CN117825714A - Auxiliary diagnosis method and system for early Alzheimer disease by using peripheral body fluid extracellular vesicle enrichment detection technology and biomarker - Google Patents

Abstract

The invention provides a peripheral body fluid extracellular vesicle enrichment detection technology and an auxiliary diagnosis method and an auxiliary diagnosis system for early Alzheimer disease by using a biomarker, wherein the biomarker is derived from AChE pathway key proteins and/or synaptic function related key proteins in peripheral body fluid extracellular vesicle EVs, the AChE pathway key proteins comprise one or more of CHT1 and CHAT proteins, and the synaptic function related key proteins comprise one or more of KA1, KA2, gluN2D (NR 2D), SLC6A12, VGAT, GAP43 and SV2A proteins. According to the invention, the AChE pathway key protein and the synaptic function related marker are jointly detected, and the multi-labeled EVs are simultaneously detected, so that the effects of avoiding obtaining cerebrospinal fluid and reacting to central nervous system change only by a small amount of peripheral body fluid samples are achieved, and early screening and auxiliary diagnosis of early Alzheimer's disease and mild cognitive impairment are realized.

Description

Auxiliary diagnosis method and system for early Alzheimer disease by using peripheral body fluid extracellular vesicle enrichment detection technology and biomarker

Technical Field

The invention relates to the technical field of detection and auxiliary diagnosis of extracellular vesicles of peripheral body fluid, in particular to an auxiliary diagnosis method and an auxiliary diagnosis system for early Alzheimer disease by using a detection technology and a biomarker for enrichment of extracellular vesicles of the peripheral body fluid.

Background

Alzheimer's Disease (AD) is the most common neurodegenerative disease and is mainly characterized by progressive cognitive impairment. AD is one of the ten thousand major diseases worldwide, and it is estimated that AD patients account for about 70% of 5000 thousand dementia patients worldwide. With the increasing population aging, it is expected that the number of patients with global senile dementia will increase three times as much as today by 2050. Furthermore, early screening of AD-derived mild cognitive impairment (mild cognitive impairment, MCI) is also a clinically significant challenge and a major challenge to address before the onset of typical AD symptoms. The "Sanzao principle" for early screening, early diagnosis and early intervention of significant brain diseases, including AD, is a global consensus for the prevention and control of such diseases. However, early warning and accurate diagnosis and treatment of AD are all the world problems and challenges puzzling the medical community, and at present, the common clinical examination means such as images, pathology and inspection cannot meet the large-scale early screening and early diagnosis work of people due to the limitations and hysteresis of the respective professions, technologies and economy. Therefore, developing more convenient, economical and accurate early disease screening and diagnosis means has become a scientific and technical problem to be solved in clinical urgent need.

In recent years, extracellular vesicles (Extracellular Vesicles, EVs) have become a research hotspot for diagnostic biomarkers. EVs consist of exosomes, microvesicles and apoptotic bodies. Almost all types of cells are capable of releasing EVs, including cells of the central nervous system, such as neurons, oligodendrocytes, astrocytes, microglia, vascular endothelial cells, and the like. Research shows that EVs are involved in pathophysiological processes such as information transmission between normal cells of the central nervous system, neuron stress response, neuroinflammation, epigenetic regulation, neural plasticity and the like. Currently, research on EVs has made some breakthroughs in the medical field such as research on neurodegenerative diseases, cancers and the like. EVs of central nervous system origin are reported to be able to cross the blood brain barrier into the peripheral blood and to be detectable in the peripheral blood. Several clinical biomarker studies have found that EVs in body fluids have important early warning and diagnostic value for neurodegenerative diseases. Liquid biopsies in particular based on EVs technology have brought new dawn for early warning and diagnosis of AD.

Therefore, focusing on key proteins in a key pathway (such as an AChE pathway) which changes at the early stage of AD and reflecting the damage of the nerve synapse function in the development process of AD, detecting relevant EVs in peripheral blood and body fluid, has the advantages of micro-invasive sampling, convenient and quick clinical detection and the like, is expected to discover potential risks of MCI or AD a few years before the symptoms of a patient appear, and realizes accurate early screening and auxiliary diagnosis of AD; thereby gaining opportunity for clinical timely intervention and having great clinical application value and social significance.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides an auxiliary diagnosis method and an auxiliary diagnosis system for early Alzheimer's disease by using a peripheral body fluid extracellular vesicle enrichment detection technology and a biomarker, which effectively screen early Alzheimer's disease and mild cognitive impairment by using peripheral body fluid EVs and carry out early screening and auxiliary diagnosis of early Alzheimer's disease and mild cognitive impairment by identifying the biomarker of which the early Alzheimer's disease is changed in peripheral body fluid.

