Method, system, composition and kit for diagnosis and differential diagnosis of alzheimer's disease based on human brain hippocampus spatial transcriptomics

Abstract

A method, system, composition and kit for diagnosis and differential diagnosis of Alzheimer's disease (AD) based on human brain hippocampus spatial transcriptomics. The present invention achieves rapid and efficient early diagnosis and differential diagnosis of AD cognitive disorder by means of one or more of CCK, Neurogranin and PMP2 carried in plasma extracellular vesicles (EVs), thereby achieving high-sensitivity and high-throughput detection of nervous system-derived EVs in peripheral blood, having the advantages of rapidness and low cost, and providing a new technical means and method for clinical application of AD cognitive disorder and large-scale screening-related accurate diagnosis work.

Description

Methods, systems, compositions and kits for diagnosing and differentially diagnosing Alzheimer's disease based on spatial transcriptomics of the human hippocampus Technical Field

[0001] The present invention relates to the technical field of precise diagnosis of Alzheimer's disease, and relates to methods, systems, compositions and kits for diagnosing and differentially diagnosing Alzheimer's disease based on spatial transcriptomics of the human hippocampus. Specifically, it relates to methods, systems, compositions and kits for diagnosing and differentially diagnosing Alzheimer's disease based on data and screening of spatial transcriptomics / single-cell sequencing of the human hippocampus, combined with exosome detection technology. Background Art

[0002] The technology for early diagnosis of AD is not yet mature, and there are relatively few related products. Due to the lack of specific and sensitive early diagnosis methods and standards in clinical practice, AD patients have already suffered a large amount of irreversible neuronal damage by the time they are diagnosed based on clinical symptoms and imaging indicators, and the disease has progressed to the middle and late stages, missing the best time for intervention. In addition, there is currently a lack of fast and efficient diagnostic methods to distinguish AD from non-AD cognitive impairment. Since the pathogenesis of AD has not yet been clarified in current research, there are no effective drugs for causal treatment of AD in clinical practice, and only symptomatic treatment can be used to alleviate patients' symptoms.

[0003] In clinical practice, imaging methods have certain value in the diagnosis of AD, but the cost of imaging examinations is extremely high. Only large general hospitals are equipped with relevant equipment. The examination process is complicated and time-consuming, and requires highly experienced imaging doctors to make the diagnosis, which is a huge economic burden for patients and society. Compared with the high cost and complex process of imaging examinations, AD biomarker testing has the advantages of low cost and short time. Cerebrospinal fluid (CSF)-related biomarkers have been found to have certain clinical value in assisting the diagnosis of AD. However, CSF collection requires high professional skills of medical staff, and patients have low acceptance of lumbar puncture, an invasive and risky operation. Therefore, CSF collection is extremely difficult in clinical diagnosis, especially in clinical screening.

[0004] As mentioned above, the early diagnosis and differential diagnosis of AD cognitive impairment have always been difficulties and research bottlenecks in clinical work and scientific research. The development of AD diagnostic biomarkers based on peripheral blood has the great advantages of being minimally invasive and easy to accept, simple to operate and easy to standardize, low in cost and easy to popularize, and has significant clinical practice and public health value.

[0005] Summary of the Invention

[0006] In order to overcome the deficiencies in the prior art, the present invention provides methods, systems, compositions and kits for diagnosing and differentially diagnosing Alzheimer's disease based on data screening developed by spatial transcriptomics / single-cell sequencing of the human hippocampus.

