Method for separating gastric juice exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions and application

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a method and application for separating gastric juice exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions, and the method comprises the following steps: obtaining a gastric juice sample of a subject; separating the exosome from the gastric juice sample; detecting the expression level of the protein CD13 in the exosome to obtain a detection value; and comparing the detection value with a reference value, and predicting the risk of the subject suffering from early gastric cancer and / or gastric precancerous lesions according to a comparison result. According to the invention, stable exosomes are separated and identified from gastric juice, and protein markers CD13 and CEACAM5, which are significantly up-regulated in the gastric juice exosomes of patients with early gastric cancer and precancerous lesions, are screened out. Clinical verification results show that precancerous lesions and early gastric cancer patients can be distinguished with high sensitivity and high specificity by detecting the CD13 in the gastric exosome and particularly combining with CEACAM5 for sequential detection, and huge clinical application potential is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method and application for isolating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors worldwide, ranking among the top in both incidence and mortality, posing a serious threat to human health. Because early-stage gastric cancer symptoms are often atypical, most patients are diagnosed at an advanced stage, missing the optimal treatment window and resulting in poor prognosis and high mortality. Currently, the gold standard for diagnosing gastric cancer is upper gastrointestinal endoscopy combined with pathological biopsy; however, this method is invasive, costly, and unsuitable for large-scale population screening.

[0003] Clinically used serum tumor markers, such as carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and carbohydrate antigen 72-4 (CA72-4), have been used for the auxiliary diagnosis and monitoring of gastric cancer, but their positive rate in early gastric cancer is extremely low, and their sensitivity and specificity are not ideal, which cannot meet the needs of early screening.

[0004] In recent years, new technologies, such as liquid biopsy, which detects circulating tumor DNA (ctDNA) and microRNA (miRNA), have provided new insights into the diagnosis of gastric cancer. However, these methods are mostly based on blood samples, which circulate throughout the body, and their results may be affected by diseases in other parts of the body, thus limiting their specificity. Furthermore, many emerging diagnostic models involve multiple indicators, resulting in complex and costly testing procedures, hindering their widespread clinical application.

[0005] Unlike gastric mucosal biopsy, which has limited sampling capabilities, and blood samples, which are mobile throughout the body, gastric juice, secreted directly by gastric epithelial cells, provides the most direct and comprehensive reflection of the pathophysiological state of the gastric microenvironment, making it an ideal source for obtaining gastric cancer-specific biomarkers. However, the highly acidic environment and abundant digestive enzymes in gastric juice rapidly degrade proteins, leading to poor stability and unreliable results from direct detection of protein biomarkers in gastric juice. Exosomes, nanoscale vesicles secreted by cells, possess a bilayer lipid membrane structure that effectively protects their encapsulated proteins, nucleic acids, and other biomolecules from degradation and transports them to the microenvironment and even distant sites to exert regulatory effects.

[0006] Therefore, developing a novel biomarker based on gastric juice samples, utilizing exosomes as a stable carrier, and possessing high sensitivity and specificity for screening and early warning of early gastric cancer and precancerous lesions has significant clinical implications and application value. Based on the above, this invention proposes a method and application for isolating gastric juice exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions. Summary of the Invention

[0007] To address the shortcomings of existing early gastric cancer screening methods, particularly the low sensitivity and specificity of current serum biomarkers, poor organ specificity of blood samples, and poor stability of direct detection of gastric juice proteins due to degradation, this invention proposes a method and application for isolating gastric juice exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions. The biomarkers, detection methods, applications, and kits of this invention for assessing the risk of early gastric cancer and precancerous lesions have high sensitivity and high specificity, which can significantly improve the early detection rate of gastric cancer and improve patient prognosis.

[0008] In a first aspect, the present invention provides a method for separating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions, employing the following technical solution: A method for isolating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions includes the following steps: S1. Obtain gastric fluid samples from the subject; S2. Isolate exosomes from gastric juice samples; S3. Detect the expression level of the protein CD13 in exosomes and obtain the detection value; S4. Compare the detected values ​​with reference values, and predict the risk of the subject developing early gastric cancer and / or precancerous lesions based on the comparison results.

[0009] Preferably, the separation in step S2 is performed using differential centrifugation and / or ultracentrifugation.

[0010] Preferably, step S3 further includes detecting the expression level of the protein CEACAM5 in exosomes.

[0011] Preferably, the detection in step S3 is performed using enzyme-linked immunosorbent assay (ELISA), Western blot, immunogold assay, magnetic bead microsphere assay, or nanoflow cytometry.

