Biomarkers for predicting risk of central nervous system lymphoid dysfunction and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular to biomarkers for predicting the risk of central nervous system paralymphoid dysfunction and application thereof. The biomarkers for predicting the risk of central nervous system paralymphoid dysfunction are astrocyte-derived migratory bodies isolated from peripheral blood. An increase in the number of peripheral blood astrocyte-derived migratory bodies can be used as a marker for predicting the risk of paralymphoid dysfunction. The peripheral blood astrocyte migratory bodies provided by the present application can be used as an index for predicting the risk of central nervous system paralymphoid dysfunction, which can help to prompt central nervous system paralymphoid dysfunction when a patient cannot complete a head magnetic resonance DTI-ALPS sequence scan. The peripheral blood astrocyte migratory bodies can help doctors to formulate appropriate prevention or treatment plans for patients in combination with clinical symptoms, medical history or other examination information in a timely manner, and have extremely important clinical application potential and value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to biomarkers for predicting the risk of lymphoid dysfunction of the central nervous system and their applications. Background Technology

[0002] Central nervous system (CNS) autoimmune diseases are a group of illnesses caused by the immune system's abnormal attack on CNS tissues. Their course is often characterized by relapse-remission or progressive exacerbation, and chronic inflammation can lead to irreversible nerve damage. Common CNS autoimmune diseases include multiple sclerosis, neuromyelitis optica spectrum disorders, acute disseminated encephalomyelitis, and diseases related to anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibodies. These diseases severely impact patients' physical and mental health and quality of life.

[0003] Recent studies have revealed that cerebrospinal fluid (CSF) can flow into the brain parenchyma along the periarterial space and exchange substances with interstitial fluid via aquaporin 4 (AQP4) on the astrocyte terminal feet, ultimately draining out through the perivenous space. This unique anatomical structure, composed of astrocyte terminal feet, is named the "glymphatic system" or "glial lymphatic system" because its function resembles that of lymphatic vessels. Its core physiological significance lies in constructing an efficient material transport network between CSF and brain tissue, participating in processes such as the clearance of metabolic waste (e.g., β-amyloid), nutrient transport, and immune regulation within the central nervous system. The glymphatic system plays a crucial role in neuroinflammation and immune responses; damage to it leads to the accumulation of metabolic waste and pro-inflammatory cytokines in the brain, thus exacerbating neuroinflammation. Existing research suggests that dysfunction of the glymphatic system may be related to the severity and prognosis of central nervous system immune diseases. Furthermore, neurofilament light chains (NfL), as the recognized gold standard blood marker reflecting irreversible axonal damage and neurodegeneration, are closely related to disease severity. If a biomarker can be found that reflects both lymphoid dysfunction and is highly correlated with NfL concentration and clinical disability scales (such as the EDSS), the accuracy of disease monitoring will be greatly improved.

[0004] Currently, the determination of lymphoid function in the central nervous system mainly relies on DTI-ALPS sequences in cranial MRI. This method is time-consuming, expensive, and requires subsequent imaging data processing and evaluation. Therefore, finding a non-invasive and simple biomarker to assess lymphoid function in the central nervous system, enabling early screening and assessment of lymphoid dysfunction in central nervous system autoimmune diseases, has become a critical issue that urgently needs to be addressed.

[0005] Migratory bodies are a newly discovered type of organelle that arises from cell migration and grows on retracted filaments at the posterior end of the cell. Migratory bodies are rich in a specific four-transmembrane protein (TSPAN4) that participates in regulating their formation. Studies have found that migratory bodies are rich in mRNA and proteins released into the extracellular matrix or translocated laterally to recipient cells, mediating intercellular communication and substance transfer. Current research on the function of migratory bodies has made initial progress both domestically and internationally, focusing primarily on immune cells. There is currently no research on whether astrocytes can produce migratory bodies, or on the detection of astrocyte migratory bodies in peripheral blood as a lymphocyte-like functional biomarker. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, one of the objectives of the present invention is to provide biomarkers for predicting the risk of lymphoid dysfunction of the central nervous system.

[0007] The biomarker is astrocyte-derived migratory organisms isolated from peripheral blood. These migratory organisms cross the blood-brain barrier from the central nervous system into the peripheral blood, and their quantity is positively correlated with the degree of lymphoid system dysfunction.

[0008] Furthermore, the migrater has the following characteristics:

[0009] Monolayer membrane vesicles with a particle size of 500-3000 nm contain smaller vesicles inside; It expresses the four-transmembrane protein TSPAN4.

