Mesenchymal stem cells, screening and identifying method of lipoprotein particles secreted by mesenchymal stem cells and application of mesenchymal stem cells

Abstract

The invention discloses mesenchymal stem cells, a screening and identifying method of lipoprotein particles secreted by the mesenchymal stem cells and application of the mesenchymal stem cells. The screening and identifying method of the mesenchymal stem cells comprises the steps of cell culture, genotype identification, multiplication capacity identification, pluripotency and self-renewal capacity identification, differentiation capacity identification, lipid droplet identification after high fat induction, APOE lipoprotein secretion capacity identification and the like. The screening and identifying method of the lipoprotein particles comprises the steps of high-fat-induced lipoprotein secretion, lipoprotein separation and purification, lipoprotein particle size identification, lipoprotein particle content identification and the like, and target cells and target lipoprotein particles can be rapidly and accurately screened through the systematic screening and identifying method. The screened target lipoprotein particles can improve the dyslipidemia of APOE4 / 4 genotype Alzheimer's disease. Compared with the prior art, the method not only can avoid the gene manipulation risk faced by APOE gene editing, but also can provide a new standard for identification of mesenchymal stem cells and lipoprotein particles secreted by the mesenchymal stem cells, and can screen wider and easier therapeutic cell sources for later application.

Description

Technical Field

[0001] The present invention belongs to the field of cell biotechnology, and in particular relates to a method for characterizing mesenchymal stem cells with different APOE genotypes and lipoprotein particles with different APOE phenotypes secreted by them. Background Art

[0002] Mesenchymal Stem Cells (MSCs) can be isolated from almost all tissues and organs. They have been widely studied and applied in the treatment of various diseases due to their low immunogenicity, good immune regulation, tissue repair and secretion of extracellular vesicles.

[0003] Mesenchymal stem cells can secrete different APOE phenotype lipoprotein particles. APOE lipoprotein particles are not only important carriers of cholesterol and triglyceride transport and metabolism in the peripheral circulatory system, but also the main carriers of cholesterol and fatty acid transport metabolism support and detoxification mechanism between neurons and glial cells in the central nervous system.

[0004] Apolipoprotein E (APOE) gene has three alleles, ε2, ε3 and ε4, which combine to form six major genotypes: APOEε2 / 2, ε2 / 3, ε2 / 4, ε3 / 3, ε3 / 4, and ε4 / 4. At the level of the overall population, the allele frequencies of APOEε2 / 2, ε2 / 3, ε2 / 4, ε3 / 3, ε3 / 4 and ε4 / 4 are 1, 22, 2, 58, 14 and 3%, respectively.

[0005] Compared with the APOEε3 / 3 (APOE3 / 3) allele type, the risk of late-onset Alzheimer's disease (LOAD) increases 3 to 4 times for carriers of one APOEε4 allele, and the risk increases 9 to 15 times for carriers of two APOEε4 (APOE4 / 4) alleles. The average age of AD development in APOEε4 gene carriers is 4 to 12 years earlier. The APOEε2 / 2 (APOEε2 / 2) gene has a protective effect against LOAD in the population.

[0006] The APOE4 / 4 alleles predispose to high cholesterol and increase the risk of cardiovascular disease. However, compared with APOE4 / 4 homozygotes, APOE3 allele carriers have an increased risk of nonalcoholic fatty liver disease and obesity, and APOE2 predisposes individuals to type III hyperlipoproteinemia. Therefore, APOE2, APOE3, and APOE4 may have protective or harmful effects in different diseases.

[0007] In the peripheral circulatory system, APOE lipoprotein particles are mainly synthesized in the liver and intestines, mediating the transport of lipids from the intestines to the liver and between the liver and peripheral cells of the body. Lipoprotein particles mediate the binding of lipoprotein particles to cell surface lipoprotein receptors through APOE, which plays an important role in the clearance of blood lipids. In plasma, most APOE is a component of a variety of lipoprotein particles, including very low-density lipoprotein particles, intermediate-density lipoprotein particles, chylomicrons, and high-density lipoprotein particles. APOE2 and APOE3 are prone to bind to cholesterol-rich high-density lipoprotein particles, while APOE4 tends to bind to triglyceride-rich very low-density lipoprotein particles and low-density lipoprotein particles.

[0008] In the central nervous system, APOE is mainly synthesized and secreted by astrocytes, and combines with cholesterol-rich high-density lipoprotein particles to form APOE lipoprotein particles, which are similar to plasma high-density lipoprotein particles and are disc-shaped. APOE lipoprotein particles serve as a metabolic regulatory link between astrocytes and neurons. On the one hand, APOE lipoprotein particles transport cholesterol, phospholipids, hydrophobic vitamins and antioxidants from astrocytes to neurons to support neuronal activity, remodeling and repair; on the other hand, overactivated neurons secrete excessive lipids and metabolic toxic products in the form of APOE lipoprotein particles, which are transported to astrocytes for decomposition and metabolism, so as not to accumulate and damage neurons, leading to neurodegeneration.

[0009] Compared with APOE4 lipoprotein particles, APOE3 lipoprotein particles have a high recycling rate and show a larger size and higher loading capacity, indicating that APOE3 lipoprotein particles may have higher transfer efficiency.

[0010] In cardiovascular diseases, APOE lipoprotein particles act as non-membrane-bound carriers to transport plasma miRNAs to receptor cells for post-transcriptional regulation of gene expression levels. In the nervous system, ApoE3 particles secreted by APOE3 astrocytes are more significantly enriched in miRNAs than ApoA1 particles; ApoE particles secreted by astrocytes are taken up by neurons through receptor-mediated endocytosis, inhibiting cholesterol synthase activity and reducing cholesterol biosynthesis in neurons, and the inhibitory effect of APOE3 particles is much greater than that of APOE4 particles. However, simply supplementing exogenous recombinant human ApoE3 protein did not change neuronal cholesterol biosynthesis, and AD symptoms were not significantly improved. This is because in addition to APOE3 protein, APOE3 particles also contain miRNA expression that is much higher than APOE4 particles.

[0011] APOE particles secreted by neurotoxic reactive astrocytes deliver very long-chain fatty acid acylphosphatidylcholine and saturated fatty acids to neurons, leading to ectopic deposition of lipids in neurons, release of inflammatory factors, and initiation of apoptotic signals, causing neuronal dysfunction and lipotoxic apoptosis. APOE particles derived from human cerebrospinal fluid are rich in specific proteins that play an important role in neuroinflammation, immune response, and neuron generation and development. This suggests that the differences in the regulation of neuronal cholesterol, fatty acid deposition and neurotrophic function by APOE particles of different sources and subtypes may be achieved through the miRNAs, lipids, and proteins they carry.

