A method for preserving gastric juice or extracellular vesicles in gastric juice

Abstract

The present invention provides a method for preserving gastric juice or extracellular vesicles in gastric juice, the method comprising diluting the gastric juice, adjusting the pH to 6-8, and storing the gastric juice at -90°C to -70°C after quick freezing with liquid nitrogen. The method can achieve long-term preservation of gastric juice or extracellular vesicles in gastric juice, and the extracellular vesicles in gastric juice can still maintain the quantity and shape integrity when the preservation time exceeds 1 month, thereby effectively preventing the degradation of the extracellular vesicles in gastric juice.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a method for preserving gastric juice or extracellular vesicles in gastric juice. Background Art

[0002] Extracellular vesicles are a general term for various vesicles with a lipid bilayer membrane structure released by cells in a resting or stressed state. The diameter of the vesicles ranges from tens of nanometers to hundreds of nanometers. Almost all living cells can release extracellular vesicles, which include exosomes or microvesicles. Extracellular vesicles are of great value in maintaining homeostasis, immune regulation, tissue repair and regeneration, and disease treatment. At present, the main method for preserving extracellular vesicles is to separate extracellular vesicles from samples and then preserve them. Preservation methods include cryopreservation, freeze-drying, or spray drying. However, extracellular vesicles are extremely unstable in vitro. Cryopreservation may cause changes in the shape and physical properties of extracellular vesicles, and may also lead to the formation and aggregation of multilamellar vesicles. Repeated freezing and thawing will cause changes in the biological characteristics, content, and marker composition of molecules on the surface of extracellular vesicles (Cheng Y, Zeng Q, Han Q, Xia W. Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes. Protein Cell. 2019 Apr;10(4):295-299.); lyoprotectants or cryoprotectants are difficult to remove in subsequent studies. It is difficult to maintain the integrity of the quantity and morphology of extracellular vesicles for a long time in vitro.

[0003] The pH value of gastric juice is about 2.0, which is highly acidic and has complex components, including gastric acid, pepsin and other digestive enzymes. The special environment of gastric juice makes it difficult to extract extracellular vesicles in gastric juice and to maintain the integrity of quantity and morphology. The study of extracellular vesicles in gastric juice is of great significance for the diagnosis and treatment of gastric diseases. At present, the extracellular vesicles in biological samples are often extracted and then preserved. However, due to their heterogeneity, extracellular vesicles from different sources have very different stability even if they are stored in the same environment (Welsh JA, Goberdhan DCI.et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. 2024 Feb;13(2):e12404.). If the extracellular vesicles in gastric juice are separated and extracted and then preserved conventionally, it is difficult to ensure that the analysis of the extracellular vesicles can effectively restore the situation of the extracellular vesicles in the gastric juice. However, there are few studies on the method of directly preserving the gastric juice containing extracellular vesicles. The long-term preservation of extracellular vesicles in gastric juice has become an urgent problem that needs to be solved. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a method for preserving gastric juice or extracellular vesicles in gastric juice, which realizes the long-term preservation of extracellular vesicles in gastric juice. The optimum temperature for short-term preservation is 4°C. Compared with -80°C for long-term preservation, the temperature span is larger, and it is difficult to directly use short-term preservation conditions as long-term preservation conditions. Furthermore, when only the preservation temperature is changed, the long-term preservation effect of extracellular vesicles in gastric juice is not ideal. The present invention also found that in the long-term preservation of gastric juice or extracellular vesicles in gastric juice, there is a mutual influence between the freezing method, pH adjustment and buffer solution involved in the preservation conditions, and the interaction between the various factors significantly affects the long-term preservation effect of extracellular vesicles in gastric juice. The preservation method provided by the present invention effectively prevents the degradation of extracellular vesicles in gastric juice stored for a long time, maintains the number and morphology of extracellular vesicles in gastric juice intact, and is of great significance for clinical application.

[0005] In a first aspect of the present invention, a method for preserving gastric juice or extracellular vesicles in gastric juice is provided, the method comprising diluting the gastric juice, adjusting the pH to 6-8, and storing the resulting gastric juice at -90°C to -70°C after quick freezing with liquid nitrogen.

[0006] The dilution multiple is 1.5-5, for example, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5; preferably, the dilution multiple is 2-4.

[0007] The dilution of gastric juice includes mixing gastric juice with a buffer.

[0008] Preferably, the dilution of gastric juice comprises mixing gastric juice and buffer in a volume ratio of (0.5-1.5):(1.5-2.5), more preferably in a volume ratio of 1:2.

[0009] The buffer solution includes one or more than two of PBS buffer solution, borate buffer solution, tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution or carbonate buffer solution.

[0010] Preferably, the buffer is 0.5×PBS buffer, 1×PBS buffer or 2×PBS buffer.

[0011] In a specific embodiment of the present invention, the dilution of gastric juice comprises mixing gastric juice with 2×PBS buffer at a volume ratio of (0.5-1.5):(1.5-2.5), preferably mixing at a volume ratio of 1:2.

[0012] The pH adjustment includes using one or more of sodium hydroxide, calcium carbonate, calcium bicarbonate, aluminum sulfate or ammonia water to adjust the pH.

[0013] The pH value is any value between 6 and 8, such as 6, 6.5, 7, 7.5, 8, etc., preferably 6.5-7.5.

