Membrane protein marker detection model for pediatric graft-versus-host disease and applications thereof

Abstract

The application discloses a kind of membrane protein marker detection model of children graft versus host disease and application thereof, belong to biological detection technical field.The application is screened by single-cell vesicle membrane proteomics PBA technology, and seven extracellular vesicle membrane proteins VEGFA, APOE, CXADR, ITGB4, EMCN, DSC3, Ly6C are obtained as the characteristic markers of children acute graft versus host disease, wherein CXADR, ITGB4, EMCN, DSC3, Ly6C are significantly down-regulated in high-risk group, and VEGFA, APOE is significantly up-regulated.The application can be used for early screening, risk classification and disease assessment of acute graft versus host disease after allogeneic hematopoietic stem cell transplantation in children, which makes up for the defects of low accuracy and low throughput of existing detection methods, provides reliable basis for early detection and early intervention in clinic, and has good clinical popularization value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a membrane protein biomarker detection model for graft-versus-host disease in children and its application. Background Technology

[0002] Allogeneic hematopoietic stem cell transplantation is a core treatment for severe diseases in children, such as hematological disorders and congenital immunodeficiency. Acute graft-versus-host disease (aGVHD) is the most common and dangerous immune-related complication after this procedure, and a major cause of death in transplant recipients. Clinical data shows that the first 100 days after transplantation are the main period for the onset of aGVHD, with a small number of children experiencing delayed onset. Studies from the Blood and Bone Marrow Transplantation Clinical Trials Network indicate that the cumulative incidence of aGVHD within 100 days can reach 62%, and after professional verification, the actual incidence rate remains as high as 49%. Disease severity directly affects the prognosis of children; the one-year overall survival rate for children with grade II aGVHD is 70%, while the one-year overall survival rate for the more severe grade III-IV patients is only 40%, with an extremely high risk of death. Although prophylactic treatment for aGVHD is now routinely performed in clinical practice, some children still progress to severe cases. Therefore, early warning and accurate risk assessment are crucial for improving survival rates and optimizing clinical treatment plans.

[0003] Acute graft-versus-host disease (AGVHD) is caused by donor immune effector cells attacking recipient tissues and organs, primarily damaging the three major target organs: skin, gastrointestinal tract, and liver. The disease develops in three phases: initiation, activation, and effector phases, mediated by a combination of various immune cells, cytokines, and intercellular signaling. Currently, clinical diagnosis of APVHD in children relies mainly on clinical symptom observation, organ damage signs assessment, and traditional cytokine detection models. These methods have significant limitations: firstly, by the time clinical symptoms appear, significant organ damage has often already occurred, missing the optimal intervention window; secondly, traditional detection techniques have low throughput and poor specificity, making it difficult to accurately grade the disease and predict risks, distinguish the roles of different immune cell subsets in the pathogenesis, or elucidate the underlying molecular communication mechanisms.

[0004] Extracellular vesicles (EVs) mediate intercellular signal transduction, and their surface membrane proteins are potential biomarkers for diseases. They can also be used to trace the cells of origin of vesicles and construct targeted delivery systems, showing great potential in the early diagnosis of tumors and immune diseases. Traditional single-vesicle detection technologies can only achieve low-throughput membrane protein detection, which cannot meet the needs of large-scale clinical sample screening. Existing research on graft-versus-host disease (GVHD) is mostly focused on the adult population, and specific detection models based on extracellular vesicle membrane proteomics for pediatric patients are still lacking. At the same time, the industry currently lacks a complete and practical system for biomarker screening, detection, model construction, and clinical application, making it difficult to accurately distinguish between high-risk and low-risk children with acute GVHD and to quantify the degree of multi-organ damage. In summary, developing a pediatric GVHD detection model based on characteristic membrane protein biomarkers to solve the problems of lagging detection, low accuracy, and insufficient specificity of existing technologies has become an urgent technical challenge to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a set of extracellular vesicle membrane protein biomarkers for detecting acute graft-versus-host disease in children and a detection model based on these biomarkers, thereby filling the gaps in existing clinical detection technologies and enabling early warning, risk grading, and disease assessment.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for detecting membrane protein biomarkers of acute graft-versus-host disease in children. The characteristic biomarkers of the method are six extracellular vesicle membrane proteins: VEGFA, APOE, CXADR, ITGB4, EMCN, and DSC3. The test sample is a peripheral body fluid sample from a child allogeneic hematopoietic stem cell transplant recipient.

[0007] Preferably, the expression levels of CXADR, ITGB4, EMCN, and DSC3 are below their corresponding thresholds, while the expression levels of VEGFA and APOE are above their corresponding thresholds, which is considered a positive expression pattern for acute graft-versus-host disease. The thresholds for CXADR are 300-310, ITGB4 is 1970-2000, EMCN is 2600-2620, and DSC3 is 640-660.

[0008] Preferably, the detection method includes exosome preparation, identification of exosome characteristic proteins, preparation of exosome membrane proteins, membrane protein difference detection unit and result determination.

[0009] Preferably, the exosome preparation adopts differential centrifugation combined with ultrafiltration to prepare exosome stock solution. The differential centrifugation includes centrifugation at 250~350×g for 8~12 min, centrifugation at 1800~2200×g for 18~22 min, and centrifugation at 9000~11000×g for 28~32 min.

[0010] Preferably, in the preparation of exosomes, an additive solution is added to a peripheral body fluid sample. The additive solution includes neopentyl lauryl maltose glycol, (diacetyl) sophorol, and 1,2-hexanediol.

[0011] Preferably, the mass-to-volume ratio of neopentyl lauryl maltose glycol to 1,2-hexanediol is 4-12 mg:1 mL, and the mass-to-volume ratio of neopentyl lauryl maltose glycol to (diacetyl)sophorol lactone is 5-20:1; the mass of the additive solution is measured by the mass of neopentyl lauryl maltose glycol therein, and the mass-to-volume ratio of neopentyl lauryl maltose glycol to the peripheral body fluid sample is 5-15 mg:100 mL.