[0010] The present invention also provides a method for isolating astrocyte-derived migratory bodies from peripheral blood, comprising the following steps: (1) Collect peripheral blood from healthy individuals or patients with central nervous system immune diseases into EDTA tubes; (2) Perform three-stage centrifugation at 4℃: ① Centrifuge at 800-1000g for 5-10 minutes, retain the supernatant, and discard the lower layer of mixed blood cell precipitate; ② Centrifuge the supernatant from step ① at 4000g for 15-20 minutes, retain the supernatant, and discard the lower layer of cell debris precipitate; ③ Centrifuge the supernatant from step ② at 18000-20000g for 40-60 minutes, and collect the precipitate as the migrating body.

[0011] The present invention also provides a method for detecting astrocyte-derived migratory bodies in peripheral blood, comprising: (1) Negative staining electron microscopy detection The isolated peripheral blood migrants were incubated with rabbit anti-GFAP antibody, washed, and centrifuged. Anti-rabbit IgG magnetic beads were mixed with antibody-migratory complexes, incubated, passed through a magnetic bead sorting column, eluted, and centrifuged to obtain GFAP. + Migratory bodies; After staining with heavy metal staining solution, the samples were observed and photographed using a transmission electron microscope. (2) Particle size detection First, separate the GFAP + The migratory organisms were resuspended in an appropriate amount of PBS and then detected and analyzed using a Nanosight NS300 system. (3) Nanoflow cytometry separation and detection The isolated peripheral blood GFAP + Migrations were co-labeled with anti-GFAP antibody, WGA or Tspan4 antibody, fluorescently labeled with flow cytometry secondary antibody, and analyzed using nanoscale flow cytometry.

[0012] Furthermore, the negative staining electron microscopy detection step specifically includes: (1) The isolated peripheral blood migrants were incubated with rabbit anti-GFAP antibody at 4-6℃ for 1-2 hours; (2) Wash with PBS at 4-6℃, centrifuge at 18000-20000g for 40-60min and discard the supernatant to obtain GFAP antibody-migratory complex; (3) Mix anti-rabbit IgG magnetic beads with GFAP antibody-migratory complex and incubate at 4-6℃ for 1-2 hours; (4) Pass through a magnetic bead separation column and elute 3-5 times with magnetic bead separation eluent; (5) GFAP + After eluting the migrates, centrifuge at 18000-20000g for 40-60 min and discard the supernatant; (6) GFAP diluted with PBS + The migrants are adsorbed onto a clean copper grid for an electron microscope. Heavy metal staining solution is added and allowed to stand for 1-2 minutes to ensure that the staining solution is in full contact with the sample. Then, the sample is placed in a dust-free environment to dry. (7) Stain, observe with transmission electron microscopy, and take pictures.

[0013] Furthermore, the nanoflow cytometry sorting and detection specifically includes: (1) The isolated peripheral blood migrants were labeled with anti-GFAP antibody, WGA or Tspan4 antibody, incubated at room temperature for 1-2 hours, washed with PBS at 4-6℃, centrifuged at 18000-20000g for 40-60 min and the supernatant was discarded. (2) Use flow cytometry secondary antibody for fluorescent labeling and incubate at room temperature for 30-60 min, rinse with PBS at 4-6℃, centrifuge at 18000-20000g for 40-60 min and discard the supernatant; (3) The nanoscale flow cytometer was calibrated using standard nanoparticles to verify the accuracy and repeatability of particle size detection; (4) Set the scattered light parameters and corresponding labeled fluorescence channel parameters to distinguish nanoparticles from background impurities, and set thresholds to exclude non-specific fluorescence signals; (5) Detection and collection of streaming data.

[0014] Furthermore, the heavy metal staining solution is phosphotungstic acid.

[0015] The present invention also provides the application of the product for detecting astrocyte-derived migrations in the preparation of diagnostic kits for central nervous system lymphoid dysfunction.

[0016] This invention also provides the application of astrocyte-derived migration bodies in constructing diagnostic models of central nervous system lymphoid dysfunction.

[0017] The present invention also provides a diagnostic model for lymphoid dysfunction of the central nervous system. The input variable of the diagnostic model is the number of astrocyte-derived migratory bodies in peripheral blood. The higher the number of astrocyte-derived migratory bodies in peripheral blood, the more severe the lymphoid dysfunction of the central nervous system.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Peripheral blood is relatively easy to obtain. It is not only stable, minimally invasive, and easy to detect, but also has more accurate quantification. This will greatly improve the sensitivity and specificity of disease diagnosis and create a new situation for the prediction of lymphoid function in the central nervous system.

