Separation and detection method of trophoblast cell-derived migration body and extracellular secretion vesicle
By isolating and detecting placental trophoblast cell-derived migratory organisms, and utilizing particle size and specific protein markers, the problem of lacking effective isolation and detection in existing technologies has been solved, thus promoting the understanding and treatment of pregnancy-related diseases.
Patent Information
- Application Number
- CN202511576576.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-31
- Publication Date
- 2026-01-23
AI Technical Summary
Current technologies lack effective methods for isolating and detecting placental trophoblast cell-derived migratory organisms, hindering research on maternal-fetal interface communication and pregnancy-related disease mechanisms.
A method is provided for the specific isolation and purification of trophoblast-derived migratory organisms, which are captured by single-layer membrane vesicles with a particle size of 300-3000 nm and specific protein markers (such as HLA-G, CK7, TSPAN4, NDST1, CPQ, PIGK), and detected by electron microscopy, nanoparticle size detection and nanoflow cytometry.
This study enabled the specific isolation and detection of placental trophoblast cell migration bodies, enriching the research on the disease mechanisms related to trophoblast dysfunction and providing new directions and clues for the treatment of pregnancy-related diseases.
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Figure CN121379938A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and relates to a method for separating and detecting a trophoblast cell-derived migrasome and an extracellular exosome. BACKGROUND
[0002] Placental trophoblast cells are fetal-derived cells that directly contact the maternal immune system, and the moderate proliferation, migration and invasion of trophoblast cells are key events for placenta formation and fetal growth. In addition to being able to migrate and invade the decidual matrix remodeling spiral arterioles to promote placental development, trophoblast cells can also communicate with various cells at the maternal-fetal interface, such as immune cells and stromal cells, to establish immune tolerance and maintain pregnancy.
[0003] During cell migration, the cell will pull out many contractile fibers behind it, and at the tips and intersections of these contractile fibers, small vesicles with a diameter of about 0.3-3 μm will be produced. These small vesicles are formed in dependence on cell migration and are called “migrasomes”. Migrasomes are a new type of organelle that is mainly formed during cell migration and is involved in intercellular communication and intracellular material transport. In recent years, research on migrasomes has been continuously deepened, and scientists have gradually revealed their important role in cell migration, development and disease. The discovery of migrasomes provides a new perspective for understanding cell-cell interactions and signal transmission, and future research will further explore their specific functions in physiological and pathological processes.
[0004] Studies have shown that extravillous trophoblasts (EVTs) at the front end of placental villi have a high migration and invasion ability, and the vesicles released by EVT cells are rich in chemotactic factors, and secrete various factors and nucleic acids and other substances, mediate information exchange between maternal and fetal tissues and organs, and maintain maternal-fetal homeostasis. There is currently no report that trophoblast cells can communicate with cells at the maternal-fetal interface by releasing migrasomes. Therefore, the present application aims to provide a method for specifically separating and purifying trophoblast cell-derived migrasomes, and to provide a research basis for the mechanism of migrasomes participating in the occurrence and development of pregnancy-related diseases. SUMMARY
[0005] The present application provides a method for specifically separating and capturing placental trophoblast cell-derived migrasomes, and also provides a method for detecting trophoblast cell-derived migrasomes and extracellular exosomes.
[0006] An extracellular exosome is a trophoblast cell-derived migrasome.
[0007] The extracellular secretion vesicle of the application has the following markers: ① vesicle with a particle size of 300-3000 nm, single-layer membrane structure; ② containing smaller vesicles inside; and ③ expressing at least one of the following proteins: HLA-G, CK7, TSPAN4, NDST1, CPQ and PIGK.
[0008] A method for separating trophoblast cell-derived migratory bodies, comprising the following steps: Step (1) collecting normal pregnancy control population and part of placental villus tissue of patients with adverse pregnancy outcome and spontaneous abortion due to social factors requiring abortion in pre-cooled PBS buffer, or selecting uterine placental tissue of pregnant mice at different gestational days in pre-cooled PBS buffer; Step (2) rinsing the placenta or villus tissue in PBS containing 1% penicillin-streptomycin double-antibiotic solution, and after sufficient rinsing, digesting into single cells to obtain cell suspension with 1640 culture medium containing type I and type IV collagenase, and neutralizing the collagenase digestion solution with 1640 culture medium containing high-temperature heat-inactivated fetal bovine serum according to 5 times volume.