To achieve the above object, the present invention provides a biomarker for aiding diagnosis of early alzheimer's disease based on a peripheral body fluid extracellular vesicle enrichment detection technique, wherein: the biomarker is derived from AChE pathway key proteins and/or synaptic function related key proteins in peripheral body fluid extracellular vesicle EVs, wherein the AChE pathway key proteins comprise one or more of CHT1 and CHAT proteins, and the synaptic function related key proteins comprise one or more of KA1, KA2, gluN2D (NR 2D), SLC6A12, VGAT and GAP43 proteins.

Preferably, the CHAT, CHT1, KA2, GAP43 proteins are used alone for the assisted diagnosis of Alzheimer's disease AD.

Preferably, the CHAT, CHT1, KA1, GAP43, NR2D proteins are used alone for the assisted diagnosis of mild cognitive impairment MCI.

Preferably, one or more of the group consisting of CHAT protein in combination with NR2D protein, CHAT protein in combination with GAP43 protein, CHT1 protein in combination with KA1 protein, CHT1 protein in combination with KA2 protein, and CHT1 protein in combination with SLC6A12 protein is/are useful for the assisted diagnosis of AD.

Preferably, one or more of the group consisting of CHAT protein in combination with KA1 protein, CHAT protein in combination with NR2D protein, CHAT protein in combination with GAP43 protein, CHT1 protein in combination with KA1 protein, CHT1 protein in combination with KA 2D protein, CHT1 protein in combination with SLC6A12 protein is/are useful for the assisted diagnosis of Mild Cognitive Impairment (MCI).

Preferably, the extracellular vesicle EVs of the peripheral body fluid are obtained by PEG8000 sedimentation and enrichment.

The invention also provides a kit for auxiliary diagnosis of early Alzheimer's disease based on a peripheral body fluid extracellular vesicle enrichment detection technology, which comprises an antibody for the biomarker of any one of claims 1-6, an antibody labeling reagent and an incubation probe DNA anchor, wherein the antibody labeling reagent comprises Alexa Fluro fluorescent labeling kit.

The invention also provides a method for detecting positive proteins based on the biomarker and the kit, which comprises the following steps,

labeling the antibody of the biomarker according to any one of claims 1-6 by using an Alexa Fluro fluorescent labeling kit, uniformly mixing the labeled antibody with the blocked exosome, then incubating overnight at 4 ℃ in a dark place, adding an incubation probe DNA anchor, and incubating at 4 ℃ in a dark place;

adding PFA into the marked sample, uniformly mixing, and incubating at room temperature in a dark place;

diluting to a proper concentration by PBS, and detecting by using CytoFLEX, wherein the number of particles per second is less than 1 ten thousand particles;

under the condition that the VSSC mode detection incubation probe DNA anchor is positive by using CytoFLEX, the positive proportion of the protein label is obtained.

Preferably, the identification is used for non-disease diagnosis and therapeutic purposes.

The invention also provides an auxiliary diagnosis system for early Alzheimer's disease based on the biomarker and the kit combined with the peripheral body fluid extracellular vesicle enrichment detection technology.

The beneficial effects of the invention are as follows:

the application aims to provide an AChE pathway key protein which can effectively identify early changes of Alzheimer's disease in peripheral body fluid, and the AChE pathway key protein is combined with a synaptic function related marker for detection, and multiple marked EVs are detected simultaneously, so that the effects of avoiding obtaining cerebrospinal fluid and reflecting central nervous system changes only by a small amount of peripheral body fluid samples are achieved.

Drawings

FIG. 1 is a graph showing the proportion of candidate biomarkers carried by plasma EVs, wherein FIG. 1A shows the labeling of key candidate biomarkers for the AChE pathway, and FIG. 1B shows the labeling of key candidate biomarkers associated with synaptic function, the abscissa representing the name of the biomarker; the ordinate represents the ratio of the proportion of relevant biomarker carried by plasma EVs to negative control IgG.

FIG. 2 is a graph showing the positive proportion of individual markers of candidate biomarkers carried by plasma EVs, wherein FIG. 2A shows the individual markers of key candidate biomarkers of AChE pathway, and FIG. 1B shows the individual markers of key candidate biomarkers associated with synaptic function, the abscissa represents the group and the ordinate represents the positive proportion of related biomarkers carried by plasma EVs; in NC group, n=5 Pool; in AD group, n=5 Pool, representing p <0.05; * P <0.01 and p <0.001.