[0012] AD phenotypes such as lipid deposition, Aβ secretion, p-Tau deposition, and neuronal synaptic loss were observed in various types of brain cells and organoids derived from APOE4 / 4 human induced pluripotent stem cells, which can be reversed by converting APOE4 to APOE3 through gene editing or structural modification. APOE3 / 3 astrocytes secrete APOE3 lipoprotein particles, which can inhibit cholesterol synthase activity, reduce cholesterol biosynthesis in neurons, and ultimately delay cognitive impairment in humanized APOE4 / 4 mice.

[0013] The therapeutic effect of MSCs is closely related to its secretion of extracellular vesicles and a variety of functionally active nucleic acids and proteins. MSCs from different tissues and genotypes have certain differences, mainly reflected in the proliferation rate of MSCs, the spectrum of secreted cytokines and immunoregulatory ability. Studies have shown that MSCs derived from fetal tissues (including umbilical cord, umbilical cord blood, amniotic membrane, amniotic fluid, placenta and other tissues) have stronger proliferation ability than adult tissues (including adult bone marrow, fat, etc.), and compared with mesenchymal stem cells from other sources, umbilical cord and amniotic membrane tissues are rich and easy to obtain, and as medical waste, they avoid ethical disputes.

[0014] As one of the cell types that has received extensive attention in the field of cell therapy, MSCs have been widely recognized for their potential in treating diseases. They have no ethical and tumorigenic safety concerns, can be cryopreserved, and can be easily and promptly applied to patients. In 2006, the International Society for Cellular Therapy gave the minimum identification standards for MSCs: ① Mesenchymal stem cells grow in a fibroblast-like morphology (spindle and fusiform) under standard culture conditions, with uniform morphology. ② Mesenchymal stem cells must express CD105, CD73, and CD90, while CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR surface molecule expression is missing. ③ Mesenchymal stem cells must be able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro. In order to better understand and describe the characteristics and functions of the heterogeneous population of MSCs, and to ensure that therapeutic research is not interfered with by their APOE genotype, however, the existing standards cannot solve these problems. Therefore. We identified and characterized MSCs with different APOE genotypes, which will help to achieve improved and highly specific therapeutic effects of MSCs in the treatment of APOE4 / 4 genotype AD and cardiovascular disease, and APOE4 / 4 genotype non-alcoholic fatty liver disease.

[0015] At present, the international identification of APOE lipoprotein particles, including APOE lipoprotein content and electron microscopy observation, lacks the characterization of lipoprotein particles with different APOE phenotypes, especially the identification and classification of the contents of APOE lipoprotein particles with different phenotypes.

[0016] Therefore, how to determine a new screening and identification method for mesenchymal stem cells and their lipoprotein particles to solve the above technical problems has long troubled those skilled in the art. Summary of the invention

[0017] In response to the above technical problems, the present invention provides the identification, characterization and application of mesenchymal stem cells with different APOE genotypes and the lipoprotein particles secreted by them. This method can not only avoid the genetic manipulation risks faced by APOE gene editing, but also provide a new standard for the identification of mesenchymal stem cells and the lipoprotein particles secreted by them, so as to screen a wider and more accessible source of therapeutic cells for later applications.

[0018] The present invention solves the above problems by the following technical means:

[0019] A method for screening and identifying mesenchymal stem cells, characterized by comprising cell culture, genotype identification, proliferation ability identification, pluripotency and self-renewal ability identification, differentiation ability identification, lipid droplet identification after high-fat induction, APOE lipoprotein secretion ability identification after high-fat induction, and fatty acid metabolism ability identification after high-fat induction, wherein:

[0020] In the process of identifying the proliferation ability, MTT and / or CCK8 methods are used to detect cell viability and / or cell proliferation ability;

[0021] In the process of identifying the pluripotency and self-renewal ability, RT-qPCR and Western blot were used to detect the mRNA and protein expressions of mesenchymal stem cell transcription factors OCT4, NANOG, and SOX2;

[0022] In the differentiation ability identification process, RT-qPCR is used to detect the mRNA expression of mesenchymal stem cell differentiation markers SOX17, FOXA2, ectoderm markers SOX1, PAX6, NES and mesoderm marker TBXT;

[0023] In the process of identifying lipid droplets after high-fat induction, a high free fatty acid environment is used to induce mesenchymal stem cells to form lipid droplets, and the area and number of lipid droplets are detected by oil red staining and / or lipid droplet-specific antibody Plin2;

[0024] In the process of identifying the secretion capacity of APOE lipoprotein after high-fat induction, a high free fatty acid environment is used to induce mesenchymal stem cells to produce APOE lipoprotein, and the ELISA method is used to detect the total APOE lipoprotein content;

[0025] In the process of identifying fatty acid metabolism ability after high-fat induction, high performance liquid chromatography-tandem mass spectrometry is used to detect the levels of 16 acylcarnitines in the culture medium.

[0026] Preferably, the high-fat induction comprises the following steps:

[0027] Mesenchymal stem cells are induced for 24 to 96 hours using 250 μM to 500 μM sodium oleate-bovine serum albumin complex and / or 125 μM to 250 μM sodium palmitate-bovine serum albumin complex.

[0028] Preferably, the method further includes result sorting and screening, wherein the result sorting and screening includes the following steps:

[0029] The cells were ranked from high to low based on the cell proliferation rate after proliferation ability identification, the expression of cell transcription factors and proteins after pluripotency and self-renewal ability identification, the amount of intracellular, mesodermal and ectodermal markers after differentiation ability identification, the number and area of ​​intracellular lipid droplets after high-fat induction, the content of secreted APOE lipoprotein after high-fat induction, and the level of acylcarnitines in the culture medium after high-fat induction;

[0030] Specifically, the six dimensions are sorted separately, and based on the sorting results, the top 50% of cells in each dimension are screened, and the intersection of the top 50% of cells in each dimension is screened as the target cells. For example: When comparing four types of MSCs cells, APOE3 / 3 genotype MSCs cells are ranked in the top two in each ranking, so APOE3 / 3 genotype MSCs cells are selected as target cells for further induction and culture.

[0031] A method for screening and identifying lipoprotein particles, characterized in that it includes high-fat induction of lipoprotein secretion, lipoprotein separation and purification, and identification of lipoprotein particle size and lipoprotein particle content, wherein:

[0032] In the process of high-fat induced lipoprotein secretion, a high free fatty acid environment is used to induce mesenchymal stem cells with different APOE genotypes to secrete different APOE lipoprotein particles. After successful high fatty acid induction, lipid vacuoles can be seen in mesenchymal stem cells.