[0012] The combination of neopentyl lauryl maltose glycol and diacetylated sophorol lactone can protect the structure of extracellular vesicles and the activity of membrane proteins, improve the purity and integrity of exosomes and the detection abundance of CD9 and CD63, and ensure the stability of subsequent membrane protein extraction and quantitative detection. Neopentyl lauryl maltose glycol, as a mild nonionic surfactant, can bind to the phospholipid layer of vesicles to improve dispersibility and prevent exosome aggregation and rupture. At the same time, it can encapsulate membrane proteins to maintain their native conformation and avoid denaturation. Diacetylated sophorol lactone is embedded in the lipid bilayer to reinforce the vesicle skeleton, enhance the ability of vesicles to withstand centrifugation and concentration, and reduce damage and impurity contamination. Combined with 1,2-hexanediol as a mild solubilizer, it can avoid the disadvantages of ethanol causing protein denaturation and vesicle damage.

[0013] Preferably, the exosome characteristic protein identification unit uses CD9 and CD63 as positive marker proteins for exosomes and Calnexin as a negative marker protein for contamination; when CD9 and CD63 are highly expressed in the sample and Calnexin is not expressed, it is judged as a qualified exosome sample.

[0014] Preferably, the detection method uses a scoring weighting method to calculate the comprehensive risk score: 1 point is awarded for abnormal expression of a single indicator, and 0 points are awarded for normal expression; the weights of CXADR, EMCN, and DSC3 are 0.17~0.19, the weight of ITGB4 is 0.15~0.17, and the weights of VEGFA, APOE, and Ly6C are 0.09~0.10; a comprehensive risk score < 0.2 is considered low risk, and a comprehensive risk score ≥ 0.2 is considered high risk.

[0015] Preferably, the detection method includes a gut-specific risk assessment mechanism, selecting three indicators: CXADR, DSC3, and Ly6C, with each indicator having a weight of 0.3 to 0.4. A gut-specific risk score < 0.2 is considered as a low risk of gut involvement, and a score ≥ 0.2 is considered as a high risk of gut involvement. Combined with the expression characteristics of biomarkers, the condition of skin and liver damage can be assessed, and the type of single organ or multiple organ involvement can be distinguished.

[0016] Preferably, the detection method is applied to one or more of the following: early screening reagents, risk grading reagents, and multi-organ involvement detection reagents for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in children.

[0017] More preferably, in the identification of exosome characteristic proteins, 5-hydroxy-2-pyrrolidone is added to the protein lysis buffer, and the mass-volume ratio of 5-hydroxy-2-pyrrolidone to the protein lysis buffer is 0.5~10 mg:4 mL.

[0018] 5-Hydroxy-2-pyrrolidone can maximally preserve the expression characteristics of markers such as VEGFA, APOE, and CXADR, ensuring the authenticity and reliability of protein expression abundance data obtained from PBA high-throughput detection and reducing experimental errors. In this detection system, the addition of this substance can significantly improve the integrity of exosome membrane proteins. It is speculated that because it has a cyclic lactam structure and active hydroxyl groups, it can bind to the active sites of endogenous proteases in the sample, effectively inhibiting protease hydrolysis activity, blocking the degradation pathway of membrane proteins, and reducing protein fragmentation loss during extraction. The hydroxyl groups in its molecule can form multiple hydrogen bonds with polar groups on the surface of membrane proteins, constructing a stable hydration layer on the outside of the protein molecule, restricting the disordered binding between protein molecules, preventing membrane proteins from denaturing, aggregating, and precipitating, and accurately maintaining the native spatial conformation of the seven target membrane proteins.

[0019] This invention also provides a method for preparing a detection model of membrane protein biomarkers for graft-versus-host disease in children, including the preparation of exosome stock solution, identification of exosome characteristic proteins, preparation of exosome membrane protein solution, initial screening of differentially expressed proteins, determination of membrane protein expression threshold, formulation of disease risk grading standards and assessment standards for multi-organ involvement.

[0020] Step 1: Preparation of exosome stock solution: Collect 1-20 mL of peripheral body fluid sample from the child using a sterile blood collection tube. Separate exosomes using differential centrifugation combined with ultrafiltration. First, centrifuge at 250-350×g for 8-12 min to remove intact cells and cell debris from the sample. Then, centrifuge at 1800-2200×g for 18-22 min to remove apoptotic bodies. Subsequently, centrifuge at 9000-11000×g for 28-32 min to remove large vesicles and impurities. Finally, collect the supernatant and concentrate it using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa to obtain the exosome stock solution.

[0021] Preferably, an additive solution is added to the peripheral body fluid sample, the additive solution comprising neopentyl lauryl maltose, (diacetyl) sophorol, and 1,2-hexanediol.

[0022] Preferably, in the additive solution, the mass-to-volume ratio of neopentyl lauryl maltose glycol to 1,2-hexanediol is 4-12 mg:1 mL, and the mass-to-volume ratio of neopentyl lauryl maltose glycol to (diacetyl)sophorol lactone is 5-20:1.

[0023] Preferably, the mass of the additive solution is measured by the mass of lauryl maltose neopentyl glycol contained therein, and the mass-to-volume ratio of lauryl maltose neopentyl glycol to the peripheral body fluid sample is 5-15 mg: 100 mL.