FIG. 3 is a graph showing the dual labeling of candidate biomarkers carried by plasma EVs, wherein FIG. 3A shows the dual labeling of CHT and key candidate biomarkers associated with synaptic function, and FIG. 3B shows the dual labeling of CHT1 and key candidate biomarkers associated with synaptic function, and the abscissa represents the group; the ordinate represents the positive proportion of relevant biomarkers carried by plasma EVs, n=5 Pool in NC group; in AD group, n=5 Pool, ×represents p <0.05, ×represents p <0.01.

FIG. 4 is a graph showing the dual labeling of candidate biomarkers carried by plasma EVs, wherein FIG. 4A shows the dual labeling of CHT and key candidate biomarkers associated with synaptic function, and FIG. 4B shows the dual labeling of CHT1 and key candidate biomarkers associated with synaptic function, with the abscissa representing the group; the ordinate represents the positive proportion of relevant biomarkers carried by plasma EVs, and fig. 4C shows ROC curves for each integrated model, n=12 in NC group; in AD group, n=12; in MCI group, n=12, representing p <0.05; * Represents p <0.01.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present application will be further described with reference to the specific examples below.

Example 1: EVs enrichment by PEG8000 sedimentation

1. Quickly thawing (within 2 min) blood plasma from a subject at 37deg.C, and vortex mixing;

2. plasma samples (> 300. Mu.L) were centrifuged at 2,000Xg for 15min at 4℃and the supernatant was taken;

3. centrifuging at 4deg.C and 12,000Xg for 30min, and collecting supernatant;

4. sucking 10 mu L of centrifuged plasma into a 1.5mL centrifuge tube, adding 70 mu L of PBS and 20 mu L of 40% PEG8000, fully blowing and mixing uniformly, and standing at room temperature for 30min;

5.12000g, centrifuging at 4 ℃ for 20min;

6. the supernatant was discarded, resuspended in 100. Mu.L PBS, and then blow-mixed, split-packed in 10. Mu.L/tube, and stored at-80℃until use.

Example 2:

1. sample closure

(1) Taking 10 μl of the above-mentioned solution to a 0.6mL centrifuge tube through the EVs of PEG 8000;

(2) 10. Mu.L of 2% BSA solution (0.22 μm membrane filtration) was added and mixed well;

(3) Blocking at room temperature (25-26 ℃) for 1 hour to remove non-specific binding;

(4) The block was terminated by dilution with 10. Mu.L PBS (0.22 μm membrane filtration).

2. Sample marking

Antibodies for detection of the biomarker are labeled using a labeling method. The reagents used were:

antibodies to CHT1, mAChRs M4, CHAT, VAChT, ACHE, and SV2A, GAP-43, neurogranin, gluN2B, gluN2C, gluN2D, gluA2, KA1, KA2, VGAT, GAT1, SLC6a12, gabaarap were labeled with Alexa fluor fluorescent labeling kit. Wherein, the reagent for Labeling the antibodies CHT1, mAChRs M4 and CHAT, VAChT, ACHE is ZenonTM Alexa FluorTM 488Labeling Kit, which is purchased from Thermo Fisher Scientific; the reagents labeled SV2A, GAP-43, neurogranin, gluN2B, gluN2C, gluN2D, gluA, KA1, KA2, VGAT, GAT1, SLC6A12, GABARAP antibodies were ZenonTM Alexa FluorTM 647IgG Labeling Kit, purchased from Thermo Fisher Scientific.

The marking steps are as follows:

(1) 1. Mu.g of the antibody of the corresponding marker was diluted to 5. Mu.L (0.2. Mu.g/. Mu.L) with PBS (0.22 μm filter);

(2) Adding 5 mu L ZenonTM Alexa FluorTM Labeling Kit A solution, mixing, and incubating at room temperature (25-26 ℃) in dark place for 20min;

(3) Adding 3 mu L (3 mu g) of ZenonTM Alexa FluorTM Labeling Kit B solution (B solution is diluted to 1 mu g/mu L) into the solution in the step (2), and incubating for 10min at room temperature (25 ℃) in a dark place after uniform mixing to quench unbound free fluorescein;

(4) PBS (0.22 μm membrane filtration) was added to dilute to a total volume of 50. Mu.L;

(5) Taking 5 mu L ZenonTM Alexa FluorTM 647Rabbit IgG Labeling Kit marked pTau217 in the sample sealed in the step 1, uniformly mixing, and then incubating at room temperature (25-26 ℃) for 5min in a dark place;

(6) Adding 3 mu L ZenonTM Alexa FluorTM 488Rabbit IgG Labeling Kit marked CHAT or NR2D to the sample in the step (5), mixing uniformly, and incubating overnight at 4 ℃ in a dark place;

3. probe incubation

The reagent is used: DNA anchor, sequence TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT; modification of 5' end: 5`6-CY3;3' -terminal modification: 3' cholestyl. The lipid probe is capable of binding to the EVs membrane, and the probe-labeled positive particles are considered to be true EVs; the method further improves the scientificity and accuracy of EVs marking. The probe incubation steps were as follows:

(1) At 2. Sample labeling, step (6) the next day of incubation overnight at 4℃in the dark, 62uL of PBS+5ul of lipid probe (lipid probe working solution concentration 10. Mu.M) (reaction volume 100ul, probe final concentration 500 nM) was added to the system and incubated at 4℃in the dark for 1h.

