A marker, detection reagent, kit and application for gastric fluid exosomes

Abstract

The present invention relates to the field of biotechnology, and specifically to a marker, a detection reagent, a kit and an application for gastric exosomes, wherein the marker of gastric exosomes is at least one of TFF2 protein and AKR1B10 protein. The present invention provides an identification basis for the diagnosis of gastric diseases based on exosomes by screening out specific biomarkers of gastric exosomes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a marker, a detection reagent, a kit and applications for gastric fluid exosomes. Background Art

[0002] Gastric juice is a digestive fluid produced in the stomach. The stomach continuously produces gastric juice at a rate of 30 mL / hour. Under different stimuli, this rate will increase to 2 times, so the stomach eventually produces 1.5-3.5 L of gastric juice per day. The main feature of gastric juice is the high H+ concentration, which is 3 million times the H+ concentration in the blood and tissues. Gastric H+ is secreted by highly specialized parietal cells located in the body of the stomach. Gastric juice also contains lipase and pepsin. The main function of gastric juice is to digest food proteins and inactivate potential pathogenic microorganisms, thereby preventing intestinal infections. Because gastric juice is in direct contact with gastric lesions, the role of gastric juice in the diagnosis of gastric diseases has been increasingly valued in recent years. Studies have reported that gastric juice analysis can be used for a variety of clinical purposes, including the detection of Helicobacter pylori infection, atrophic gastritis, intestinal metaplasia, and cancer.

[0003] Exosomes are nano-scale vesicles with a lipid bilayer structure secreted by cells, with a diameter of <200 nm. They can encapsulate important substances such as nucleic acids, proteins, and lipids. They are effective carriers of intercellular communication and participate in various physiological and pathological processes. The contents of exosomes can reflect the pathological conditions of the cells from which they originate, and their lipid bilayer structure makes the encapsulated contents difficult to degrade, so they are considered to be a new diagnostic marker with great potential.

[0004] Exosomes are widely present in a variety of body fluids, such as blood, urine, pleural effusion, and ascites. At present, blood-derived exosomes are the most commonly used for screening disease biomarkers, but the proportion of exosomes derived from lesion tissues in blood exosomes is low. In contrast, since gastric fluid can directly contact gastric lesion tissues and has strong tissue specificity, gastric fluid exosomes can more closely reflect the composition of biomarkers in gastric lesion tissues. At present, there are few studies on gastric fluid exosomes, mainly focusing on their role in the diagnosis of gastric cancer. The content of bacterial exosomes derived from Helicobacter pylori (H.pylori) in the gastric fluid of patients with gastric adenocarcinoma increases, mainly by specifically targeting gastric epithelial cells to induce inflammation. Methylation analysis of BARHL2 using gastric fluid-derived exosome DNA can be used for early detection of gastric cancer that is not dependent on Helicobacter pylori. In addition, gastric fluid exosomes also play an important role in functional dyspepsia. hsa-miR-933 in gastric fluid exosomes may be a potential biomarker for functional dyspepsia. However, the function of gastric fluid exosomes has remained largely unexplored.

[0005] Exosome biomarkers are important indicators for the isolation and identification of exosomes. Currently, tetraspanins CD9, CD63, and CD81 are the most commonly used exosome biomarkers. However, these markers have heterogeneity in their ability to identify exosomes from different cell types. Our previous studies also found that in exosomes derived from pleural effusion, the percentage of CD81-positive particles was the highest, while the percentage of CD63-positive particles was the lowest. These studies show that the currently commonly used tetraspanins have limited effects in identifying exosomes from different cell sources.

[0006] In addition, exosomes from different tissues and body fluids have their own specific biomarkers. Ldb3 was identified as a potential marker for cardiac exosomes, and compared with other tissues, Ldb3 was almost exclusively detected in the heart of neonatal rats; compared with cardiac fibroblasts, it was detected in cardiomyocytes. Therefore, it is desirable to find specific biomarkers for gastric fluid exosomes. Summary of the invention

[0007] The purpose of the present invention is to provide a marker, a detection reagent, a kit and an application for gastric exosomes. The specific biomarkers of gastric exosomes provide an identification basis for the diagnosis of gastric diseases based on exosomes.