[0014] The temperature of the liquid nitrogen quick freezing is -250°C to -100°C, for example -250°C, -240°C, -230°C, -220°C, -210°C, -200°C, -199°C, -198°C, -197°C, -196°C, -195°C, -190°C, -180°C, -170°C, -160°C, -150°C, -140°C, -130°C, -120°C, -110°C, -100°C. Preferably, the temperature of the liquid nitrogen quick freezing is -200°C to -150°C. Further preferably, the temperature of the liquid nitrogen quick freezing is -196°C.

[0015] The liquid nitrogen quick-freezing time is 1 min-30 min. Preferably, the liquid nitrogen quick-freezing time is 5 min-30 min, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min.

[0016] Further preferably, the liquid nitrogen quick freezing time is 5 minutes.

[0017] After quick freezing with liquid nitrogen, the mixture is stored at -90°C to -70°C, for example, -90°C, -88°C, -86°C, -84°C, -82°C, -80°C, -78°C, -76°C, -74°C, -72°C, or -70°C. Preferably, the mixture is stored at -85°C to -70°C after quick freezing with liquid nitrogen.

[0018] In a specific embodiment of the present invention, the liquid nitrogen is quick-frozen and then stored at -80°C.

[0019] The median diameter of the extracellular vesicles is 30-1000 nm, preferably, the median diameter of the extracellular vesicles is 50-800 nm, further preferably, the median diameter of the extracellular vesicles is 50-500 nm, and further preferably, the median diameter of the extracellular vesicles is 50-250 nm. In some specific embodiments, the median diameter of the cell vesicles is 130-230 nm.

[0020] The extracellular vesicle markers include one or more of FLOT1, Alix, ANXA1 or TSG101.

[0021] The storage method is long-term storage, and the long-term storage includes a storage period of more than 3 days. Preferably, the storage period is more than 10 days, and more preferably, the storage period is more than 30 days.

[0022] Preferably, the storage method is to mix gastric juice and 2×PBS buffer in a volume ratio of 1:2, adjust the pH to 7 (preferably with NaOH), and then quickly freeze the mixture with liquid nitrogen and store it at -80°C.

[0023] A second aspect of the present invention provides a method for extracting extracellular vesicles from gastric juice, the method comprising obtaining gastric juice and then performing differential centrifugation.

[0024] The gastric juice includes gastric juice preserved by the preservation method described in the first aspect or fresh gastric juice;

[0025] The method includes:

[0026] Step 1: Centrifuge the gastric juice at 0-30°C;

[0027] Step 2: Mix the supernatant obtained by centrifugation in step 1 with the buffer solution and centrifuge at 0-30°C;

[0028] Step 3: Filter the supernatant obtained by centrifugation in step 2, and mix the filtrate with a buffer solution;

[0029] Step 4: The mixed solution obtained in step 3 is subjected to ultracentrifugation at 0-30°C, and the precipitate is the extracellular vesicles.

[0030] The buffer solution includes one or more than two of PBS buffer solution, borate buffer solution, tris(hydroxymethylaminomethane)-hydrochloric acid buffer solution or carbonate buffer solution.

[0031] In step one, the gastric juice is centrifuged at 0-30°C, for example, the gastric juice is centrifuged at 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, or 30°C, preferably the gastric juice is centrifuged at 0-10°C.

[0032] The centrifugal force of the centrifugation in the step 1 is 1000xg-2000xg. Preferably, the centrifugal force of the centrifugation is 1200xg-2000xg, for example, 1000xg, 1100xg, 1200xg, 1300xg, 1400xg, 1500xg, 1600xg, 1700xg, 1800xg, 1900xg, 2000xg.

[0033] The centrifugation time in the step one is 20 min-40 min. Preferably, the centrifugation time is 25 min-40 min, for example, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, and 40 min.

[0034] In a specific embodiment of the present invention, step one comprises centrifuging the gastric juice at 1500 x g for 30 min at 4°C.

[0035] The concentration of the buffer in step 2 includes 0.5× buffer, 1× buffer or 2× buffer. Preferably, the concentration of the buffer in step 2 is 1× buffer.

[0036] The centrifugal force of the centrifugation in the step 2 is 3000xg-4000xg. Preferably, the centrifugal force of the centrifugation is 3200xg-4000xg, for example, 3000xg, 3100xg, 3200xg, 3300xg, 3400xg, 3500xg, 3600xg, 3700xg, 3800xg, 3900xg, and 4000xg.

[0037] The centrifugation time in the step 2 is 20 min-40 min. Preferably, the centrifugation time is 25 min-40 min, for example, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, and 40 min.

[0038] In a specific embodiment of the present invention, step 2 comprises mixing the supernatant in step 1 with 1× buffer and centrifuging at 3500×g for 30 min at 4°C.

[0039] The filtration in step three includes filtering using a filter, and the filter includes a polyethersulfone filter membrane, a cellulose acetate membrane, a polypropylene filter membrane, a polyvinylidene fluoride membrane or a mixed cellulose ester filter membrane.

[0040] The pore size of the filter is 0.1μm-1μm. Preferably, the pore size of the filter is 0.2μm-0.5μm, for example, 0.1μm, 0.15μm, 0.2μm, 0.22μm, 0.24μm, 0.26μm, 0.28μm, 0.3μm, 0.35μm, 0.4μm, 0.45μm, 0.5μm, 0.55μm, 0.6μm, 0.7μm, 0.8μm, 0.9μm, and 1μm.