[0019] (2) Peripheral blood migrations have a clear origin. In the early stage of demyelinating diseases of the central nervous system, they mainly originate from astrocytes. Therefore, they have a certain accuracy and specificity as biomarkers.

[0020] (3) The present invention found that the increased number of peripheral blood astrocytic cell-derived migration bodies can serve as a marker of lymphoid dysfunction. The peripheral blood astrocytic cell migration bodies provided can serve as an indicator for predicting central nervous system lymphoid dysfunction. This can help patients who cannot complete the DTI-ALPS sequence of cranial magnetic resonance imaging to indicate the risk of central nervous system lymphoid dysfunction. It can serve as a clinical auxiliary indicator and help physicians to formulate appropriate prevention or treatment plans for patients in a timely manner in combination with clinical symptoms, medical history or other examination information. It has extremely important clinical application potential and value.

[0021] (4) This invention establishes various animal models of central nervous system inflammation, such as LPS-induced encephalitis, EAE, and NMOSD models, to demonstrate in vivo that astrocyte terminal foot retraction and detachment is the direct cause of elevated peripheral blood migration bodies. Combined with in vivo experiments using cerebrospinal fluid fluorescent tracers, it confirms that the concentration of peripheral blood migration bodies is significantly correlated with the influx and outflow of substances from the lymphoid system of the central nervous system. This provides solid biological mechanism support and direct animal-level evidence for the clinical application of this biomarker. Attached Figure Description

[0022] Figure 1 Identification of astrocyte-derived migratory organisms, including negative staining electron microscopy, particle size potential, and Western blot analysis results; A shows the typical negative-stain electron microscopy structure of the migrates obtained from the patient's peripheral blood; B is a negative-stained electron microscope image of migratory organisms derived from animal peripheral blood and brain tissue; C represents the particle size distribution and potential distribution of the migrating particles; D represents the expression of specific marker proteins such as TSPAN4, TSPAN9, CPQ, and NDST1 in astrocyte cell bodies and migration bodies, detected by Western blotting.

[0023] Figure 2 The procedure for detecting asteroids in peripheral blood and their correlation with lymphoid system function (ALPS score); A is a flowchart for peripheral blood migration detection; B is a representative diagram of nanoflow cytometry based on TSPAN4 positive gate; C represents the plasma GFAP levels in healthy controls, patients with neuromyelitis optica, and patients with multiple sclerosis. + Statistical comparison of the number of migrants; D represents GFAP in plasma. + Correlation analysis between the number of migrants and ALPS scores.

[0024] Figure 3 To assess the diagnostic efficacy of peripheral blood astrocytes for disease states and their correlation with neurological disability and axonal injury; A represents the ROC curve (AUC comparison) for differentiating patients with idiopathic inflammatory demyelinating diseases by migration bodies and ALPS scores. B represents the correlation analysis and Steiger's Z-test results between the number of migrations and ALPS scores and the neurological disability status (EDSS). C represents the correlation analysis and Steiger's Z-test results between the number of migratory bodies and ALPS scores and neural axonal injury markers (NfL).

[0025] Figure 4 The dynamic evolution of peripheral blood astrocytes in three animal models of neuroinflammation; A represents the dynamic changes in the number of plasma transporters in the lipopolysaccharide (LPS) induced model; B represents the dynamic changes in the passive transfer model of neuromyelitis optica (NMOSD); C represents the dynamic changes in the active immunization model of multiple sclerosis (EAE).

[0026] Figure 5 To verify the direct correlation between the number of peripheral blood astrocytes and the inflow and outflow functions of the lymphoid system in animal models; A shows a representative fluorescence image of dextran permeation in animal brain slices and quantitative data on the infiltration area; B represents plasma GFAP. + Correlation analysis between the number of migrants and the area of ​​dextran inflow; C represents plasma GFAP. + Correlation analysis of the number of migrants with the fluorescence intensity (efflux function) of dextran recovered from plasma. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] If the manufacturer of the reagents or instruments used is not specified, they are considered to be conventional products that can be purchased on the market.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example 1 I. Sample Collection 1. Subjects: 15 healthy controls and 30 patients with central nervous system immune diseases (such as multiple sclerosis and neuromyelitis optica spectrum disorders).

[0034] 2. Sample type: 5ml of peripheral blood, collected in a blood collection tube containing EDTA anticoagulant.

[0035] 3. Clinical data: (1) Record the patient’s baseline information (age, gender, course of disease, etc.); (2) Collect EDSS scale scores: The Expanded Disability Status Scale (EDSS) is based on the evaluation of eight functional systems of the central nervous system (pyramidal motor, cerebellum, brainstem, sensory, gut and bladder, vision, cerebrum, and others). It primarily assesses the severity of neurological dysfunction and disease, with a score range of 0-10. Higher scores indicate more severe neurological deficits.