[0008] The present invention solves the technical problem by adopting the following technical solutions:

[0009] In a first aspect, the present invention provides a marker for gastric exosomes, wherein the marker is at least one of TFF2 protein and AKR1B10 protein.

[0010] In the study of exosomes from various body fluids, it is first necessary to identify the separation and purification effect of exosomes. Currently, the commonly used identification methods mainly include the identification of exosome particle size and morphology, as well as the identification of universal markers of exosomes. Studies have reported that exosomes from different body fluids have their own specific biomarkers. This study confirmed that the TFF2 molecule is a specific marker for gastric fluid-derived exosomes, and the expression of TFF2 in exosomes can be detected by Western blotting, nanoflow cytometry and other methods ( Fig.15 , 19 , 21, 24, 28, 32, 36), thereby using TFF2 to identify the separation effect of gastric fluid exosomes, laying the foundation for research based on gastric fluid exosomes.

[0011] In some embodiments of the present invention, the marker is TFF2 protein.

[0012] In some embodiments of the present invention, the gastric exosomes are gastric exosomes prepared by ultracentrifugation (UC).

[0013] In a second aspect, the present invention provides a detection reagent for gastric exosomes, wherein the detection reagent comprises the above-mentioned marker.

[0014] In a third aspect, the present invention provides a detection kit for gastric fluid exosomes, wherein the detection kit comprises the above-mentioned marker, or the detection kit comprises the above-mentioned detection reagent.

[0015] In a fourth aspect, the present invention provides use of the above-mentioned marker in preparing a diagnostic kit for gastric diseases.

[0016] In some embodiments of the present invention, the marker is TFF2 protein.

[0017] In some embodiments of the present invention, the gastric disease includes at least one of atrophic gastritis, intestinal metaplasia, and gastric cancer.

[0018] In some embodiments of the present invention, the gastric disease is gastric cancer.

[0019] The present invention has the following beneficial effects: the present invention provides an identification basis for the diagnosis of gastric diseases based on exosomes by screening out specific biomarkers of gastric fluid exosomes. In particular, TFF2 protein, as a specific marker of gastric fluid-derived exosomes, can be used for the identification of gastric fluid exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The separation and purification processes of gastric fluid exosomes were shown in three methods.

[0021] Figure 2 The concentration and particle size of gastric exosomes isolated by three methods were tested by NTA.

[0022] Figure 3 Transmission electron microscopy was used to observe the morphology of gastric fluid exosomes separated by three methods.

[0023] Figure 4 The expression of commonly used exosome markers was detected by WB.

[0024] Figure 5 The total protein quantification of the samples was detected by WB.

[0025] Figure 6 NanoFCM was used to detect the expression of commonly used exosome membrane markers.

[0026] Figure 7 Identification of gastric fluid exosome proteins isolated by three methods.

[0027] Figure 8 PCA was used to analyze the repeatability between methods and samples.

[0028] Fig. 9 The numbers of gastric fluid exosome core proteins isolated by three methods.

[0029] Fig.10 Comparison of gastric fluid exosome proteins separated by three methods with the public exosome database.

[0030] Fig.11 Annotation of gastric exosome proteins isolated by three methods for GO cellular component analysis.

[0031] Fig.12 The expression of high-abundance proteins in gastric fluid exosomes and gastric tissue-specific proteins. The matching proteins are shown in green boxes.

[0032] Fig.13 The expression of candidate molecule TFF2 in gastric tissue was analyzed for the GEPIA database.

[0033] Fig.14 The expression of candidate molecule AKR1B10 in gastric tissue was analyzed for the GEPIA database.