[0009] Step (3) the following centrifugation operations are all carried out at 4℃: 1000 rpm centrifugation for 5 minutes to obtain cell precipitate; 1000 g centrifugation for 10 minutes to remove larger fragments, and the supernatant is continuously centrifuged at 4000 g for 20 minutes to remove cell fragments; the supernatant is continuously centrifuged at 20,000 g for 30-60 minutes to obtain the precipitate as crude migratory bodies; the supernatant is transferred to a new centrifuge tube, and the centrifugation is continuously carried out at 160,000 g for 2-3 hours to obtain the precipitate as exosomes; using a 4℃ pre-cooled high-speed centrifuge, 1000 g centrifugation is carried out for 10 min, the precipitate is discarded, and the supernatant is recovered, then 4000 g centrifugation is carried out for 20 min, the precipitate is discarded, and the supernatant is recovered, then 20000 g centrifugation is carried out for 30 min, and the precipitate is the crude migratory bodies obtained by separation; Step (4) using Optiprep as a density medium, a density gradient of 40% (1 ml), 35% (1 ml), 30% (1 ml), 25% (1 ml), 20% (1 ml), 15% (1 ml), 10% (1 ml) and 5% (1 ml) and sample (5%, 1.5 ml) is established, a horizontal rotor is used on a high-speed centrifuge, 150,000 g slow speed is used at 4℃, and the centrifugation is carried out for 4 hours, the target sample is enriched between 10%-25%, the precipitate is washed with PBS and centrifuged at 20,000 g for 30 minutes again to obtain the trophoblast cell-derived migratory bodies.
[0010] A detection method of trophoblast cell-derived migratory bodies, comprising one of electron microscope detection, nano-particle size detection and nano-flow analysis detection, and being used for judging the existence of the migratory bodies.
[0011] The electron microscope detection method of the application comprises the following steps: Step (1) The separated migration body is placed in a 2.5% glutaraldehyde solution and fixed overnight in a 4°C refrigerator; Step (2) The sample is washed with PBS at 4°C; Step (3) The sample is fixed again with a 1% osmium tetroxide solution at 4°C; Step (4) The sample is dehydrated in an ethanol and acetone solution at 4°C; Step (5) The sample is soaked in acetone and resin; Step (6) The sample is embedded with pure resin at 60°C overnight; Step (7) Ultrathin sectioning and nickel mesh sectioning; Step (8) Soaking in 1% NaIO4 or 1% H2O2 for 10 min and washing with water for 3 times; Step (9) Transmission electron microscope observation.
[0012] The nanoparticle size detection method of the application comprises the following steps: Step (1) The separated migration body is filtered through a 0.45 μm filter, and an appropriate amount of PBS is sucked with a syringe to flush the filter in the reverse direction to obtain migration bodies larger than 0.45 μm; Step (2) The migration bodies obtained after flushing are detected by a nanoparticle size analyzer to detect the particle size range as shown in Figure 3 .
[0013] The nanoflow analysis detection method of the application comprises the following steps: sample staining: fixing the migration body with paraformaldehyde for 30 min, blocking the sample with a 0.1%-triton-100 3%-BSA solution for 30 min, diluting the specific surface marker with PBS, resuspending the prepared migration body with a primary antibody diluent, and staining at 4°C for 1 hour; stopping the staining with 2 times the volume of PBS, centrifuging at 20,000 g for 30 min, and discarding the supernatant; incubating with a fluorescent conjugated antibody, avoiding light at 4°C for 30 min, stopping the staining with 2 times the volume of PBS, centrifuging at 20,000 g for 30 min, discarding the supernatant, and resuspending in 200 ul PBS and then transferring to a flow tube.
[0014] Beneficial effects: The application first provides a group of membrane proteins specifically expressed by placental villous trophoblast-derived migration bodies, and provides a method for specifically capturing placental trophoblast-derived migration bodies based on the same, which can enrich the new direction of studying the mechanism of trophoblast dysfunction-related diseases.
[0015] (1) The migration body described in the application can have one or more characteristics, so that it can be separated, detected or characterized. The characteristics of the components can distinguish it from other organelles or other extracellular vesicles.