[0024] Step 2: Identification of exosome characteristic proteins: Add protein lysis buffer to the exosome stock solution, lyse on ice for 25-35 min, centrifuge at 11000-13000×g for 13-17 min, collect the supernatant to obtain protein samples; prepare 11%-13% SDS-PAGE gel, load 13-17 μL / well, electrophoresis at 75-85V for 28-32 min, adjust the voltage to 110-130V and continue electrophoresis until bromophenol blue reaches the bottom of the gel; use wet transfer method for membrane transfer, transfer current 280-320 kJ / min. At mA, the transfer time is 85-95 min to transfer the protein to a PVDF membrane. After the transfer, the membrane is blocked with 4%-6% skim milk at room temperature for 1.5-2.5 h. Then, CD9, CD63, and Calnexin primary antibodies are added and incubated overnight at 4°C. The next day, the membrane is washed 2-4 times with TBST buffer for 4-6 min each time, and horseradish peroxidase-labeled secondary antibody is added and incubated at room temperature for 0.8-1.2 h. After washing with TBST again, ECL chemiluminescence developing solution is added, and images are acquired using a gel imaging system.

[0025] Preferably, the volume ratio of exosome stock solution to protein lysis buffer is 1:0.8~1.2.

[0026] Preferably, the primary antibody dilution ratio is 1:800~1200.

[0027] Preferably, the dilution ratio of the secondary antibody is 1:4000~6000.

[0028] Preferably, the criteria for judging the detection results are that the exosome characteristic proteins CD9 and CD63 are highly expressed, and the endoplasmic reticulum marker protein Calnexin is not expressed, then the sample is judged to be a qualified exosome.

[0029] More preferably, 5-hydroxy-2-pyrrolidone is added to the protein lysis buffer, and the mass-to-volume ratio of 5-hydroxy-2-pyrrolidone to the protein lysis buffer is 0.5~10 mg:4 mL.

[0030] Step 3: Preparation of exosome membrane protein solution: Add qualified exosome stock solution to a special membrane protein extraction reagent, vortex to mix for 0.8-1.2 min, and let stand on ice for 18-22 min, vortexing once every 4-6 min during this period; centrifuge at 11000-13000×g for 18-22 min at 4℃, and aspirate the supernatant, which is the enriched exosome membrane protein solution. Use a BCA protein quantification kit to determine the protein concentration, and adjust the protein concentration of all samples to 0.8-1.2 μg / μL to obtain the exosome membrane protein solution.

[0031] Preferably, the volume ratio of exosome stock solution to membrane protein extraction reagent is 1:3.5~4.5.

[0032] Step 4: Initial Screening of Differential Proteins: Using the single-cell vesicle membrane proteomics PBA detection platform, the total volume of the detection system was set to 190-210 μL. 8-12 μL of membrane protein sample, 170-190 μL of detection buffer, and 8-12 μL of specific antibody mixture were added to each well. The detection program was: incubation at 35-39℃ for 1.8-2.2 h, followed by single-particle signal acquisition, simultaneously detecting the expression abundance of hundreds of membrane proteins in the sample. Based on the membrane protein expression data, the fold change in expression and the P-value were used as screening criteria to initially screen membrane proteins with significant differences in expression between groups.

[0033] Step 5: Determination of membrane protein expression threshold: Combining quantitative data from PBA detection, scatter plot analysis, and significance test results, the expression ranges of each protein in the high-risk and low-risk groups are statistically analyzed, and the optimal critical threshold is determined by combining the ROC curve analysis results.

[0034] Preferably, the CXADR threshold is 300~310.

[0035] Preferably, the ITGB4 threshold is 1970~2000.

[0036] Preferably, the EMCN threshold is 2600~2620.

[0037] Preferably, the DSC3 threshold is 640~660.

[0038] Preferably, when the expression levels of CXADR, ITGB4, EMCN, DSC3, and Ly6C in the sample are lower than the corresponding thresholds, and the expression levels of VEGFA and APOE are higher than the corresponding thresholds, it is determined to be an aGVHD positive expression pattern.

[0039] Step Six: Establishment of Disease Risk Grading Standards: If the measured expression levels of VEGFA and APOE are higher than the corresponding thresholds, and the measured expression levels of CXADR, ITGB4, EMCN, and DSC3 are lower than the corresponding thresholds, it is judged that a single indicator shows an abnormal expression pattern of acute graft-versus-host disease and is scored as 1 point; otherwise, it is scored as 0 points. Then, the diagnostic efficacy of each protein is assigned a weight according to the ROC curve, with CXADR, EMCN, and DSC3 each having a weight of 0.17~0.19, ITGB4 having a weight of 0.15~0.17, and VEGFA and APOE each having a weight of 0.09~0.10. The comprehensive risk score is obtained by multiplying the scores of each indicator by their corresponding weights and summing them. CXADR and DSC3 indicators, which are highly correlated with intestinal damage, are selected, and the weight of each is set to 0.3~0.4. The intestinal-specific risk score is calculated according to the same scoring rules.

[0040] Preferably, a comprehensive risk score of <0.2 is considered as low risk of acute graft-versus-host disease, and a comprehensive risk score of ≥0.2 is considered as high risk of acute graft-versus-host disease.

[0041] Preferably, a gut-specific risk score of <0.2 indicates a low risk of gut involvement, and a score of ≥0.2 indicates a high risk of gut involvement.

[0042] Step 7: Development of assessment criteria for multi-organ involvement: Based on the expression characteristics of membrane proteins and the clinical sites of involvement, target organ damage is differentiated; intestinal involvement is determined by abnormal expression of intestinal-associated membrane proteins, skin involvement is determined by skin characteristic proteins and markers, and liver involvement is determined by liver-associated proteins, which can accurately distinguish between single-organ involvement and multi-organ involvement.

