Serum transporter identification method

Abstract

The invention discloses a serum migration body identification method which comprises the following steps: extracting a serum migration body from serum by performing multiple centrifugal treatment on the serum; carrying out immunofluorescence staining treatment on the serum migration body to obtain an immunofluorescence stained serum migration body; and carrying out imaging treatment on the serum migration body subjected to immunofluorescence staining by utilizing a super-resolution fluorescence microscope SIM to identify the serum migration body. Whether an extracellular vesicle in serum is a migration body or not is identified from the two aspects of biochemistry and shape, so that the defect that the serum migration body is identified only from the shape in the prior art is overcome.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for identifying serum migrants that allows for the observation of high-resolution serum migrants. Background Technology

[0002] At present, the identification of migratory bodies mainly relies on electron microscopy to observe their morphology. Generally, vesicles with a diameter between 0.5 and 3 μm and with a small tail are defined as migratory bodies.

[0003] Chinese patent CN118910266A discloses a method for the extraction and identification of serum migrants, including:

[0004] (1) Extraction and purification of serum migratory bodies: Blood samples from normal individuals, breast cancer patients, and BCBM patients were collected in coagulation-promoting vacuum tubes using standard intravenous puncture procedures; serum was extracted by centrifugation at 3000 rpm for 10 min; 5 ml of cell-free serum was required for each purification. Centrifugation was performed at 1000 g for 10 min, followed by centrifugation at 4000 g for 20 min to remove large fragments and retain the supernatant; centrifugation was performed at 20000 g for 30 min to collect coarse particles, which were then washed with PBS; the precipitate was collected with 400 μl of extraction buffer, and the migratory bodies were purified by density gradient centrifugation using Optiprep as the density medium (Sigma-Aldrich, LYSISO1). First, a stepwise gradient was established, starting with 27% (800 μl), followed by 22.5% (1000 μl), 19% (1000 μl), 16% (1000 μl), 12% (900 μl), and 8% (300 μl). The crude extract of the miracules (4% Diluted Optiprep Fraction) was centrifuged at 150,000 × g for 4 h at 4 °C. Samples were collected from top to bottom (500-700 μl per fraction). Each fraction was mixed with an equal volume of PBS, centrifuged at 20,000 g for 30 min, and the precipitate was collected; this was the purified serum miracules.

[0005] (2) Transmission electron microscopy (TEM): The migratings were resuspended in 20 μl PBS, gently blown, and an equal volume of TEM fixative was added. The mixture was fixed at room temperature in the dark for 2 h and then placed in a 4℃ refrigerator for later use. The copper mesh with the support membrane was brought into contact with the sample solution surface and allowed to stand for 5 min. The copper mesh was then removed, and excess suspension was blotted off with filter paper. The copper mesh was then transferred to sterile distilled water and rinsed once. Excess water was blotted off with filter paper. The copper mesh was then transferred to 1% uranium acetate staining solution and stained for 2 min. The remaining dye was blotted off with filter paper and then rinsed once with distilled water. Excess water was blotted off with filter paper and the copper mesh was dried before being observed on the TEM.

[0006] (3) Western blot: Migratory proteins were extracted using RIPA reagent (Beyotime, Beijing, China). Protein concentration was determined using a BCA protein assay kit (Beyotime, Beijing, China); proteins were separated by 10–15% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane; the membrane was then blocked with 5% milk and incubated overnight at 4°C with primary antibodies against PIGK (1:1000, #ab201693, Abcam, USA), TSPAN4 (1:1000, #PA5-69344, Thermo Fisher, USA), and NDST1 (1:1000, #SAB1307040, Sigma, MO, USA); after washing the membrane with TBST, it was incubated with enzyme-labeled secondary antibodies (HRP-labeled goat anti-rabbit IgG (H+L), #A0208, Beyotime; HRP-labeled goat anti-mouse IgG (H+L), #A0216, Beyotime) at room temperature for 1 hour, followed by incubation with an enhanced chemiluminescence reagent (Thermo Fisher Scientific). The broodstock was incubated at Fisher Scientific, Waltham, MA, USA, and visualized using a chemiluminescence imaging system (Tanon, Shanghai, China).

[0007] Transmission electron microscopy confirmed that the size of the migratory bodies was approximately 500-3000 nm, indicating that the serum migratory bodies had been successfully purified. PIGK, NDST1, and TSPAN4 are protein markers in the serum migratory bodies. Western blotting experiments showed that these three marker proteins were detected in the serum migratory bodies of normal individuals, breast cancer patients, and BCBM patients.