(2) Adding 100 mu L of 4% PFA (0.22 mu m filter membrane filtration) into the marked sample, uniformly mixing, and incubating at room temperature (25-26 ℃) for 20min in a dark place;

(3) Dilution with PBS to appropriate concentration was put on machine, standard: the number of particles per second was less than 1 ten thousand particles as measured by CytoFLEX. 50000 particles were collected under the lipid probe gate.

4. Detection of

The positive proportion of markers was detected using the VSSC mode of CytoFLEX.

Example 3

AChE pathway key proteins and synaptic function-related key proteins potentially useful for diagnosis and differential diagnosis of early Alzheimer's disease and mild cognitive impairment are screened.

The key candidate biomarkers of the ache pathway are: CHT1, mAChRs M4, CHAT, VAChT, ACHE;

key candidate biomarkers associated with synaptic function are: SV2A, GAP43, neurogranin, gluN2B, gluN2C, gluN2D, gluA2, KA1, KA2, VGAT, GAT1, SLC6a12, gabaarap.

2. The candidate biomarkers described above were initially screened in a plasma sample of clinical Pool.

To ensure stability and reliability of the primary screening results, clinical Pool plasma samples were used in this example, each Pool being mixed from 10 plasma samples to reduce the instability factor due to individual plasma differences. The healthy control group (NC group) contained 5 Pool plasma samples, and the alzheimer's disease subjects (AD group) contained 5 Pool plasma samples.

First, the proportion and content of candidate biomarkers carried by plasma EVs were examined. Experiments were performed as in examples 1 and 2 using NC group plasma samples with samples of 5 Pool.

The results are shown in FIG. 1. We selected for further investigation a marker with a ratio of relevant biomarkers carried by plasma EVs to negative control IgG of greater than 2, in which case we considered that relevant biomarkers carried by EVs could be reliably detected. Thus, excluding ACHE, gluN2B, GABARAP, ACHE pathway key proteins were selected: CHT1, mAChRs M4, CHAT, VAChT; key proteins associated with synaptic function: further studies were performed on SV2A, GAP, neurogranin, gluN B (NR 2B), gluN2C (NR 2C), gluN2D (NR 2D), gluA2, KA1, KA2, VGAT, GAT1, SLC6A12.

Next, we performed experiments according to examples 1 and 2 in NC group 5 Pool plasma samples and AD group 5 Pool plasma samples. Further screening, it was examined whether there was a difference in the proportion of positive particles in the NC and AD group EVs for the candidate biomarkers.

The results are shown in FIG. 2. AChE pathway key protein: CHT1, mAChRs M4, CHAT, VAChT. The proportion of CHAT and CHT1 positive EVs was significantly reduced when AD occurred compared to NC; while the ratio of mAChRs M4 and VAChT positive EVs did not differ significantly between NC and AD groups. The AChE pathway key proteins CHAT and CHT1 were therefore selected for further investigation. Synaptic function-related key proteins: SV2A, GAP, neurogranin, gluN B (NR 2B), gluN2C (NR 2C), gluN2D (NR 2D), gluA2, KA1, KA2, VGAT, GAT1, SLC6A12. When AD occurs, the proportion of KA1, KA2, gluN2D (NR 2D), SLC6a12, VGAT, GAP43 positive EVs is significantly reduced compared to NC group; while the proportions of Neurogranin, gluN B (NR 2B), gluN2C (NR 2C), gluA2, GAT1, SV2A positive EVs were not significantly different between NC and AD groups. Thus, key proteins KA1, KA2, gluN2D (NR 2D), SLC6A12, VGAT, GAP43 related to synaptic function were selected for the next study.

The AChE pathway changes at the early stages of AD; in addition, the development and progression of AD is accompanied by impairment of the function of the neurites. The simultaneous detection of the AChE pathway changes and the synaptic function related protein changes may improve the sensitivity and specificity of early diagnosis of AD and differential diagnosis with MCI. Thus, in the following, we further performed a combination double labeling of AChE pathway key proteins carried by EVs and synaptic function related key proteins, examining the ability of the related marker combinations in distinguishing NC and AD groups. As shown in FIG. 3, AChE pathway candidate key proteins CHAT and CHT1 are combined with synaptic function-related key proteins KA1, KA2, gluN2D (NR 2D), SLC6A12, VGAT and GAP43 to form a double label, which is stable and consistent with the single labeling trend. Clinical queue detection may be performed.