[0036] All patients with central nervous system autoimmune diseases (multiple sclerosis, neuromyelitis optica spectrum disorders) participating in the study underwent assessment using the EDSS scale by experienced clinical neurologists. A double-blind reassessment was implemented in 20% of the sample, with a second neurologist independently assessing the patients, and the within-group correlation coefficient (ICC) was calculated between the two scores.

[0037] II. Isolation of Peripheral Blood Migrations 1. Collect peripheral blood from healthy controls and patients with central nervous system immune diseases (such as multiple sclerosis and neuromyelitis optica spectrum disorders) into 5ml EDTA tubes.

[0038] 2. All the following steps shall be performed at 4-6℃.

[0039] Centrifuge at 800-1000g for 10-15 min using a high-speed centrifuge pre-cooled at 4-6℃, discard the precipitate and recover the supernatant. Then centrifuge at 4000g for 15-20 min, discard the precipitate and recover the supernatant. Finally centrifuge at 18000-20000g for 40-60 min. The precipitate is the separated migrating body.

[0040] III. Detection of peripheral blood migration bodies 1. Negative staining electron microscopy detection (1) The isolated peripheral blood migrants were incubated with rabbit anti-GFAP antibody at 4-6℃ for 1-2 hours.

[0041] (2) Rinse with PBS at 4-6℃, centrifuge at 18000-20000g for 40-60min and discard the supernatant.

[0042] (3) Mix anti-rabbit IgG magnetic beads with GFAP antibody-migratory complex and incubate at 4-6℃ for 1-2 hours.

[0043] (4) Pass the sample through a magnetic bead separation column and elute 3-5 times with magnetic bead separation eluent. The eluent is Miltenyi autoMACS® Washing Solution, catalog number: 130-092-987.

[0044] (5) Combined GFAP + After eluting the migratings, centrifuge at 18000-20000g for 40-60 minutes and discard the supernatant.

[0045] (6) GFAP diluted with PBS + The migrants are adsorbed onto a clean electron microscope copper grid. A heavy metal staining solution (2% phosphotungstic acid solution) is added and allowed to stand for 1-2 minutes to allow the staining solution to fully contact the sample. The electron microscope copper grid (support grid) is then placed in a dust-free environment to dry.

[0046] (7) Observe and photograph using a transmission electron microscope.

[0047] GFAP obtained by negative staining transmission electron microscopy for sorting magnetic beads + Morphological observation of the migratory bodies revealed typical migratory body structures. Figure 1 The images clearly show retraction fibers and vesicle-like organelles of varying sizes attached to their ends. These structures exhibit a typical membrane-bound vesicle morphology. Further measurements revealed that the diameters of these vesicle-like structures are primarily distributed between 500 nm and 3000 nm, consistent with the typical size range of migratory organisms, thus confirming that the isolated vesicles are migratory organisms. Furthermore, Figure 1The migration structures in A-1B are intact with clear membrane structures, further verifying the successful isolation of high-purity migration structures from patient peripheral blood, animal peripheral blood, and brain tissue using magnetic bead sorting.

[0048] 2. Particle size detection (1) Separation of GFAP + The migration process is the same as described in point two above. The separated GFAPs... + The migratory cells were resuspended in an appropriate amount of PBS; (2) The GFAP that was resuspended + The migrated particles were detected and analyzed using a Nanosight NS300 system. The sample particle size was mainly concentrated in the range of 500 nm to 3000 nm, with a peak around 1000 nm. Figure 1 C). This distribution range perfectly matches the 500-3000 nm diameter of the migratory bodies observed by electron microscopy, quantitatively confirming that the separated vesicles are migratory bodies. The particle size distribution map shows a single-peaked and relatively narrow distribution characteristic, indicating that the migratory body population obtained by magnetic bead sorting is relatively uniform in size, with fewer impurities (such as cell debris or excessively small exosomes), and high separation purity. Zeta potential (dynamic potential) is a key indicator for measuring its surface charge and colloidal stability. The analysis results show that the zeta potential of the migratory bodies is mainly distributed between -20 mV and -30 mV ( Figure 1 C). High negative charge is one of the typical electrical properties of migratory particles. This negative charge helps the migratory particles maintain good dispersibility and stability in solution and prevents non-specific aggregation between particles.

[0049] 3. Nanoflow cytometry detection (1) The isolated peripheral blood migrants were labeled with anti-GFAP antibody, WGA or Tspan4 antibody, incubated at room temperature for 1-2 hours, washed with PBS at 4-6℃, centrifuged at 18000-20000g for 40-60 min and the supernatant was discarded.

