CN114660298A - Application of neutral amino acid transporter-based expression in exosome in aging detection - Google Patents
Application of neutral amino acid transporter-based expression in exosome in aging detection Download PDFInfo
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- CN114660298A CN114660298A CN202210056751.XA CN202210056751A CN114660298A CN 114660298 A CN114660298 A CN 114660298A CN 202210056751 A CN202210056751 A CN 202210056751A CN 114660298 A CN114660298 A CN 114660298A
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Abstract
The invention discloses an application of neutral amino acid transporter-based expression in exosomes in senescence detection, and relates to the field of biological senescence detection. The invention utilizes the quantitative detection of protein products of gene SLC1A5 with increased expression in exosome in the aging process, and the like, is applied to the determination of the aging state of organism cells, can be used for the detection of the effectiveness of anti-aging intervention, and has the advantages of simple and convenient sampling, relatively fast detection flow, good repeatability, high reliability and the like compared with the existing aging determination method.
Description
Technical Field
The invention relates to the field of biological aging detection, in particular to a method for determining cell aging degree based on exosomes obtained by extraction.
Background
Aging is a physiological process in which an organism undergoes functional decline with the increase of life time, and is closely related to the incidence and mortality of all types of diseases. Therefore, research on aging has been extensively conducted, and it is desired to develop a technology that interferes with the aging process. Whether the research on the biological mechanism of aging or the development of technical means for intervening aging, a method for detecting the speed and degree of aging of the organism is needed as a technical basis. At present, the detection of aging degree mainly surrounds external detection means such as natural age or human body function test. However, since there is a difference in aging rate between individuals due to factors such as genetics and lifestyle habits, the true aging degree cannot be objectively reflected by the above-mentioned conventional external detection means.
In recent years, molecular biological and biochemical means are increasingly applied to the detection of the degree of aging. However, these inherent detection methods still have some problems. For example, IL-6, TNF-a, and C-reactive protein (CRP) in blood can be used to measure the degree of aging, but the sample is easily degraded after leaving the body to cause inaccurate data, and thus does not well reflect the actual degree of aging. In addition, DNA damage, telomere length, and DNA methylation are also used in the detection of the degree of senescence, but since these methods are complicated to implement, sampling is relatively inconvenient. Therefore, it is necessary to develop a new and easy internal detection means with high reliability.
Exosomes are membrane vesicles secreted by cells with diameters between 40-160nm, and contain bioactive molecules such as lipids, proteins, nucleic acids, etc., which play an important role in mediating intercellular communication. In terms of disease diagnosis, exosome has been tried to be used for early diagnosis or prognosis of diseases such as tumor, because exosome can protect the contents from degradation and the source and method for extracting exosome are convenient. This suggests that we also have potential for exosomes to be used to detect the aging process.
The SLC1A5 (solvent Carrier Family 1Member 5) gene encodes the production of three neutral amino acid transporters located in the cell membrane, namely neutral amino acid transporter B (0) isoform 1, neutral amino acid transporter B (0) isoform 2 and neutral amino acid transporter B (0) isoform 3, which are widely expressed in many types of normal tissues. In a model of cell aging, the expression level of the protein product of SLC1A5 is obviously increased, and the protein product has good specificity and repeatability. Even more remarkably, the protein product of SLC1a5 could also be detected in exosomes from a variety of sources.
Disclosure of Invention
The invention aims to solve the technical problem of providing an application of a neutral amino acid transporter-based expression in exosomes in senescence detection, and solving the problems of inconvenient implementation and inaccurate detection structure of the conventional senescence detection.
In order to solve the technical problems, the technical scheme of the invention is as follows:
an application of the neutral amino acid transporter in the expression of its secretion in the senility test is disclosed, which features that the secretion with a certain concentration is obtained from the body fluid of human body, and the neutral amino acid transporter generated after the SLC1A5 gene in said secretion is encoded, expressed and translated is detected by immunological test method.
Preferably, the exosomes are extracted from a body fluid of a body.
Preferably, the body fluid is at least one of blood, cerebrospinal fluid, tears, saliva, sputum, pericardial fluid, thoracic cavity fluid, abdominal cavity fluid, gastric fluid, pancreatic fluid, biliary fluid, prostatic fluid, joint cavity fluid, lymph fluid, urine, semen, vaginal secretion, amniotic fluid or sweat.
Preferably, the method for obtaining exosomes at a certain concentration from body fluid of a body uses at least one of ultracentrifugation, density gradient centrifugation, filtration, immunoaffinity separation, precipitation, microfluidic separation or size exclusion chromatography.
Preferably, the exosomes have a diameter in the range of 40-160 nm.