Example 4

The differential diagnostic ability of the above biomarkers is tested in clinical samples. Plasma samples of 12 healthy controls (NC group) and 12 subjects with alzheimer's disease (AD group) and 12 subjects with mild cognitive impairment (MCI group) were obtained, and experiments were performed by the methods of examples 1 and 2.

The proportion of EVs positive for the double fluorescent markers CHAT, CHT1, KA2, gluN2D (NR 2D), SLC6A12, VGAT, GAP43 single channel fluorescent markers, CHAT and KA1, CHAT and KA2, CHAT and GluN2D (NR 2D), CHAT and SLC6A12, CHAT and VGAT, CHAT and GAP43, CHT1 and KA1, CHT1 and KA2, CHT1 and GluN2D (NR 2D), CHT1 and SLC6A12, CHT1 and VGAT, CHT1 and GAP43 were measured using the VSSC mode of CytoFLEX, respectively, the proportion of EVs positive for the lipid probe marker was calculated, and the ability to distinguish NC, AD, MCI was examined.

The results are shown in FIG. 4. The combined dual labeling results for CHAT and synaptic function-related proteins are as follows,

at dual labeling of CHAT with KA1, the proportion of CHAT protein positive EVs to total EVs in AD group plasma was significantly reduced compared to NC group (×p < 0.01); the proportion of CHAT protein positive EVs to total EVs in AD group plasma was also significantly reduced compared to MCI group (x, p < 0.01) (fig. 4A). The proportion of KA1 protein positive EVs in AD group plasma to total EVs was significantly reduced compared to NC group (< 0.05); the proportion of KA1 protein positive EVs to total EVs was also significantly reduced in AD group plasma compared to MCI group (< 0.05) (fig. 4A); there was a downward trend in the AD group for the proportion of CHAT protein and KA1 protein double positive EVs to total EVs number in plasma compared to NC group, but the differences were not statistically significant; the proportion of CHAT protein and KA1 protein double positive EVs to total EVs was significantly reduced in AD group plasma compared to MCI group (< 0.05) (fig. 4A).

At the dual labeling of CHAT and KA2, the proportion of CHAT protein positive EVs in total EVs in AD group plasma was significantly reduced compared to NC group (p < 0.05); there was a decrease in the proportion of CHAT protein positive EVs to total EVs in AD group plasma compared to MCI group, but no statistical difference was reached (fig. 4A). Compared with NC group, the proportion of KA2 protein positive EVs in the blood plasma of AD group to total EVs tends to be reduced, but statistical difference is not achieved; the proportion of KA2 protein positive EVs in the plasma of AD group to total EVs was reduced compared to MCI group, but statistical differences were not reached (fig. 4A); there was a downward trend in the AD group for the proportion of CHAT protein and KA2 protein double positive EVs to total EVs number in plasma compared to NC group, but the differences were not statistically significant; there was a decreasing trend in the ratio of CHAT protein and KA2 protein double positive EVs to total EVs in AD group plasma compared to MCI group, but the differences were not statistically significant (fig. 4A).

At the dual labeling of CHAT and GluN2D (NR 2D), the proportion of CHAT protein positive EVs in total EVs in AD group plasma was significantly reduced compared to NC group (x, p < 0.05); there was a decrease in the proportion of CHAT protein positive EVs to total EVs in AD group plasma compared to MCI group, but no statistical difference was reached (fig. 4A). Compared with NC group, the ratio of NR2D protein positive EVs in the plasma of AD group to total EVs tends to be reduced, but statistical difference is not achieved; the proportion of NR2D protein positive EVs to total EVs in MCI plasma was reduced compared to NC group, but statistical differences were not reached (fig. 4A); the proportion of CHAT protein and NR2D protein double positive EVs to total EVs number in plasma was significantly reduced in AD group compared to NC group (< 0.05); the proportion of CHAT protein and NR2D protein double positive EVs to total EVs was significantly reduced in MCI plasma compared to NC group (< 0.05) (fig. 4A).

At the time of dual labeling of CHAT and SLC6a12, the proportion of CHAT protein positive EVs in AD group plasma to total EVs was reduced compared to NC group, but statistical difference was not reached; the proportion of CHAT protein positive EVs to total EVs was significantly reduced in AD group plasma compared to MCI group (< 0.05) (fig. 4A). The ratio of SLC6a12 protein positive EVs to total EVs was not significantly different in plasma for AD and MCI compared to NC (fig. 4A); the ratio of the double positive EVs for CHAT protein and SLC6a12 protein in plasma to the total EVs was not significantly variable in both AD and MCI groups compared to NC group (fig. 4A).

