham, MA, USA, #C34572) / 7-AAD flow cytometry was used to detect the cytotoxicity of NK cells on tumor cells. The specific steps were as follows: NK or CD73 + NK cells were mixed with far-red stained target cells at different E:T ratios in V-bottom 96-well plates, and each E:T ratio sample was mixed in three independent wells, followed by incubation for 4 hours. After incubation, the incubated cells in each of the three independent wells of each sample were collected into 1.5 mL centrifuge tubes, and the cells were resuspended in FACS buffer (PBS + 2% FBS) containing 7-AAD. The percentage of target cell lysis was analyzed using a CytoFLEX flow cytometer and FlowJo software, and the percentage of specific lysis was calculated based on the fluorescence measurements of the fluorescent dye relative to the fluorescence of the cancer cells alone.

[0043] 1.6, Karyotyping NK or CD73 cells were analyzed by standard G-banding. + NK cells were analyzed by karyotyping.

[0044] 1.7, Experimental animals Thirty 4-week-old male C57BL / 6 mice were purchased from Guangdong Animal Center. The mice were raised in an environment with 12-hour light and dark alternation, with an ambient temperature of 20±2℃ and a humidity of 50±5%, and had free access to food and water. When the mice grew to 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 operation procedures were approved by the Experimental Animal Ethics Committee of the Second People's Hospital of Shenzhen and followed the "Guide for the Care and Use of Laboratory Animals" (NIH publication No. 85-23, revised in 1985) of the U.S. National Institutes of Health.

[0045] The animal model was established and the experimental design was as follows: All mice were anesthetized by intraperitoneal injection of 5% hydroxyl chloride (0.1 mL / 10g) and fixed on a brain stereotaxic instrument. After surface disinfection, the scalp was incised. The Bregma point was located, and the coordinates of the bilateral ventricles were drilled using an electric skull drill (coordinates: 1.0 mm left and right of the Bregma point, 0.5 mm posterior, and 2.5 mm deep). Then, the mice in the STZ group were injected with STZ (streptozotocin) into the lateral ventricle through an automatic sampler (injection dose: 0.3 mg / kg, injection speed: 0.5 μL / min, injection volume per side: 1 μL, STZ dissolved in ACSF for standby). After the injection was completed, the needle was left for 5 min, and then the needle was removed. The mouse scalp was sutured, and an appropriate amount of normal saline was injected into the abdominal cavity, and then the mouse was placed in an electric heating blanket until it woke up, and then placed in a cage.

[0046] After a recovery period of 1 month, all mice were divided into 4 groups, including: a normal control group, an AD group, an AD+NK group and an AD+CD73 + NK group, 6 mice per group. The right lateral ventricle of the mouse was cannulated using a stereotaxic instrument and a drug delivery catheter (62003, 62102, RWD). The normal control group was not treated, the mice in the AD group were injected with STZ into the lateral ventricle, the mice in the AD+NK group were injected with 20 μL of NK cells (10 6 NK cells dissolved in 100 μL PBS) through the tail vein every day, and the mice in the AD+CD73 + NK group were injected with 20 μL of CD73 + NK cells (10 6 CD73 +NK cells were lysed in 100 μL PBS). All mice received 5 days of dosing. Following dosing, a series of behavioral tests and related experiments were performed on all mice.

[0047] 1.8. Immunofluorescence Staining (IF) A subset of mice from each group were randomly selected for cardiac perfusion with PBS until no blood was exiting, then perfusion was switched to 4% paraformaldehyde, and perfusion was restarted after the whole body was in spasm for 3 minutes. The whole brain of each mouse was completely removed and fixed in 4% paraformaldehyde solution overnight at room temperature, then immersed in 30% sucrose solution for dehydration until the bottom. The whole brain was placed in a disposable embedding box, filled with OCT embedding medium, and frozen overnight at -80°C. A 30 μm thick section was prepared using a microtome, attached to a glass slide, and dried for 10 minutes in a fume hood after recovering to room temperature. The tissue section was labeled using an immunohistochemical pen, and blocked with goat serum for 30 minutes at room temperature. After removing the liquid, the section was incubated 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) overnight at 4°C in the dark. After washing with PBST, the section was incubated with goat anti-rabbit FITC or goat anti-rabbit Cy3 for 1 hour at 37°C. After washing with PBST again, anti-fluorescence quenching mounting medium was added dropwise for mounting. The plate containing DAPI was placed on a laser scanning confocal fluorescence microscope for imaging.