[0012] This invention discovers that CD13 and CEACAM5 are both located on the exosome membrane, making them highly suitable for detection methods based on the antigen-antibody binding principle.

[0013] Preferably, the reference value in step S4 is a statistical value (e.g., mean or median) of the expression level of protein CD13 in gastric exosomes of healthy individuals or patients with chronic superficial gastritis, or a preset cut-off value determined by receiver operating characteristic (ROC) curve analysis.

[0014] Preferably, in step S4, when the comparison result is that the detected value is higher than the reference value, it indicates that the subject has an increased risk of early gastric cancer and / or precancerous lesions of the stomach.

[0015] Preferably, the precancerous lesions in step S4 include gastric mucosal atrophy, intestinal metaplasia, and / or dysplasia.

[0016] Secondly, the present invention provides an application of CD13 in the preparation of an in vitro diagnostic reagent for predicting early gastric cancer and precancerous lesions, using the following technical solution: Application of CD13 in the preparation of in vitro diagnostic reagents for predicting early gastric cancer and precancerous lesions Thirdly, this invention provides an application of CD13 combined with CEACAM5 in the preparation of an in vitro diagnostic reagent for predicting early gastric cancer and precancerous lesions, using the following technical solution: Application of CD13 combined with CEACAM5 in the preparation of an in vitro diagnostic reagent for predicting early gastric cancer and precancerous lesions.

[0017] Fourthly, this invention provides a kit for predicting the risk of early gastric cancer and precancerous lesions, employing the following technical solution: A kit for predicting the risk of early gastric cancer and precancerous lesions includes a first detection reagent for specifically recognizing and binding to the protein CD13 derived from gastric exosomes.

[0018] Preferably, the first detection reagent is an anti-CD13 antibody.

[0019] Preferably, the kit further comprises a second detection reagent for specifically recognizing and binding to the protein CEACAM5 derived from gastric exosomes.

[0020] Preferably, the second detection reagent is an anti-CEACAM5 antibody.

[0021] In summary, the present invention has the following beneficial effects: 1. High sample source specificity: This invention uses gastric juice as the test sample, which comes directly from the diseased environment of the stomach. Compared with systemic samples such as blood, it can more accurately and specifically reflect the pathological state of the gastric mucosa and reduce the interference of other organ diseases.

[0022] 2. Good biomarker stability: This invention detects protein biomarkers in exosomes and utilizes the natural membrane structure of exosomes to protect proteins from degradation by strong gastric acid and proteases, ensuring the stability and reliability of the detection results.

[0023] 3. High sensitivity and specificity for early lesions: The biomarker CD13 and its combination with CEACAM5 provided by this invention have extremely high diagnostic efficacy for early gastric cancer and precancerous lesions (such as dysplasia). In particular, the combined detection of the two biomarkers has significantly better diagnostic performance (area under the ROC curve 0.84, specificity 94.12%) than a single biomarker and far superior to traditional serum tumor markers, enabling effective screening and early warning for high-risk populations.

[0024] 4. Non-invasive and convenient, easy to promote in clinical practice: Gastric fluid samples can be routinely collected during gastroscopy, causing no additional trauma to patients. The detection method can use the mature ELISA technology, which is simple to operate, low in cost, and suitable for promotion and application in medical institutions at all levels.

[0025] In summary, this invention successfully isolated and identified stable exosomes from gastric juice, and through proteomics screening, identified the protein markers CD13 and CEACAM5, which are significantly upregulated in gastric juice exosomes from patients with early gastric cancer and precancerous lesions. Clinical validation results show that detecting CD13 in gastric juice exosomes, especially in conjunction with sequential detection of CEACAM5, can distinguish between precancerous lesions and early gastric cancer patients with high sensitivity and specificity, demonstrating significant clinical application potential. Attached Figure Description

[0026] Figure 1 The images show the identification results of exosomes in the gastric fluid of a patient in Example 1 of this invention. A is a transmission electron microscope image, and B is a particle size analysis diagram.

[0027] Figure 2 The results of Western blot analysis of the expression levels of marker proteins CD63, TSG101, and CD81 in gastric exosomes from patients with different gastric mucosal diseases in Example 1 of this invention.

[0028] Figure 3 This is a transmission electron microscope image showing the effect of the gastrointestinal defoaming agent on the integrity of exosomes secreted by in vitro cultured cells in Example 2 of the present invention.