In the case of dual labeling of CHAT and VGAT, the proportion of CHAT protein positive EVs in AD plasma to total EVs was reduced compared to NC, but statistical differences were not reached; there was a decrease in the proportion of CHAT protein positive EVs to total EVs in AD group plasma compared to MCI group, but no statistical difference was reached (fig. 4A). The ratio of VGAT protein positive EVs to total EVs was not significantly different in plasma for AD and MCI compared to NC (fig. 4A); there was a downward trend in the AD group for the ratio of CHAT protein and VGAT protein double positive EVs to total EVs number in plasma compared to NC group, but the differences were not statistically significant; there was a decreasing trend in the ratio of CHAT protein and VGAT protein double positive EVs to total EVs in AD group plasma compared to MCI group, but the differences were not statistically significant (fig. 4A).

At the dual labeling of CHAT and GAP43, the proportion of CHAT protein positive EVs in total EVs in AD group plasma was significantly reduced compared to NC group (p < 0.05); there was a decrease in the proportion of CHAT protein positive EVs to total EVs in AD group plasma compared to MCI group, but no statistical difference was reached (fig. 4A). The proportion of GAP43 protein positive EVs in AD group plasma to total EVs was significantly reduced compared to NC group (×p < 0.01); the proportion of GAP43 protein positive EVs to total EVs was significantly reduced in MCI group plasma compared to NC group (×p < 0.05) (fig. 4A); the proportion of CHAT protein and GAP43 protein double positive EVs to total EVs number in plasma was significantly reduced in AD group compared to NC group (×p < 0.01); the proportion of CHAT protein and GAP43 protein double positive EVs to total EVs was significantly reduced in MCI plasma compared to NC group (×p < 0.01) (fig. 4A).

The combined double labeling results of CHT1 and synaptic function-related proteins are as follows,

at double labeling of CHT1 and KA1, the proportion of CHT1 protein positive EVs in AD group plasma to total EVs was significantly reduced compared to NC group (< 0.05); there was a decreasing trend in the proportion of CHT1 protein positive EVs to total EVs in AD group plasma compared to MCI group, but the differences were not statistically significant (fig. 4B). The proportion of KA1 protein positive EVs in AD group plasma to total EVs number was significantly reduced compared to NC group (×p < 0.01); there was a downward trend in the proportion of KA1 protein positive EVs to total EVs in AD group plasma compared to MCI group, but the differences were not statistically significant (fig. 4B); the proportion of CHT1 protein and KA1 protein double-positive EVs in plasma to total EVs number was significantly reduced in AD group compared to NC group (×p < 0.01); the proportion of CHT1 protein and KA1 protein double positive EVs in the MCI plasma to total EVs number was significantly reduced compared to NC group (< 0.05) (fig. 4B).

At double labeling of CHT1 and KA2, the proportion of CHT1 protein positive EVs in AD group plasma to total EVs was significantly reduced compared to NC group (< 0.05); the proportion of CHT1 protein positive EVs in the plasma of AD group to total EVs was a decreasing trend compared to MCI group, but no statistical difference was reached (fig. 4B). The proportion of KA2 protein positive EVs in AD group plasma to total EVs was significantly reduced compared to NC group (< 0.05); the proportion of KA2 protein positive EVs in the plasma of AD group to total EVs was reduced compared to MCI group, but statistical differences were not reached (fig. 4B); the proportion of CHT1 protein and KA2 protein double-positive EVs in plasma to total EVs number was significantly reduced in AD group compared to NC group (×p < 0.01); the proportion of CHT1 protein and KA2 protein double positive EVs in the MCI plasma to total EVs number was significantly reduced compared to NC group (< 0.05) (fig. 4B).

At the time of double labeling of CHT1 and NR2D, compared with NC group, the proportion of CHT1 protein positive EVs in the plasma of AD group to the total EVs tends to be reduced, but statistical difference is not achieved; the proportion of CHT1 protein positive EVs to total EVs was significantly reduced in AD group plasma compared to MCI group (×p < 0.05) (fig. 4B). Compared with NC group, the ratio of NR2D protein positive EVs in the plasma of AD group to total EVs tends to be reduced, but statistical difference is not achieved; the proportion of NR2D protein positive EVs to total EVs was significantly reduced in AD group plasma compared to MCI group (×p < 0.05) (fig. 4B); the proportion of CHT1 protein and NR2D protein double positive EVs in plasma to total EVs number was a decreasing trend in AD group compared to NC group, but not statistically different; the proportion of CHT1 protein and NR2D protein double positive EVs to total EVs was significantly reduced in AD group plasma compared to MCI group (< 0.05) (fig. 4B).

