CN114457002B - Method for separating extracellular vesicle subgroup and application thereof - Google Patents
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Abstract
The invention discloses a method for separating extracellular vesicle subgroup and application thereof, wherein the vesicle subgroup is TCR-CD3 dimer vesicle subgroup, and the separation method is that EVs in a sample are captured by double-aptamer modified nano gold (Au@Apts); centrifugally separating to obtain a nano gold-EVs compound; and (3) reacting the separated nano gold-EVs complex with a complementary strand C1, centrifuging, re-suspending the precipitate, reacting with a complementary strand C2, and centrifuging to obtain the successfully separated TCR-CD3 dimer vesicle subgroup. And experiments show that dCD3+EVs can reflect the immune state of the parent T cells more accurately. As a marker for evaluating the immunological status change of the parent cells, the method effectively plays a role in diagnosing rejection reaction after organ transplantation. Aiming at the defects of difficult diagnosis of organ rejection, multiple interference factors of extracellular vesicle detection and the like, a brand-new, accurate and universal disease diagnosis marker is provided.
Description
Technical Field
The invention belongs to the technical field of nucleic acid nanotechnology, and particularly relates to a method for separating extracellular vesicle subpopulations and application thereof.
Background
Extracellular vesicles (Extracellular vesicles, EVs) are vesicles with a phospholipid bilayer secreted by most cells of the body. Its size is generally 30 to 200nm. EVs contain a variety of complex RNAs and proteins, and are widely present and distributed in various body fluids. Studies have shown that EVs are actively involved in intercellular communication by shuttling and transmitting signaling molecules between cells, with surface receptor recognition on EVs and plasma membranes being a prerequisite for cellular communication. The surface receptors of EVs originate from their parent cells, which can be targeted and captured by different receptor cells, which in turn trigger the EVs to deliver their carried proteins, lipids and even genetic information to the receptor cells. Thus, it has recently been proposed that EVs are more complex mixtures than previously recognized and that they can be further divided into different sub-populations according to their biological origin, particle size, molecular composition and characteristics. Subtle molecular differences between different subpopulations may lead to significant changes in biological function, and therefore, isolation and analysis of different extracellular vesicle subpopulations is critical for elucidating their characteristic expression patterns, identifying biomolecules that are preferentially enriched in certain subpopulations, and discovering potential disease markers.
The interaction of surface receptor proteins plays an important role in intracellular signal transduction, where dimerization and subsequent intracellular domain phosphorylation of enzyme-coupled receptors are key steps in triggering a series of intracellular downstream signaling cascades, including receptor tyrosine kinases, T cell receptor-CD 3 complexes, etc., which are involved in the development of physiological processes such as cell activation, proliferation, secreted proteins, apoptosis, etc. Taking the T cell receptor-CD 3 complex (TCR-CD 3) as an example, it initiates T cell activation by binding to the antigenic peptide of the Major Histocompatibility Complex (MHC) expressed on antigen presenting cells to induce TCR aggregation, conformational changes in CD3, and phosphorylation of immune receptor tyrosine-dependent activation motifs.
It has been found that TCR-CD3 is also expressed on membranes of EVs secreted by T cells, making EVs a powerful carrier for targeted delivery of signaling molecules to cells with the correct peptide/MHC complex. Thus, the CD3 protein on the surface of EVs is a potential disease marker that can be used to assess the degree of immune activation of the parental T cells. Several research methods for disease diagnosis and assessment based on CD3 positive expressed EVs (cd3+ EVs) have been reported so far, for example: in urine samples from kidney transplant patients, CD3+ EVs showed significantly higher levels in patients with organ rejection; the number of cd3+ EVs was observed to be greater in the plasma of tumor patients than in healthy donors. However, the accuracy of disease change is assessed by only the difference in the number of cd3+ EVs in body fluid, which is affected by factors such as individual differences of patients, sampling mode, storage conditions, EVs separation method, etc. Therefore, there is a need to develop a more practical and accurate method to quantify the variability of the expression of CD3 on the surface of EVs, i.e., the difference in the aggregation state of TCR-CD3, thereby eliminating the interference of the sample condition changes with the detection results.
TCR-CD3 exists predominantly in monomeric form on the cell membrane in the unactivated state, but is susceptible to aggregation and conformational changes following initial triggering of the antigenic peptide, a process necessary for T cell activation. Thus, the difference in surface CD3 abundance of EVs secreted by T cells of varying degrees of activation may be related to the aggregation state of TCR-CD3, and a new subset of EVs based on the change in the aggregation state of TCR-CD3 may be present to more accurately reflect the immune state of the parent T cell.