[0016] (2) The placental villus tissue is relatively easy to obtain as medical waste in the abortion curettage operation, and is large in quantity and easy to detect.
[0017] (3) The migration body of the placental villus tissue is mainly derived from the trophoblast cells, and has certain accuracy and specificity as a biomarker.
[0018] The present application aims to provide a method for separating and detecting the placental trophoblast-derived migration body and extracellular secretory vesicle, and by specifically capturing the trophoblast-derived migration body and accurately analyzing the same, new thinking direction and clues can be provided for the treatment of pregnancy-related diseases. The migration body is used as a biomarker to predict early damage of the trophoblast cells in early pregnancy. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 Double immunofluorescence staining diagram of the migration body marker protein TSPAN4 and the specific dye WGA Figure 2 Transmission electron microscope diagram for separating the migration body in the placental villus tissue.
[0020] Figure 3 Distribution diagram of the particle size of the migration body detected by the Nanosight.
[0021] Figure 4 Flow cytometry analysis diagram of HLA-G, CK7 and E-cadherin of the placental trophoblast migration body of the normal control population detected by the Nanosight.
[0022] Figure 5 Flow cytometry diagram and statistical analysis diagram of TSPAN4 of the placental villus migration body of the normal person and the spontaneous abortion patient detected by the Nanosight. DETAILED DESCRIPTION
[0023] The technical solutions of the present application will be described in detail below with reference to the drawings: An extracellular secretory vesicle, wherein the extracellular vesicle is a migration body derived from a trophoblast cell.
[0024] The marker is: ① a vesicle with a particle size of 300-2000 nm and a single-layer membrane structure; ② containing a smaller vesicle inside; and ③ expressing proteins HLA-G, CK7, TSPAN4, NDST1, CPQ and PIGK.
[0025] The present application proposes that the expression of the specific proteins of the migration body of the normal person and the patient with an adverse pregnancy outcome is analyzed, so as to judge the change of the migration body in different pregnancy outcomes.
[0026] A separation method of a migration body derived from a trophoblast cell, comprising the following steps: Step (1) Collecting the placental villi tissues of normal pregnancy with social factors requiring abortion and the patients with adverse pregnancy outcomes and spontaneous abortion in pre-cooled PBS buffer solution, or selecting the uterine placental tissues of pregnant mice at different gestational days in pre-cooled PBS buffer solution; Step (2) Rinsing the placental or villi tissues in PBS with 1% penicillin-streptomycin double-antibiotic solution, and after sufficient rinsing, digesting into single cells to obtain cell suspension with 1640 culture medium containing type I and type IV collagenase, and diluting and neutralizing the collagenase digestion solution with 1640 culture medium containing high-temperature heat-inactivated fetal bovine serum according to 5 times volume.
[0027] Step (3) The following centrifugation operations are all carried out at 4℃: 1000 rpm centrifugation for 5 minutes to obtain cell precipitate; 1000 g centrifugation for 10 minutes to remove larger fragments, and the supernatant is continuously centrifuged at 4000 g for 20 minutes to remove cell fragments; the supernatant is continuously centrifuged at 20,000 g for 30-60 minutes to obtain the precipitate as crude migratory bodies; the supernatant is transferred to a new centrifuge tube, and the supernatant is continuously centrifuged at 160,000 g for 2-3 hours to obtain the precipitate as exosomes; using a 4℃ pre-cooled high-speed centrifuge, 1000 g centrifugation for 10 min, discarding the precipitate and recovering the supernatant, then 4000 g centrifugation for 20 min, discarding the precipitate and recovering the supernatant, then 20000 g centrifugation for 30 min, and the precipitate is the crude migratory bodies obtained by separation; Step (4) Using Optiprep (Sigma-Aldrich) as a density medium, a density gradient of 40% (1 ml), 35% (1 ml), 30% (1 ml), 25% (1 ml), 20% (1 ml), 15% (1 ml), 10% (1 ml) and 5% (1 ml) and sample (5%, 1.5 ml) is established, using a horizontal rotor on a high-speed centrifuge, slowly increasing and decreasing speed at 150,000 g at 4℃, centrifuging for 4 hours, and the target sample is enriched between 10%-25%, and the precipitate is washed with PBS and centrifuged at 20,000 g for 30 minutes to obtain the migratory bodies derived from the trophoblast cells.