[0034] Fig.15 WB was used to verify the expression of TFF2 in gastric fluid exosomes.

[0035] Fig.16 A TFF2-expressing cell line was constructed in gastric mucosal cells GES1.

[0036] Fig.17 The concentration and particle size of exosomes derived from GES1-NC and GES1-TFF2 cells were identified for NTA.

[0037] Fig.18 Transmission electron microscopy was used to observe the morphology of exosomes from the two cell lines.

[0038] Fig.19 WB was used to detect the expression of exosome markers in the two cell lines.

[0039] Fig. 20 This is the total protein quantification of the sample in WB detection.

[0040] Fig.21 To detect the expression of commonly used exosome membrane markers and target molecule TFF2 by latex microbead-based flow cytometry.

[0041] Fig. 22 The concentration and size of exosomes derived from mouse gastric fluid were identified for NTA.

[0042] Fig.23 Transmission electron microscopy was used to observe the morphology of exosomes in mouse gastric fluid.

[0043] Fig.24WB was used to detect the expression of common markers and TFF2 in mouse gastric fluid exosomes.

[0044] Fig.25 The total protein quantification of samples in mouse gastric fluid exosomes was detected by WB.

[0045] Fig.26 Identify the concentration and size of exosomes from human gastric fluid for NTA.

[0046] Fig. 27 Transmission electron microscopy was used to observe the morphology of exosomes in human gastric fluid.

[0047] Fig.28 WB was used to detect the expression of common markers and TFF2 in human gastric fluid exosomes.

[0048] Fig.29 The total protein quantification of samples in human gastric fluid exosomes was detected by WB.

[0049] Fig.30 The concentration and size of exosomes derived from mouse intestinal fluid and plasma were characterized for NTA.

[0050] Fig.31 Transmission electron microscopy was used to observe the morphology of exosomes in mouse intestinal fluid and plasma.

[0051] Fig.32 WB was used to detect the expression of common markers and TFF2 in mouse gastric juice, intestinal fluid and plasma exosomes.

[0052] Fig.33 WB was used to detect the total protein quantification of samples in mouse gastric juice, intestinal juice and plasma exosomes.

[0053] Fig.34 Characterize the concentration and size of exosomes from human intestinal fluid and plasma for NTA.

[0054] Fig.35 Transmission electron microscopy was used to observe the morphology of exosomes in human intestinal fluid and plasma.

[0055] Fig.36 Western blot was used to detect the expression of common markers and TFF2 in exosomes in human gastric juice, intestinal juice and plasma.

[0056] Fig.37 WB was used to detect the total protein quantification of samples in human gastric juice, intestinal juice and plasma exosomes.

[0057] Unless stated to the contrary, the terms used in the specification and claims have the following meanings.

[0058] As used herein, the term "prevention" refers to preventing the occurrence of a disease and / or preventing the recurrence of a disease. DETAILED DESCRIPTION

[0059] The present invention specification describes the specific implementation scheme in detail. Those skilled in the art should recognize that the following implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the protection scope of the claims of the present invention. The following examples specifically illustrate the beneficial effects of the present invention.

[0060] Experimental design

[0061] (1) Purification of gastric fluid-derived exosomes

[0062] Gastric fluid-derived exosomes were isolated and purified by ultracentrifugation (UC), ultracentrifugation and size exclusion chromatography (UC-SEC), and ultrafiltration and size exclusion chromatography (UF-SEC).

[0063] (2) Verify whether gastric exosomes are successfully isolated

[0064] The diameter and concentration of exosomes were analyzed by nanoparticle tracking analysis (NTA); the morphology of exosomes was observed by transmission electron microscopy (TEM); and the expression of exosome markers was identified by Western blotting (WB) and nano-flow cytometry (NanoFCM). The above experiments verified whether the gastric fluid exosomes were successfully isolated.