At double labeling of CHT1 and SLC6a12, the proportion of CHT1 protein positive EVs in AD group plasma to total EVs was significantly reduced compared to NC group (< 0.05); the proportion of CHT1 protein positive EVs in the plasma of AD group to total EVs was a decreasing trend compared to MCI group, but no statistical difference was reached (fig. 4B). The ratio of SLC6a12 protein positive EVs to total EVs was not significantly different in plasma for AD and MCI compared to NC (fig. 4B); the proportion of CHT1 protein and SLC6a12 protein double-positive EVs in AD group plasma to total EVs number was significantly reduced compared to NC group (< 0.05); the proportion of CHT1 protein and SLC6a12 protein double positive EVs to total EVs was significantly reduced in AD group plasma compared to MCI group (< 0.05) (fig. 4B).

At double labeling of CHT1 and VGAT, the proportion of CHT1 protein positive EVs in AD group plasma to total EVs was significantly reduced compared to NC group (< 0.05); the proportion of CHT1 protein positive EVs in the plasma of AD group to total EVs was a decreasing trend compared to MCI group, but no statistical difference was reached (fig. 4B). The ratio of VGAT protein positive EVs to total EVs was not significantly different in plasma for AD and MCI compared to NC (fig. 4B); there was a downward trend in the AD group for the proportion of CHT1 protein and VGAT protein double positive EVs to total EVs number in plasma compared to NC group, but the differences were not statistically significant; there was a trend of decreasing the ratio of CHT1 protein and VGAT protein double positive EVs to total EVs in AD group plasma compared to MCI group, but the differences were not statistically significant (fig. 4B).

At the time of double labeling of CHT1 and GAP43, the proportion of CHT1 protein positive EVs in AD group plasma to total EVs was decreased compared to NC group, but statistical difference was not reached; the proportion of CHT1 protein positive EVs in the plasma of AD group to total EVs was a decreasing trend compared to MCI group, but no statistical difference was reached (fig. 4B). The proportion of GAP43 protein positive EVs in AD group plasma to total EVs was reduced compared to NC group, but statistical differences were not reached; the proportion of GAP43 protein positive EVs in AD group plasma to total EVs was reduced compared to MCI group, but statistical differences were not achieved (fig. 4B); the proportion of double positive EVs of CHT1 protein and GAP43 protein in the plasma to total EVs compared to NC group was a decreasing trend in AD group, but did not reach statistical difference; there was a decrease in the proportion of CHT1 protein and GAP43 protein double positive EVs in the plasma of AD group to total EVs compared to MCI group, but no statistical difference was achieved (fig. 4B).

Based on the above results, an integration model was established using a logistic regression analysis Enter method to incorporate Age (Age) and related analysis index, diagnostic efficiency was evaluated by subject work characteristic curve (receiver operating characteristic curve, ROC) analysis, and the area under the ROC curve (AUC) of each group was obtained as shown in table 1.

TABLE 1 area under ROC curve for each integrated model

Note that: ROC: a subject work profile; AUC: area under the curve; NC: healthy controls; AD: alzheimer's disease; MCI: mild cognitive impairment.

As can be seen from table 1, the double-labeled data of Age, cht1+ka1 and cht1+ka2 were included using the logistic regression analysis Enter method, auc=0.98 (NC VS AD); auc=0.944 (NC VS MCI); auc=0.689 (AD VS MCI) (fig. 4C); the end method was used to incorporate the Age, chat+nr2d and cht1+nr2d double-labeled data, auc=0.875 (NC VS AD); auc=0.958 (NC VS MCI); auc=0.861 (AD VS MCI) (fig. 4C); the end method was used to incorporate the Age, chat+gap43 and cht1+gap43 double-labeled data, auc=0.979 (NC VS AD); auc=0.965 (NC VS MCI); auc=0.757 (AD VS MCI) (fig. 4C); double labeling data of Age, chat+ka2, and cht1+ka2 were included using logistic regression analysis Enter method, auc=0.939 (NC VS AD); auc=0.944 (NC VS MCI); auc=0.652 (AD VS MCI) (fig. 4C).