[0050] (2) Use flow cytometry secondary antibody for fluorescent labeling and incubate at room temperature for 30-60 min, rinse with PBS at 4-6℃, centrifuge at 18000-20000g for 40-60 min and discard the supernatant.

[0051] (3) Use standard nanoparticles to calibrate the instrument and verify the accuracy and repeatability of particle size detection.

[0052] (4) Set the scattered light parameters and corresponding labeled fluorescence channel parameters to distinguish nanoparticles from background impurities, and set thresholds to exclude non-specific fluorescence signals.

[0053] (5) Go to the computer and collect streaming data.

[0054] (6) Data Analysis: The raw data was filtered using the flow cytometry software FlowJo. Non-specific events such as cell debris and aggregates were excluded using FSC-SSC scatter plots. Target nanoparticles were screened within a particle size range (500-3000 nm). The scatter plots were used to distinguish between positive and negative particles labeled with GFAP, WGA, or Tspan4 (migratory markers), and the number of positive particles was calculated. Figure 2 B). GFAP levels per 200 μL of peripheral plasma + Positive granules were used to assess the number of migrations originating from astrocytes, and a histogram was obtained. Figure 2 C), used to compare differences between healthy controls and patients with central nervous system autoimmune diseases. First, based on the location of the migrates within the region, the intensity of the FITC-labeled GFAP signal clearly distinguishes migrates from different origins. The universal marker TSPAN4 for migrates is used to delineate the migrating population, ensuring the accuracy of the analysis. The weak GFAP signal in healthy controls indicates a very low number of astrocyte-derived migrates in their peripheral blood. In contrast, samples from patients with multiple sclerosis (MS) and neuromyelitis optica (NMOSD) showed significantly enhanced GFAP signals, indicating a considerable number of astrocyte fragments from the central nervous system—i.e., GFAP-positive migrates—in their peripheral blood. A bar chart quantitatively analyzed and compared the number of migrates in the three groups, with clear results: the number of GFAP-positive migrates in the peripheral blood of the healthy control group remained at a very low level. The number of GFAP-positive migrates in the disease groups was significantly increased. Notably, the number of migrates in the neuromyelitis optica (NMOSD) patient group was significantly higher than that in the multiple sclerosis (MS) patient group. This suggests that the number of GFAP-positive migrations is not only related to disease status, but may also have the potential to differentiate between different central nervous system immune diseases.

[0055] 4. Western Blot Detection (1) Collect the isolated migratory bodies and corresponding astrocyte cell bodies, add RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors to extract total protein.

[0056] (2) After protein quantification using the BCA method, proteins were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane.

[0057] (3) Block the membrane with 5% skim milk (prepared with TBST) at room temperature for 1-2 hours to reduce non-specific binding. After blocking, place the membrane in a 1:500 diluted primary antibody (including migration marker antibodies TSPAN4, TSPAN9, CPQ, NDST1, exosome marker antibody TSG101, and internal control GAPDH antibody) and incubate overnight at 4°C.

[0058] (4) After washing the membrane with TBST, add horseradish peroxidase (HRP) labeled secondary antibody and incubate at room temperature for 1 hour. Finally, use ECL luminescent solution for development and image acquisition.

[0059] In the identification of migratory organisms, in addition to morphological ( Figure 1 A, Figure 1 B) and particle size potential ( Figure 1 In addition to the features described in C), this invention further detected specific marker proteins of the migratory organism using Western blotting. For example... Figure 1 As shown in D, the isolated astrocyte migratory bodies were highly enriched with characteristic migratory body markers TSPAN4, TSPAN9, CPQ, and NDST1, but lacked traditional exosome markers TSG101 and intracellular quality control protein GAPDH. This molecularly confirmed that the extracted vesicles were high-purity migratory bodies, eliminating interference from cell debris or exosomes.

[0060] IV. ALPS Score 1. Acquisition and data analysis of cranial MRI DTI-ALPS sequences: Healthy controls and patients with central nervous system immune diseases (multiple sclerosis, neuromyelitis optica spectrum disorders) underwent cranial magnetic resonance imaging using a 3.0 T MR scanner at the Third Affiliated Hospital of Sun Yat-sen University. The sequences included in the analysis are as follows: T1-weighted images (T1WI), T2-weighted images (T2WI), T2 fluid attenuation inversion recovery sequence (FLAIR), and diffusion tensor imaging (DTI). The imaging parameters are shown in Table (1.1).