[0007] This application is mainly based on human brain spatial transcriptomics / single-cell sequencing data, combined with the exosome database, to screen candidate targets, and detect brain region-specific extracellular vesicles (EVs) of nervous system origin in peripheral blood, in order to achieve early diagnosis and differential diagnosis of AD. The present invention is expected to be applied to clinical practice and large-scale surveys due to its advantages of minimally invasive, easy to accept, low cost and easy to popularize. The key biomarkers in this application are differential markers discovered by human brain spatial transcriptomics. Because they have the characteristics of disease specificity and brain region specificity, they can more directly reflect AD-related central nervous system-specific pathological changes, thereby achieving the diagnosis and differential diagnosis of AD. This application combines all differentially expressed genes (A2M, ABCA2, ABI1, AC009879.3, AC091167.2, AC092143.1, AC093330.1, APOE, AC093512.2, ACADVL, ACAT2, ACTB, ACTG1, ACTR1A, ADD3, ADGRB3, ADGRG1, ADIRF, AEBP1, AHNAK, C9orf16, AHSA1, AL0498, etc.) in the spatial transcriptomics / single-cell sequencing data of the human hippocampus. 39.2, AL121594.1, AL365205.1, ANGPTL4, ANK2, AQP1, AP2M1, C3, AP2S1, APLNR, APOC1, ARL1, ARL6IP1, ARPC2, ARPC5L , CALM1, ATF4, CABP7, CADM2, ATP1A1, ATP1B1, BHLHE22, ATP2A2, ATP5F1A, ATP5F1B, CALM3, ATP5F1D, ATP5F1E, CADM3, ATP5MC1, ATP5MC2, ATP5MC3, ATP5MF, CACYBP, ATP5MG, ATP5MPL, CALM2, ATP5PB, ATP6AP2, ATP6V0E2, ATP6V1D, ATP6V1 E1, ATP6V1F, ATP6V1H, BEX4, ATP8A1, AUTS2, AUXG01000058.1, B2M, B4GAT1, BAIAP3, BCYRN1, BEX1, C1orf216, BEX2, B EX3, BRK1, C1QB, BSCL2, BTBD3, BTF3, CALY, CAMK1, CAMK2B, CAMK2N1, CAPS, CBR1, CCT5, CCT8, CD14, CD151, CD44, CD59 , CD63, CD74, CEBPD, CELF4, CELSR2, CHGB, CHI3L1, CFL1, CHCHD2, CELF2, CD81, CD9, CDK2AP1, CDKN1A, CIRBP, CLSTN1,CLSTN2, CNN3, CKB, COMT, CLDN5, CLU, CNBP, CLASP2, CLDN11, CNDP2, COPA, DLG2, COX4I1. COX6C, COX5A, COX5B, COX6A1, COX6B1, COX7A2, COX7C, CRYAB, CSMD1, CSMD3, CYCS, DBI, C