[0043] This invention employs single-cell vesicle membrane proteomics (PBA) technology to screen seven extracellular vesicle membrane proteins—VEGFA, APOE, CXADR, ITGB4, EMCN, DSC3, and Ly6C—as characteristic biomarkers. A detection model is constructed by combining standardized exosome isolation, membrane protein extraction procedures, and a quantitative scoring system, thus offering the following advantages: First, leveraging the high-throughput detection advantages of PBA, this invention can simultaneously perform quantitative analysis of multiple membrane proteins, significantly improving detection efficiency and shortening the single-sample testing time compared to traditional single-vesicle detection and cytokine detection technologies, thus meeting the needs of large-scale clinical sample screening; second, the seven differentially expressed membrane proteins can be used for diagnosis... This invention boasts excellent performance, with an AUC value of 0.96 for CXADR, EMCN, and DSC3, and an overall detection accuracy exceeding 90%. Its sensitivity and specificity are outstanding, enabling early warning of acute graft-versus-host disease (APH) in children. Furthermore, it can establish comprehensive risk scoring and intestinal-specific risk scoring standards based on membrane protein expression characteristics, accurately stratifying disease risk. It can also assess the involvement of multiple organs, including the skin, intestines, and liver, by combining biomarker correlations, overcoming the limitations of existing technologies in quantifying organ damage. In addition, the entire experimental process has been optimized through multiple examples and comparative studies, resulting in high exosome purity, membrane protein integrity, strong detection system stability, and good reproducibility. Therefore, this invention provides a membrane protein biomarker detection model with high accuracy, high throughput, and strong practicality, suitable for early screening, risk stratification, and multi-organ involvement assessment of APH in children, possessing significant clinical application and promotional value. Attached Figure Description

[0044] Figure 1 This is a schematic diagram showing the results of exosome purity and concentration detection.

[0045] Figure 2 This is a schematic diagram showing the microstructural characterization results of exosomes.

[0046] Figure 3 This is a schematic diagram showing the results of the identification of characteristic proteins of exosomes.

[0047] Figure 4 This is a schematic diagram of the results of the validation experiment on the difference in membrane protein expression between high- and low-risk groups.

[0048] Figure 5 This is a schematic diagram of the subject screening process.

[0049] Figure 6 This is a diagram illustrating the specific inclusion and exclusion criteria. Detailed Implementation

[0050] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0052] Example 1: This example provides a method for preparing a detection model of membrane protein biomarkers for graft-versus-host disease in children. The main steps include preparation of exosome stock solution, identification of exosome characteristic proteins, preparation of exosome membrane protein solution, initial screening of differentially expressed proteins, determination of membrane protein expression threshold, formulation of disease risk grading criteria and assessment criteria for multi-organ involvement, as detailed below.

[0053] Step 1: Preparation of Exosome Stock Solution: 5 mL of peripheral body fluid was collected from the child using a sterile blood collection tube. Exosomes were separated using differential centrifugation combined with ultrafiltration. First, centrifugation at 300×g for 10 min removed intact cells and cell debris. Then, centrifugation at 2000×g for 20 min removed apoptotic bodies. Subsequently, centrifugation at 10000×g for 30 min removed large vesicles and impurities. Finally, the supernatant was collected and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa to obtain the exosome stock solution. The peripheral body fluid samples were from children aged 1-5 years who had received allogeneic hematopoietic stem cell transplantation and had three underlying diseases: chronic granulomatous disease, myelodysplastic syndrome, and aplastic anemia. The samples were divided into a high-risk group and a low-risk group for aGVHD (acute graft-versus-host disease).

[0054] Step 2: Identification of exosome characteristic proteins: Add protein lysis buffer to the exosome stock solution, lyse on ice for 30 min, centrifuge at 12000×g for 15 min, collect the supernatant to obtain protein samples; prepare a 12% SDS-PAGE gel, load 15 μL / well, electrophoresis at 80V for 30 min, adjust the voltage to 120V and continue electrophoresis until bromophenol blue reaches the bottom of the gel; use wet transfer method, transfer current 300mA, transfer time 90 min, transfer protein to PVDF membrane; after transfer, block with 5% skim milk at room temperature for 2 h, then add CD9, CD63, and Calnexin primary antibodies, and incubate overnight at 4℃; the next day, wash 3 times with TBST buffer, 5 min each time, add horseradish peroxidase-labeled secondary antibody, and incubate at room temperature for 1 h; wash again with TBST, add ECL chemiluminescence developing solution, and acquire images using a gel imaging system. The volume ratio of exosome stock solution to protein lysis buffer was 1:1, the primary antibody was diluted 1:1000, and the secondary antibody was diluted 1:5000. The criteria for judging the test results were that the exosome characteristic proteins CD9 and CD63 were highly expressed, and the endoplasmic reticulum marker protein Calnexin was not expressed. If the results were judged to be qualified exosomes, the sample was considered to be qualified.

[0055] Step 3: Preparation of exosome membrane protein solution: Add qualified exosome stock solution to the special membrane protein extraction reagent, vortex to mix for 1 min, and incubate on ice for 20 min, vortexing once every 5 min during this period; centrifuge at 12000×g for 20 min at 4℃, and aspirate the supernatant, which is the enriched exosome membrane protein solution. Determine the protein concentration using a BCA protein quantification kit, and adjust the protein concentration of all samples to a uniform 1 μg / μL to obtain the exosome membrane protein solution. The volume ratio of exosome stock solution to membrane protein extraction reagent is 1:4.

[0056] Step 4: Initial Screening of Differential Proteins: The single-cell vesicle membrane proteomics PBA detection platform was used, with a total detection volume of 200 μL. 10 μL of membrane protein sample, 180 μL of detection buffer, and 10 μL of specific antibody mixture were added to each well. The detection procedure was: incubation at 37℃ for 2 hours, followed by single-particle signal acquisition, simultaneously detecting the expression abundance of hundreds of membrane proteins in the samples. Ten samples from both the high-risk and low-risk aGVHD groups were tested in parallel, with three biological replicates and three technical replicates for each group. Based on the membrane protein expression data, fold change and P-value were used as screening criteria to initially screen membrane proteins with significant differences in expression between groups. In this embodiment, a total of 260 characteristic exosomal membrane proteins were detected, of which 25 proteins were upregulated and 17 proteins were downregulated in the high-risk group compared with the low-risk group. Further, based on the expression difference magnitude and statistical significance, 7 core differentially expressed membrane proteins were initially identified: VEGFA, APOE, CXADR, ITGB4, EMCN, DSC3, and Ly6C.