[0061] Table 1.1 Magnetic Resonance Parameter Table

[0062] Note: MRI parameters: repetition time (TR) (ms) / echo time (TE) (ms) / field-of-view (Fov) (mm) 2 Layer thickness (mm) Diffusion tensor imaging analysis along the perivascular space (DTI-ALPS) is an emerging neuroimaging technique based on DTI that can non-invasively assess the function of the brain's lymphatic system. DTI assesses the diffusion capacity of water molecules by applying gradient magnetic fields in different directions and measuring changes in water molecule signals between adjacent gradients.

[0063] The DTI-ALPS method primarily uses diffusion tensor to measure water molecule diffusivity to assess the movement of water molecules in the perivascular space (PVS). At the level of the lateral ventricle body, the PVS is perpendicular to the projection fibers (z-axis) and the connecting fibers (y-axis). This anatomical feature reduces the influence of water molecules diffusing along the main white matter fibers. A region of interest (ROI) is placed on both the projection and connecting fibers of each unilateral cerebral hemisphere, and the diffusivity in the x, y, and z axes is extracted to calculate the ALPS index. This index is represented by the ratio of the two diffusivity values ​​perpendicular to the dominant fibers in the tissue, calculated using the following formula:

[0064] In the formula, mean (Dyproj, Dzassoc) is the average of the sum of the diffusivity of the projecting fibers along the y-axis and the diffusivity of the connecting fibers along the z-axis, and mean (Dxproj, Dxassoc) is the average of the sum of the diffusivity of the projecting fibers along the x-axis and the diffusivity of the connecting fibers along the x-axis. The larger the ALPS index, the stronger the diffusion ability of water molecules along the PVS, and the higher the activity of the lymphoid system.

[0065] Figure 2 The scatter plot (D) shows the association between the number of migratory cells and the ALPS score (an indicator of lymphoid system function). The plot reveals a clear negative correlation: as the number of GFAP-positive migratory cells in peripheral blood increases, the patient's ALPS score decreases. This result has significant pathophysiological implications, suggesting a possible association between astrocyte damage (release of migratory cells) and impaired lymphoid system function. The lymphoid system is a crucial pathway for clearing metabolic waste from the brain, and its dysfunction is a common feature of many neurological diseases.

[0066] V. Methods for Detection and Statistical Evaluation of Plasma Neurofilament Light Chain (NfL) Concentration The concentration of neurofilament light chains (NfL) in patient plasma was detected using an enzyme-linked immunosorbent assay (ELISA) kit. The specific procedures were strictly followed according to the kit instructions. The absorbance (OD value) was measured at 450 nm using an ELISA reader, and the sample concentration was calculated.

[0067] VI. Data Preprocessing and Correlation Analysis The distribution characteristics of the migration body number and ALPS score data were examined, and the Shapiro-Wilk test was used to determine whether the data conformed to a normal distribution. If the data were normally distributed, the Pearson correlation coefficient was used to analyze the correlation between the two; if the data did not conform to a normal distribution, the Spearman rank correlation coefficient was used. The receiver operating characteristic (ROC) curve was used to assess the ability of the biomarkers to distinguish disease states, and the area under the curve (AUC) was calculated. Steiger's Z-test was used. Z This study directly compared the correlation coefficients (rs values) between the number of astrocytic migratory cells and the ALPS index with clinical scores (EDSS, NfL) to assess the efficacy of different biomarkers in reflecting disease severity. Correlation analysis was performed using GraphPad Prism, and scatter plots were generated to show the relationship between the number of peripheral blood astrocytic migratory cells and the ALPS score. Correlation coefficients (r values) and their 95% confidence intervals were calculated, and significance tests were performed (P < 0.05 was considered statistically significant).

[0068] Clinical diagnostic efficacy of peripheral blood astrocyte migration bodies and their deep association with nerve axon injury. To evaluate the efficacy of peripheral blood migration bodies as a clinical diagnostic biomarker, this invention performed ROC curve analysis (…). Figure 3 A). The results showed that in a cohort of patients with idiopathic inflammatory demyelinating disease, peripheral blood astrocyte migrations exhibited extremely high disease state discrimination ability (AUC=0.8305), significantly superior to the traditional magnetic resonance imaging indicator ALPS score (AUC=0.6916), demonstrating its superiority as a highly sensitive "liquid biopsy" indicator. More importantly, in assessing disease severity, this invention directly compared the efficacy of migration number and ALPS score in reflecting the Clinical Disability Scale (EDSS) and neuronal axonal injury marker (NfL) using Steiger's Z-test. Figure 3 BC). The results showed that the number of peripheral blood astrocytes was significantly positively correlated with both EDSS score (rs=0.8080) and plasma NfL concentration (rs=0.7403); in contrast, the correlation between ALPS score and the above indicators was weaker, and the differences were statistically significant. Z The values ​​were 3.95 and 2.94, respectively (P < 0.001). This strongly suggests that, compared with the hydrodynamic changes measured by imaging, the massive release of peripheral blood astrocytes is more directly and closely associated with irreversible nerve axonal injury (NfL) and clinical limb disability (EDSS) in patients.