Preferably, the immunological detection method comprises at least one of a fluorescence immunological technique, a radioimmunoassay, an enzyme-linked immunosorbent assay, a chemiluminescent immunological technique or an immunogold labeling technique.
Preferably, the neutral amino acid transporter is assayed quantitatively or semi-quantitatively.
By adopting the technical scheme, the quantitative detection of protein products of genes such as SLC1A5 with increased expression in exosome in the aging process is utilized, the method is applied to the determination of the aging state of body cells, can be used for detecting the effectiveness of anti-aging intervention, and has the advantages of simple and convenient sampling, relatively fast detection flow, good repeatability, high reliability and the like compared with the existing aging determination method.
Detailed Description
The following further describes embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
Establishing an aging cell model; and extracting the exosome. The specific scheme is as follows:
(1-1) adding adriamycin into a culture medium of a human embryonic kidney HEK293 cell line, and determining the aging state of the treated cell model by using an SA-beta galactosidase kit after 24 hours of treatment;
(1-2) when the cell fusion degree reaches about 90%, collecting cell supernatant, filling the cell supernatant into a sterile 50ml centrifuge tube, sealing the centrifuge tube by a sealing film, centrifuging the cell supernatant, discarding cell precipitates, collecting the supernatant into a new sterile 50ml centrifuge tube, and performing sterile packaging at-80 ℃ for storage;
(1-3) ultracentrifugation for exosome extraction: centrifuging at low speed for 10min, centrifuging at medium speed for 10min, centrifuging at high speed for 30min, collecting supernatant, discarding precipitate, performing ultracentrifugation twice with ultracentrifuge, performing ultracentrifugation for 70min for the first time, discarding supernatant, and washing precipitate with large amount of PBS; then carrying out second ultracentrifugation for 70min, wherein the precipitate obtained after the second ultracentrifugation is the exosome precipitate, and the centrifugation steps are all carried out at 4 ℃;
(1-4) the pellet was dissolved in PBS and 50. mu.l of each tube was dispensed, and the pellet was stored at-80 ℃.
Example 2
The exosome is extracted from blood of an aged volunteer, and the specific scheme is as follows:
(2-1) extracting 50mL of peripheral blood from veins of old volunteers, centrifuging at low speed for 10min, discarding cell precipitate, collecting supernatant, placing into a new sterile 50mL centrifuge tube, aseptically packaging, storing at-80 deg.C,
(2-2) extracting extracellular vesicles by a kit method:
(2-2-1) collecting fresh supernatant or dissolving the supernatant stored at-80 ℃ at 4 ℃, keeping the temperature at 4 ℃, centrifuging at medium speed for 15min to remove cell debris, and transferring the supernatant to a new 50ml centrifuge tube;
(2-2-2) adding equal volume of Buffer XBP, slightly reversing the centrifuge tube for 5 times to mix the materials, placing the mixture in a test tube rack, and raising the temperature to room temperature;
(2-2-3) adding 10mL of Buffer XBP mixed solution on an exo easy centrifugal column membrane, centrifuging at low speed for 1 minute, discarding the effluent, putting the centrifugal column membrane into the same centrifuge tube, and repeating the step if the volume of the sample-Buffer XBP mixed solution is more than 10mL, so that 64mL of mixed solution is filtered by each centrifugal column membrane at most when all the liquid passes through the centrifugal column membrane;
(2-2-4) centrifuging the centrifugal column membrane by adding 10mL of Buffer XWP 5000g for 5 minutes, discarding the effluent, putting the centrifugal column membrane into a new centrifuge tube, adding 400 muL-1 mL of Buffer XE, incubating for 1 minute, centrifuging at low speed for 5 minutes, recovering the eluent, adding the eluent into the centrifugal column membrane, incubating for 1 minute, centrifuging at medium speed for 5 minutes, collecting the eluent to obtain a solution containing the exosome, and centrifuging at 4 ℃.
Example 3
The transmission electron microscope identifies exosome, and the specific scheme is as follows:
(3-1) preparing 2PSCs-EVs solution and a clean sealing film, dripping 20 mu l of extracellular vesicle solution onto the sealing film, and soaking a copper mesh in the extracellular vesicle solution for 1 min;
(3-2) absorbing the floating liquid by using filter paper, dropping uranyl acetate on a copper mesh for precipitation for 1min for counterstaining, absorbing the floating liquid by using the filter paper, and drying for 30min at normal temperature;
(3-3) transmission electron microscopy (TEM-1400plus) identification imaging is carried out at 80kv, and the shape of extracellular vesicles is observed, and the diameter is measured and photographed.