[0008] Current techniques cannot accurately define whether an extracellular vesicle in serum is a migratory body based on its shape. The size and shape of migratory bodies can change after various centrifugation and pipetting processes. Relying on shape to determine migratory bodies has many drawbacks, and new technologies are urgently needed to define migratory bodies. Summary of the Invention

[0009] The purpose of this invention is to provide a method for identifying serum migratory organisms. This invention identifies whether an extracellular vesicle in serum is a migratory organism from both biochemical and morphological perspectives, thereby overcoming the shortcomings of existing technologies that only identify serum migratory organisms based on morphology.

[0010] A method for identifying serum transporters to achieve the above-mentioned objective includes:

[0011] Serum migrants were extracted from the serum by multiple centrifugation processes.

[0012] The serum migrants were subjected to immunofluorescence staining to obtain immunofluorescently stained serum migrants;

[0013] The serum migration bodies were identified by imaging the immunofluorescence-stained serum migration bodies using super-resolution fluorescence microscopy (SIM).

[0014] Preferably, the multiple centrifugation process for serum includes: performing multiple strong centrifugations on the serum in the tube, discarding the supernatant in the tube, and then performing density centrifugation and strong centrifugation on the serum in the tube.

[0015] Preferably, the process of performing multiple strong centrifugation treatments on the serum in the vacuum tube includes: performing a first strong centrifugation treatment on the serum in the tube; after the first strong centrifugation treatment on the serum in the tube, discarding the supernatant in the tube, resuspending it with PBS, and then performing a second strong centrifugation treatment on the serum in the tube.

[0016] Preferably, the density centrifugation and high-intensity centrifugation treatment includes: resuspending the cells in 10% sucrose suspension, adding centrifugation solutions of different sucrose density gradients one by one into centrifuge tubes, and centrifuging at 150,000g for 4 hours in an ultracentrifuge; taking the vesicles in the layer containing the migratory bodies, resuspending them in PBS, and then performing a third high-intensity centrifugation treatment; discarding the supernatant to obtain the purified serum migratory bodies.

[0017] Preferably, the immunofluorescence staining treatment of the serum migrants to obtain immunofluorescence-stained serum migrants includes: staining the serum migrant marker proteins with four primary antibodies; and treating the stained serum migrants with corresponding secondary antibodies carrying detectable labels to obtain immunofluorescence-stained serum migrants.

[0018] Preferably, staining serum migration marker proteins with four primary antibodies includes:

[0019] The migratory bodies were stained with primary antibodies against EOGT, PIGK, CPQ, and NDST1 marker proteins; and

[0020] The stained migratory bodies were subjected to a fourth strong centrifugation, then resuspended in PBS and washed twice.

[0021] Preferably, staining of the migratory body with primary antibodies against EOGT, PIGK, CPQ, and NDST1 marker proteins includes:

[0022] Anti-EOGT antibodies are used to bind to EOGT marker proteins that migrate within the body;

[0023] Anti-PIGK antibodies are used to bind to PIGK-marking proteins that migrate within the body;

[0024] Anti-CPQ antibodies are used to bind to CPQ marker proteins that migrate within the body;

[0025] Anti-NDST1 antibodies were used to bind to NDST1 marker proteins in the body.

[0026] Preferably, the treatment of the stained serum migratory bodies with a secondary antibody carrying a detectable label includes: resuspending the migratory bodies with the secondary antibody and incubating at room temperature for 30 min; then performing a fifth strong centrifugation, resuspending in PBS, and washing twice.

[0027] Preferably, the first, second, third, fourth, and fifth strong centrifugation treatments are all performed by centrifuging at 20,000g centrifugal force for 30 minutes in a centrifuge at 4 degrees Celsius.

[0028] This invention identifies serum migratory organisms by combining immunofluorescence staining with super-resolution imaging. By staining four marker proteins of migratory organisms—EOGT, PIGK, CPQ, and NDST1—and combining them with super-resolution imaging, the success rate of serum migratory organism identification is improved by identifying whether an extracellular vesicle in the serum is a migratory organism from both biochemical and morphological perspectives. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the process for identifying serum migration bodies using immunofluorescence staining combined with super-resolution imaging according to the present invention;

[0030] Figure 2 This is a comparison image of serum migrations observed using the method of this invention and serum migrations observed using electron microscopy with existing technology.