[0029] Figure 4 This is a clustering heatmap (A) and a KEGG pathway enrichment analysis diagram (B) of differentially expressed proteins in gastric exosomes from patients in the early gastric cancer group and gastritis group in Example 3 of the present invention.

[0030] Figure 5 This is a volcano diagram of differentially expressed proteins in gastric exosomes from patients in the early gastric cancer group and the gastritis group in Example 3 of the present invention.

[0031] Figure 6 The results of immunohistochemical staining of some differentially expressed proteins (CD13 and FN1) in gastric cancer and gastritis tissues in Example 4 of this invention are shown.

[0032] Figure 7 The immunohistochemical staining results show the expression level of CD13 in tissues at different stages of gastric mucosal disease development (chronic gastritis, dysplasia, and early gastric cancer) in Example 4 of this invention.

[0033] Figure 8 This is the Western blot verification and immunogold localization result of CD13 expression level in gastric exosomes of the gastric cancer group and gastritis group in Example 4 of the present invention.

[0034] Figure 9 This is a receiver operating characteristic (ROC) curve of CD13 combined with CEACAM5 in gastric exosomes in Example 5 of the present invention for predicting precancerous lesions and early gastric cancer. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the embodiments.

[0036] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0037] Example 1: Gastric fluid sample collection and exosome verification 1. Sample Collection: This invention collects gastric fluid from patients with early-stage gastric cancer undergoing painless gastroscopy or treatment. Patients fasted for at least 8 hours and abstained from water for at least 4 hours before the examination. When the endoscope is inserted into the esophagus and reaches the bottom of the stomach, negative pressure suction is activated, a disposable suction catheter is connected, and 10 mL of residual fluid from the gastric cavity is aspirated through the biopsy channel. The fluid is immediately placed on ice and transferred to a -80°C freezer for later storage within 30 minutes.

[0038] 2. Exosome Extraction: After thawing the frozen gastric fluid sample at 4°C, centrifuge at 3000 g for 10 min at 4°C, discarding the precipitate (cells and large cell debris). Centrifuge the supernatant at 15000 g for 30 min at 4°C, discarding the precipitate (bacteria, etc.). Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100000 g for 120 min at 4°C. Carefully discard the supernatant; the precipitate is the crude exosome extract. Resuspend the precipitate in 10 mL of pre-chilled PBS at 4°C, and centrifuge again at 100000 g for 120 min at 4°C to wash away residual proteins. After discarding the supernatant, finally resuspend the precipitate in 100 μL of sterile PBS (pH 7.4) to obtain the exosome suspension.

[0039] 3. Exosome verification: Morphological observation: 50 μL of exosome suspension was incubated with 4% osmium tetroxide at 4℃ for 30 min, then dropped onto a copper grid that had undergone glow discharge treatment. The grid was allowed to stand for 2 min to allow adsorption. Excess liquid was blotted away with filter paper, and 1% phosphotungstic acid solution (pH 7.0) was added for negative staining for 1 min. The grid was then blotted dry again with filter paper. After the copper grid was completely dry, it was observed and photographed using a JEM-1400 transmission electron microscope (TEM) at an accelerating voltage of 60 kV. The results are as follows: Figure 1 As shown in Figure A, a clear and intact exosome structure is visible. Particle size analysis: Particle size distribution was determined using a Zetasizer NanoZS dynamic light scattering (Malvern Instruments, UK) particle size analyzer or a nano-particle tracking analysis (NTA) system. Results are as follows... Figure 1 As shown in B, the particle size of the exosomes conforms to the common exosome particle size.

[0040] Biomarker detection: To further investigate whether there are differences in gastric exosomes among patients with different gastric mucosal diseases, 10 mL of gastric juice was collected from patients with chronic gastritis, gastric polyps, dysplasia, and gastric cancer. Exosomes were extracted according to the method in Example 1 and analyzed by Western blot. The results are as follows: Figure 2 As shown in the figure, #1, #2, #3, and #4 are gastric exosome samples from patients with chronic gastritis, gastric polyps, dysplasia, and gastric cancer, respectively. It can be seen that the abundance of the exosome marker CD63 increases sequentially in each disease. At the same time, the expression of common exosome marker proteins TSG101 and CD81 differs in different samples, suggesting that there are differences in the abundance and protein expression of exosomes in gastric juice from patients with different gastric mucosal diseases. Protein molecules in gastric exosomes may serve as diagnostic and early warning markers for the occurrence and development of different gastric mucosal diseases.