[0065] (3) Screening of potential biomarkers of gastric exosomes

[0066] The protein expression profiles in gastric exosomes obtained by three methods of non-label quantitative proteomics analysis, combined with bioinformatics analysis, are expected to screen out specific markers of gastric exosomes.

[0067] (4) In vitro experimental verification of potential markers

[0068] The potential markers of gastric exosomes screened out were verified by in vitro experiments: TFF2 overexpression and control cell lines were constructed by lentiviral transfection in gastric mucosal cells GES-1. Cell exosomes were separated by ultracentrifugation, and WB and latex bead-based flow cytometry were used to detect whether TFF2 was specifically expressed in exosomes.

[0069] (5) In-vitro experimental verification of potential markers

[0070] The specificity of TFF2 expression in exosomes was verified in mice and clinical specimens: exosomes from mouse and human gastric fluid were separated by ultracentrifugation, and the expression of TFF2 in gastric fluid, ultracentrifugation supernatant, and exosomes was detected by WB to analyze the specificity of TFF2 expression in exosomes.

[0071] (6) TFF2 specific expression verification experiment

[0072] The tissue specificity of TFF2 expression in gastric exosomes was verified in mice and clinical specimens: gastric fluid, intestinal fluid (closer to the tissue source of gastric fluid), and plasma specimens were collected from mice and humans, and exosomes from various body fluids were separated by ultracentrifugation. WB was used to detect the expression of TFF2 in three body fluids, three body fluid ultracentrifugation supernatants, and three body fluid-derived exosomes, and the specificity of TFF2 expression in gastric exosomes was analyzed.

[0073] Example 1

[0074] Three methods for isolating and purifying gastric fluid-derived exosomes

[0075] Gastric fluid-derived exosomes were isolated and purified by ultracentrifugation (UC), ultracentrifugation and size exclusion chromatography (UC-SEC), and ultrafiltration and size exclusion chromatography (UF-SEC).

[0076] Ultracentrifugation (UC) to separate gastric exosomes

[0077] 1) Prepare sodium phosphate buffer (PBS) (PH=7.4) and sterilize by high pressure. After cooling, filter with a 0.22μm filter membrane in a biosafety cabinet to remove impurities and particles larger than 200μm in PBS as much as possible.

[0078] 2) Precool the ultracentrifuge to 4°C in advance, take out the gastric lavage fluid stored at -80°C and thaw it, gently mix it and take 10 mL of sample, balance it with sterile filtered PBS and put it into the ultracentrifuge, set the ultracentrifuge temperature to 4°C, draw the vacuum and centrifuge at 110,000g for 70 minutes, take out the centrifuge tube after the centrifuge stops and releases the vacuum, remove the supernatant in the biosafety cabinet, carefully blow the precipitate with sterile filtered PBS, gently suspend it and transfer the liquid to a new centrifuge tube.

[0079] 3) Precool the ultracentrifuge to 4°C again, balance the centrifuge tube with sterile filtered PBS, centrifuge at 110,000g for 70 min, retain the precipitate, remove the centrifuge tube and transfer it to a biosafety cabinet, resuspend it with PBS and make up to 500 μL, then transfer the sample to a 1.5 mL EP tube, divide the sample into 5 equal parts, and store it at -80°C.

[0080] Isolation of gastric exosomes by ultracentrifugation combined with size exclusion chromatography (UC-SEC)

[0081] 1) Take 10 mL of the naturally thawed sample for ultracentrifugation. Centrifuge at 110,000 g for 70 min at 4°C and retain the precipitate. Remove the centrifuge tube and transfer it to a biosafety cabinet and suspend it with PBS to make the volume 500 μL. Transfer the sample to a 1.5 mL EP tube and store it at 4°C for subsequent use.

[0082] 2) A 35 nm qEV size exclusion chromatography column (IZON) was used to separate gastric exosomes. The column was equilibrated with 20 mL of PBS before use. When PBS stopped dripping, 500 μL of the sample was added to the column and eluted with PBS.