[0028] After the migratory bodies are stained with WGA, incubated with TSPAN4 antibody overnight, washed and centrifuged with PBS, stained with Alexa-488 fluorescent secondary antibody for 1 hour, and then observed and photographed under a fluorescence microscope to observe the staining of the migratory bodies. Figure 1 As shown in the figure, it can be seen that the signal of TSPAN4 and the signal of WGA are almost completely coincided, proving that the migratory bodies can be separated from the placental villi tissues according to the application.
[0029] A detection method of trophoblast cell-derived migratory bodies, comprising one of electron microscope detection, nanoparticle size detection and nanoflow analysis detection, for judging the presence of the migratory bodies.
[0030] The electron microscope detection method of the present application comprises the following steps: Step (1) placing the separated migratory bodies in a 2.5% glutaraldehyde solution and fixing in a 4℃ refrigerator overnight; Step (2) PBS washing of the sample at 4℃; Step (3) 1% osmium tetroxide solution 4℃ re-fixing of the sample; Step (4) placing the sample in an ethanol and acetone solution for 4℃ dehydration; Step (5) acetone and resin soaking of the sample; Step (6) pure resin 60℃ embedding of the sample overnight; Step (7) ultrathin sectioning and nickel mesh sectioning; Step (8) 1% NaIO4 or 1% H2O2 soaking for 10 min and water washing for 3 times; Step (9) transmission electron microscope observation and photographing as shown in Figure 2 It can be seen from the photograph that the morphological characteristics of the separated and purified migratory bodies of the present application conform to the morphological definition of the migratory bodies in related research.
[0031] The nanoparticle size detection method of the present application comprises the following steps: Step (1) filtering the separated migratory bodies with a 0.45μm filter, sucking an appropriate amount of PBS with a syringe and injecting it in the reverse direction to flush the filter, so as to obtain migratory bodies greater than 0.45μm; Step (2) using a nanoparticle size analyzer to detect the particle size range of the migratory bodies obtained after flushing. As shown in Figure 3 It can be seen from the photograph that the particle size of the separated and purified migratory bodies of the present application conforms to the standard for the particle size of the migratory bodies in related research.
[0032] The nanoflow analysis detection method of the present application comprises the following steps: sample staining: fixing the migratory bodies with paraformaldehyde for 30 min, blocking the sample with a 0.1%-triton-100 3%-BSA solution for 30 min, diluting a specific surface marker with PBS, resuspending the prepared migratory bodies with a primary antibody diluent and staining at 4℃ for 1 hour; stopping the staining with 2 times the volume of PBS, centrifuging at 20,000g for 30 min and discarding the supernatant; incubating with a fluorescent conjugated antibody at 4℃ for 30 min, stopping the staining with 2 times the volume of PBS, centrifuging at 20,000g for 30 min and discarding the supernatant, and transferring 200 ul of the resuspended PBS to a flow tube. As shown in Figure 4 and Figure 5As shown in the figure, it can be seen that the migration body after separation expresses HLA-G, CK7, E-cadherind and TSPAN4 marker proteins; the expression amount of TSPAN4 of the placenta of the abortion patient is significantly lower than that of the normal pregnancy placenta.
[0033] The protection scope of the present application is not limited to the above-mentioned embodiments. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.
Claims
1. An extracellularly secreted vesicle, characterized in that The extracellular secretory vesicle is a trophoblast cell-derived migratory body.
2. Extracellularly secreted vesicle according to claim 1, characterized in that The marker is: ① vesicles with a particle size of 300-3000 nm, a single-layer membrane structure; ② containing smaller vesicles inside; ③ expressing at least one of the following proteins: HLA-G, CK7, TSPAN4, NDST1, CPQ, and PIGK.