[0041] In a specific embodiment of the present invention, the pore size of the filter is 0.22 μm or 0.45 μm.

[0042] The buffer concentration in step three is 1× buffer.

[0043] The centrifugal force of the ultracentrifugation in the step 4 is 100000xg-200000xg. Preferably, the centrifugal force of the ultracentrifugation is 120000xg-180000xg, for example, 100000xg, 110000xg, 120000xg, 130000xg, 140000xg, 150000xg, 160000xg, 170000xg, 180000xg, 190000xg, and 200000xg.

[0044] The ultracentrifugation time in step 4 is 1.5h-5h. Preferably, the ultracentrifugation time is 2h-4h, for example, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h.

[0045] In a specific embodiment of the present invention, step four comprises ultracentrifuging the mixed solution obtained in step three at 4°C, with a centrifugal force of 150,000 xg and a centrifugal time of 3.5 h, and the precipitate is the extracellular vesicles.

[0046] The method further comprises adding protease to the mixed solution of the filtrate obtained in step 3 and the buffer.

[0047] The protease includes one or more of pepsin, proteinase K or trypsin. Preferably, the protease is proteinase K.

[0048] The median diameter of the extracellular vesicles is 30-1000 nm, preferably, the median diameter of the extracellular vesicles is 50-800 nm, further preferably, the median diameter of the extracellular vesicles is 50-500 nm, and further preferably, the median diameter of the extracellular vesicles is 50-250 nm. In some specific embodiments, the median diameter of the cell vesicles is 130-230 nm.

[0049] The extracellular vesicle markers include one or more of FLOT1, Alix, TSG101 or ANXA1.

[0050] The third aspect of the present invention provides a method for preventing the degradation of extracellular vesicles in gastric juice or a method for maintaining the number and structural integrity of extracellular vesicles in gastric juice, the method comprising diluting the gastric juice, adjusting the pH to 6-8, quick-freezing with liquid nitrogen, and storing at -90°C to -70°C.

[0051] The dilution multiple is 1.5-5, for example, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5; preferably, the dilution multiple is 2-4.

[0052] The dilution of gastric juice includes mixing gastric juice with a buffer.

[0053] Preferably, the dilution of gastric juice comprises mixing gastric juice and buffer in a volume ratio of (0.5-1.5):(1.5-2.5), more preferably in a volume ratio of 1:2.

[0054] The buffer comprises one or more of PBS buffer, borate buffer, tris-hydrochloric acid buffer or carbonate buffer. Preferably, the buffer is 0.5×PBS buffer, 1×PBS buffer or 2×PBS buffer.

[0055] In a specific embodiment of the present invention, the dilution of gastric juice comprises mixing gastric juice with 2×PBS buffer at a volume ratio of 1:2.

[0056] The pH adjustment includes using one or more of sodium hydroxide, calcium carbonate, calcium bicarbonate, aluminum sulfate or ammonia water to adjust the pH.

[0057] The pH value is any value between 6 and 8, such as 6, 6.5, 7, 7.5, 8, etc., preferably 6.5-7.5.

[0058] The temperature of the liquid nitrogen quick freezing is -250°C to -100°C, for example -250°C, -240°C, -230°C, -220°C, -210°C, -200°C, -199°C, -198°C, -197°C, -196°C, -195°C, -190°C, -180°C, -170°C, -160°C, -150°C, -140°C, -130°C, -120°C, -110°C, -100°C. Preferably, the temperature of the liquid nitrogen quick freezing is -200°C to -150°C. Further preferably, the temperature of the liquid nitrogen quick freezing is -196°C.

[0059] The liquid nitrogen quick-freezing time is 1 min-30 min. Preferably, the liquid nitrogen quick-freezing time is 5 min-30 min, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min.

[0060] Further preferably, the liquid nitrogen quick freezing time is 5 minutes.

[0061] After quick freezing with liquid nitrogen, the mixture is stored at -90°C to -70°C, for example, -90°C, -88°C, -86°C, -84°C, -82°C, -80°C, -78°C, -76°C, -74°C, -72°C, or -70°C. Preferably, the mixture is quick frozen with liquid nitrogen and stored at -85°C to -70°C.

[0062] In a specific embodiment of the present invention, the liquid nitrogen is quick-frozen and then stored at -80°C.

[0063] The median diameter of the extracellular vesicles is 30-1000 nm, preferably, the median diameter of the extracellular vesicles is 50-800 nm, further preferably, the median diameter of the extracellular vesicles is 50-500 nm, and further preferably, the median diameter of the extracellular vesicles is 50-250 nm. In some specific embodiments, the median diameter of the cell vesicles is 130-230 nm.

[0064] The extracellular vesicle markers include one or more of FLOT1, Alix, ANXA1 or TSG101.

[0065] In a fourth aspect of the present invention, a method for preserving extracellular vesicles is provided, the method comprising diluting gastric juice, adjusting the pH to 6-8, adding extracellular vesicles, and then quick-freezing with liquid nitrogen and storing at -90°C to -70°C.

[0066] The extracellular vesicles are derived from gastric juice, urine, tumor cell supernatant, bile, cerebrospinal fluid, pleural effusion, ascites, blood or saliva.