[0033] In the lipoprotein separation and purification process, APOE2, APOE3 or APOE4 antibody-labeled magnetic beads are used to separate and purify different APOE lipoprotein particles in the culture medium;

[0034] In the process of identifying the particle size and content of the lipoprotein particles, the ELISA method is used to detect the difference in particle content, the particle morphology is observed using a transmission electron microscope and the particle size is analyzed, and the miRNA expression profiles of different lipoprotein particles are compared.

[0035] Preferably, the identification of the lipoprotein particle size and the lipoprotein particle content comprises the following steps:

[0036] Using a transmission electron microscope to observe whether the APOE protein particles are aggregated and / or fused, and to detect whether the average particle size of the APOE protein particles is within a range of 8 nm to 20 nm;

[0037] The APOE protein content on the surface of non-aggregated, non-fused lipoprotein particles with an average particle size of 8nm to 20nm was ranked from high to low in terms of concentration and miRNA content;

[0038] For different applications, separate sorting is performed in two dimensions. Based on the sorting results, the top 50% of APOE lipoproteins are screened out in each dimension, and the intersection lipoproteins of the top 50% of APOE lipoproteins in each dimension are screened as target lipoproteins.

[0039] For example: in the ranking of four different APOE phenotype lipoprotein particles, APOE3 / 3 lipoprotein particles ranked first in average particle size and APOE protein content on the particle surface, showing a larger size and higher loading capacity. The APOE protein on the particle surface is rich and easily absorbed and utilized by cells, and the miRNA expression spectrum shows that miRNA target genes are mostly enriched in the lipid metabolism process. Therefore, APOE3 / 3 lipoprotein particles are the first choice for the treatment of APOE4 genotype AD and cardiovascular diseases. In view of the different identification characteristics of APOE4 / 4 lipoprotein particles and APOE3 / 3 lipoprotein particles, APOE4 / 4 lipoprotein particles are mostly used as inducers of APOE4 genotype AD and cardiovascular disease models or reference agents that reflect the dose-dependent effects of APOE.

[0040] A method for applying lipoprotein particles, characterized in that different APOE genotype mesenchymal stem cells are induced to secrete different APOE lipoprotein particles in a high fatty acid environment, and APOE3 / 3 and APOE4 / 4 lipoprotein particles are screened out therefrom, and applied to improve abnormal lipid metabolism in APOE4 / 4 genotype Alzheimer's disease.

[0041] Preferably, the APOE3 / 3 and APOE4 / 4 lipoprotein particles are applied to neurons of APOE4 / 4 genotype mice, wherein: the APOE3 / 3 and APOE4 / 4 lipoprotein particles downregulate the intracellular cholesterol content by inhibiting the expression of cholesterol synthase.

[0042] Preferably, the APOE3 / 3 and APOE4 / 4 lipoprotein particles are used in an APOE4 / 4 neuron-astrocyte co-culture system, wherein: the APOE3 / 3 and APOE4 / 4 lipoprotein particles promote the delivery of excess fatty acids in neurons to astrocytes.

[0043] Preferably, the APOE3 / 3 and APOE4 / 4 lipoprotein particles are used in an APOE4 / 4 neuron-astrocyte co-culture system, wherein: the APOE3 / 3 and APOE4 / 4 lipoprotein particles promote mitochondrial β-oxidation of fatty acids transferred into astrocytes.

[0044] Preferably, the APOE3 / 3 and APOE4 / 4 lipoprotein particles are used at a concentration of 5-10 ng / mL.

[0045] Preferably, APOE3 / 3 and APOE4 / 4 lipoprotein particles can inhibit the expression of cholesterol synthase in APOE4 / 4 genotype neurons, reduce cholesterol accumulation in neurons, and promote neuron-astrocyte lipid coupling, transfer excess fatty acids in neurons to astrocytes, and activate mitochondrial β-oxidation metabolism of fatty acids in astrocytes, thereby improving neuronal structure and function.

[0046] The screening and identification method of mesenchymal stem cells and their lipoprotein particles has the following beneficial effects:

[0047] Through the systematic screening and identification method, target cells and target lipoprotein particles can be quickly and accurately screened out. The screened target lipoprotein particles can improve the abnormal lipid metabolism of APOE4 / 4 genotype Alzheimer's disease. Compared with existing methods, this method can not only avoid the genetic manipulation risks faced by APOE gene editing, but also provide new standards for the identification of mesenchymal stem cells and their secreted lipoprotein particles, and screen a wider and more accessible source of therapeutic cells for later applications.

[0048] In the research and development process of mesenchymal stem cell products, because different researchers have different sources of mesenchymal stem cell tissues (such as bone marrow, adipose tissue, umbilical cord and placenta) and culture methods, even if they have the same morphology, cell surface markers and differentiation characteristics, their biological functional properties may still have significant differences. Therefore, it is necessary to formulate appropriate identification and characterization standards to ensure product quality. The identification standards established according to the mechanism of action of mesenchymal stem cells with different APOE genotypes and their secreted lipoprotein particles can also provide data for researchers and regulators to compare the efficacy of mesenchymal stem cells in different clinical trials. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0050] Figure 1 This is a schematic diagram of the characterization of MSCs with different APOE genotypes and the separation, purification and identification of the lipoprotein particles they secrete.

[0051] Figure 2 This is a schematic diagram of the structure of spherical APOE lipoprotein particles.

[0052] Figure 3It is the evaluation of the proliferation, multipotency, self-renewal and differentiation ability of MSCs with different APOE genotypes. A: Growth curve of proliferation kinetics of MSCs with different APOE genotypes; B: Western blot detection of OCT4, NANOG and SOX2 expression; C: Western blot analysis of gray value statistical histogram; D: RT-qPCR detection of OCT4, SOX2, NANOG mRNA expression; E: RT-qPCR detection of endoderm markers SOX17, FOXA2, neuroectoderm markers SOX1, PAX6, NES and mesoderm marker TBXT mRNA expression; (*P<0.05, **P<0.01, ****P<0.0001, compared with APOE3 / 3genetypeMSCs).

[0053] Figure 4 It is the characterization of MSCs with different APOE genotypes after high-fat induction. A: Microscopic observation of the formation of lipid droplets in MSCs with different APOE genotypes after high-fat induction; Scale bars, 200μm. B: Oil Red O staining to detect neutral lipid droplets in MSCs with different APOE genotypes after high-fat induction; Scale bars, 100μm; C: Quantification of the number of lipid droplets in MSCs stained with Oil Red O. The bar graph represents the average Oil Red O count per image for each individual (E2 / 3, n=4; E3 / 3, n=7; E3 / 4, n=4; E2 / 4, n=3). Each point represents the average Oil Red O count for 3×10 images per individual; D: Quantification of lipid droplet area in MSCs stained with Oil Red O; E: Elisa detection of APOE protein secretion in the culture medium of MSCs with different APOE genotypes after high-fat induction; F: LC-MS / MS measurement of acylcarnitine levels in the culture medium of MSCs with different APOE genotypes after high-fat induction. (*P<0.05, **P<0.01, ***P<0.0001, ****P<0.0001, compared with APOE3 / 3genetype MSCs).