[0069] Example 2: Animal Model Confirmation of the Relationship between Peripheral Blood Astrocyte Migrations and Lymphoid System Function To further verify the reliability of peripheral blood astrocyte-derived migration bodies (ACMs) as biomarkers of lymphoid system dysfunction from a biological mechanism perspective, this embodiment constructed various animal models of central nervous system inflammation and demyelination, and conducted direct lymphoid system function assessments.

[0070] I. Establishment of animal models and dynamic monitoring of peripheral blood metastases 1. Model Building: (1) LPS-induced acute neuroinflammation model: acute blood-brain barrier disruption and neuroinflammation were induced by injecting lipopolysaccharide (LPS, 10 µg / animal) into the cerebellomedullary cistern.

[0071] (2) NMOSD passive transfer model: mice were injected intraperitoneally with purified patient-derived NMO-IgG (4 mg / mouse / day) to mimic the aquaporin 4 (AQP4) targeting damage in astrocytes.

[0072] (3) Multiple sclerosis (EAE) model: Chronic autoimmune encephalomyelitis was induced in mice by active immunization with MOG35-55 peptide.

[0073] 2. Quantitative analysis of peripheral blood migrations (nanoflow cytometry): The peripheral blood of the model animals was analyzed using the same separation and detection methods as in Example 1 (nanoflow cytometry based on GFAP and TSPAN4 dual labeling). Nanoflow cytometry results ( Figure 4 The results showed that in a lipopolysaccharide (LPS)-induced acute encephalitis model ( Figure 4 A) Passive transfer model of neuromyelitis optica (NMOSD) Figure 4 B) and the active immunization model of multiple sclerosis (EAE) Figure 4 In C), GFAP in animal peripheral blood + The number of peripheral blood migration bodies surged significantly during the acute phase or peak of the disease, and then declined as the disease progressed into the chronic or recovery phase. These results confirm, at the in vivo level, that inflammation of the central nervous system and astrocyte damage are the direct sources of elevated peripheral blood migration bodies, and demonstrate the universality and sensitivity of this marker in tracking acute neurological injury.

[0074] II. Fluorescence Tracing Method for Assessing Lymphoid Function and Correlation Analysis 1. Assessment of Lymphoid System Inflow and Outflow Function: To directly assess the efficiency of the lymphoid system in exchanging substances with cerebrospinal fluid / interstitial fluid, this invention uses a low molecular weight fluorescent tracer (3 kDa Texas Red-dextran) injected into the cerebellomedullary cistern. Thirty minutes after injection, samples were collected and the tracer coverage area in the brain parenchyma and spinal cord parenchyma was quantified to assess "inflow function"; the tracer fluorescence intensity in peripheral plasma was quantified to assess "outflow / clearance function". Figure 5 A).

[0075] 2. Mechanism-related verification: GFAP from animal peripheral blood was analyzed. + Linear regression analysis was performed on the concentration of the migrant and the functional data obtained from the above fluorescent tracers.

[0076] The correlation analysis results directly confirmed ( Figure 5 BC): In three major animal models, peripheral blood GFAP + The number of migratory bodies was significantly negatively correlated with the area of ​​tracer penetration in brain slices (lymphatic system inflow function); at the same time, its number was also significantly negatively correlated with the fluorescence intensity of dextran recovered from plasma and deep cervical lymph nodes (lymphatic system efflux function).

[0077] Conclusion: In vivo animal experiments confirmed that elevated concentrations of astrocyte migration bodies in peripheral blood directly reflect severe lymphoid system transport arrest at a fundamental biological level. This model data provides a solid pathophysiological basis for the clinical application of this invention, demonstrating that peripheral blood GFAP... + Migratory bodies are highly sensitive and well-understood quantitative biomarkers that reflect dysfunction of the lymphoid system.

[0078] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. Biomarkers for predicting the risk of lymphoid dysfunction in the central nervous system, characterized by: The biomarker is a migratory organism derived from astrocytes isolated from peripheral blood.

2. The biomarker for predicting the risk of central nervous system lymphoid dysfunction according to claim 1, characterized in that, The migrater has the following characteristics: Monolayer membrane vesicles with a particle size of 500-3000 nm contain smaller vesicles inside; It expresses the four-transmembrane protein TSPAN4.