SRP1, CST3, CTSB, CTSD, CTXND1, CX3CL1, DGKG, DHCR24, DHCR7, DKK3, DCLK1, DDIT4, DEGS 1.DNAJA1,DNAJA4,DNPH1,DSE,DTD1,DUSP1,DYNLL1,EEF1A1,EEF2,EEF1G,EFHD1,EID1E IF4A1, EIF4A2, EIF5, ELOB, ENC1, ENO1, ENO2, FBXO2, EPDR1, EPHA4, EPHA5, ERBIN, ERC2 ERH, FABP3, FABP5, FAM107A, FAM162A, FAM3C, FAU, FBXW5, FEZ1, FHL1, FIS1, FKBP1B, FKB P2, FKBP5, FOS, FSCN1, FTH1, FTL, FTX, GALNT15, FXYD6, GABARAP, GABARAPL1, GABARAPL2 GADD45B, GAPDH, GDF1, GDI1, GLS, GLUL, GNAI2, GNAO1, GNAS, GNB2, GOT1, GPM6B, GPRC5B. GRB2, GRIA1, GSN, GSTP1, GUK1, H2AFZ, H3F3B, HACD3, HINT1, HLA-B, HLA-DRB1, HMGCR, HMGCS1, HNRNPC, HNRNPDL, HNRNPK, HO PX, HS6ST3, HSP90AA1, HSP90AB1, HSPA4, HSPA8, HSPB1, HSPB8, HSPE1, HSPH1, HTRA1, ICAM5, ID4, IDI1, ISCU, ISG15, ITGAV. IDS, ITGB4, ITM2B, ITM2C, JUN, ITPKB, IGFBP5, IGFBP7, IRS2, JUNB, JUND, KCNIP4, KCNMB4, KIAA0408, KIF1A, KIF1B, KLF9, K LHL2, LARP6, LDHA, LDHB, LGALS1, MAPK8IP3, MIR7-3HG, MICAL2, MGST3, MAP4K4, LINC00844, MARCKSL1, MFSD6, MFSD4A, LMO4.<h2 style=";text-align:left;direction:ltr">LRP1B、LSAMP、MAL、MALAT1、MAOB、MGST1、MAP1LC3A、MDH2、MAP1LC3B、MAP2K 1、MDH1、MEG3、MLLT11、MMD、MRFAP1、MRPL41、MRPL51、MRPS21、MSMO1、MT1E、M T2A、MTATP6P1、MTCH1、MT-CO2、MT-CO3、MT-CYB、MTLN、MT-ND1、MT-ND2、MT- ND4、MT-ND6、MTPN、MT-RNR1、MT-RNR2、MYL12B、NDUFA11、MYL6、NDUFA13、NAA 60、NDUFA4、NACA、NAP1L5、NAPB、NCAM2、NCDN、NDRG1、NDRG3、NDRG4、NDUFA1 、NDUFA8、NDUFB8、NDUFAB1、NDUFS6、NDUFAF8、NDUFB4、NDUFV1、NEAT1、NECAB 1、NENF、NLRP1、NME1、NME1-NME2、NOP56、NORAD、NPC2、NPM1、NPTN、NPTXR、N RIP3、NRN1、NRXN1、NRXN3、NTRK2、NUCKS1、NUPR1、OAZ1、OLFM1、OXCT1、PREPL 、PAK3、PARK7、PCDH8、PCDH9、PCP4、PDHA1、PEBP1、PEX5L、PFKP、PGAM1、PGK1 、PGRMC1、PHC2、PHPT1、PI4KA、PIP4K2A、PITHD1、PKM、PLCB1、PLEC、PLEKHA2、 PLLP、PRDX5、POLR2I、PPIA、PRDX1、PPP2CA、PPP2R2B、PPP3CA、PPP3R1、PPT1 、PREX1、PSMA7、PRKAR1B、PRKCA、PRXL2A、PSMA4、PSAP、PSMB4、PSMB5、PSMB7、 PSMC3、PSMD1、PSMD8、PTMA、PTMS、PTP4A2、PTPRD、PTTG1IP、QDPR、QKI、RAB3 1、RABAC1、RACK1、RPL18A、RAMP1、RAN、RANGAP1、RASD1、RASGRP1、RBM3、RBX1 、RFK、、RGMA、RGS14、RPL18、RHBDD2、RNASE1、RNASET2、RPL10、RPL17-C18or f32、RPL10A、RPL11、RPL13、RPL13A、RPL15、RPL17-C18orf32、RPL19、RPL23、<h2 