[0057] Step 5: Determination of Membrane Protein Expression Thresholds: Combining the quantitative data from PBA detection, scatter plot analysis, and significance test results, the expression ranges of each protein in the high-risk and low-risk groups were statistically analyzed. Based on ROC curve analysis, the optimal critical thresholds were determined as follows: CXADR: 306.7387; ITGB4: 1979.2075; EMCN: 2601.7398; DSC3: 642.5453. When the expression levels of CXADR, ITGB4, EMCN, DSC3, and Ly6C in a sample are below their corresponding thresholds, and the expression levels of VEGFA and APOE are above their corresponding thresholds, the sample is considered to have an aGVHD positive expression pattern.

[0058] Step Six: Establishment of Disease Risk Grading Criteria: If the measured expression levels of VEGFA and APOE are higher than the corresponding thresholds, and the measured expression levels of CXADR, ITGB4, EMCN, DSC3, and Ly6C are lower than the corresponding thresholds, it is judged that a single indicator shows an abnormal expression pattern of acute graft-versus-host disease and is scored as 1 point; otherwise, it is scored as 0 points. Then, the diagnostic efficacy of each protein is assigned a weight based on the ROC curve, with CXADR, EMCN, and DSC3 each having a weight of 0.18, ITGB4 having a weight of 0.16, and VEGFA, APOE, and Ly6C having a weight of 0.16. Each indicator score was assigned a weight of 0.10. The scores of each indicator were multiplied by their corresponding weights and then summed to obtain the comprehensive risk score. A comprehensive risk score < 0.2 was considered low risk of acute graft-versus-host disease (APVD), and ≥ 0.2 was considered high risk of APVD. Three indicators highly correlated with intestinal injury, namely CXADR, DSC3, and Ly6C, were selected, each with a weight of 0.3333. The intestinal-specific risk score was calculated according to the same scoring rules: an intestinal-specific risk score < 0.2 was considered low risk of intestinal involvement, and ≥ 0.2 was considered high risk of intestinal involvement.

[0059] Step 7: Development of assessment criteria for multi-organ involvement: Based on the expression characteristics of membrane proteins and the clinical sites of involvement, target organ damage is differentiated; intestinal involvement is determined by abnormal expression of intestinal-associated membrane proteins, skin involvement is determined by skin characteristic proteins and markers, and liver involvement is determined by liver-associated proteins, which can accurately distinguish between single-organ involvement and multi-organ involvement.

[0060] Example 2: Compared with Example 1, the only difference in this example is in the preparation steps of the exosome stock solution. In Example 2, an additive solution is added to the peripheral body fluid sample. The additive solution includes neopentyl lauryl maltose glycol, (diacetyl)sophorol, and 1,2-hexanediol. In the additive solution, the mass-to-volume ratio of neopentyl lauryl maltose glycol to 1,2-hexanediol is 8 mg:1 mL, and the mass ratio of neopentyl lauryl maltose glycol to (diacetyl)sophorol is 10:1. The mass of the additive solution is measured by the mass of neopentyl lauryl maltose glycol in it, and the mass-to-volume ratio of neopentyl lauryl maltose glycol to the peripheral body fluid sample is 8 mg:100 mL.

[0061] Example 3: The only difference between this example and Example 2 is in the preparation steps of the exosome stock solution. In Example 3, the mass-volume ratio of lauryl maltose neopentyl glycol to peripheral body fluid sample was adjusted from 8 mg:100 mL to 12 mg:100 mL.

[0062] Example 4: The only difference between this example and Example 3 is in the identification of exosome characteristic proteins. In Example 4, 5-hydroxy-2-pyrrolidone was added to the protein lysis buffer. The mass-volume ratio of 5-hydroxy-2-pyrrolidone to the protein lysis buffer was 1 mg: 4 mL.

[0063] Comparative Example 1: This comparative example provides a traditional acute graft-versus-host disease cytokine detection model. The subjects are the same as in Example 1. Enzyme-linked immunosorbent assay (ELISA) is used to detect the level of cytokines in the peripheral blood of the children. The reagent kit is prepared according to the instructions, the sample dilution ratio is 1:50, the incubation temperature is 37°C, and the incubation time is 1.5h. The disease risk is determined by the cytokine concentration threshold.

[0064] Comparative Example 2: This comparative example used traditional single vesicle detection technology. The detection subjects were the same as in Example 1, with samples divided into high-risk and low-risk groups for acute graft-versus-host disease (APV). The sample source, pretreatment, protein concentration, and experimental replication settings were all kept consistent. The experiment used conventional flow cytometry single vesicle detection equipment and reagents. First, membrane protein samples with a uniform concentration of 1 μg / μL were diluted and mixed with flow cytometry buffer. Then, fluorescently labeled antibodies corresponding to the single target membrane protein were added. Incubation and blocking were completed under light-protected conditions. Finally, the samples were placed in the flow cytometer to collect fluorescence signals.

[0065] Comparative Example 3: The only difference between this comparative example and Example 2 is in the preparation steps of the exosome stock solution. In Comparative Example 3, an additive solution was added to the peripheral body fluid sample. The additive solution included neopentyl lauryl maltose glycol and 1,2-hexanediol. The mass-to-volume ratio of neopentyl lauryl maltose glycol to 1,2-hexanediol in the additive solution was 8 mg:1 mL. The mass of the additive solution was measured by the mass of neopentyl lauryl maltose glycol in it, and the mass-to-volume ratio of neopentyl lauryl maltose glycol to the peripheral body fluid sample was 8 mg:100 mL.