Example 4
The specific scheme for identifying exosome by using the nanoparticle tracking analysis method is as follows:
(4-1) adding a PSCs-EVs solution into a PBS solution, and diluting by 100 times;
(4-2) extracellular vesicle size distribution and concentration were measured using Malvern NanoSight LM14, which was repeated 3 times for each measurement and averaged.
Example 5
The western blot experiment identifies the expression of the exosome marker proteins CD9, CD63 and CD81, and the specific scheme is as follows:
(5-1) cleavage: adding a proper amount of cell lysate into the extracted exosomes for resuspension, placing on ice for cracking for 30min, and shaking on a vortex instrument once every 10min to ensure that the exosomes are fully cracked;
(5-2) BCA assay for protein concentration: carrying out protein concentration determination according to the BCA protein quantification kit specification, preparing a working solution with a proper volume (solution A: solution B is 50:1), diluting a BSA standard substance with a PBS solution, preparing a standard substance solution with a certain concentration gradient, adding the standard substance solution with each concentration into a 96-well plate, making three multiple wells with 10 mu l of each well, adding 2 mu l of the protein sample to be determined into each well, supplementing 10 mu l of the PBS solution (diluting the protein sample by 5 times), making three multiple wells with 200 mu l of the working solution, fully mixing, incubating in a 37 ℃ incubator for 30min, measuring the absorbance of each well at 562nm of a microplate reader, and calculating the protein concentration of the sample to be determined according to a standard curve;
(5-3) electrophoresis: installing an electrophoresis apparatus, pouring the prepared electrophoresis solution into an electrophoresis tank, filling the inner tank, carefully pulling out a comb, adding a colored protein marker and a protein sample to be detected into glue holes of the prefabricated glue, adding 5 mu l of 1 × Loading buffer into the glue holes on two sides, initially setting the electrophoresis parameters to be constant voltage of 80V, changing the constant voltage of 120V after about 45min when the protein sample enters separation glue, and stopping electrophoresis after about 1h phenol blue runs to the bottom of a glass plate;
(5-4) film transferring: taking a protein Maker as a reference, cutting a PDVF membrane with proper size and cutting gel according to the molecular weight of a target protein, soaking the cut PDVF membrane in methanol for activation for 3min, then transferring the PDVF membrane into a membrane transferring solution by using tweezers to prevent the membrane from drying, soaking a sponge and filter paper in the membrane transferring solution in advance, placing a membrane transferring clamp into a membrane transferring groove correctly from bottom to top according to the conditions that a positive pole of the membrane transferring plate → the sponge → 3 layers of filter paper → the PDVF membrane → protein sample gel to be detected → 3 layers of filter paper → the sponge → a negative pole of the membrane transferring plate (no air bubbles are generated in the process), pouring the membrane transferring solution into the membrane transferring groove carefully to ensure that the liquid does not pass through the PDVF membrane, placing the membrane transferring solution into an ice box, placing the whole membrane transferring groove into a container containing ice blocks, and setting membrane transferring parameters as constant currents of 200mA and 2 h;
(5-5) washing the membrane: after the membrane transfer is finished, putting the PDVF membrane into an antibody incubation box, putting the PDVF membrane on a shaker, and washing the PDVF membrane for 6min and 3 times by using a TBST solution;
(5-6) sealing: pouring off the TBST solution, and sealing each PDVF membrane for 90min by using a proper volume of skimmed milk powder solution;
(5-7) washing the membrane: after the sealing is finished, cleaning the PDVF membrane by using TBST solution for 3 times, 6min each time;
(5-8) blocking primary antibody: diluting the primary antibody by using an antibody diluent as a solvent according to an antibody specification, pouring out a TBST solution, sucking out residual liquid by using a liquid transfer machine, adding a proper amount of the primary antibody into an antibody incubation box to enable the primary antibody to be submerged in a corresponding PDVF membrane, and then putting the antibody incubation box into a shaking table at 4 ℃ for overnight incubation;
(5-9) washing the membrane: recovering primary antibody, storing at 4 deg.C, and cleaning PDVF membrane with TBST solution for 6min each time for 3 times;
(5-10) blocking secondary antibody: diluting the secondary antibody with skimmed milk powder according to the antibody specification, adding the diluted secondary antibody into a PDVF membrane, incubating for 90min at room temperature by using a shaking table, recovering the secondary antibody, and storing at-20 ℃ for washing the membrane by TBST;
(5-11) developing: the developing solution (solution A: solution B ═ 1:1) is prepared in situ under the dark condition, the PDVF film is placed on a developing platform, the developing solution is uniformly dropped, and the developing result is observed by a developing instrument.