[0067] Beneficial effects:

[0068] (1) In the present invention, gastric juice is mixed with 2×PBS buffer at a volume ratio of 1:2, the pH is adjusted to 7, and the mixture is quickly frozen with liquid nitrogen and stored at -80°C. After one month of storage, the extracellular vesicles in the gastric juice still maintain their morphological integrity, and the markers of the extracellular vesicles are successfully detected, indicating that the storage effect is good and long-term storage of gastric juice and the extracellular vesicles in the gastric juice is achieved.

[0069] (2) The present invention verifies the effects of cryopreservation method, pH and dilution buffer on the preservation effect. Among the different cryopreservation methods, quick freezing with liquid nitrogen followed by storage at -80°C resulted in a higher concentration of extracellular vesicles, more markers detected, and intact extracellular vesicle morphology and a more complete structure than direct storage at -80°C and gradient cooling to -80°C. This indicates that even if the final storage temperature is the same, the difference in storage method has a significant effect on the preservation effect.

[0070] In the study of the preservation effect of extracellular vesicles in gastric juice at different pH values, the concentration of particles in gastric juice stored at pH 7 was more than 40% higher than that at pH 2 when the gastric juice was adjusted to pH 7 compared to that at pH 2, indicating that direct use of gastric juice to preserve extracellular vesicles in gastric juice is not suitable. Even at pH 6 or pH 8, which is only 1 pH value lower than the neutral condition, the concentration of particles in the stored gastric juice is nearly 30% lower, and at pH 2 or 4, the markers of extracellular vesicles are almost undetectable. It can be seen that a slight change in pH will have a great impact on the preservation effect of extracellular vesicles in gastric juice.

[0071] In the study of the preservation effect of buffer on extracellular vesicles in gastric juice, the particle concentration of the group with added buffer was 39% higher than that of the group without added buffer, and the morphology of extracellular vesicles in the group with added buffer was also more complete. In the group with added buffer, the effect of adding different concentrations of buffer on the preservation effect was also very significant. Compared with the group with added 0.5× buffer, the group with added 2× PBS buffer detected more extracellular vesicle markers and had better effect. Therefore, whether to add buffer and the concentration of buffer have a great influence on the preservation effect of extracellular vesicles in gastric juice.

[0072] In summary, a single factor significantly affects the preservation of extracellular vesicles in gastric fluid, and changes in any factor may change the number and morphological integrity of extracellular vesicles in long-term preserved gastric fluid.

[0073] (3) In the single factor validation experiment of the present invention, for example, in the study of different freezing methods, compared with quick freezing in liquid nitrogen and then storing at -80°C, the particle concentration decreased by 17% when directly stored at -80°C, and by 11% when gradually cooled to -80°C; in the study of different pH values, compared with pH 7, the particle concentration decreased by 41% at pH 2, and by 32% at pH 4; compared with adding buffer, the particle concentration decreased by 39% when no buffer was added. However, in the orthogonal experiment, the effects of three factors on the preservation of gastric juice were evaluated by orthogonal design. It was found that, for example, compared with group 1 and group 2, the preservation methods and pH values ​​were different. Theoretically, the particle concentration of group 2 should be 49% lower than that of group 1 (the superposition of 17% and 32%), but the actual result was 24%. For example, compared with group 1 and group 3, the particle concentration of group 3 should be 52% lower than that of group 1 (the superposition of 11% and 41%), but the actual result was 29%. For example, Group 1 and Group 7 have different pH values ​​and different additions of PBS buffer. Theoretically, the particle concentration in Group 7 should be 71% lower than that in Group 1 (the sum of 32% and 39%), but the actual result is 27%. This shows that there is an interaction between the storage method, pH and PBS buffer. When a single factor is at the optimal value, it will reduce the impact of other adverse factors on extracellular vesicles in gastric juice.

[0074] In summary, the three single factors interact with each other, causing them to work together to preserve extracellular vesicles in gastric juice. A slight change in any one factor will cause a change in the interaction between them and affect the final preservation effect.

[0075] (4) The present invention verifies the preservation effect of extracellular vesicles in gastric juice at different temperatures, and finds that in the short-term preservation, -80℃ is obviously not as effective as 4℃. However, as the preservation time increases, in the detection of extracellular vesicle markers, the markers detected in the -80℃ environment are significantly more than those in the 4℃ environment. This significant contradiction also proves that the conditions for short-term preservation of gastric juice are not suitable for long-term preservation of gastric juice. To achieve long-term preservation of gastric juice or extracellular vesicles in gastric juice, it is not possible to simply apply the short-term preservation conditions, nor can it be obtained by optimizing the conditions. In addition, even if the gastric juice is stored in an environment of -80℃, the degradation of extracellular vesicles in the gastric juice cannot be prevented. After 10 days of preservation, the particle concentration is 36% lower than that of the control group, which further proves that the loss of extracellular vesicles in the gastric juice caused by the high salt ion concentration and strong acidic environment in the gastric juice cannot be solved by simply changing the preservation temperature. While optimizing the preservation temperature, the present invention further optimizes the freezing preservation method, the buffer solution, and the pH adjustment. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 :Flowchart for extracting extracellular vesicles from gastric juice;

[0077] Figure 2 : Diameter distribution of extracellular vesicles in fresh gastric juice;

[0078] Figure 3 :The morphology of extracellular vesicles in fresh gastric juice;