[0066] Comparative Example 4: The only difference between this comparative example and Example 2 is in the preparation step of the exosome stock solution. In Comparative Example 4, an additive solution was added to the peripheral body fluid sample. The additive solution included (diacetyl)sophorol and 1,2-hexanediol. The mass-to-volume ratio of (diacetyl)sophorol to 1,2-hexanediol in the additive solution was 0.8 mg:1 mL. The mass of the additive solution was measured by the mass of (diacetyl)sophorol in it. The mass-to-volume ratio of (diacetyl)sophorol to the peripheral body fluid sample was 0.8 mg:100 mL.

[0067] Comparative Example 5: The only difference between this example and Example 3 is in the identification of exosome characteristic proteins. In Comparative Example 5, 2-pyrrolidone was added to the protein lysis buffer. The mass-volume ratio of 2-pyrrolidone to the protein lysis buffer was 1 mg: 4 mL.

[0068] Experimental Example 1: Detection of the purity and concentration of exosomes.

[0069] Test sample: Exosome stock solution prepared in Example 1.

[0070] Test method: Take 10 μL of exosome stock solution and perform serial dilution with sterile PBS buffer at a dilution ratio of 1:115200. After thorough mixing, take 1 mL of the diluted solution and add it to the nanoparticle tracking and detection instrument for detection. Detection parameter settings: ambient temperature 25℃, 5 detection fields, acquisition time of 60s for each field. Record the exosome concentration after sample dilution, stock solution concentration, average particle size, and particle volume, and count the proportion of particles in the 30~200nm range.

[0071] The purity and concentration detection results of the exosomes prepared in this invention are as follows: Figure 1 As shown, the concentration of exosomes after dilution was 7.1 × 10⁻⁶. 7 The exosome concentration was 8.2 × 10⁶ cells / mL. 12 The exosomes prepared by this invention have a particle size of 119.3 nm, an average particle size of 188.0 ± 87.6 nm, and a particle size of 188.0 ± 87.6 nm. The proportion of particles in the 30 ~ 200 nm range is 99.3%, indicating that the purity of the exosomes prepared by this invention meets the detection requirements.

[0072] Experimental Example 2: Microstructural characterization of exosomes.

[0073] Test sample: Exosome stock solution prepared in Example 1.

[0074] Test method: Take 5 μL of diluted exosome sample and drop it onto a copper mesh carrier film. Let it stand at room temperature for 5 min to absorb the adsorption. Use filter paper to remove excess liquid. Add 2% phosphotungstic acid staining solution for negative staining for 2 min. Remove the staining solution again and let it air dry at room temperature for 30 min. Place the copper mesh into a nano transmission electron microscope and set the accelerating voltage to 120 kV to observe the microstructure of exosomes.

[0075] The microstructure characterization results prepared by this invention are as follows: Figure 2 As shown, the qualified exosomes prepared by this invention exhibit a typical saucer-like, double-membrane structure and appear white after negative staining.

[0076] Experimental Example 3: Identification of characteristic proteins of exosomes.

[0077] Test sample: Exosome stock solution prepared in Example 1.

[0078] Test method: The expression of exosome marker proteins CD9 and CD63 and endoplasmic reticulum contamination marker protein Calnexin was measured by Western blotting.

[0079] The results of the identification of characteristic proteins of exosomes are as follows: Figure 3 As shown in the Western blot results, CD9 and CD63 were highly expressed in the samples, while Calnexin was not expressed. CD9 and CD63 are classic surface characteristic proteins of exosomes, and normal qualified exosome samples should highly express these two proteins. Calnexin is located in the endoplasmic reticulum and is a characteristic marker of impurities such as cell debris and endoplasmic reticulum vesicles. Its absence indicates that the exosomes isolated and prepared in this study were not contaminated by endoplasmic reticulum components and cell debris.

[0080] Experimental Example 4: Validation of differences in membrane protein expression between high-risk and low-risk groups.

[0081] Test sample: Exosome stock solution prepared in Example 1.

[0082] Test method: High-throughput quantitative detection of membrane proteins was performed using PBA technology. Difference analysis was conducted on the 260 detected membrane proteins, and statistical significance was tested. The high-risk group of aGVHD was designated as group A, and the low-risk group of aGVHD was designated as group B.

[0083] The results of the validation experiment on the difference in membrane protein expression between high-risk and low-risk groups are as follows: Figure 4As shown, a total of 7 membrane proteins with statistically significant differences were screened from the two groups of samples (P < 0.05). Among them, CXADR, ITGB4, EMCN, DSC3, and Ly6C were downregulated in the high-risk group of aGVHD, while VEGFA and APOE were upregulated in the high-risk group of aGVHD. This expression pattern can be used as the core basis for distinguishing between high-risk and low-risk children with acute graft-versus-host disease, proving that the differences between the selected marker groups are significant.

[0084] Experimental Example 5: ROC curve validation test of diagnostic efficacy of membrane protein markers.

[0085] Test sample: Quantitative detection data of high- and low-risk membrane proteins in Experiment Example 2.

[0086] Test method: ROC curves were plotted based on quantitative data of membrane protein expression. The area under the curve (AUC), optimal critical threshold, true positive rate (TPR), false positive rate (FPR), sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of each protein were calculated to comprehensively evaluate the diagnostic efficacy of the biomarkers.

[0087] The results of the ROC curve validation test for the diagnostic efficacy of membrane protein biomarkers are shown in Table 1.

[0088] Table 1. Results of ROC curve validation test for diagnostic efficacy of membrane protein biomarkers

[0089] The test results showed that the AUC values ​​of CXADR, EMCN, and DSC3 were all 0.96, and the AUC value of ITGB4 was 0.92. Under the preferred threshold, the detection sensitivity of each protein can reach 100%, the specificity can reach 100%, and the overall accuracy reaches 90%, which proves that the membrane protein biomarkers screened in this invention have excellent diagnostic efficacy, and the detection model constructed based on them has extremely high reliability for clinical application.