Example 6
The enzyme linked immunosorbent assay is used for detecting the amount of the SCL1A5 coding product in the exosome, and the specific scheme is as follows:
(6-1) coating: diluting the antibody with 0.05M PH9. carbonate coated buffer solution until the protein content is 1-10 μ g/mL, adding 0.1mL into each reaction well of polystyrene plate, standing overnight at 4 deg.C, discarding the solution in the well the next day, washing 3 times with washing buffer solution each time for 3 min;
(6-2) sample application: adding 0.1mL of diluted exosome lysate to be detected into the coated reaction hole, placing the reaction hole at 37 ℃ for incubation for 1 hour, and then washing (simultaneously making blank holes, negative control holes and positive control holes);
(6-3) adding an enzyme-labeled antibody: adding a fresh diluted enzyme-labeled antibody (the dilution after titration) into each reaction hole, incubating for 0.5-1 hour at 37 ℃, and washing;
(6-4) adding a substrate liquid for color development: adding a TMB substrate solution prepared temporarily into each reaction hole, and incubating for 10-30 minutes at 37 ℃;
(6-5) termination of the reaction: adding 0.05mL of 2M sulfuric acid into each reaction hole;
(6-6) preliminary judgment of the result: the results can be observed directly with the naked eye on a white background: the darker the color in the reaction hole, the stronger the positive degree, the colorless or extremely light negative reaction, which is indicated by a "+" or "-" sign according to the color depth;
(6-7) OD value measurement: the OD value of each well was measured after zeroing the blank control well at 450nm using a microplate reader.
The results of the assay showed that the amount of SLC1a5 protein product in exosomes from senescent cell models and blood of older volunteers was significantly higher than that of exosomes from normal cells and young volunteers.
The invention utilizes the quantitative detection of protein products of gene SLC1A5 with increased expression in exosome in the aging process, and the like, is applied to the determination of the aging state of organism cells, can be used for the detection of the effectiveness of anti-aging intervention, and has the advantages of simple and convenient sampling, relatively fast detection flow, good repeatability, high reliability and the like compared with the existing aging determination method.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in the embodiments without departing from the principles and spirit of the invention, and these embodiments are still within the scope of the invention.
Claims (7)
1. Use of a neutral amino acid transporter-based expression in exosomes in an aging assay, characterized by: obtaining exosome with a certain concentration from body fluid of an organism, and determining neutral amino acid transporter produced after SLC1A5 gene coding expression translation in the exosome by using an immunological detection method.
2. Use of a neutral amino acid transporter-based expression in exosomes according to claim 1 in senescence assay, wherein: the exosome is extracted from a body fluid of the body.
3. Use of a neutral amino acid transporter-based expression in exosomes according to claim 1 in senescence assay, wherein: the body fluid is at least one of blood, cerebrospinal fluid, tears, saliva, sputum, pericardial fluid, thoracic cavity fluid, abdominal cavity fluid, gastric juice, pancreatic juice, biliary fluid, prostatic fluid, joint cavity fluid, lymph fluid, urine, semen, vaginal secretion, amniotic fluid or sweat.
4. Use of a neutral amino acid transporter-based expression in exosomes according to claim 1 in senescence assay, wherein: the method for obtaining exosome with a certain concentration from body fluid of organism adopts at least one of ultracentrifugation separation method, density gradient centrifugation separation method, filtration method, immunoaffinity separation method, precipitation method, microfluid separation method or size exclusion chromatography.
5. Use of a neutral amino acid transporter-based expression in exosomes according to claim 1 in senescence assay, wherein: the exosome diameter ranges from 40-160 nm.
6. Use of a neutral amino acid transporter-based expression in exosomes according to claim 1 in senescence assay, wherein: the immunological detection method comprises at least one of fluorescence immunoassay, radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay or immunogold labeling.
7. Use of a neutral amino acid transporter-based expression in exosomes according to claim 1 in senescence assay, wherein: a quantitative or semi-quantitative determination of the neutral amino acid transporter is performed.
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| CN117024556A (en) * | 2023-10-10 | 2023-11-10 | 天津外泌体科技有限公司 | Application of SLC1A5 as extracellular vesicle scaffold protein, extracellular vesicle, preparation method and application thereof |
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| CN117024556A (en) * | 2023-10-10 | 2023-11-10 | 天津外泌体科技有限公司 | Application of SLC1A5 as extracellular vesicle scaffold protein, extracellular vesicle, preparation method and application thereof |
| CN117024556B (en) * | 2023-10-10 | 2024-01-30 | 天津外泌体科技有限公司 | Application of SLC1A5 as extracellular vesicle scaffold protein, extracellular vesicle, preparation method and application thereof |
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