3. A method for isolating trophoblast cell-derived migratory bodies, characterized by The method comprises the following steps: Step (1) Collecting normal pregnancy control population and part of placental villus tissue of adverse pregnancy outcome spontaneous abortion patients due to social factors requiring abortion in pre-cooled PBS buffer, or selecting pregnant mice at different gestational days to remove uterine placental tissue in pre-cooled PBS buffer; Step (2) Rinsing the placenta or villus tissue in PBS containing 1% penicillin-streptomycin double-antibiotic solution, sufficiently rinsing, digesting into single cells to obtain cell suspension with type I and type IV collagenase-containing 1640 culture medium, and diluting and neutralizing the collagenase digestion solution with 1640 culture medium containing high-temperature heat-inactivated fetal bovine serum according to 5 times volume; Step (3) The following centrifugal operations are all carried out at 4 DEG C: 1000 rpm centrifugation for 5 minutes to obtain cell precipitate; 1000 g centrifugation for 10 minutes to remove larger fragments, and leaving the supernatant to continue 4000 g centrifugation for 20 minutes to remove cell fragments; 20,000 g centrifugation for 30-60 minutes to obtain the precipitate as crude migratory bodies; the supernatant is transferred to a new centrifugal tube, and 160,000 g centrifugation for 2-3 hours is continued to obtain the precipitate as exosomes; using a 4 DEG C pre-cooled high-speed centrifuge, 1000 g centrifugation for 10 min is carried out, the precipitate is discarded, and the supernatant is recovered; then 4000 g centrifugation for 20 min is carried out, the precipitate is discarded, and the supernatant is recovered; then 20000 g centrifugation for 30 min is carried out, and the precipitate is the crude migratory bodies obtained by separation; Step (4) Using Optiprep as a density medium, a density gradient of 40% (1 ml), 35% (1 ml), 30% (1 ml), 25% (1 ml), 20% (1 ml), 15% (1 ml), 10% (1 ml) and 5% (1 ml) and sample (5%, 1.5 ml) is established, a horizontal rotor is used on a high-speed centrifuge, 150,000 g slow speed is used for speed-up and speed-down at 4 DEG C, and 4 hours of centrifugation is carried out, the target sample is enriched between 10%-25%, the precipitate is washed with PBS and 20,000 g centrifugation for 30 minutes is carried out again, and the trophoblast cell-derived migratory body is obtained.
4. A method for detecting trophoblast cell-derived migratory bodies, characterized by The method comprises one of electron microscope detection, nanoparticle size detection and nanoflow analysis detection, and is used for judging the existence of the migratory body.
5. The detection method of claim 4, wherein The electron microscope detection comprises the following steps: Step (1) Placing the separated migratory body in a 2.5% glutaraldehyde solution and fixing overnight in a 4 DEG C refrigerator; Step (2) PBS rinsing of the sample at 4 DEG C; Step (3) 1% osmium tetroxide solution 4 DEG C fixing of the sample again; Step (4) Dehydration of the sample in an ethanol and acetone solution at 4 DEG C; Step (5) Acetone and resin soaking of the sample; Step (6) Pure resin 60 DEG C embedding of the sample overnight; Step (7) Ultrathin sectioning and nickel mesh sectioning. Step (8) 1% NaIO4 or 1% H2O2 soak for 10 min, water wash 3 times; Step (9) Transmission electron microscope observation.
6. The method of claim 4, wherein The nanoparticle size detection includes the following steps: Step (1) filtering the separated migration body through a 0.45 μm filter, using a syringe to suck an appropriate amount of PBS, and injecting the filter in the reverse direction to wash it, so as to obtain migration bodies larger than 0.45 μm; Step (2) using a nanoparticle size analyzer to detect the particle size range of the migration bodies obtained after washing, as shown in FIG.
3.
7. The method of claim 4, wherein The nanoflow analysis detection includes the following steps: sample staining: fixing the migration body with paraformaldehyde for 30 min, blocking the sample with a 0.1%-triton-100 3%-BSA solution for 30 min, diluting a specific surface marker with PBS, resuspending the prepared migration body with a primary antibody diluent, and staining at 4°C for 1 hour; stopping the staining with 2 times the volume of PBS, centrifuging at 20,000 g for 30 minutes, and discarding the supernatant; again incubating with a fluorescent conjugated antibody, avoiding light at 4°C for 30 minutes, stopping the staining with 2 times the volume of PBS, centrifuging at 20,000 g for 30 minutes, discarding the supernatant, and resuspending in 200 ul PBS before transferring to a flow tube.