[0090] Experimental Example 6: Clinical sample validation of a pediatric graft-versus-host disease membrane protein biomarker detection model.

[0091] Test samples: the membrane protein biomarker detection model prepared in Example 1 and the conventional cytokine model prepared in Comparative Example 1.

[0092] Test method: 15 pairs of paired aGVHD clinical specimens were collected according to the inclusion and exclusion criteria. Inclusion criteria: pediatric patients aged 1 to 14 years undergoing allogeneic hematopoietic stem cell transplantation, who voluntarily signed the informed consent form; exclusion criteria: pediatric patients with inconsistent clinical symptoms and evaluation results of the traditional cytokine model, lost to follow-up, or refusing to cooperate with sampling. The specimens were divided into a diseased group and a treatment-improved group, with 15 cases in each group. The exosome isolation and identification of 15 pairs of specimens were completed according to the method of Example 1, and then the expression levels of 7 core membrane proteins were quantitatively detected according to the PBA detection method of Example 2, with 2 parallel tests set for each sample. The risk stratification and organ involvement results obtained by the detection model of the present invention were compared pairwise with the results of clinical physical examination and the traditional cytokine model, and the sensitivity, specificity and coincidence rate of the model were counted to complete the validity verification of the model. The subject screening process is as Figure 5 shown, and the specific inclusion and exclusion criteria are as Figure 6 shown.

[0093] The overall accuracy of the traditional cytokine model prepared in Comparative Example 1 is about 72%, the single-sample detection time is about 2.5 hours, it can only detect a single index, and cannot distinguish the involvement of multiple organs such as skin, intestine and liver; the overall accuracy of the detection model of the present invention reaches more than 90%, relying on PBA technology to realize simultaneous high-throughput detection of multiple proteins, the single-sample detection time is shortened to 1 hour, and the risk of involvement of different target organs can be accurately evaluated. Compared with the traditional technology, the present invention has obvious advantages in terms of accuracy, detection efficiency and functional dimension.

[0094] Experimental Example 7: Performance comparison test of different vesicle detection technologies.

[0095] Test samples: extracellular vesicle membrane protein solutions prepared in Example 1 and Comparative Example 2.

[0096] Test method: The single-cell vesicle membrane proteomics PBA detection platform supporting the present invention was used to carry out detection according to the established experimental system and reaction conditions, and the number of membrane proteins that can be detected simultaneously in a single run, the detection type and the overall time consumption of a single sample were counted. The traditional single-vesicle detection technology was adopted, and the conventional flow single-vesicle detection equipment was used to perform parallel tests on the same sample under the same environmental conditions, and the detection throughput, detection attributes and single-sample detection time of this technology were recorded.

[0097] Traditional single-vesicle detection can only make qualitative judgments based on the intensity of fluorescence signals, and cannot accurately quantify the abundance of membrane protein expression or determine the critical threshold for protein expression. The entire process of detecting 7 target proteins in a single sample takes about 4 hours, resulting in low overall throughput and a long detection cycle. This technology can only simply determine the protein expression status and cannot build a quantitative assessment model, nor can it achieve risk stratification of acute graft-versus-host disease in children or assess the extent of multi-organ involvement. Overall, its detection efficiency and clinical applicability are poor. PBA technology can achieve high-throughput, simultaneous quantitative detection of multiple proteins in a single particle, detecting hundreds of membrane proteins in a single run, with a single sample detection time of 1.2 hours. PBA technology perfectly solves the shortcomings of traditional technologies, such as low throughput and inability to accurately quantify, and is suitable for the needs of large-scale clinical sample screening.

[0098] Experiment 8: Validation of the detection efficiency of exosome characteristic proteins.

[0099] Test samples: Exosomal membrane protein solutions prepared in each example and comparative example.

[0100] Test method: Western blotting was used to detect the relative expression abundance of CD63, a characteristic protein of exosomes, in each group of samples. The entire process was carried out at room temperature of 25℃. Three parallel experiments were set up for each group, and the average value of the parallel experiments was taken as the test result.

[0101] The results of the detection efficiency verification of exosome characteristic proteins are shown in Table 2.

[0102] Table 2. Validation results of the detection efficiency of exosome characteristic proteins

[0103] The detection protocol in Example 1, with its standardized and stable exosome isolation and membrane protein preparation process, can normally detect CD63 characteristic protein, meeting the basic requirements for detecting membrane proteins in pediatric graft-versus-host disease. Comparative Example 3 only added dodecyl-β-D-maltose, and Comparative Example 4 only added (diacetized) sophorolactone. Compared to Example 1, the relative expression abundance of CD63 only slightly increased, indicating that using (diacetized) sophorolactone alone can only slightly optimize the lipid environment of exosomes, with limited protective effect on membrane proteins. Example 2 combined dodecyl-β-D-maltose with (diacetized) sophorolactone, and the CD63 expression level... For groups where the expression abundance was higher than that of either substance alone; Example 3 adjusted the addition ratio of dodecyl-β-D-maltodextrin based on Example 2, further optimizing the exosome pretreatment system, and the CD63 expression abundance continued to increase; Example 4 added 5-hydroxy-2-pyrrolidone to the mature system of Example 3, and the relative expression abundance of CD63 reached the highest value among all groups, and the whole system achieved the best protective effect on exosome structure and membrane proteins; Comparative Example 5 only replaced the additive in the protein cleavage stage with 2-pyrrolidone, and the exosome pretreatment system was consistent with Example 3, and the CD63 expression level was slightly lower than that of Example 3.

[0104] Experimental Example 9: Evaluation of Exosome Membrane Protein Integrity.

[0105] Test samples: Exosomal membrane protein solutions prepared in each example and comparative example.