[0083] 3) During elution, place 13 EP tubes at the bottom to collect the liquid, 1 mL aliquot, a total of 13 aliquots, and store the eluate at -80°C.

[0084] Isolation of gastric exosomes by ultrafiltration coupled with size exclusion chromatography (UF-SEC)

[0085] 1) Add sterile filtered PBS to an ultrafiltration tube (4 ml, 3KD). The PBS should completely cover the filter membrane. The filter membrane in the ultrafiltration tube should be oriented in the direction of centrifugation to better balance and clean the ultrafiltration tube. Centrifuge at 4,000 g for 5 min. Then align the filter membrane on the other side of the ultrafiltration tube with the centrifugal direction. Centrifuge at 4,000 g for another 5 min. Aspirate and discard the liquid above and below the ultrafiltration tube.

[0086] 2) Take out 10mL of the sample and thaw it naturally at 4℃. Divide it into two equal parts and add them to two ultrafiltration tubes respectively. Adjust the centrifuge temperature to 4℃ and centrifuge at 4,000g for 30-60min. Stop the centrifugation when the upper layer of liquid in each tube is about 250μL, and aspirate the liquid in the upper concentration tube.

[0087] 3) Size exclusion separation was performed using a 35 nm qEV size exclusion column. The separation and collection methods of each component were the same as above. A total of 13 components were collected and packaged, and the samples were stored in a -80 °C refrigerator.

[0088] Exosomes from gastric fluid were successfully isolated and obtained by three methods. Figure 1 The separation and purification process of gastric exosomes separated by three methods is shown below. Figure 2 To test the concentration and particle size of gastric exosomes separated by three methods using NTA, Figure 3 Transmission electron microscopy was used to observe the morphology of gastric fluid exosomes separated by three methods.

[0089] The NTA results showed that the particle concentration obtained by the UC method was about 1*10^12 particles / mL, and the peak particle concentrations obtained by the UC-SEC method and the UF-SEC method appeared in the 4th elution component, which were about 7*10^11 particles / mL and 9*10^11 particles / mL, respectively; and the diameters of the exosomes obtained by the three methods were all less than 150nm, which was consistent with the particle size of exosomes.

[0090] The morphological characteristics of gastric fluid exosomes were observed by transmission electron microscopy, and the results showed that the typical characteristics of exosomes can be observed in the particles obtained by the three methods: a lipid bilayer membrane structure with a diameter of about 100nm. The background of gEVs separated by UC method is relatively clean, and the exosomes separated by UC-SEC and UF-SEC methods contain more non-exosome particles, such as lipid and protein aggregates.

[0091] The above NTA and TEM detection structures suggest that all three methods can successfully isolate and obtain gastric fluid-derived exosomes, among which the exosomes obtained by the UC method have the highest purity.

[0092] Example 2

[0093] Screening of gastric exosome markers

[0094] The expression of commonly used exosome markers was detected by WB and NanoFCM to further identify the isolation and purification of gastric fluid exosomes.

[0095] First, the expression of four commonly used exosome markers (Alix, CD63, CD9, and TSG101) and a common exosome contaminating protein, Calnexin, was detected by WB. Figure 4The expression of common exosome markers detected by WB. The results showed that the expression of four exosome markers could be detected in the gastric fluid exosomes obtained by the three methods, while the expression of contaminating proteins was not significantly detected.

[0096] Since there is a lack of appropriate internal reference molecules for detecting exosomal proteins using WB, we used the total protein quantification method to standardize the sample loading amount. Figure 5 The total protein quantification of the sample was detected by WB. In the UF-SEC method, although the total protein content reached a peak in the 8th to 12th elution fractions, the expression peak of the exosome marker still appeared in the 4th fraction, indicating that the UF-SEC method can remove contaminating proteins in gastric fluid exosomes to a large extent.