[0054] Figure 5It is the characterization and identification of lipoprotein particles secreted by MSCs with different APOE genotypes after high-fat induction. A: Elisa detects the APOE content of APOE lipoprotein particles secreted by MSCs with different genotypes; B: Co-IP detects the expression of APOE protein in APOE particles after magnetic bead separation and purification; C: Transmission electron microscopy detects the morphology of APOE3 lipoprotein particles and APOE4 lipoprotein particles and performs particle size analysis. D: WB detection of APOE3 and APOE4 protein expression in APOE particles derived from conditioned medium (CM), E3 / hAMSCs and E4 / hAMSCs; E: Cluster heat map of miRNA expression in APOE2 / 3, APOE3 / 3 and APOE3 / 4 lipoprotein particles (Log2(E3 / 3 / E2 / 3)≥2, Log2(E3 / 3 / E3 / 4)≥2, p<0.05); F: Enrichment bubble map of kegg_disease, the target gene of differentially expressed miRNA in lipoprotein particles; G: Enrichment bubble map of kegg_pathway, the target gene of differentially expressed miRNA in lipoprotein particles.

[0055] Figure 6 The effect of APOE3 / 3 and APOE4 / 4 lipoprotein particles on cholesterol and fatty acid accumulation in primary neurons and astrocytes of APOE4 / 4 genotype mice. A: Determination of cholesterol content in neurons; B: Immunofluorescence co-staining of BODIPY-cholesterol and neuronal marker MAP2; C: Determination of free fatty acids in astrocytes; D: Immunofluorescence co-staining of BODIPY-C12 and astrocyte marker GFAP. (*P<0.05, **P<0.01, ****P<0.0001, compared with APOE3 / 3 lipoprotein particles.

[0056] Figure 7 The effect of APOE3 and APOE4 lipoprotein particles on the expression of cholesterol synthase in primary neurons of APOE4 / 4 mice. A: RT-qPC detected the mRNA expression of neuronal lipoprotein particle uptake receptors (LDLR, LRP1, VLDLR and Apoer2) and cholesterol synthase (HMGCS1, HMGCR, IDI1, FDFT1, LSS, CYP51, DHCR7 and DHCR24). B: Western blot detected the protein levels of neuronal synthases HMGCS1, HMGCR and CYP51; C: Western blot analysis of gray value statistical histogram. (*P<0.05, compared with APOE3 / 3 lipoprotein particles).

[0057] Figure 8APOE3 / 3 lipoprotein particles promote lipid coupling of APOE4 / 4 genotype neurons and astrocytes. AB: Fatty acid deposition in APOE4 / 4 neurons (A) and astrocytes (B) before co-culture (Scale bar: 20μm); C: Schematic diagram of neuron-astrocyte co-culture system; D: After co-culture, mitochondrial fission and fusion of APOE4 / 4 genotype astrocytes and mRNA expression of fatty acid β-oxidation-related proteins; E: Fatty acid deposition in APOE4 / 4 genotype neurons after co-culture; F: Immunofluorescence co-staining of mitochondrial markers Tomm 20 and BODIPY-C12 in APOE4 / 4 genotype astrocytes. DETAILED DESCRIPTION

[0058] In the description of the present invention, it should be understood that the terms "center", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. The terms "first" and "second" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0059] The following will be combined with the attached Figures 1 to 8 The present invention is described in detail.

[0060] Example 1) MSCs with different APOE genotypes were obtained using maternal umbilical cord and placental tissues.

[0061] Under the premise of passing ethical review and obtaining informed consent, maternal umbilical cord and placental tissues were collected for primary culture of MSCs. MSCs with good growth status at the 3rd to 5th generations were cultured at 5.0×10 4 cells / cm 2The cells were inoculated in a 6-well plate at a high density. When the cells covered about 80% of the bottom area of ​​the well plate, the cell DNA was extracted using a cell genomic DNA extraction kit. The extracted DNA was measured for DNA concentration and purity using an ultraviolet spectrophotometer. The A260 / 280 of the DNA was between 1.8 and 2.0. The DNA of the sample to be tested (10 ng / μL), the weak positive control, and the blank control were added to the reaction tubes containing the three APOE2, APOE3, and APOE4 PCR reaction solutions, respectively, for PCR amplification. The APOE genotype was determined based on the fluorescence signals collected by the FAM and VIC channels.

[0062] Example 2) Identification of differences in proliferation capacity of MSCs with different APOE genotypes.

[0063] The 3rd to 5th generation MSCs with different APOE genotypes were grown in good condition at 1.0×10 4 cells / cm 2 The cells were inoculated at a density in a 96-well plate, and the plate was placed in an incubator for 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. The culture medium was removed by suction, and 100 μL of culture medium (including 10 μL of CCK-8 solution) was added to each well. The cells were incubated in the incubator for another 2 h. The absorbance at 450 nm was measured with an enzyme-labeled instrument to calculate the cell proliferation rate = (A 96h -A 空白对照 ) / (A 24h -A 空白对照 ). The proliferation rates of APOE2 / 4, APOE3 / 4, APOE2 / 3, and APOE3 / 3 genotype MSCs were 2.52, 2.64, 2.79, and 3.05, respectively. The order of cell proliferation rate was APOE2 / 3-MSCs>APOE3 / 3-MSCs>APOE3 / 4-MSCs>APOE2 / 4-MSCs, indicating that APOE3 / 3-MSCs had the fastest proliferation rate and the strongest vitality, followed by APOE3 / 4 and APOE2 / 3-MSCs, and APOE2 / 4-MSCs had the slowest proliferation rate and the weakest vitality.

[0064] Example 3) Identification of the multipotency and self-renewal ability of MSCs with different APOE genotypes.

[0065] MSCs of different APOE genotypes at passages 3 to 5 were cultured at 1.0×10 6 cells / cm 2 Density inoculation 75cm 2In the culture flask, cells were collected, and RT-qPCR and Western blot were used to detect the mRNA and protein expressions of OCT4, NANOG, and SOX2 transcription factors that regulate MSCs multipotency and self-renewal; compared with APOE2 / 4-MSCs, APOE3 / 4-MSCs, and APOE2 / 3-MSCs, the mRNA expression of APOE3 / 3-MSCs increased significantly by 4 times, 3 times, and 1.2 times, respectively. The order of cell multipotency and self-renewal ability was APOE2 / 3-MSCs>APOE3 / 3-MSCs>APOE3 / 4-MSCs>APOE2 / 4-MSCs. Therefore, the multipotency and self-renewal ability of APOE3 / 3 and APOE2 / 3 genotype MSCs were stronger than those of APOE3 / 4 and APOE2 / 4 genotypes.