[0048] Nissl staining was performed as follows: The ventricular section was delipidated with xylene, then stained with Nissl solution to a deep blue color, washed with distilled water, and a suitable amount of Nissl Differentiation differentiation solution (MCE) was added dropwise for 4-8 seconds until most of the staining was removed. Then, the section was dehydrated with anhydrous ethanol, delipidated with xylene again, and finally mounted with neutral resin. The section was dried in a fume hood for 2 hours, and then observed under a microscope and images were collected.

[0049] Thiamine S staining was performed as follows: The section was fixed with 95% ethanol, then covered with 1% thiamine S for 1 hour, and the section was differentiated with 50% ethanol before collecting images under a fluorescence microscope.

[0050] 1.9. Western Blot For detection of 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.

[0051] For detection of mouse hippocampal protein expression, 3 mice per group were selected immediately after cervical dislocation and sacrificed. Brain tissues were dissected immediately and hippocampal tissues were isolated and stored in liquid nitrogen. Western blot method described in previous study was used to lyse the tissues with RIPA lysis buffer containing protease and phosphatase inhibitors and protein concentration in each sample was determined using BCA protein quantification kit. Protein loading buffer and protein samples were mixed and heated in boiling water for 10 minutes. After cooling, samples were loaded into 12.5% SDS-PAGE gel. After electrophoresis, membrane transfer was performed and proteins were quickly blocked. Primary antibodies were incubated overnight at 4°C with the following primary antibody dilution ratios: anti-Abeta 1-42 (1 : 1000), 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-1 beta (1 : 500), anti-IL-6 (1 : 1000), anti-TNF-alpha (1 : 1000), and anti-beta-actin (1 : 1000). The cell membrane was then treated with the corresponding HRP-labeled secondary antibody according to the protein of interest. Protein levels were analyzed using ImageJ and normalized to the internal reference beta-actin.

[0052] 1.10. Quantitative Real-Time PCR (qPCR) Quantitative Real-Time Polymerase Chain Reaction (qPCR) was used to determine the levels of IL-1 beta mRNA, IL-6 mRNA, and TNF-alpha mRNA in all experimental groups. Partial hippocampal tissues from 3 mice per group were used to extract RNA. The extraction steps were as follows: Trizol was used to lyse the tissues, chloroform was added, and the sample was incubated before centrifugation. The supernatant was mixed thoroughly with isopropanol and precipitated at -20°C overnight. After centrifugation, the supernatant was discarded and the precipitate was resuspended with 75% ethanol. After centrifugation again, the supernatant was discarded and the precipitate was resuspended with nuclease-free water and RNA quantification was performed. After quantification, reverse transcription was performed using Evo M-MLV RT Premix, and real-time fluorescence quantification was performed using cDNA, upstream primer, downstream primer, nuclease-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.

[0053] 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.

[0054] 1.11 Statistical Analysis 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.

[0055] 2. Experimental Results 2.1 Generating NK from different origins of UCB 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).

[0056] 2.2 Comparison of NK cells from different sources in umbilical cord blood 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 the initial 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 2The expression of all three types of NK was similar across all surface markers (F). Figure 2 (F in the text)

[0057] 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 3 G in ).

[0058] 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.

[0059] 2.3 CD73 differentiated from NK cells of different origins in umbilical cord blood + NK cell comparison 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 the third week, 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 4 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 5 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.

[0060] 2.4, CD73 + NK cells inhibited the activation of microglia in the hippocampus of STZ-injected mice. Figure 6 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.

[0061] 2.5, CD73 + NK cells inhibited the activation of astrocytes in the hippocampus of STZ-injected mice. Figure 6 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.

[0062] 2.6, CD73 + NK cells reduced neuroinflammation in the hippocampus of AD mice. 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. ​D, E). IF results showed that the Aβ positive and p-Tau positive fluorescence intensity in the hippocampus of model group mice was significantly higher than that of control group mice 1-42 The fluorescence intensity of AD+CD73 group was significantly higher than that of AD group, and the fluorescence intensity of AD+CD73+NK group was significantly lower than that of AD+CD73 group + The Aβ positive and p-Tau positive fluorescence intensity in the hippocampus of NK group was significantly lower than that of AD group, and there was no significant change in AD+NK group ​ F, G).

[0063] The above detailed description is made to the embodiments of the present application, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. CD73 + Application of NK cells in the preparation of drugs for treating Alzheimer's disease.

2. The application according to claim 1, characterized in that, The CD73 + NK cells are derived from CD34 cells in umbilical cord blood. + cell.

3. The application according to claim 2, characterized in that, 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.

4. The application according to claim 3, characterized in that, The RPMI 1640 medium contained 10% 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 MPidotimod, 1 mM 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.

5. The application according to claim 2, characterized in that, 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.

6. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, 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, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, antioxidants, adsorbents, filter aids, and release inhibitors.

8. The application according to claim 6, characterized in that, The dosage forms of the drug include tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories.

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