[0097] In addition, common exosome membrane surface markers CD9, CD81, and CD63 were detected by NanoFCM. Figure 6 NanoFCM was used to detect the expression of commonly used exosome membrane markers. The results showed that the three membrane markers could be detected in the gastric fluid exosomes obtained by the three methods, but the expression levels of the three markers were different. Among them, the proportion of CD63-positive particles was the highest, followed by CD9-positive particles, while CD81-positive particles were almost undetectable, indicating that the expression levels of commonly used exosome markers in gastric fluid exosomes were heterogeneous. The above exosome marker detection structure suggests that the three methods can successfully isolate and obtain gastric fluid-derived exosomes, but the expression of different markers in gastric fluid exosomes is heterogeneous. Therefore, we want to further screen new specific gastric fluid exosome markers through proteomics.

[0098] Example 3

[0099] Proteomics analysis of gastric exosome protein expression profiles

[0100] (1) Label free LC-MS / MS was used to analyze the protein expression profile of gastric fluid exosomes. Figure 7 The number of proteins identified in gastric fluid exosomes isolated by three methods. The results showed that the number of proteins identified in gastric fluid exosomes obtained by UC method was the largest (1772±197), followed by UC-SEC method (1572±133) and UF-SEC method (1448±111).

[0101] (2) The reproducibility of various methods and biological samples was evaluated by principal component analysis (PCA). Figure 8 The results show that the protein expression profiles of gastric exosomes obtained by UC and UC-SEC methods are more similar, and gastric exosomes from different samples also have good reproducibility.

[0102] (3) To study the protein expression profile characteristics of gastric fluid exosomes obtained by each method, we defined the proteins identified in all samples as “core proteins” through Venn analysis. Fig. 9 The number of core proteins of gastric fluid exosomes isolated by three methods. The results showed that 1529 core proteins were identified by UC method, 1274 core proteins were identified by UC-SEC method, and 1074 core proteins were identified by UF-SEC method. Among them, 921 core proteins were identified in gastric fluid exosomes obtained by three methods.

[0103] (4) We further compared the gastric fluid exosome protein expression profiles obtained by the three methods with the exosome database. The ExoCarta database is a comprehensive database covering exosome proteins, RNA, liposomes, and biomarkers. We first compared the identified gastric fluid exosome proteins in this database. The results showed that 82.15% (1256 / 1529) of the gastric fluid exosome core proteins obtained by the UC method matched the ExoCarta database, and 82.81% (1055 / 1274) and 84.08% (903 / 1074) of the core proteins in the UC-SEC method and the UF-SEC method matched the database, respectively.

[0104] Fig.10 The comparison of gastric fluid exosome proteins isolated by three methods with the public exosome database. 85.12% (784 / 921) of the core proteins identified by the three methods matched the exosome proteins reported by ExoCarta.

[0105] Fig.11 The annotation of gastric exosome proteins isolated by three methods for GO cell component analysis. GO cell component analysis showed that the gastric exosome proteins identified by the three methods were mainly enriched in "extracellular exosomes".

[0106] The above proteomic results indicate that all three methods can successfully obtain gastric fluid-derived exosomes.

[0107] Example 4

[0108] Screening of specific markers for gastric exosomes based on proteomic analysis

[0109] (1) The Human Protein Atlas

[0110] In order to screen out efficient gastric exosome-specific markers, based on quantitative proteomics, we first listed the top 20 proteins expressed in gastric exosomes obtained by each method, and then compared these highly expressed proteins with human gastric tissue-specific proteins in the Human Protein Atlas database. Fig.12 The expression of high-abundance proteins in gastric exosomes and gastric tissue-specific proteins, and the matching proteins are shown in green boxes. The results show that among the core proteins of gastric exosomes obtained by the three methods, two protein molecules, TFF2 and AKR1B10, match the human gastric tissue proteins.