[0066] Example 4) Identification of the differentiation ability of MSCs with different APOE genotypes.

[0067] MSCs of different APOE genotypes at passages 3 to 5 were cultured at 1.0×10 6 cells / cm 2 Density inoculation 75cm 2 Cells were collected from the culture flasks, cell RNA was extracted by Trizol, and RT-qPCR was used to detect the mRNA expression of endoderm markers SOX17 and FOXA2, ectoderm markers SOX1, PAX6, NES, and mesoderm marker TBXT. Compared with APOE2 / 4-MSCs, APOE3 / 4-MSCs, and APOE2 / 3-MSCs, the mRNA expression of endodermal, ectoderm, and mesoderm markers in APOE3 / 3-MSCs increased significantly by 5 times, 3 times, and 1.5 times, respectively. Therefore, the differentiation ability of APOE3 / 3 and APOE2 / 3 genotype MSCs is stronger than that of APOE3 / 4 and APOE2 / 4 genotypes.

[0068] Example 5) After high fatty acid induction of MSCs with different APOE genotypes, the number and area of ​​lipid droplets in MSCs were evaluated.

[0069] As the organelles for storing neutral lipids, lipid droplets are the center of intracellular lipid metabolism and play a vital role in maintaining lipid homeostasis of MSCs. 5 cells / cm 2The cells were seeded in a 24-well plate at a high density. After culturing for 24 hours, the serum-free low-glucose DMEM medium containing 250 μM sodium oleate-bovine serum albumin complex was replaced and cultured for 72 hours. The medium was discarded, 4% paraformaldehyde was added to fix at 37°C for 10 minutes, rinsed twice with PBS, stained with 5 μg / mL Oil Red O solution at room temperature for 6 hours, rinsed with 75% isopropanol to remove excess dye, rinsed twice with PBS, and counterstained with hematoxylin for 5 minutes. The staining results were observed under a microscope, and the area and number of lipid droplets in different APOE MSCs were measured with FiJi software. The percentage (%) and number of lipid droplets in MSCs of different APOE genotypes were APOE2 / 4 (2.4, 213), APOE3 / 4 (3.3, 300), APOE2 / 3 (5.3, 349), and APOE3 / 3 (7.0, 411), respectively. The order of the area and number of lipid droplets in cells was APOE3 / 3-MSCs>APOE2 / 3-MSCs>APOE3 / 4-MSCs>APOE2 / 4-MSCs. This indicates that APOE3 / 3-MSCs have the largest area and number of lipid droplets and have the strongest tolerance after high fatty acid induction.

[0070] Example 6) After high fatty acid induction of MSCs with different APOE genotypes, the secretion of APOE protein by MSCs was identified.

[0071] After high fatty acid induction of MSCs, APOE protein was mainly secreted in the form of lipoprotein particles. The potential of MSCs to secrete lipoprotein particles was evaluated by measuring the APOE protein content of MSCs. 5 cells / cm 2 The cells were inoculated in a 24-well plate at a density of 1.5 × 10 cells / mL and cultured in an incubator for 12 h. The culture medium was replaced with serum-free low-glucose DMEM medium containing 250 μM sodium oleate-bovine serum albumin complex and cultured for another 72 h. The culture medium was collected and centrifuged at 3000 g for 15 min to remove cell debris. The culture supernatant was ultrafiltered quickly through a 10,000 Dalton molecular weight cutoff filter (UFC9010, Millipore), centrifuged at 4000 g for 30 min, and the concentrate was collected from the sample reservoir of the filtration device. The APOE protein content was determined according to the instructions of the human APOE enzyme-linked immunosorbent assay kit (Elabscience, E-EL-H0470). The APOE protein content secreted by MSCs of different APOE genotypes was APOE2 / 4 (124 ng / mL), APOE3 / 4 (152 ng / mL), APOE2 / 3 (246 ng / mL), and APOE3 / 3 (208 ng / mL). The APOE protein content in the culture medium of MSCs of different APOE genotypes was APOE2 / 3>APOE3 / 3>APOE2 / 4>APOE3 / 4, indicating that APOE2 / 3-MSCs secreted the most APOE protein and had the strongest ability to secrete APOE lipoprotein particles.

[0072] Example 7) After high-fat induction of MSCs with different APOE genotypes, LC-MS / MS was used to measure the level of acylcarnitines in the culture medium to evaluate the fatty acid metabolism capacity of MSCs with different APOE genotypes.

[0073] Carnitine acts as a carrier to transport long-chain fatty acids from outside the mitochondrial membrane to inside the membrane, promoting the β-oxidation of fatty acids and thus promoting fat metabolism. The fatty acid metabolism capacity of MSCs with different APOE genotypes is evaluated by detecting the level of acylcarnitine.

[0074] The above-mentioned concentrated culture medium of MSCs with different APOE genotypes was dropped onto a dry filter paper and dried naturally. The levels of free carnitine, acetyl, propionyl, butyrylcarnitine and other 16 acylcarnitines in the culture medium were detected according to the amino acid and carnitine detection kit method of BGI Biotechnology Co., Ltd. The main steps included extraction, derivatization, re-dissolution and on-machine detection, Waters triple quadrupole liquid chromatography-mass spectrometry detection system (ACQUITYUPLCI-CLASS / Xevo TQ-S micro IVD System), and the scanning mode was parent ion scanning. The contents of free carnitine C0 (31.3 μM) and acylcarnitine C2 (10.8 μM), C4 (0.226 μM), C6 (0.053 μM), C8 (0.04 μM), C10 (0.037 μM), C12 (0.037 μM), C14 (0.11 μM), C16 (0.537 μM), and C18 (0.14 μM) in the culture medium of APOE3 / 3-MSCs were significantly higher than those in the culture medium of APOE2 / 4 and APOE3 / 4-MSCs. Compared with APOE2 / 3 genotype MSCs, only the contents of C0, C2, C4, and C8 increased in the culture medium of APOE3 / 3-MSCs. The order of fatty acid oxidation metabolism ability of MSCs with different APOE genotypes is: APOE3 / 3>APOE2 / 3>APOE3 / 4, APOE2 / 4, indicating that APOE3 / 3-MSCs have the strongest fatty acid metabolism ability, followed by APOE2 / 3-MSCs, and APOE2 / 4-MSCs and APOE3 / 4-MSCs are relatively weak.

[0075] Example 8) Isolation and purification of APOE lipoprotein particles secreted by MSCs with different APOE genotypes.