style=";text-align:left;direction:ltr">RPL21、RPL27A、RPL27、RPL28、RPL24、RPL26、RPL3、RPL30、RPL31、RPL34、RPL35、RPL35A、RPL36、RP L37A、RPL38、RPL4、RPL41、RPL5、RPL8、RPL9、RPLP0、RPLP1、RPS10、RPS11、RPS12、RPS13、RPS14、RPS 15、RPS15A、RPS16、RPS19、RPS2、RPS20、RPS21、RPS23、RPS27、RPS27A、RPS29、RPS3、RPS3A、RPS4X、 RPS6、RPS7、RPS8、RPSA、RTN3、RTN4、RYR2、S100A10、S100A6、SAP18、SAT1、SCAMP5、SCD、SCG3、SCGN、 SCOC、SCRG1、SDHA、SEC61B、SELENOP、SELENOW、SEM1、SEMA3B、SEMA5A、SERF2、SERINC1、SHTN1、SIK3、S KP1、SLC22A17、SLC24A3、SLC25A3、SLC25A4、SLC2A3、SLC38A2、SLC44A1、SLC7A11、SLIT1、SNRPN、SOD1 、SOD2、SORBS1、SPARC、SPARCL1、SPOCK1、SPP1、STXBP1、SUB1、SUMO2、SUN2、SUSD4、SYNE1、SYNM、SYS1- DBNDD2、SYT11、SYT13、TAGLN3、TALDO1、TBCA、TCEAL2、TCEAL3、TCEAL4、TCEAL5、TCF4、TCP1、TENM2、TF、 THY1, TIMP2, TIMP3, TKT, TM2D3, TM9SF2, TMA7, TMBIM1, TMEM130, TMEM35A, TMSB10, TMSB4X, TMTC1, TNRC6C, TOLLIP, TOMM34, TOMM7, TPI1, TPPP, TPT1, TRIM2, TRMT112, TSC22D1, TSPAN7, TSPYL4, TUBA1A, T UBA4A, TUBB2A, TXN, TXNIP, TXNL1, UBB, UBC, UBE2N, UBL5, UBQLN1, UCHL1, UQCR10, UQCR11, UQCRB, UQCRH, UQCRQ, USP11, USP22, VAMP2, VAPA, VDAC2, VIM, VSTM2L, WBP2, XRCC6, YJEFN3, YWHAB, YWHAG, YWHAHThe genes that changed significantly in the occurrence and development of AD were screened out: YWHAQ, YWHAZ, ZCCHC12, ZCCHC24, ZFAND5, ZFP36, ZFP36L1, ZFP36L2, ZMAT2) and exosome databases, and the following genes (AK5, ALDOC, AMER, APC2, APOD, ATP1A3, BAALC, BCAN, C1orf61, CABP1, CACNG3, CACNG8, CAMKV, CCK, CHN1, CNKSR2, CHN1, CNDP1, CNTNAP4, CREG2, CTXN1, DLGAP1, DNAJC6, ENHO, GABRA5, GAD1, GAP43, GFAP, GNG3, GPM6A, GRIK2, GRIN1, GRIN2A, GRIN2B, HEPACAM, HPCA, KCNJ10, KCNQ3, KCTD16, KIF5C, LAMP5, MAP2, MBP, MOBP, MTURN, MLC1, MT3, MTURN, NEFL, NEFM, NEUROD2, NKX6-2, OMG, NPTX1, NRGN, OLIG1, OPALIN, PAQR6, PDYN, PHYHIP, PIANP, PLANP, PLEKHB1, PLP1, PMP2, PMP22, PNMA2, POLR2F, PPKCG, PTPRZ1, RAB3A, RGS4, RTN1, S100B, SCN2B, SEZ6L, SLC1A2, SLC17A7, SLC1A3, SLC24A2, SLC2A1, SLC4A10, SLC6A7, SNAP25, SNCB, STMN4, STMN2, SULT4A1, SYN2, SYNPR, SYT1, TTC9B, TUBB2B, TUBB4A). This data is reliable and can further improve the reliability of diagnosis and differential diagnosis.