[0111] (2) Database (http: / / gepia.cancer-pku.cn / )

[0112] The tissue specificity of TFF2 and AKR1B10 proteins was further verified in another database (http: / / gepia.cancer-pku.cn / ). Fig.13 The expression of candidate molecule TFF2 in gastric tissue was analyzed for the GEPIA database. The results showed that TFF2 was specifically expressed in gastric tissue of healthy individuals, while AKR1B10 was expressed in both gastric and esophageal tissues of healthy individuals, and also in other tissues such as liver and lung of cancer patients, suggesting that TFF2 was more specifically expressed in gastric tissue.

[0113] The expression level of TFF2 in gastric exosomes was further verified by WB. Fig.14 Analyze the expression of candidate molecule AKR1B10 in gastric tissue for GEPIA database, Fig.15 WB was used to verify the expression of TFF2 in gastric fluid exosomes. The results showed that a high level of TFF2 expression could be detected in gastric fluid exosomes obtained by the three methods.

[0114] The above results suggest that TFF2 may be a potential specific marker for gastric exosomes.

[0115] Example 5

[0116] In vitro experiments verified that TFF2 molecules are specifically expressed in exosomes of gastric mucosal cells

[0117] TFF2 is a member of the trefoil factor (TFF) family. It is mainly expressed in the gastric epithelium and duodenal gland together with mucin MUC6 and is a small secretory peptide involved in gastrointestinal protection and repair. Given that TFF2 is a small secretory peptide, we further analyzed whether the TFF2 detected by previous protein profiling and Western blotting was derived from soluble peptides in gastric juice or specifically from gastric juice exosomes.

[0118] First, we constructed a gastric mucosal cell line overexpressing TFF2 and a control cell line (GES1-NC / TFF2). Fig.16 A TFF2-expressing cell line was constructed in gastric mucosal cells GES1. Fig.17 The concentration and particle size of exosomes derived from GES1-NC and GES1-TFF2 cells were identified for NTA. Fig.18 Transmission electron microscopy was used to observe the morphology of exosomes from the two cell lines. Fig.19 WB was used to detect the expression of exosome markers in the two cell lines. Fig. 20 This is the total protein quantification of the sample in WB detection. Fig.21 To detect the expression of commonly used exosome membrane markers and target molecule TFF2 by latex microbead-based flow cytometry.

[0119] Exosomes were isolated from cell culture supernatant by UC method. NTA, TEM and WB results showed that exosomes were successfully obtained. Compared with the quantitative results of total protein ( Fig. 20 ), TFF2 was mainly expressed in exosomes, but not in the supernatant of ultracentrifugation ( Fig.19 ), and the results of latex microbead-based flow cytometry showed that in the exosomes of gastric mucosal cells overexpressing TFF2, the percentage of TFF2-positive particles was as high as 24%, which was much higher than the currently commonly used exosome membrane markers CD9 (10%), CD81 (6%) and CD63 (5%) ( Fig.21 ).

[0120] The above results indicate that TFF2 is mainly expressed in exosomes derived from gastric mucosal cells, rather than expressed as a soluble peptide in cell culture supernatant, and that the efficiency of TFF2 in labeling gastric mucosal cell exosomes is much higher than that of the currently commonly used exosome membrane markers, suggesting that TFF2 is a potential and highly effective gastric fluid exosome-specific marker.

[0121] Example 6

[0122] Validation of TFF2 expression specificity in exosomes in mice and clinical specimens

[0123] Gastric fluid of healthy mice was collected and exosomes were isolated by UC method. NTA, TEM and WB. Fig. 22 The concentration and size of exosomes derived from mouse gastric fluid were identified for NTA. Fig.23 Transmission electron microscopy was used to observe the morphology of exosomes in mouse gastric fluid. Fig.24 WB was used to detect the expression of common markers and TFF2 in mouse gastric fluid exosomes. Fig.25 The total protein quantification of samples in mouse gastric fluid exosomes was detected by WB. Fig.26 Identify the concentration and size of exosomes from human gastric fluid for NTA. Fig. 27 Transmission electron microscopy was used to observe the morphology of exosomes in human gastric fluid. Fig.28 WB was used to detect the expression of common markers and TFF2 in human gastric fluid exosomes. Fig.29 The total protein quantification of samples in human gastric fluid exosomes was detected by WB.