3. A method for isolating astrocyte-derived migratory bodies from peripheral blood, characterized in that... Includes the following steps: (1) Collect peripheral blood from healthy individuals or patients with central nervous system immune diseases into EDTA tubes; (2) Perform three-stage centrifugation at 4℃: ① Centrifuge at 800-1000g for 5-10 minutes, retain the supernatant, and discard the lower layer of mixed blood cell precipitate; ②The supernatant from step ① is centrifuged at 4000g for 15-20 minutes, the supernatant is retained and the lower layer of cell debris precipitate is discarded; ③ The supernatant from step ② is centrifuged at 18000-20000g for 40-60 minutes, and the precipitate is collected as the migrating body.

4. A method for detecting astrocyte-derived migratory bodies in peripheral blood, characterized in that... Includes the following steps: (1) Negative staining electron microscopy detection The isolated peripheral blood migrants were incubated with rabbit anti-GFAP antibody, washed, and centrifuged. Anti-rabbit IgG magnetic beads were mixed with antibody-migratory complexes, incubated, passed through a magnetic bead sorting column, eluted, and centrifuged to obtain GFAP. + Migratory bodies; After staining with heavy metal staining solution, the samples were observed and photographed using a transmission electron microscope. (2) Particle size detection First, separate the GFAP + The migratory organisms were resuspended in an appropriate amount of PBS and then detected and analyzed using a Nanosight NS300 system. (3) Nanoflow cytometry separation and detection The isolated peripheral blood GFAP + Migrations were co-labeled with anti-GFAP antibody, WGA or Tspan4 antibody, fluorescently labeled with flow cytometry secondary antibody, and analyzed using nanoscale flow cytometry.

5. The method for detecting astrocyte-derived migratory bodies in peripheral blood according to claim 3, characterized in that, The specific steps for negative staining electron microscopy detection are as follows: (1) The isolated peripheral blood migrants were incubated with rabbit anti-GFAP antibody at 4-6℃ for 1-2 hours; (2) Wash with PBS at 4-6℃, centrifuge at 18000-20000g for 40-60min and discard the supernatant to obtain GFAP antibody-migratory complex; (3) Mix anti-rabbit IgG magnetic beads with GFAP antibody-migratory complex and incubate at 4-6℃ for 1-2 hours; (4) Pass through a magnetic bead separation column and elute 3-5 times with magnetic bead separation eluent; (5) GFAP + After eluting the migrates, centrifuge at 18000-20000g for 40-60 min and discard the supernatant; (6) GFAP diluted with PBS + The migrants are adsorbed onto a clean copper grid for an electron microscope. Heavy metal staining solution is added and allowed to stand for 1-2 minutes to ensure that the staining solution is in full contact with the sample. Then, the sample is placed in a dust-free environment to dry. (7) Stain, observe with transmission electron microscopy, and take pictures.

6. The method for detecting astrocyte-derived migratory bodies in peripheral blood according to claim 3, characterized in that, Its characteristics are, The nanoflow cytometry sorting detection is as follows: (1) The isolated peripheral blood migrants were labeled with anti-GFAP antibody, WGA or Tspan4 antibody, incubated at room temperature for 1-2 hours, washed with PBS at 4-6℃, centrifuged at 18000-20000g for 40-60 min and the supernatant was discarded. (2) Use flow cytometry secondary antibody for fluorescent labeling and incubate at room temperature for 30-60 min, rinse with PBS at 4-6℃, centrifuge at 18000-20000g for 40-60 min and discard the supernatant; (3) The nanoscale flow cytometer was calibrated using standard nanoparticles to verify the accuracy and repeatability of particle size detection; (4) Set the scattered light parameters and corresponding labeled fluorescence channel parameters to distinguish nanoparticles from background impurities, and set thresholds to exclude non-specific fluorescence signals; (5) Detection and collection of streaming data.

7. The use of the product containing astrocyte-derived migration bodies as described in claim 1 in the preparation of a diagnostic kit for central nervous system lymphoid dysfunction.

8. To test the use of the astrocyte-derived migration bodies of claim 1 in constructing a diagnostic model for predicting the risk of lymphoid dysfunction of the central nervous system.

9. A diagnostic model for predicting the risk of lymphoid dysfunction in the central nervous system, characterized by: The input variable of the diagnostic model is the number of astrocyte-derived migratory bodies in peripheral blood as described in claim 1. The more astrocyte-derived migratory bodies there are in peripheral blood, the more severe the central nervous system lymphoid dysfunction is judged to be.

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