[0008] To achieve the above objectives, the present invention provides a composition for diagnosing and differentially diagnosing Alzheimer's disease developed based on spatial transcriptomics / single-cell sequencing of the human hippocampus, which is one or more of CCK, Neurogranin (NRGN) and PMP2 carried by plasma extracellular vesicles EVs.

[0009] Preferably, CCK, Neurogranin and PMP2 carried by the plasma extracellular vesicles EVs are enriched by PEG8000 sedimentation.

[0010] The present invention provides a detection kit, which includes a reagent Zenon labeled antibody TM Alexa Fluor TM647 Rabbit IgG Labeling Kit, lipid probes, and antibodies;

[0011] The lipid probe has a sequence of SEQ ID NO: 1: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, a 5' end modification: 5' 6-CY3, a 3' end modification: 3' Cholesteryl; the antibody comprises one or more of CCK, Neurogranin and PMP2 antibodies.

[0012] The present invention provides an application of CCK protein in differential diagnosis of Alzheimer's disease and non-AD dementia.

[0013] The present invention provides a method for detecting CCK, Neurogranin and PMP2 positive proteins, which comprises the following steps:

[0014] CCK, Neurogranin, and PMP2 antibodies were labeled with the Alexa Fluor fluorescent labeling kit. The labeled antibodies were mixed with the blocked exosomes and incubated at 4°C in the dark overnight. The lipid probe was then added and incubated at 4°C in the dark.

[0015] Add PFA to the labeled sample, mix well, and incubate at room temperature in the dark;

[0016] Dilute with PBS to an appropriate concentration and load onto the instrument. Use CytoFLEX or similar nanoflow cytometry technology to detect the particle count, with less than 10,000 particles per second.

[0017] When the lipid probe is positive using CytoFLEX or similar nanoflow cytometry technology in VSSC mode, the positive ratio of protein markers can be obtained.

[0018] Preferably, the method is used for identification purposes other than disease diagnosis and treatment.

[0019] The beneficial effects of the present invention are as follows:

[0020] The core detection technology used in this application is nanoflow cytometry, which enables highly sensitive and high-throughput detection of EVs from the central nervous system in peripheral blood, and has the advantages of being fast and low-cost, providing technical support for clinical applications and large-scale screening. This application focuses on the clinical and scientific challenges of early diagnosis and differential diagnosis of AD cognitive impairment. By leveraging internationally leading spatial transcription and single-cell sequencing and new nanoflow cytometry technologies, the resources of the Chinese brain, and innovative discovery of EV markers from brain regions and cell-specific sources in the central nervous system, this application achieves rapid and efficient early diagnosis and differential diagnosis of AD cognitive impairment, providing new technical means and methods for the clinical diagnosis of AD cognitive impairment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is an identification diagram of plasma EVs extracted in Example 1 of the present invention. Transmission electron microscopy detected that the EVs had a double-layer membrane structure and were within the particle size range of EVs;

[0022] Figure 2 shows the particle size distribution and concentration analysis results of EVs enriched by PEG8000 sedimentation in Example 1 of the present invention, where the abscissa represents the particle size distribution and the ordinate represents the EVs concentration;

[0023] Figure 3 shows the specificity and stability test results of nanoflow cytometry of plasma EVs extracted in Example 1 of the present invention. A shows that the percentage of EVs containing CCK, Neurogranin, and PMP2 in peripheral plasma is significantly higher than that in the isotype IgG control group; BC shows that the labeling percentages of EVs remain stable under different dilutions (B) and incubation times (C) of CCK, Neurogranin, and PMP2 antibodies, respectively.

[0024] FIG4 is a screening of the differential diagnostic ability of some potential biomarkers according to the present invention, wherein AB is the statistical analysis and ROC results of the CCK protein detection results, CD is the statistical analysis and ROC results of the Neurogranin protein detection results, EF is the statistical analysis and ROC results of the PMP2 protein detection results, GH is the statistical analysis and ROC results of the CREG2 protein detection results, IJ is the statistical analysis and ROC results of the CPLX2 protein detection results, KL is the statistical analysis and ROC results of the NEFM protein detection results, MN is the statistical analysis and ROC results of the STMN4 protein detection results, and OP is the statistical analysis and ROC results of the STMN protein detection results;

[0025] Figure 5 is an analysis diagram of the verification cohort results of Example 3 of the present invention, wherein Figure 5A shows that compared with the NC group, the ratio of CCK protein-positive EVs to the total number of EVs in the plasma of the AD group was significantly reduced (****, p<0.000), and compared with the NAD group, the ratio of CCK protein-positive EVs to the total number of EVs in the plasma of the AD group was also significantly reduced (***, p<0.001); compared with the NC group, the ratio of CCK protein-positive EVs to the total number of EVs in the plasma of the NAD group was also significantly reduced (**, p<0.01), and in Figure 5B, compared with the NC group, the ratio of Neurogranin protein-positive EVs to the total number of EVs in the plasma of the AD group was significantly reduced (****, p<0.0001), and compared with the NC group, the ratio of Neurogranin protein-positive EVs to the total number of EVs in the plasma of the NAD group was The ratio of nin protein-positive EVs to the total number of EVs was also significantly decreased (***, p<0.001). In Figure 5C, compared with the NC group, the ratio of PMP2 protein-positive EVs to the total number of EVs in the plasma of the AD group was significantly decreased (****, p<0.0001). Compared with the NC group, the ratio of PMP2 protein-positive EVs to the total number of EVs in the plasma of the NAD group was also significantly decreased (**, p<0.01). In Figure 5D, the CCK, Neurogranin and PMP2 data were included in the Enter method using logistic regression analysis, and the AUC was 0.92 (NCVSAD). In Figure 5E, the CCK, Neurogranin and PMP2 data were included in the Enter method using logistic regression analysis, and the AUC was 0.83 (ADVSNAD). DETAILED DESCRIPTION