[0076] hAMSCs of passages 3-5 were seeded in 150 cm 2 When the cells grew to about 80% of the bottom area of ​​the culture flask, replace the serum-free low-glucose DMEM medium containing 250 μM sodium oleate-bovine serum albumin complex and continue culturing for 72 h. The culture medium was collected and centrifuged at 3000 g for 15 min to remove cell debris. The culture supernatant was rapidly ultrafiltered through a 10,000 Dalton molecular weight cutoff filter (UFC9010, Millipore) and centrifuged at 4000 g for 30 min, and the concentrate was collected from the sample reservoir of the filtration device. TMM-280Tosylactivated, APOE particle separation: ① resuspend the washed magnetic beads in 100μg anti-APOE2 or APOE3 or APOE4 antibody, and incubate at 37℃ for 16h; ② place the test tube on a magnetic rack, remove the supernatant, add 0.5% BSA solution, incubate at 37℃ for 1h, and remove the supernatant; ③ add 1mL 0.1% BSA solution, vortex to mix, place on a magnetic rack to stand for 2min, remove the supernatant, repeat this step once, and resuspend the magnetic beads with 500μL 0.1% BSA; ④ add 1mL of the above concentrate to 50uL ligand-coupled magnetic beads, incubate at room temperature for 1h to capture the target protein; ⑤ place the test tube on a magnetic rack to stand, remove the supernatant, add 1mL 0.1% BSA solution, vortex to mix, and repeat this step 4 times; ⑥ use 3M NaSCN was shaken at room temperature for 10 min to elute APOE lipoprotein particles, and the supernatant containing APOE lipoprotein particles was dialyzed with PBS at 4°C overnight to collect APOE lipoprotein particles.

[0077] Example 9) Detect the APOE protein expression in the APOE particle-free conditioned medium (CM) and APOE lipoprotein particles obtained after magnetic bead separation using the above-mentioned immunoprecipitation method.

[0078] It was proved that MSCs secrete APOE protein mainly in the form of lipoprotein particles. APOE lipoprotein particles and conditioned medium BCA protein quantification, the sample amount was 20μg, SDS-PAGE gel electrophoresis, running at 100V for 1-2h, semi-dry transfer (constant current 1.3A) for 8min, blocking the membrane with 5% milk blocking buffer for 1h at room temperature, incubating with primary antibody (APOE2, APOE3 or APOE4 antibody, 1:1000) in blocking buffer at 4℃ overnight, washing the membrane 3 times with TBST for 5 minutes each time, incubating the membrane with conjugated secondary antibody in blocking buffer at room temperature for 1h, washing the membrane 3 times with TBST for 5 minutes each time, adding chemiluminescent agent, and exposing. It was found that APOE3 / 3 or APOE4 / 4 lipoprotein particles highly expressed APOE3 or APOE4 protein, respectively, but not expressed in conditioned medium.

[0079] Example 10) Identification of APOE protein content in lipoprotein particles of different APOE phenotypes.

[0080] Evaluation of the content of lipoprotein particles of different APOE phenotypes. APOE lipoprotein particles secreted by MSCs of different APOE genotypes were resuspended in PBS, and the APOE protein level was determined according to the instructions of the human APOE enzyme-linked immunosorbent assay kit (Elabscience, E-EL-H0470). The concentrations of APOE2 / 4 lipoprotein particles (148 ng / mL), APOE3 / 4 lipoprotein particles (206 ng / mL), APOE2 / 3 lipoprotein particles (394 ng / mL), and APOE3 / 3 lipoprotein particles (323 ng / mL) showed that the APOE protein content of lipoprotein particles of different APOE phenotypes was APOE2 / 3>APOE3 / 3>APOE2 / 4>APOE3 / 4, indicating that the concentration of APOE2 / 3 lipoprotein particles was the highest.

[0081] Example 11) Transmission electron microscopy was used to measure the particle size and morphology of lipoproteins of different APOE phenotypes.

[0082] APOE lipoprotein particles of different phenotypes were distinguished by their size. APOE lipoprotein particles were resuspended in PBS and the morphology of APOE particles was observed by negative staining under transmission electron microscopy. It was observed that APOE3 / 3 or APOE4 / 4 lipoprotein particles were spherical, isolated, and without aggregation and / or fusion. The results of particle size and concentration analysis showed that the concentration of APOE4 / 4 lipoprotein particles was 4.16×10 10 ±1.98×10 9 The average particle size was 13.3±2.1nm, and the APOE3 / 3 lipoprotein particle concentration was 4.06×10 10 ±2.16×10 9 The average particle size is 15.9±1.4nm.

[0083] Example 12) High-throughput sequencing was used to detect the expression of miRNA in APOE2 / 3, APOE3 / 3 and APOE3 / 4 lipoprotein particles.

[0084] By detecting the miRNA content and expression profile in APOE lipoprotein particles, APOE lipoprotein particles of different phenotypes were distinguished. The expression profiles of mature miRNAs in APOE2 / 3, APOE3 / 3, and APOE3 / 4 lipoprotein particles were analyzed. Compared with APOE2 / 3 lipoprotein particles, 73 miRNAs were increased and 64 were decreased in APOE3 / 3 lipoprotein particles; compared with APOE3 / 4 lipoprotein particles, 69 miRNAs were increased and 53 were decreased in APOE3 / 3 lipoprotein particles. The order of miRNA content in APOE lipoprotein particles of different phenotypes was: APOE3 / 3 lipoprotein particles>APOE2 / 3 lipoprotein particles>APOE3 / 4 lipoprotein particles. The top 48 differentially expressed miRNAs (Log2(E3 / 3 / E2 / 3)≥2, Log2(E3 / 3 / E3 / 4)≥2, p<0.05) were selected for miRNA target gene kegg_disease enrichment analysis. The top 10 diseases with the highest enrichment included periodic paralysis, mitochondrial fatty acid oxidation disorder, chronic granulomatous disease, avascular necrosis of the femoral head, Liddle syndrome, Beckwith-Wiedemann syndrome, short QT syndrome, bronchiectasis, adrenal cancer, and diabetic retinopathy. The kegg_pathway enrichment analysis of miRNA target genes showed that the top 10 pathways with the highest enrichment levels included cGMP-PKG, calcium ions, neuroactive ligand-receptor interactions, cell adhesion molecules, fluid shear stress and atherosclerosis, hypertrophic cardiomyopathy, chemical carcinogen-receptor activation, cAMP, fatty acid degradation, and lysosomal signaling pathways; it verified that miRNAs targeting mitochondrial fatty acid oxidation were highly expressed in APOE3 / 3 lipoprotein particles, and may have an effective effect on the treatment of diseases related to the nervous system and cardiovascular system.

[0085] Example 13) APOE3 / 3 or APOE4 / 4 lipoprotein particles were used to treat neurons of APOE4 / 4 genotype mice, and the intracellular cholesterol content was significantly reduced.