[0031] Figure 3 This is a schematic diagram of the composition of serum-migrating proteins obtained by the method of this invention, based on protein proteometry. Detailed Implementation

[0032] See Figure 1 The serum migration identification method of the present invention mainly includes:

[0033] Step 1: Centrifuge the serum at 20000g for 30 minutes at 4 degrees Celsius.

[0034] Step 2: Discard the supernatant, resuspend in PBS, and repeat Step 1.

[0035] Step 3: Discard the supernatant, resuspend the sample in 10% sucrose suspension, and add the sample into centrifuge tubes one by one according to density. Centrifuge at 150,000g for 4 hours in an ultracentrifuge.

[0036] Step 4: Take the vesicles from the layer containing the migratory bodies, resuspend them in PBS, and centrifuge at 20000g for 30 minutes.

[0037] Step 5: Discard the supernatant and stain the migratory bodies with primary antibodies against marker proteins such as EOGT, PIGK, CPQ, and NDST1 at room temperature for one hour.

[0038] Step 6: Centrifuge at 20000g for 30 minutes, resuspend in PBS, and wash twice;

[0039] Step 7: Resuspend the corresponding secondary antibody in the migratory cells and incubate at room temperature for 30 min;

[0040] Step 8: Centrifuge at 20000g for 30 minutes, resuspend in PBS, and wash twice;

[0041] Step 9: Image using super-resolution SIM (super-resolution fluorescence microscopy).

[0042] Specifically, a method for identifying serum migrants according to the present invention includes: extracting serum migrants from serum by performing multiple centrifugation treatments on serum, that is, purifying serum migrants to obtain purified serum migrants;

[0043] The serum migrants were subjected to immunofluorescence staining to obtain immunofluorescently stained serum migrants;

[0044] The serum migration bodies were identified by imaging the immunofluorescence-stained serum migration bodies using super-resolution fluorescence microscopy (SIM).

[0045] The method of the present invention for multiple centrifugation of serum includes: performing multiple strong centrifugations on the serum in the tube, discarding the supernatant in the tube, and then performing density centrifugation and strong centrifugation on the serum in the tube.

[0046] The method of the present invention for performing multiple strong centrifugation treatments on serum in a tube includes: performing a first strong centrifugation treatment on the serum in the tube; after the first strong centrifugation treatment on the serum in the tube, discarding the supernatant in the tube, resuspending it with PBS, and then performing a second strong centrifugation treatment on the serum in the tube.

[0047] In this invention, density centrifugation and high-intensity centrifugation include: resuspending the cells in 10% sucrose suspension, adding centrifugation solutions of different sucrose density gradients one by one into centrifuge tubes, and centrifuging at 150,000g for 4 hours in an ultracentrifuge; taking the vesicles in the layer containing the migratory cells, resuspending them in PBS, and then performing a third high-intensity centrifugation; discarding the supernatant to obtain the purified serum migratory cells.

[0048] The method of the present invention for immunofluorescence staining of serum migrants to obtain immunofluorescence-stained serum migrants includes: staining four marker proteins of serum migrants with four primary antibodies; and treating the stained serum migrants with four marker proteins with secondary antibodies carrying detectable labels to obtain immunofluorescence-stained serum migrants.

[0049] The method of this invention uses four primary antibodies to stain serum migratory bodies, including: staining serum migratory bodies with primary antibodies against EOGT, PIGK, CPQ, and NDST1 marker proteins; and performing a fourth strong centrifugation on the stained migratory bodies, followed by resuspending in PBS and washing twice.

[0050] Specifically, staining serum migrants with primary antibodies against EOGT, PIGK, CPQ, and NDST1 marker proteins includes: binding anti-EOGT antibody to EOGT marker proteins in migrants; binding anti-PIGK antibody to PIGK marker proteins in migrants; binding anti-CPQ antibody to CPQ marker proteins in migrants; and binding anti-NDST1 antibody to NDST1 marker proteins in migrants.

[0051] The method of the present invention utilizes a secondary antibody carrying a detectable label to treat stained serum migratory bodies, including: resuspending the migratory bodies with the secondary antibody and incubating at room temperature for 30 min; then performing a fifth strong centrifugation, resuspending in PBS, and washing twice.

[0052] The first, second, third, fourth, and fifth strong centrifugation treatments of this invention are all performed by centrifuging at 20,000g centrifugal force for 30 minutes in a centrifuge at 4 degrees Celsius.