[0026] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present application will be further described below in conjunction with specific embodiments.

[0027] Example 1

[0028] EVs enrichment by PEG8000 sedimentation

[0029] 1. Plasma from the subject was rapidly thawed at 37°C (within 2 minutes) and vortexed to mix;

[0030] 2. Centrifuge plasma samples (>300 μL) at 2,000 × g for 15 min at 4°C and collect the supernatant.

[0031] 3. Centrifuge at 12,000 × g for 30 min at 4°C and collect the supernatant.

[0032] 4. Pipette 10 μL of centrifuged plasma into a 1.5 mL centrifuge tube, add 70 μL of PBS and 20 μL of 40% PEG8000, mix thoroughly by pipetting, and let stand at room temperature for 30 minutes;

[0033] 5. Centrifuge at 12000g, 4°C, for 20 min.

[0034] 6. Discard the supernatant, add 100 μL PBS to resuspend, mix thoroughly by pipetting, aliquot into 10 μL / tube, and store at -80℃ until use.

[0035] Example 2

[0036] 1. Sample sealing

[0037] (1) Transfer 10 μL of EVs enriched by PEG8000 sedimentation using the above method (Example 1) to a 0.6 mL centrifuge tube;

[0038] (2) Add 10 μL of 2% BSA solution (filtered through a 0.22 μm membrane) and mix thoroughly;

[0039] (3) Block at room temperature (25-26°C) for 1 hour to remove nonspecific binding;

[0040] (4) Add 10 μL of PBS (filtered with a 0.22 μm membrane) to dilute and terminate blocking.

[0041] 2. Sample labeling

[0042] The antibodies used to detect biomarkers were labeled using a labeling method. Reagents used: CCK, Neurogranin, and PMP2 antibodies were labeled using the Alexa Fluro fluorescent labeling kit. The antibody labeling reagent was Zenon TM Alexa Fluor TM 647 Rabbit IgG Labeling Kit was purchased from Thermo Fisher Scientific. The lipid-labeled probe had the sequence SEQ ID NO: 1: TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, 5'-end modification: 5`6-CY3, 3'-end modification: 3`Cholesteryl.

[0043] (1) Take 1 μg of the antibody of the corresponding marker and dilute it to 5 μL (0.2 μg / μL) with PBS (filtered with a 0.22 μm membrane);

[0044] (2) Add 5 μL Zenon TM Alexa Fluor TM Mix 647 Rabbit IgG Labeling Kit A and incubate at room temperature (25-26°C) in the dark for 20 minutes.

[0045] (3) Add 3 μL (3 μg) of Zenon to the solution in step (2).TM Alexa Fluor TM 647 Rabbit IgG Labeling Kit Solution B (Solution B diluted to 1 μg / μL), mix well, and incubate at room temperature (25°C) in the dark for 10 minutes to quench unbound free fluorescein;

[0046] (4) Add PBS (filtered with a 0.22 μm membrane) and dilute to a total volume of 50 μL;

[0047] (5) Take 3 μL Zenon TM Alexa Fluor TM Add the antibody labeled with the 647 Rabbit IgG Labeling Kit to the sample blocked in step 1, mix well, and incubate overnight at 4°C in the dark.