[0086] Primary APOE4 / 4 genotype neurons were cultured at 1.0×10 6 cells / cm 2 Density seeding was performed in 24-well plates. After 8-10 days of culture, APOE3 / 3 or APOE4 / 4 lipoprotein particles were added for 24 hours. Neuronal cells were collected and plated at a ratio of 1x10 60.1 ml of lysis buffer was added to each cell, the mixture was shaken and mixed, and the mixture was allowed to stand for 10 min. The mixture was centrifuged at 2000 g at room temperature for 5 min, and 10 μL of supernatant was transferred to a 96-well plate. 190 μL of enzyme working solution (Applygen Technologies, E1015) was added. The mixture was reacted at 37°C for 20 min. The absorbance was measured at 550 nm using a microplate reader, and the cholesterol content was corrected based on the concentration of each mg of protein. Compared with the blank control group (0.37 μM / mg protein) and the APOE4 / 4 lipoprotein particle group (0.35 μM / mg protein), the total cholesterol content in neurons of the APOE3 / 3 lipoprotein particle group (0.22 μM / mg protein) was significantly reduced. Further immunofluorescence co-staining of BODIPY-cholesterol (MCE, HY-125746) and the neuronal marker MAP2 revealed that the distribution of cholesterol in the perinuclear and axonal regions of neurons in the APOE3 / 3 lipoprotein particle group was also reduced. This indicates that the APOE3 / 3 lipoprotein particle group reduces the cholesterol content in nerve cells, and the effect is stronger than that of APOE4 / 4 lipoprotein particles.

[0087] Example 14) After APOE3 / 3 or APOE4 / 4 lipoprotein particles were used to treat astrocytes of APOE4 / 4 genotype mice, the intracellular fatty acid content was significantly reduced.

[0088] The third generation of APOE4 / 4 genotype astrocytes were cultured at 1.0×10 6 cells / cm 2 Density seeding 24-well plates, cultured for 1 day, added APOE3 / 3 lipoprotein particles or APOE4 / 4 lipoprotein particles for 24 hours, collected cells, added 0.1 ml of lysis buffer containing 5% isopropanol and 5% Triton X-100, oscillated and mixed, let stand for 10 minutes, centrifuged at 2000g for 5 minutes at room temperature, took 10 μL of supernatant and transferred it to a 96-well plate, and added 90 μL of fluorescence working solution (Sigma-Aldrich, MAK466), reacted at room temperature for 30 minutes, and the fluorescence intensity was measured at 570 nm by a microplate reader. Compared with the control group (4.12nM / mg protein) and the APOE4 / 4 lipoprotein particle group (3.59nM / mg protein), after the action of APOE3 / 3 lipoprotein particles, the intracellular fatty acid content (2.76nM / mg protein) was significantly reduced, and BODIPY-C12 and astrocyte GFAP immunofluorescence co-staining showed that the intracellular free fatty acid content was also significantly reduced. This indicates that the APOE3 / 3 lipoprotein particle group reduces the fatty acid level in astrocytes, and the effect is stronger than that of APOE4 / 4 lipoprotein particles.

[0089] Example 15) APOE3 / 3 or APOE4 / 4 lipoprotein particles were used to treat neurons of APOE4 / 4 genotype mice, and the neurons took up lipoprotein particles and expressed receptor mRNA.

[0090] Primary APOE4 / 4 genotype neurons were cultured at 1.0×10 6 cells / cm 2 The cells were inoculated at a high density in 24-well plates. After 8-10 days of culture, APOE3 / 3 or APOE4 / 4 lipoprotein particles were added for 24 hours, and the neurons were collected. The cell RNA was extracted by Trizol method, and the mRNA expression of LDLR, LRP1, VLDLR and Apoer2, which mediate the uptake of APOE lipoprotein particles, was detected by RT-qPCR. Compared with the control group and the APOE4 / 4 lipoprotein particle group, the mRNA expression of LDLR, LRP1, VLDLR and Apoer2 increased after the action of APOE3 / 3 lipoprotein particles, indicating that the potential of neurons of APOE4 / 4 genotype mice to take up APOE3 / 3 lipoprotein particles is stronger than that of APOE3 / 3 lipoprotein particles.

[0091] Example 16) APOE3 / 3 or APOE4 / 4 lipoprotein particles were used to treat neurons of APOE4 / 4 genotype mice, and the mRNA and protein expression of cholesterol synthase in neurons.

[0092] Primary APOE4 / 4 genotype neurons were cultured at 1.0×10 6 cells / cm 2 The cells were inoculated in 24-well plates at a high density. After 8-10 days of culture, APOE3 / 3 or APOE4 / 4 lipoprotein particles were added for 24 hours. The neurons were collected, and the cell RNA was extracted by Trizol method. The mRNA expression of intracellular cholesterol synthase HMGCR, HMGCS1, IDI1, FDFT1, LSS, CYP51, DHCR7 and DHCR24 was detected by RT-qPCR. Compared with the control group and the APOE4 / 4 lipoprotein particle group, the mRNA expression of HMGCR, HMGCS1, IDI1, FDFT1, LSS, CYP51, DHCR7 and DHCR24 was significantly reduced after the APOE3 / 3 lipoprotein particles were applied, and the protein expression of HMGCR, HMGCS1 and CYP51 was also significantly decreased. This indicates that APOE3 / 3 lipoprotein particles downregulate the intracellular cholesterol content by inhibiting the expression of cholesterol synthase.

[0093] Example 17) APOE3 / 3 lipoprotein particles promote APOE4 / 4 neuron-astrocyte lipid coupling.

[0094] Before neuron-astrocyte co-culture, the fatty acid dye BODIPY-C12 was used for co-staining with anti-MAP2 and anti-GFAP immunofluorescence, and it was found that fatty acid deposition could be observed in APOE4 / 4 neurons and astrocytes, and the fatty acid deposition in neurons was more than that in astrocytes.

[0095] A neuron-astrocyte co-culture system was established, and APOE3 / 3 lipoprotein particles were treated for 72 hours. By co-staining with BODIPY-C12 and anti-MAP2 immunofluorescence, it was observed that the fatty acids in APOE4 / 4 neurons were reduced, and the length, number of branches and area of ​​neuronal axons were significantly increased.

[0096] In the neuron-astrocyte co-culture system, BODIPY-C12 was used to track and label fatty acids in neurons to observe whether they were delivered to mitochondria after treatment with APOE3 / 3 lipoprotein particles. It was found that 6 hours after APOE3 / 3 lipoprotein particle intervention, BODIPY-C12-labeled fatty acids appeared in astrocyte mitochondria, indicating that APOE3 / 3 lipoprotein particles can promote the delivery of excess fatty acids in neurons to astrocytes.