[0053] This invention utilizes a combination of biochemical analysis and super-resolution optical imaging. Super-resolution imaging technology can overcome the weakness of immunofluorescence staining signals for serum migratory organisms, thereby enabling the identification of serum migratory organisms.

[0054] This invention is characterized by its accuracy, speed, and convenience, and it is the first of its kind for the accurate identification of serum migratory organisms.

[0055] Figure 2 b shows serum migratory bodies observed using electron microscopy with existing technology. Figure 2 c shows serum migrants observed using the method of the present invention; Figure 2 d shows serum migratory organisms observed using the method of the present invention. Figure 2 b and Figure 2 c and Figure 2 Compared to d, the present invention can significantly increase the success rate of serum migration identification. In other words, the method of the present invention can accurately identify whether vesicles in serum are migration bodies.

[0056] Furthermore, the mass spectrometry detection of the migrants identified by this invention better reflects the true situation of the migrants, see [link to related document]. Figure 3 e and Figure 3 f.

[0057] Although the present invention has been described in detail above, it is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the protection scope of the present invention.

Claims

1. A method for identifying serum migrants, comprising: Serum migrants were extracted from the serum by multiple centrifugation processes. The serum migrants were subjected to immunofluorescence staining to obtain immunofluorescently stained serum migrants; The serum migration bodies were identified by imaging the immunofluorescence-stained serum migration bodies using super-resolution fluorescence microscopy (SIM).

2. The method for identifying serum migrants according to claim 1, wherein the serum undergoes multiple centrifugation treatment, comprising: After performing multiple strong centrifugations on the serum in the tube, the supernatant in the tube was discarded. Then, the serum in the tube was subjected to density centrifugation and strong centrifugation.

3. The method for identifying serum migrants according to claim 2, wherein the serum in the tube undergoes multiple strong centrifugation treatments, comprising: Perform a first strong centrifugation on the serum in the tube; After the serum in the tube is subjected to the first strong centrifugation, the supernatant in the tube is discarded, the serum is resuspended in PBS, and then the serum in the tube is subjected to a second strong centrifugation.

4. The method for identifying serum migrants according to claim 3, wherein the density centrifugation and strong centrifugation treatments include: Resuspend the contents in a 10% sucrose suspension and add centrifuged liquids of different sucrose density gradients into centrifuge tubes one by one. Centrifuge at 150,000g for 4 hours in an ultracentrifuge. Take the vesicles from the layer containing the migratory bodies, resuspend them in PBS, and then perform a third strong centrifugation. The supernatant was discarded, and the purified serum migrations were obtained.

5. A method for identifying serum migrants according to any one of claims 1-4, wherein immunofluorescence staining is performed on the serum migrants to obtain immunofluorescently stained serum migrants, comprising: Four primary antibodies were used to stain four marker proteins of serum migratory cells; Serum migrants stained with four marker proteins were treated with secondary antibodies carrying detectable labels to obtain immunofluorescently stained serum migrants.

6. The method for identifying serum migrants according to claim 5, comprising staining serum migrants with four primary antibodies, including: Serum migrants were stained with primary antibodies against EOGT, PIGK, CPQ, and NDST1 marker proteins. as well as The stained migratory bodies were subjected to a fourth strong centrifugation, then resuspended in PBS and washed twice.

7. The method for identifying serum migratory organisms according to claim 6, comprising staining serum migratory organisms with primary antibodies against EOGT, PIGK, CPQ, and NDST1 marker proteins, including: Anti-EOGT antibodies are used to bind to EOGT marker proteins that migrate within the body; Anti-PIGK antibodies are used to bind to PIGK-marking proteins that migrate within the body; Anti-CPQ antibodies are used to bind to CPQ marker proteins that migrate within the body; Anti-NDST1 antibodies were used to bind to NDST1 marker proteins in the body.

8. The method for identifying serum migratory organisms according to claim 5, comprising treating the stained serum migratory organisms with a secondary antibody carrying a detectable marker, comprising: The secondary antibody was resuspended in the transporter and incubated at room temperature for 30 min. Then perform a fifth strong centrifugation, resuspend in PBS, and wash twice.

9. A method for identifying serum migratory organisms according to claim 3, 4, 6, or 8, wherein the first, second, third, fourth, and fifth strong centrifugation treatments are all performed by centrifuging at 20,000g for 30 minutes at 4 degrees Celsius.

Images