[0041] Example 2: Effect of defoaming agents on exosome stability in gastroscopy Because the endoscopic field of view is relatively small, and gastric mucus can easily affect the intraoperative view, patients need to take an antifoaming agent before the actual gastroscopy procedure. To avoid the antifoaming agent interfering with the structure and integrity of exosome vesicles, we investigated whether the antifoaming agent affects the stability of exosomes through in vitro cell experiments.

[0042] The culture media of the immortalized gastric epithelial cell line GES-1 and the gastric cancer cell line BGC823, which had previously been treated with endoscopic defoaming agent (mainly composed of simethicone and gastrin), were designated as the treatment group, while those without the endoscopic defoaming agent were designated as the untreated group. Both groups were treated at 37°C for 1 h. The culture supernatant was collected, and exosomes were extracted according to the method in Example 1. The exosome structure was examined by transmission electron microscopy. The results are as follows: Figure 3As shown, the exosomes in the right-hand treatment group had intact morphology and structure, with no significant difference from the left-hand untreated group. This indicates that the gastroscopy defoaming agent does not affect the integrity of the exosome structure, and the collection of gastric fluid samples does not affect normal endoscopic examination and treatment procedures, making it convenient and feasible.

[0043] Example 3: Proteomics Analysis of Gastric Juice Exosomes and Screening of Core Biomarkers Gastric fluid was collected from 8 patients with early gastric cancer who underwent painless gastroscopy or treatment, and gastric fluid was collected from 7 patients with chronic gastritis who were matched by age and gender. Exosomes were extracted according to the method in Example 1. After quality inspection, proteomics methods were used to detect and analyze the differential proteins between the two groups.

[0044] Differential protein analysis: based on a fold change > 2 and P A value <0.05 was used as the screening criterion, and a total of 53 differentially expressed proteins were identified, of which 21 were upregulated and 32 were downregulated. Figure 4 A). Volcano map ( Figure 5 The distribution of differentially expressed proteins was visually demonstrated, among which aminopeptidase N (CD13) was significantly upregulated by 7.45-fold in the gastric cancer group. P =0.043), carcinoembryonic antigen-associated cell adhesion molecule 5 (CEACAM5) was significantly upregulated by 29.83-fold ( ). P =0.011), these results may suggest that, compared with normal gastric mucosa, the expression levels of proteins such as CD13 and CEACAM5 are significantly upregulated during the early development and progression of gastric cancer, and may play an important regulatory role in tumor cells and tumor microenvironment through increased exosome secretion.

[0045] Functional enrichment analysis: Further KEGG pathway enrichment analysis showed that differentially expressed proteins were mainly enriched in glucose and lipid metabolism pathways, immune regulation pathways, and PPAR signaling pathways. Figure 4 B), these pathways are closely related to the occurrence and development of tumors.

[0046] Example 4: Validation and localization of core biomarkers in tissues and exosomes 1. In situ validation: Immunohistochemical staining (IHC) was used to verify whether the expression levels of differentially expressed proteins in gastric exosomes in situ on the gastric mucosa were consistent with those in gastric exosomes. The results are as follows: Figure 6 As shown, the expression levels of CD13 and FN1 in the gastric mucosa of patients with gastric cancer were significantly higher than those in the gastric mucosa of patients with gastritis. The differential protein expression levels in gastric exosomes were consistent with the in situ tissue results, which can reflect the in situ protein expression levels in tissues.

[0047] The expression level of CD13 in different stages of gastric mucosal diseases was further explored using immunohistochemical (IHC) staining. The results are as follows: Figure 7As shown, CD13 expression is low in patients with chronic superficial gastritis, and gradually increases with the development of gastric mucosal diseases. It is significantly higher in the dysplasia and early gastric cancer stages than in the chronic superficial gastritis stage, with statistically significant differences. While CD13 expression further increased between the dysplasia and early gastric cancer groups, there was no statistically significant difference. This suggests that CD13 can effectively screen for precancerous lesions and early gastric cancer, enabling diagnosis at an early stage of tumor development.

[0048] 2. Exosome verification and localization: Western blot validation: Gastric juice was collected from patients with chronic superficial gastritis and patients with early gastric cancer. Exosomes were extracted according to the method in Example 1, and the expression of CD13 in the gastric juice exosomes was detected by Western blot. The results are as follows: Figure 8 As shown in Figure A, the expression level of CD13 in gastric exosomes of the gastric cancer group was significantly higher than that of the gastritis group, further validating the proteomics results.