[0079] Figure 4 : Morphology of extracellular vesicles in fresh gastric juice, where A is a representative extracellular vesicle morphology with a diameter of 100 nm, and B is a representative extracellular vesicle morphology with a diameter of 200 nm;

[0080] Figure 5 : Immunoblotting of extracellular vesicle markers in 6 fresh gastric juice samples;

[0081] Figure 6 : Massive mucin image;

[0082] Figure 7 : filamentous mucin diagram;

[0083] Figure 8 : The concentration and diameter of extracellular vesicles in gastric fluid after storage at 4℃, -20℃ and -80℃ for 1 day (A, D), 3 days (B, E) and 10 days (C, F);

[0084] Fig. 9 : Immunoblotting of extracellular vesicle markers in gastric juice after storage at 4°C, -20°C and -80°C for 1 day (A), 3 days (B) and 10 days (C);

[0085] Fig.10 :The morphology of extracellular vesicles in gastric juice at different storage temperatures;

[0086] Fig.11 : Images of floccules precipitated from gastric juice after freeze-thawing at 4℃ (A), -20℃ (B) and -80℃ (C);

[0087] Fig.12 :Concentration (A) and diameter (B) of extracellular vesicles in gastric fluid after different freezing treatments;

[0088] Fig.13 : Immunoblotting of extracellular vesicle markers in gastric juice after different freezing treatments;

[0089] Fig.14 :The morphology of extracellular vesicles in gastric juice after different freezing treatments;

[0090] Fig.15:Concentration (A) and diameter (B) of extracellular vesicles under different pH conditions;

[0091] Fig.16 : Immunoblotting of extracellular vesicle markers under different pH conditions;

[0092] Fig.17 :The morphology of extracellular vesicles under different pH conditions;

[0093] Fig.18 : Immunoblotting of extracellular vesicle markers in gastric juice when different concentrations of PBS buffer were added and the pH was adjusted to 7 using NaOH;

[0094] Fig.19 : The concentration (A) and diameter (B) of extracellular vesicles when different concentrations of PBS buffer were added and the pH was adjusted to 7 using NaOH;

[0095] Fig. 20 : Morphological images of extracellular vesicle markers in gastric juice when different concentrations of PBS buffer were added and the pH was adjusted to 7 using NaOH;

[0096] Fig.21 : Immunoblotting of extracellular vesicle markers in gastric juice of each group in the orthogonal experiment;

[0097] Fig. 22 :The concentration (A) and diameter (B) of extracellular vesicles in gastric juice of each group in the orthogonal experiment;

[0098] Fig.23 : Morphological images of extracellular vesicles in gastric juice of each group in orthogonal experiment. DETAILED DESCRIPTION

[0099] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0100] The experimental methods involved in the embodiments are as follows:

[0101] 1. Extraction of EVs from gastric juice by ultracentrifugation

[0102] The specific process of extracting extracellular vesicles from gastric juice is as follows Figure 1As shown. Take 3mL of gastric juice and centrifuge at 1500xg for 30 minutes at 4°C to remove mucus and cell debris. The resulting supernatant is then mixed with 6mL of 1× PBS buffer and centrifuged at 3500xg for 30 minutes at 4°C, and the supernatant is taken. The supernatant is then filtered through a 0.45 micron filter to remove large particles and then diluted with 25mL of 1× PBS. The subsequent procedure depends on the intended application of the sample: when performing transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), 20μL of proteinase K is usually added to the sample to help break down proteins to prevent them from interfering with the analysis. However, if the sample has been frozen, it is not necessary to add proteinase K. In addition, if the sample is used to identify extracellular vesicle (EV)-specific protein markers, the addition of proteinase K should also be omitted to prevent these proteins from being degraded. Subsequently, the samples should be centrifuged at 150,000 x g for 3.5 h at 4 °C (ultracentrifugation), the supernatant removed, and the pellet containing the extracellular vesicles (EVs) resuspended in 200 μl 1× PBS for storage or subsequent analysis.

[0103] 2. Western blot analysis of extracellular vesicles (WB)

[0104] During the western blot analysis of extracellular vesicles, the samples were first prepared by adding 5-fold concentration of SDS-PAGE loading buffer to the EVs suspension, and then the samples were boiled in boiling water for 15 minutes to completely denature the proteins. The denatured samples were then loaded onto a 10% SDS-PAGE gel and electrophoresed at 200V for 35 minutes in electrophoresis buffer. After electrophoresis, the proteins were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was then blocked with 5% skim milk at room temperature. To identify specific target proteins, such as Alix, TSG101, ANXA1, and FLOT1, the samples were incubated with specific primary antibodies selected from Proteintech at a dilution of 1:10,000 overnight at 4°C. After washing the PVDF membrane to remove unbound proteins, it was incubated with anti-mouse IgG-HRP secondary antibody (1:20,000 dilution) at room temperature for 90 minutes to amplify the detection signal by the HRP-labeled secondary antibody. Super Signal West Pico (Thermo) Chemiluminescent Substrate is an enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate that generates a signal upon interaction with an HRP-conjugated secondary antibody, allowing visualization of protein bands.

[0105] 3. Transmission electron microscopy (TEM) analysis

[0106] EVs samples were incubated on a 200-mesh carbon support membrane for 30 minutes and fixed with 4% paraformaldehyde and 1% glutaraldehyde. Subsequently, EVs on the carbon support membrane were stained with 2% uranyl acetate for 30 seconds to enhance contrast and aid visualization. The morphology of EVs was then examined using transmission electron microscopy, which provides high-resolution images for detailed analysis of the morphology and structure of EVs.