[0124] The results showed that mouse gastric fluid exosomes were successfully isolated and obtained ( Figure 22-23 Compared with the quantitative results of total protein ( Fig.24 ), TFF2 is mainly expressed in gastric exosomes, but hardly expressed in the ultracentrifugation supernatant of gastric fluid ( Fig.23 ). Consistent with this, TFF2 was also detected in exosomes derived from human gastric fluid at a higher level; however, compared with the quantitative results of total protein, TFF2 was expressed at a lower level in the ultracentrifugation supernatant of human gastric fluid. The above results indicate that TFF2 is specifically expressed in gastric fluid exosomes of mice and humans, suggesting that TFF2 may be a specific biomarker for gastric fluid-derived exosomes.

[0125] Example 7

[0126] Verification of tissue specificity of TFF2 expression in gastric exosomes in mice and clinical specimens

[0127] To further evaluate the tissue specificity of TFF2 as a biomarker of gastric exosomes, we first isolated exosomes from mouse gastric fluid, intestinal fluid and plasma by UC method, and identified the exosomes from the three body fluid sources by NTA, TEM and WB.

[0128] Fig.30 The concentration and size of exosomes derived from mouse intestinal fluid and plasma were characterized for NTA. Fig.31 Transmission electron microscopy was used to observe the morphology of exosomes in mouse intestinal fluid and plasma. Fig.32 WB was used to detect the expression of common markers and TFF2 in mouse gastric juice, intestinal fluid and plasma exosomes. Fig.33 WB was used to detect the total protein quantification of samples in mouse gastric juice, intestinal juice and plasma exosomes. Fig.34 Characterize the concentration and size of exosomes from human intestinal fluid and plasma for NTA. Fig.35 Transmission electron microscopy was used to observe the morphology of exosomes in human intestinal fluid and plasma. Fig.36 Western blot was used to detect the expression of common markers and TFF2 in exosomes in human gastric juice, intestinal juice and plasma. Fig.37 WB was used to detect the total protein quantification of samples in human gastric juice, intestinal juice and plasma exosomes.

[0129] The results showed that exosomes were successfully obtained from mouse gastric juice, intestinal juice and plasma. Compared with the total protein quantification results, TFF2 was mainly expressed in mouse gastric juice exosomes, but almost not expressed in the ultracentrifugation supernatant of gastric juice, that is, it was not expressed as a soluble peptide in gastric juice. At the same time, TFF2 was only expressed in gastric juice exosomes, but not in exosomes derived from intestinal juice and plasma. Consistent with this, in human gastric juice, intestinal juice and plasma specimens, TFF2 was also mainly expressed in exosomes derived from gastric juice, but almost not in exosomes derived from intestinal juice and plasma. The above results show that TFF2 is a specific biomarker for gastric juice exosomes.

[0130] In summary, proteomic analysis showed that TFF2 was highly expressed in gastric exosomes isolated by various methods. After ultracentrifugation, TFF2 was only expressed in exosomes, but not in the supernatant of gastric ultracentrifugation. In addition, TFF2 was specifically expressed in gastric exosomes, but not in exosomes derived from intestinal fluid and plasma. The above results indicate that TFF2 is a specific biomarker for gastric exosomes, providing a new idea for the identification of gastric exosomes.

[0131] The specification of the present invention describes the specific implementation scheme in detail. Those skilled in the art should recognize that the above implementation scheme is exemplary and cannot be understood as limiting the present invention. For those skilled in the art, without departing from the principle of the present invention, by making several improvements and modifications to the present invention, the technical solutions obtained by these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of TFF2 protein as a marker for identifying gastric exosomes.

Images