[0048] 3. Probe incubation reagents: Synthetic DNA anchor with the sequence TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 1); 5'-end modification: 5'-6-CY3; 3'-end modification: 3'-Cholesteryl. This lipid probe binds to the EV membrane, and particles positively labeled by the probe are considered authentic EVs. This method further improves the scientific and accurate labeling of EVs.

[0049] (1) The next day, add 62 μL PBS + 5 μL lipid probe (lipid probe working solution concentration 10 μM) (reaction volume 100 μL, probe final concentration 500 nM) and incubate at 4°C in the dark for 1 h.

[0050] (2) Add 100 μL of 4% PFA (filtered through a 0.22 μm membrane) to the labeled sample, mix well, and incubate at room temperature (25-26°C) in the dark for 20 min;

[0051] (3) Dilute with PBS to an appropriate concentration for loading onto the instrument. Standard: Detection by CytoFLEX with a particle count of less than 10,000 particles per second. Collect 50,000 particles under the lipid probe gate (the total number of particles in this case is approximately 100,000; in other words, the particles obtained in Example 1 were screened using the lipid probe, of which approximately 50% were genuine EVs).

[0052] 4. Detection

[0053] The VSSC mode of CytoFLEX was used to detect the positive ratio of related protein markers when the lipid probe was positive.

[0054] Example 3

[0055] Screening of the differential diagnostic capacity of some of the above biomarkers in a small cohort

[0056] Plasma samples were obtained from 15 age- and sex-matched healthy controls (NC group), 15 Alzheimer's disease subjects (AD group), and 15 non-AD-dementia subjects (NAD group), and experiments were performed using the methods of Examples 1 and 2 above.

[0057] The VSSC mode of CytoFLEX was used to detect the number of EVs labeled with CCK, Neurogranin, PMP2, CREG2, CPLX2, NEFM, STMN4, and STMN2, respectively, and their proportion to the number of EVs positively labeled with lipid probes was calculated to test its ability to distinguish NC, AD, and NAD. The results are shown in Figure 4.

[0058] Compared with the NC group, the proportion of CCK protein-positive EVs in the plasma of the AD group was significantly reduced (****, p < 0.0001). Compared with the NC group, the proportion of CCK protein-positive EVs in the plasma of the NAD group was significantly reduced (****, p < 0.0001) (4A), ROC = 0.86 (4B);

[0059] Compared with the NC group, the proportion of Neurogranin protein-positive EVs in the AD group to the total number of EVs in the plasma was significantly decreased (****, p < 0.0001). Compared with the NC group, the proportion of Neurogranin protein-positive EVs in the NAD group to the total number of EVs in the plasma was significantly decreased (**, p < 0.01) (4C), ROC = 0.84 (4D);

[0060] Compared with the NC group, the proportion of PMP2 protein-positive EVs in the plasma of the AD group was significantly decreased (***, p < 0.001). Compared with the NC group, the proportion of PMP2 protein-positive EVs in the plasma of the NAD group was significantly decreased (**, p < 0.01) (4E), ROC = 0.80 (4F);

[0061] Compared with the NC group, the proportion of CREG-2 protein-positive EVs in the total number of EVs in the AD group was significantly decreased (**, p < 0.01) (4G), ROC = 0.76 (4H);

[0062] Compared with the NC group, the proportion of CPLX2 protein-positive EVs in the plasma of the AD group was significantly decreased (****, p < 0.0001). Compared with the NC group, the proportion of CPLX2 protein-positive EVs in the plasma of the NAD group was significantly decreased (****, p < 0.0001) (4I), ROC = 0.79 (4J);

[0063] Compared with the NC group, there was no significant difference in the proportion of NEFM protein-positive EVs to the total number of EVs in the AD and NAD groups (4K), ROC = 0.79 (4L);

[0064] Compared with the NC group, there was no significant difference in the proportion of STMN4 protein-positive EVs to the total number of EVs in the AD and NAD groups (4M), ROC = 0.69 (4N);

[0065] Compared with the NC group, there was no significant difference in the proportion of STMN2 protein-positive EVs to the total number of EVs in the AD and NAD groups (4O), ROC = 0.64 (4P).