[0097] Example 18) After APOE4 / 4 genotype neuron-astrocyte co-culture, APOE3 / 3 lipoprotein particles further promoted the expression of astrocyte mitochondrial β-oxidation-related protein mRNA.

[0098] After neuron-astrocyte co-culture, APOE3 / 3 lipoprotein particles were applied for 72 hours, and RT-qPCR detected the mRNA expression of mitochondrial fission and fusion-related proteins Drp1 and OPA1 and fatty acid β-oxidation-related proteins CPT1, CROT, MCAD, Echs1, Had1 and ACAA in astrocytes. It was found that APOE3 / 3 lipoprotein particles could upregulate the mRNA expression of mitochondrial fission and fusion-related proteins and fatty acid β-oxidation-related proteins, indicating that APOE3 / 3 lipoprotein particles can promote the mitochondrial β-oxidation of fatty acids transferred into astrocytes.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening and identifying mesenchymal stem cells, characterized in that: Including cell culture, genotype identification, proliferation ability identification, pluripotency and self-renewal ability identification, differentiation ability identification, lipid droplet identification after high-fat induction, APOE lipoprotein secretion ability identification after high-fat induction, fatty acid metabolism ability identification after high-fat induction, including: In the process of identifying the proliferation ability, MTT and / or CCK8 methods are used to detect cell viability and / or cell proliferation ability; In the process of identifying the pluripotency and self-renewal ability, RT-qPCR and Western blot were used to detect the mRNA and protein expressions of mesenchymal stem cell transcription factors OCT4, NANOG, and SOX2; In the differentiation ability identification process, RT-qPCR is used to detect the mRNA expression of mesenchymal stem cell differentiation markers SOX17, FOXA2, ectoderm markers SOX1, PAX6, NES and mesoderm marker TBXT; In the process of identifying lipid droplets after high-fat induction, a high free fatty acid environment is used to induce mesenchymal stem cells to form lipid droplets, and the area and number of lipid droplets are detected by oil red staining and / or lipid droplet-specific antibody Plin2; In the process of identifying the secretion capacity of APOE lipoprotein after high-fat induction, a high free fatty acid environment is used to induce mesenchymal stem cells to produce APOE lipoprotein, and the ELISA method is used to detect the total APOE lipoprotein content; In the process of identifying fatty acid metabolism ability after high-fat induction, high performance liquid chromatography-tandem mass spectrometry is used to detect the levels of 16 acylcarnitines in the culture medium.

2. The method for screening and identifying mesenchymal stem cells according to claim 1, characterized in that: The high-fat induction comprises the following steps: Mesenchymal stem cells are induced for 24 to 96 hours using 250 μM to 500 μM sodium oleate-bovine serum albumin complex and / or 125 μM to 250 μM sodium palmitate-bovine serum albumin complex.

3. The method for screening and identifying mesenchymal stem cells according to claim 1, characterized in that: It also includes result sorting and screening, which includes the following steps: The cells were ranked from high to low based on the cell proliferation rate after proliferation ability identification, the expression of cell transcription factors and proteins after pluripotency and self-renewal ability identification, the amount of intracellular, mesodermal and ectodermal markers after differentiation ability identification, the number and area of ​​intracellular lipid droplets after high-fat induction, the content of secreted APOE lipoprotein after high-fat induction, and the level of acylcarnitines in the culture medium after high-fat induction; The six dimensions were sorted separately, and based on the sorting results, the top 50% of cells in each dimension were screened out, and the intersection cells of the top 50% of cells in each dimension were screened as target cells.

4. A method for screening and identifying lipoprotein particles, characterized in that: It includes high-fat induced lipoprotein secretion, lipoprotein separation and purification, lipoprotein particle size and lipoprotein particle content identification, including: In the process of high-fat induced lipoprotein secretion, a high free fatty acid environment is used to induce mesenchymal stem cells with different APOE genotypes to secrete different APOE lipoprotein particles. After successful high fatty acid induction, lipid vacuoles can be seen in mesenchymal stem cells. In the lipoprotein separation and purification process, APOE2, APOE3 or APOE4 antibody-labeled magnetic beads are used to separate and purify different APOE lipoprotein particles in the culture medium; In the process of identifying the particle size and content of the lipoprotein particles, the ELISA method is used to detect the difference in particle content, the particle morphology is observed using a transmission electron microscope and the particle size is analyzed, and the miRNA expression profiles of different lipoprotein particles are compared.

5. A method for screening and identifying lipoprotein particles according to claim 4, characterized in that: The identification of the lipoprotein particle size and the lipoprotein particle content comprises the following steps: Using a transmission electron microscope to observe whether the APOE protein particles are aggregated and / or fused, and to detect whether the average particle size of the APOE protein particles is within a range of 8 nm to 20 nm; The APOE protein content on the surface of non-aggregated, non-fused lipoprotein particles with an average particle size of 8nm to 20nm was ranked from high to low in terms of concentration and miRNA content; For different applications, separate sorting is performed in two dimensions. Based on the sorting results, the top 50% of APOE lipoprotein particles are screened out in each dimension, and the intersection lipoprotein particles of the top 50% of APOE lipoprotein particles in each dimension are screened as target lipoproteins.

6. A method for applying lipoprotein particles, characterized in that: In a high fatty acid environment, mesenchymal stem cells with different APOE genotypes were induced to secrete different APOE lipoprotein particles, and APOE3 / 3 and APOE4 / 4 lipoprotein particles were screened out and applied to improve the abnormal lipid metabolism in APOE4 / 4 genotype Alzheimer's disease.

7. The method for using lipoprotein particles according to claim 6, characterized in that: The APOE3 / 3 and APOE4 / 4 lipoprotein particles are applied to neurons of APOE4 / 4 genotype mice, wherein: APOE3 / 3 and APOE4 / 4 lipoprotein particles downregulate intracellular cholesterol content by inhibiting the expression of cholesterol synthase.

8. The method for using lipoprotein particles according to claim 7, characterized in that: The APOE3 / 3 and APOE4 / 4 lipoprotein particles are used in an APOE4 / 4 neuron-astrocyte co-culture system, wherein: the APOE3 / 3 and APOE4 / 4 lipoprotein particles promote the delivery of excess fatty acids in neurons to astrocytes.

9. The method for using lipoprotein particles according to claim 7, characterized in that: The APOE3 / 3 and APOE4 / 4 lipoprotein particles are used in an APOE4 / 4 neuron-astrocyte co-culture system, wherein: the APOE3 / 3 and APOE4 / 4 lipoprotein particles promote the mitochondrial β-oxidation of fatty acids transferred into astrocytes.

10. The method for using lipoprotein particles according to claim 7, characterized in that: The APOE3 / 3 and APOE4 / 4 lipoprotein particles are used at a concentration of 5-10 ng / mL.

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