[0106] Test method: The membrane protein integrity of each group of samples was comprehensively evaluated by combining Western blotting and quantitative protein detection methods. During the experiment, all experimental conditions, such as temperature, reaction time, and reagent ratio, were strictly controlled to remain consistent. Each group was tested five times, and the average value was taken as the final test data.

[0107] The results of the exosome membrane protein integrity evaluation test are shown in Table 3.

[0108] Table 3. Results of the exosome membrane protein integrity evaluation test

[0109] Example 1 demonstrates a stable extraction and preparation process, providing a reliable foundation for detecting membrane proteins in pediatric graft-versus-host disease. Comparative Example 4, with only (diacetyl)sophorol, showed lower membrane protein integrity than Example 1. This is because this component only acts on the lipid bilayer structure of exosomes and cannot interfere with the hydrolytic degradation process of membrane proteins during lysis and extraction; its use alone cannot adequately maintain the intact conformation of membrane proteins. Comparative Example 3, with only neopentyl lauryl maltose glycol, showed slightly higher membrane protein integrity than Example 1. This component only reduces exosome damage by improving vesicle dispersion, indirectly reducing membrane protein loss but not directly preventing protein denaturation. Example 2, by simultaneously introducing both neopentyl lauryl maltose glycol and (diacetyl)sophorol, improved membrane protein integrity compared to Comparative Examples 3 and 4, which used single-component additions. Example 3 further adjusted the amount of neopentyl lauryl maltose glycol, resulting in a slight increase in membrane protein integrity compared to Example 2. Example 4, based on the pretreatment system of Example 3, added 5-hydroxy-2-pyrrolidone to the protein lysis buffer, resulting in the highest membrane protein integrity among all groups. This substance can bind to the active sites of proteases in the sample to inhibit protein hydrolysis, and simultaneously form hydrogen bonds with polar groups of membrane proteins through hydroxyl groups, maintaining the native spatial structure of the proteins and reducing aggregation and precipitation. Comparative Example 5 replaced the lysis buffer additive with 2-pyrrolidone, while keeping other conditions consistent with Example 3. The final membrane protein integrity was lower than that of Example 4, demonstrating that 5-hydroxy-2-pyrrolidone has a better protective effect on membrane proteins than 2-pyrrolidone.

[0110] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0111] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for detecting membrane protein markers of acute graft-versus-host disease in children, characterized in that: The characteristic biomarkers of the detection method are six extracellular vesicle membrane proteins: VEGFA, APOE, CXADR, ITGB4, EMCN, and DSC3. The detection sample is a peripheral body fluid sample from pediatric allogeneic hematopoietic stem cell transplant recipients. In pediatric samples at high risk of acute graft-versus-host disease, the expression levels of CXADR, ITGB4, EMCN, and DSC3 are downregulated, while the expression levels of VEGFA and APOE are upregulated.

2. The detection method according to claim 1, characterized in that: The expression levels of CXADR, ITGB4, EMCN, and DSC3 are below the corresponding thresholds, which are judged as positive expression patterns of acute graft-versus-host disease; the thresholds for CXADR are 300-310, ITGB4 is 1970-2000, EMCN is 2600-2620, and DSC3 is 640-660.

3. The detection method according to claim 1, characterized in that: The detection method includes exosome preparation, identification of exosome characteristic proteins, preparation of exosome membrane proteins, membrane protein difference detection unit and result determination.

4. The detection method according to claim 3, characterized in that: The exosome preparation method employs differential centrifugation combined with ultrafiltration to prepare exosome stock solution. The differential centrifugation includes centrifugation at 250~350×g for 8~12 min, centrifugation at 1800~2200×g for 18~22 min, and centrifugation at 9000~11000×g for 28~32 min.

5. The detection method according to claim 4, characterized in that: In the preparation of the exosomes, an additive solution is added to the peripheral body fluid sample. The additive solution includes lauryl maltose neopentyl glycol, (diacetyl) sophorol, and 1,2-hexanediol.

6. The detection method according to claim 5, characterized in that: The mass-to-volume ratio of neopentyl lauryl maltose glycol to 1,2-hexanediol is 4-12 mg:1 mL, and the mass-to-volume ratio of neopentyl lauryl maltose glycol to (diacetyl)sophorol lactone is 5-20:1; the mass of the additive solution is measured by the mass of neopentyl lauryl maltose glycol in it, and the mass-to-volume ratio of neopentyl lauryl maltose glycol to peripheral body fluid sample is 5-15 mg:100 mL.

7. The detection method according to claim 3, characterized in that: The exosome characteristic protein identification unit uses CD9 and CD63 as positive marker proteins for exosomes and Calnexin as a negative marker protein for contamination. When CD9 and CD63 are highly expressed in a sample and Calnexin is not expressed, it is judged as a qualified exosome sample.

8. The detection method according to claim 1, characterized in that: The detection method uses a scoring and weighting method to calculate the comprehensive risk score: 1 point is awarded for abnormal expression of a single indicator, and 0 points are awarded for normal expression; the weights of CXADR, EMCN, and DSC3 are 0.17~0.19, the weight of ITGB4 is 0.15~0.17, and the weights of VEGFA and APOE are 0.09~0.10; a comprehensive risk score <0.2 is considered low risk, and a comprehensive risk score ≥0.2 is considered high risk.

9. The detection method according to claim 1, characterized in that: The detection method includes an intestinal-specific risk assessment mechanism, selecting CXADR and DSC3 indicators, each with a weight of 0.3~0.4; an intestinal-specific risk score <0.2 is considered as low risk of intestinal involvement, and a score ≥0.2 is considered as high risk of intestinal involvement; combined with biomarker expression characteristics, the condition of skin and liver damage can be assessed, and the type of single organ or multiple organ involvement can be distinguished.

10. The application of the detection method according to any one of claims 1 to 9, characterized in that: The detection method is applied to the preparation of one or more of the following: early screening reagents, risk grading reagents, and multi-organ involvement detection reagents for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in children.

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