[0066] Example 4

[0067] The differential diagnostic ability of the above biomarkers was tested in a clinical standardized cohort

[0068] Plasma samples were obtained from 66 age- and sex-matched healthy controls (NC group), 45 Alzheimer's disease subjects (AD group), and 45 non-AD-dementia subjects (NAD group), and experiments were performed using the methods of Examples 1 and 2 above.

[0069] The VSSC mode of CytoFLEX was used to detect the number of EVs labeled with CCK, Neurogranin, and PMP2, and their proportion to the number of EVs positively labeled with lipid probes was calculated to test its ability to distinguish NC, AD, and NAD. The results are shown in Figure 5.

[0070] Compared with the NC group, the proportion of CCK protein-positive EVs in the AD group to the total number of EVs in the plasma was significantly reduced (****, p < 0.0001). Compared with the NAD group, the proportion of CCK protein-positive EVs in the AD group to the total number of EVs in the plasma was also significantly reduced (**, p < 0.01). Both differences were significant (Figure 5A).

[0071] Compared with the NC group, the proportion of Neurogranin protein-positive EVs in the plasma of the AD group was significantly decreased (****, p<0.0001) (Figure 5B); compared with the NC group, the proportion of PMP2 protein-positive EVs in the plasma of the AD group was significantly decreased (****, p<0.0001) (Figure 5C).

[0072] Logistic regression analysis using the Enter method, incorporating CCK, Neurogranin, and PMP2 data, yielded AUCs of 0.92 (NC vs. AD) (Figure 5D) and 0.83 (AD vs. NAD) (Figure 5E). This analysis demonstrated that the proportion of CCK-, Neurogranin-, and PMP2-positive EVs relative to the total number of EVs not only effectively differentiated NC from AD but also effectively diagnosed AD from NAD.

[0073] The above description is merely a preferred embodiment of the present application and does not constitute any other limitation to the present application. Any person skilled in the art may utilize the above-disclosed technical content and the disclosed targets to modify or modify them into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the technical solution of the present application.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A composition for diagnosing and differentially diagnosing Alzheimer's disease based on spatial transcriptomics of human hippocampus, Features: It is one or more of CCK, Neurogranin and PMP2 carried by plasma extracellular vesicles EVs.

2. The composition for diagnosis and differential diagnosis of Alzheimer's disease based on spatial transcriptomics / single-cell sequencing of human hippocampus according to claim 1, Features: The CCK, Neurogranin and PMP2 carried by the plasma extracellular vesicles EVs were enriched by PEG8000 sedimentation.

3. A detection kit based on the composition according to claim 1 or 2, Features: Including reagents for labeling antibodies Zenon TM Alexa Fluor TM 647Rabbit IgG Labeling Kit, lipid probes, and antibodies; The lipid probe sequence SEQ ID NO: 1 is TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT, 5′ end modification: 5′ 6-CY3, 3′ end modification: 3′ Cholesteryl; The antibodies include one or more of CCK, Neurogranin and PMP2.

4. Application of CCK protein in the differential diagnosis of Alzheimer's disease and non-AD-dementia.

5. A method for detecting positive protein based on the composition according to claim 1 or 2 or the kit according to claim 3, Features: The following steps are included: Use Alexa Fluro fluorescent labeling kit to label CCK, Neurogranin, and PMP2 antibodies, take the labeled antibodies and mix them with the blocked exosomes, incubate them at 4°C in the dark overnight, add lipid probes, and incubate them at 4°C in the dark; Add PFA to the labeled sample, mix well and incubate at room temperature in the dark; Dilute with PBS to the appropriate concentration and load on the machine. Use CytoFLEX to detect, the number of particles per second is less than 10,000 particles; When the lipid probe is positive when the VSSC mode of CytoFLEX is used, the positive ratio of the protein marker can be obtained.

6. The method according to claim 4, Features: Identification for non-disease diagnosis and treatment purposes.

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