[0107] 4. Nanoparticle Tracking Analysis (NTA)

[0108] NTA uses light scattering as a method to track the movement of individual particles undergoing Brownian motion. The size and composition of vesicles are assessed by analyzing the trajectories of vesicles of different sizes. To ensure accurate measurements, EV samples are diluted with phosphate-buffered saline (PBS) to achieve a detection range of 50-200 particles per frame. Subsequently, the number and diameter of extracellular vesicles are determined using a PMX 110 instrument (ZetaView).

[0109] Example 1: Extraction of EVs from gastric fluid using ultracentrifugation

[0110] Fresh gastric juice was collected from 20 patients with superficial gastritis and atrophic gastritis by ultracentrifugation, EVs were extracted, and the isolated EVs suspension was analyzed.

[0111] Nanoparticle tracking analysis (NTA) was used to assess the size and concentration of EVs, and the results showed that the median diameter was approximately 216 nm and the concentration ranged from 5.9 × 10 per ml. 10 to 4.3×10 11 Particles ( Figure 2 and Table 1).

[0112] Table 1: Statistical analysis of the diameter and concentration of extracellular vesicles in fresh gastric juice

[0113]

[0114] Transmission electron microscopy (TEM) observations showed that EVs exhibited a “cup or saucer-shaped” morphology and size ( Figure 3 and Figure 4 ), which is consistent with the NTA results.

[0115] Specific markers in EVs were detected by immunoblotting to confirm the presence and nature of EVs. The selected markers include ANXA1, FLOT1, TSG-101 and Alix, among which ANXA1 is a marker for large vesicles (150-1000nm) and FLOT1 is a marker for small vesicles (30-150nm). TSG-101 and Alix are components of the ESCRT (exosomal sorting complex) dependent pathway, and FLOT1 and ANXA1 are known components of cell membrane microdomains. Six samples were randomly selected for immunoblotting to detect specific markers of EVs, such as Figure 5 As shown, the markers were successfully detected. The above results indicate that ultracentrifugation can efficiently extract EVs from gastric juice, and also reflect the stability of EVs in gastric juice.

[0116] During the extraction of fresh gastric juice, proteinase K needs to be added before the ultracentrifugation step to reduce mucin contamination. If proteinase K is not added, EVs extracted at 4°C are easily contaminated by mucin. Figure 6 It is a mass of mucin. Figure 7 It is filamentous mucin.

[0117] Example 2: Effects of different temperatures on the preservation of EVs in gastric juice

[0118] In order to examine the effects of different temperatures on EVs in gastric juice, gastric juice samples from 20 patients with superficial gastritis and atrophic gastritis were first obtained by gastroscopy and combined to prepare standardized gastric juice samples. Afterwards, these standardized gastric juice samples were directly stored at 4°C, -20°C and -80°C, and EVs were isolated for evaluation 1 day, 3 days and 10 days after storage. Before the experiment, EVs were also isolated from standardized gastric juice samples as a control group. The EVs separated by ultracentrifugation were analyzed in detail by WB, NTA and TEM to determine the effects of different temperature conditions and storage time on EVs in gastric juice. NTA results ( Figure 8 ) showed that under 4°C storage conditions, after 10 days of storage, the number of EVs changed the least, only decreasing by 7%. This shows that 4°C is a relatively stable short-term storage environment. However, under -80°C storage conditions, after 1 day of storage, not only did the number of EVs decrease by 20% during the first freeze-thaw process, but the number of EVs particles continued to decrease over time. This phenomenon indicates that directly freezing gastric juice samples at -80°C may cause EVs to degrade over time.

[0119] WB analysis results ( Fig. 9) further verified the above results. After 1 day of storage, the storage effect at 4°C was significantly better than that at -20°C and -80°C. However, as the storage time increased, the storage effect at 4°C gradually became worse than that at -80°C, especially in the case of long-term storage.

[0120] Electron microscopy analysis results ( Fig.10 ) revealed the effect of storage temperature on the morphology of EVs. Under 4°C and -20°C storage conditions, most of the EVs in gastric juice maintained a good cup or saucer shape and contained a higher proportion of large vesicles (>200nm). In contrast, when stored at -80°C, the EVs morphology was folded and the number of large vesicles decreased. Although the morphology of EVs at various temperatures did not change significantly over time, the distribution density observed under the microscope decreased.

[0121] In addition, after the gastric juice undergoes freeze-thaw treatment, especially under low temperature conditions, a large amount of floccules will precipitate ( Fig.11 ). It is speculated that the acidic environment in gastric juice may cause a large amount of mucin and other proteins to precipitate. These precipitated proteins may wrap EVs and cause them to precipitate, thereby affecting the recovery rate of EVs.

[0122] In summary, 4°C is the best storage condition for EVs in a short period of time (≤3 days). However, long-term storage (more than 10 days), whether at 4°C, -20°C or -80°C, is difficult to prevent the gradual degradation and morphological changes of EVs. This finding emphasizes the importance of finding more suitable long-term storage methods to maintain the integrity of EVs in gastric fluid.