[0049] Through electron microscopy and immunogold staining, we found that CD13 is mainly expressed on the gastric exosome membrane, while previous studies have confirmed that CEACAM5 is expressed on the exosome membrane. This lays an important foundation for its subsequent detection by direct antigen-antibody recognition.

[0050] Immunogold localization: The results of the immunogold staining experiment under electron microscopy are as follows... Figure 8 As shown in B, CD13 is mainly expressed on the gastric exosome membrane, while previous studies have confirmed that CEACAM5 is expressed on the exosome membrane. This lays an important foundation for its subsequent detection by direct antigen-antibody recognition.

[0051] Example 5: Clinical diagnostic efficacy assessment of CD13 combined with CEACAM5 in a validation cohort Based on the localized expression of CD13 on exosome membranes, the detection method was optimized. This invention collected gastric fluid from patients undergoing gastroscopy to form a validation cohort; baseline data for the validation cohort are shown in Table 1. After processing using a freeze-thaw method, CD13 and CD63 were detected using an ELISA kit.

[0052] Table 1. Verification Queue Baseline Information The results are as follows Figure 9As shown, CD13 levels were significantly elevated in both the precancerous lesion and early gastric cancer groups, showing a significant difference compared to the chronic superficial gastritis group. ROC curve calculations using CD63 to standardize CD13 indicated good diagnostic value for precancerous lesions and early gastric cancer, with an area under the curve (AUC) of 0.78, a cut-off value of 35.41, and a sensitivity of 90.91%. Sequential screening using CEACAM5 in high-risk groups for precancerous lesions and early gastric cancer yielded an AUC of 0.84, a cut-off value of 35.42, and a specificity of 94.12%. In conclusion, sequential screening using CD13 combined with CEACAM5 demonstrates high sensitivity and specificity for patients with precancerous lesions and early gastric cancer, and this targeted screening approach has significant screening value and economic benefits.

[0053] In summary, this invention successfully isolated and identified stable exosomes from gastric juice, and through proteomics screening, identified the protein markers CD13 and CEACAM5, which are significantly upregulated in gastric juice exosomes from patients with early gastric cancer and precancerous lesions. Clinical validation results show that detecting CD13 in gastric juice exosomes, especially in conjunction with sequential detection of CEACAM5, can distinguish between precancerous lesions and early gastric cancer patients with high sensitivity and specificity, demonstrating significant clinical application potential.

[0054] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for separating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions, characterized in that, Includes the following steps: S1. Obtain gastric fluid samples from the subject; S2. Isolate exosomes from gastric juice samples; S3. Detect the expression level of the protein CD13 in exosomes and obtain the detection value; S4. Compare the detected values ​​with reference values, and predict the risk of the subject developing early gastric cancer and / or precancerous lesions based on the comparison results.

2. The method for separating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions according to claim 1, characterized in that, In step S2, the separation is performed using differential centrifugation and / or ultracentrifugation.

3. The method for separating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions according to claim 1, characterized in that, Step S3 also includes detecting the expression level of the protein CEACAM5 in exosomes.

4. The method for separating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions according to claim 1, characterized in that, The detection in step S3 is performed using enzyme-linked immunosorbent assay (ELISA), Western blot, immunogold assay, magnetic bead microsphere assay, or nanoflow cytometry.

5. The method for separating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions according to claim 1, characterized in that, In step S4, the reference value is the statistical value of the expression level of protein CD13 in gastric exosomes of healthy individuals or patients with chronic superficial gastritis, or a preset threshold value.

6. The method for separating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions according to claim 1, characterized in that, In step S4, when the comparison result is higher than the reference value, it indicates that the subject has an increased risk of early gastric cancer and / or precancerous lesions of the stomach.

7. The method for separating gastric exosomes and detecting CD13 to predict early gastric cancer and precancerous lesions according to claim 1, characterized in that, The precancerous lesions in step S4 include gastric mucosal atrophy, intestinal metaplasia, and / or dysplasia.

8. The use of CD13 according to any one of claims 1-7 in the preparation of an in vitro diagnostic reagent for predicting early gastric cancer and precancerous lesions.

9. The use of CD13 combined with CEACAM5 as described in any one of claims 1-7 in the preparation of an in vitro diagnostic reagent for predicting early gastric cancer and precancerous lesions.

10. A kit for predicting the risk of early gastric cancer and precancerous lesions, characterized in that, The kit contains a first detection reagent for specifically recognizing and binding to the protein CD13 derived from gastric exosomes and / or a second detection reagent for specifically recognizing and binding to the protein CEACAM5 derived from gastric exosomes.

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