In summary, we have found that based on the pathogenesis of AD and MCI (i.e., AChE pathway changes in early stages of AD; furthermore, the development and progression of AD is accompanied by impairment of neuronal synaptic function), changes in AChE pathway and changes in synaptic function-related proteins are detected in plasma EVs, and several groups of marker combinations with remarkable effects are obtained: (1) The ratio of Age, CHT1+KA1 and CHT1+KA2 double-positive EVs to the total EVs; (2) The ratio of Age, chat+nr2d and cht1+nr2d double positive EVs to total EVs number; (3) The ratio of Age, chat+gap43 and cht1+gap43 double positive EVs to total EVs number; (4) The ratio of Age, CHAT+KA2 and CHT1+KA2 double-positive EVs to the total EVs; by means of the marker combination, NC and AD patients can be well distinguished; NC and MCI can also be effectively distinguished. Wherein, the combination of the ratio of Age, CHAT+NR2D and CHT1+NR2D double positive EVs to total EVs can realize the distinction between the AD and the MCI while distinguishing the NC and the AD patients and the NC and the MCI patients.

In short, the research based on the plasma EVs provides a high-efficiency and rapid means for realizing the early screening of MCI and the early and accurate diagnosis of AD, and has higher transformation prospect and application value.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. The biomarker for auxiliary diagnosis of early Alzheimer disease based on the detection technology of extracellular vesicle enrichment of peripheral body fluid is characterized by comprising the following components: the biomarker is derived from AChE pathway key proteins and/or synaptic function related key proteins in peripheral body fluid extracellular vesicle EVs, wherein the AChE pathway key proteins comprise one or more of CHT1 and CHAT proteins, and the synaptic function related key proteins comprise one or more of KA1, KA2, gluN2D (NR 2D), SLC6A12, VGAT, GAP43 and SV2A proteins.

2. The biomarker for aiding diagnosis of early alzheimer's disease based on the peripheral body fluid extracellular vesicle enrichment detection technique of claim 1, characterized in that: the CHAT, CHT1, KA2, GAP43 proteins can be used alone for the assisted diagnosis of Alzheimer's disease AD.

3. The biomarker for aiding diagnosis of early alzheimer's disease based on the peripheral body fluid extracellular vesicle enrichment detection technique of claim 1, characterized in that: CHAT, CHT1, KA1, GAP43, NR2D proteins alone can be used for the assisted diagnosis of mild cognitive impairment MCI.

4. The biomarker for aiding diagnosis of early alzheimer's disease based on the peripheral body fluid extracellular vesicle enrichment detection technique of claim 1, characterized in that: one or more groups of CHAT protein combined with NR2D protein, CHAT protein combined with GAP43 protein, CHT1 protein combined with KA1 protein, CHT1 protein combined with KA2 protein and CHT1 protein combined with SLC6A12 protein can be used for auxiliary diagnosis of Alzheimer disease AD.

5. The biomarker for aiding diagnosis of early alzheimer's disease based on the peripheral body fluid extracellular vesicle enrichment detection technique of claim 1, characterized in that: one or more groups of CHAT protein combined KA1 protein, CHAT protein combined NR2D protein, CHAT protein combined GAP43 protein, CHT1 protein combined KA1 protein, CHT1 protein combined KA2 protein, CHT1 protein combined NR2D protein and CHT1 protein combined SLC6A12 protein can be used for auxiliary diagnosis of Mild Cognitive Impairment (MCI).

6. The biomarker for aiding diagnosis of early alzheimer's disease based on the peripheral body fluid extracellular vesicle enrichment detection technique of claim 1, characterized in that: the extracellular vesicle EVs of the peripheral body fluid are obtained through PEG8000 sedimentation and enrichment.

7. The kit for auxiliary diagnosis of early Alzheimer's disease based on the peripheral body fluid extracellular vesicle enrichment detection technology is characterized by comprising the following components: an antibody comprising the biomarker of any of claims 1-6, an antibody labelling reagent comprising Alexa fluorescent labelling kit, and an incubation probe DNA anchor.

8. A method for positive protein detection based on the biomarker of any of claims 1 to 6, the kit of claim 7, characterized in that: comprises the steps of,

labeling the antibody of the biomarker according to any one of claims 1-6 by using an Alexa Fluro fluorescent labeling kit, uniformly mixing the labeled antibody with the blocked exosome, then incubating overnight at 4 ℃ in a dark place, adding an incubation probe DNA anchor, and incubating at 4 ℃ in a dark place;

adding PFA into the marked sample, uniformly mixing, and incubating at room temperature in a dark place;

diluting to a proper concentration by PBS, and detecting by using CytoFLEX, wherein the number of particles per second is less than 1 ten thousand particles;

under the condition that the VSSC mode detection incubation probe DNA anchor is positive by using CytoFLEX, the positive proportion of the protein label is obtained.

9. The method according to claim 8, wherein: identification for non-disease diagnostic and therapeutic purposes.

10. An auxiliary diagnostic system for early alzheimer's disease based on the biomarker of any of claims 1-6, the kit of claim 7 in combination with a peripheral body fluid extracellular vesicle enrichment detection technique.