[0123] Example 3: Effects of different cooling methods on the preservation of EVs in gastric juice

[0124] To investigate the effects of different cryopreservation methods on EVs in gastric juice, three cryopreservation methods were used: directly freezing standardized gastric juice samples at -80°C (control group), quick freezing with liquid nitrogen and then storing at -80°C (quick freezing group), and gradually cooling the environment to -80°C (gradient cooling group). After one day of cryopreservation, EVs were isolated by ultracentrifugation for analysis. The preservation status of EVs was evaluated using WB, NTA, and TEM.

[0125] EVs are membrane-bound vesicles released by cells, and their stability under different freezing conditions is the focus of this study.

[0126] NTA Results ( Fig.12 ) showed that different cryopreservation methods had no significant effect on the diameter (median diameter) of EVs. However, in terms of preserving the number of EVs, liquid nitrogen quick freezing was significantly superior to other methods.

[0127] The results of WB ( Fig.13 ) is consistent with the NTA analysis. After ultracentrifugation, the gastric juice samples preserved by liquid nitrogen quick freezing can obtain a precipitate containing more EVs markers, which further verifies the effectiveness of liquid nitrogen quick freezing in protecting EVs.

[0128] Electron microscopy analysis results ( Fig.14 ) further revealed the specific effects of various preservation methods on the structure of EVs. In the control group, the structure of most EVs was damaged. In contrast, most EVs in the quick-freeze group maintained a typical "cup or saucer"-like structure, indicating that their structural integrity was well protected. In the gradient cooling group, although smaller vesicles were able to maintain their morphology, the morphology of larger vesicles was poor.

[0129] Based on the above results, it is inferred that compared with direct cryopreservation or gradient cooling cryopreservation of cells, the use of liquid nitrogen quick freezing is more beneficial for protecting EVs in gastric juice and maintaining their morphology.

[0130] Example 4: Effects of different pH levels on extracellular vesicles

[0131] To investigate the effects of different pH levels on EVs released from tissue cells, we isolated EVs from the culture supernatant of gastric cancer cells (AGS cells) by ultracentrifugation. Subsequently, these EVs were resuspended in PBS buffer and divided into six equal portions. Five EVs were added to gastric juice from which EVs had been removed and the pH values ​​were adjusted to 2, 4, 6, 7, and 8, respectively, and stored at 4°C for seven days. After the storage period, EVs were isolated again by ultracentrifugation for further analysis. The remaining EV suspension was used as a control group, from which EVs were isolated by ultracentrifugation immediately after the addition of PBS buffer.

[0132] In this study, EVs extracted from the supernatant of AGS cells were added to gastric juice from which EVs had been removed and adjusted to different pH values, and stored at 4°C for 7 days to explore the effect of pH on the stability of EVs.

[0133] The results of NTA showed that only 41% of EVs could be detected after storage in a strong acidic environment ( Fig.15 ). This is lower than what was observed in a neutral environment. 82% of EVs in a neutral environment were still detectable after storage. WB analysis also showed that most of the biomarkers of EVs were degraded in an acidic environment ( Fig.16 ), while in a neutral environment, the degradation of protein markers was minimal, indicating that a neutral environment is conducive to the stable preservation of EVs.

[0134] Electron microscopy results further support this finding ( Fig.17). Compared with the neutral environment, the number of EVs stored in the acidic environment was significantly reduced, and the structural integrity of EVs was also poor. In contrast, the structure of EVs in gastric fluid stored in a neutral environment was more intact.

[0135] These results indicate that EVs released by gastric mucosal cells into gastric fluid are more likely to be degraded more rapidly in the acidic environment of gastric fluid, and that extracellular vesicles in gastric fluid are also degraded more rapidly when stored in gastric fluid. This finding is of great significance for understanding and optimizing the stability of EVs in different biological environments.

[0136] Example 5: Effects of different concentrations of PBS buffer on extracellular vesicles in gastric juice

[0137] In order to reduce the degradation and consumption of EVs in gastric juice during long-term storage, the particle concentration was adjusted by adding different concentrations of PBS buffer (0.5×, 1× and 2×) to gastric juice and buffer at a volume ratio of 1:2 and adjusting the pH value of gastric juice with NaOH solution. Different concentrations of PBS buffer were added to the gastric juice sample (the same as the standardized gastric juice sample in Example 2) at a ratio of 1:2, and the mixed solution was directly placed in a -80°C environment for 10 days. The gastric juice was directly placed in a -80°C environment for 10 days as a control group. EVs were separated by ultracentrifugation, and the preservation effect of different concentrations of PBS buffer on EVs in gastric juice was evaluated. The condition of EVs was evaluated by using WB and NTA to determine the most effective buffer concentration and pH for long-term storage of gastric juice. According to existing experimental results, the high salt ion concentration and acidic environment in gastric juice will cause loss of EVs in gastric juice, and even direct freezing at -80°C cannot prevent the degradation of EVs. To address this problem, EVs in gastric juice were protected by reducing the salt ion concentration in gastric juice and adjusting the acidity of gastric juice using NaOH.

[0138] The results of WB analysis showed that ( Fig.18 ), adding 2× PBS buffer to the gastric juice and adjusting the pH to 7 with NaOH solution was the best preservation method. This method was not only better than adding 1× PBS buffer, but also significantly better than the control group where the samples were directly placed at -80°C.

[0139] Secondly, the results of NTA showed that ( Fig.19 ), the number of particles detected in all samples with added buffer was higher than that in the control group. And the median diameter of EVs was also greater than that in the control group, indicating that adding buffer may be more conducive to the preservation of larger vesicles.

[0140] Electron microscopy analysis results ( Fig. 20) showed that EVs in the control group were significantly damaged, while EVs in the buffer group maintained a typical "cup or saucer" structure, indicating that their structural integrity was well protected.

[0141] In summary, by adding 2× PBS buffer to the gastric juice at a volume ratio of 1:2 and adjusting the pH of the mixed solution to 7, the preservation effect of EVs in gastric juice was significantly improved compared with the method of directly placing it at -80°C.

[0142] Example 6: Orthogonal experiment to study the best preservation method of gastric juice

[0143] An orthogonal experimental design (OED) was used to investigate the optimal storage method for gastric juice, covering three factors and three levels, as shown in Table 2. These factors included PBS buffer concentration, cryopreservation method, and target pH adjustment, and each factor had three levels, representing different experimental conditions. The specific experimental design was based on previous research results and is shown in detail in Table 3.

[0144] Table 2: Orthogonal experiment table

[0145]

[0146] Table 3: Optimal experimental design

[0147]

[0148] An orthogonal experimental design was used to systematically evaluate the effects of different factors on the preservation of gastric juice samples (the same as the standardized gastric juice samples in Example 2) and select the best preservation method. By combining the results of the previous experiments, the optimal combination of conditions was determined as Group 1 for experimental design.

[0149] After one month of storage, WB results showed ( Fig.21 ), the marker levels of groups 7-9 were all low, the preservation effects of groups 1, 2, and 4 were significantly better than those of the other groups, and the preservation effect of group 1 was the most significant among all 9 groups of experiments, which was consistent with the expectations of the early experiments. In addition, it was noted that after the addition of the buffer, the flocs precipitated from the gastric juice after freeze-thaw treatment were significantly reduced. This phenomenon is likely because the buffer reduces the salt ion concentration in the gastric juice, thereby reducing the denaturation and precipitation of proteins.

[0150] The results of NTA further confirmed the advantages of groups 1, 2, and 4 ( Fig. 22 ), showing that not only was the number of EVs in its gastric fluid the largest, but the diameter of the EVs (median diameter) was also closest to the state before preservation.

[0151] In electron microscopy observation ( Fig.23), Group 1 showed the largest distribution density, and the EVs extracted from the gastric juice of Groups 1 and 3 had the most ideal morphology. On the contrary, in the samples of Groups 7, 8, and 9, the density of EVs under the microscope was extremely low and the morphology appeared irregular. Based on the above results, Group 1 had the best effect.

[0152] In summary, the preservation of EVs in gastric fluid samples was significantly improved by combining the buffer and other related conditions. This not only verifies the effectiveness of the orthogonal experimental design, but also reflects the key role of the buffer in stabilizing EVs in samples.

[0153] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for preserving gastric juice or extracellular vesicles in gastric juice, characterized in that: The storage method comprises diluting the gastric juice, adjusting the pH to 6-8, and storing it at -90°C to -70°C after quick freezing with liquid nitrogen; The gastric juice dilution includes mixing the gastric juice with a buffer solution, wherein the buffer solution is 0.5×PBS buffer solution, 1×PBS buffer solution or 2×PBS buffer solution; the dilution multiple is 1.5-5.

2. The storage method according to claim 1, characterized in that: The dilution multiple is 2-4.

3. The storage method according to claim 1, characterized in that: The gastric juice dilution comprises mixing the gastric juice with 2×PBS buffer at a volume ratio of (0.5-1.5): (1.5-2.5).

4. The storage method according to claim 1, characterized in that: The median diameter of the extracellular vesicles is 50-250 nm.

5. The storage method according to claim 1, characterized in that: The extracellular vesicle markers include one or more of FLOT1, Alix, TSG101 or ANXA1.

6. The storage method according to any one of claims 1 to 5, characterized in that: The storage method further comprises extracting the extracellular vesicles in the stored gastric juice; The extraction comprises: Step 1: centrifuging the gastric juice at 0-30°C, with the centrifugal force of 1000xg-2000xg; Step 2: Mix the supernatant obtained by centrifugation in step 1 with a buffer solution, and centrifuge at 0-30° C., wherein the centrifugal force of the centrifugation is 3000×g-4000×g; Step 3: Filter the supernatant obtained by centrifugation in step 2, and mix the filtrate with a buffer solution; Step 4: ultracentrifuge the mixed solution obtained in step 3 at 0-30°C, wherein the centrifugal force of the ultracentrifugation is 100000xg-200000xg, and the precipitate is the extracellular vesicles.

7. The storage method according to claim 6, characterized in that: The filtration in step three includes filtering using a filter, and the filter includes a polyethersulfone filter membrane, a cellulose acetate membrane, a polypropylene filter membrane, a polyvinylidene fluoride membrane or a mixed cellulose ester filter membrane.

8. The storage method according to claim 7, characterized in that: The pore size of the filter is 0.1 μm-1 μm.

9. The storage method according to claim 8, characterized in that: The pore size of the filter is 0.2 μm-0.5 μm.

10. The storage method according to claim 6, characterized in that: The extraction further comprises adding protease to the mixed solution of the filtrate obtained in step 3 and the buffer solution.

11. The storage method according to claim 10, characterized in that: The protease includes one or more of pepsin, proteinase K or trypsin.

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