Disclosure of Invention
It is a first object of the present invention to provide a subset of vesicles.
It is a second object of the present invention to provide a method for isolating a subset of vesicles as described above.
A third object of the present invention is to provide the use of a substance for detecting a subset of vesicles according to the first aspect of the invention for the preparation of a product for assessing immune responses.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention there is provided a subset of vesicles which are a subset of TCR-CD3 dimerized vesicles (dcd3+evs). In a second aspect of the invention there is provided a method of isolating a subpopulation of vesicles according to the first aspect of the invention comprising the steps of:
s1: capturing EVs in a sample by double-aptamer modified nano-gold (Au@Apts); centrifugally separating to obtain a nano gold-EVs compound;
s2: and (3) reacting the separated nano gold-EVs complex with a complementary strand C1, centrifuging, re-suspending the precipitate, reacting with a complementary strand C2, and centrifuging to obtain the vesicle subgroup.
In some embodiments of the invention, the method for preparing the dual aptamer modified nanogold comprises the following steps: forming a double-aptamer structure through a nucleic acid aptamer, a connecting chain and a C5 chain, and then reacting with the nano gold particles; wherein the sequence of the nucleic acid aptamer is:
a1 sequence: 5'-GCCGCGGGGTGGGTCTAGTGTGGATGTTTAGGGGGCGGCCTACATCATCTCGATGGC-3';
a2 sequence: 5'-GCCGCGGGGTGGGTCTAGTGTGGATGTTTAGGGGGCGGCATCTATGAAAAGTATCTA-3';
the sequence of the connecting chain is as follows:
5’-SH-(CH 2 ) 6 -TAGTAGAGCTACCGAAGCCATCGAGATGATGTAGTAGTTACCATCCTACCATTGATGATGTGTTGTTAGATACTTTTCATAGATTGTTGT-3’；
the C5 sequence is as follows:
5’-CATCATCAATGGTAGGATGG-3’。
in some embodiments of the invention, the method of preparing the dual aptamer is: SH-T1, A1, A2 and C5 are mixed and placed at 35-38 ℃ to react for 1.5-2.5 h.
In some embodiments of the invention, the SH-T1, A1, A2, C5 concentration ratio is (0.5-1.5): 1.
In some preferred embodiments of the invention, the SH-T1, A1, A2, C5 concentration ratio is 1:1:1:1.
In some embodiments of the invention, the double-aptamer and the nano-gold are mixed and then reacted for 12-18 hours at 4 ℃, the free aptamer is removed by centrifugation, and then the gold surface which is not combined with the proper ligand is respectively subjected to sealing treatment by using mercaptohexitol and bovine serum albumin, so as to prevent nonspecific adsorption in the process of capturing EVs by the nano-gold.
In some embodiments of the invention, the concentration of the diadaptomic ligand is 1 to 3 μm; the concentration of the nano gold is 4-6 mg/mL; the dosage ratio of the double aptamer to the nano gold is (0.5-1.5): 1; preferably 1:1.
In some embodiments of the invention, the sequence of the complementary strand is:
c1 sequence: 5'-ACAACAATCTATGAAAAGTATCTAACAACA-3';
c2 sequence: 5'-TAACTACTACATCATCTCGATGGCTTCGGT-3'.
In some embodiments of the invention, the conditions for both the C1 and C2 reactions are from 35 to 38deg.C for 1.5 to 2.5 hours.
In a fourth aspect of the invention there is provided the use of a substance for detecting a subpopulation of vesicles as described in the first aspect of the invention in the manufacture of a product for assessing immune responses.
In immune activation, the proportion of the vesicle subgroup in the invention can be increased, so that the immune state of the sample can be evaluated.
After organ transplantation, diagnosis of rejection after organ transplantation can be made by detecting the proportion of vesicle subpopulations.
The beneficial effects of the invention are as follows:
the present invention uses double aptamer modified nanogold (Au@Apts) to capture CD3+ EVs in plasma and successfully isolate a new EVs subset based on the change of TCR-CD3 aggregation state, namely TCR-CD3 dimerized EVs subset (dCD3+ EVs). And experiments show that dCD3+EVs can reflect the immune state of the parent T cells more accurately. As a marker for evaluating the immunological status change of the parent cells, the method effectively plays a role in diagnosing rejection reaction after organ transplantation. Aiming at the defects of difficult diagnosis of organ rejection, multiple interference factors of extracellular vesicle detection and the like, a brand-new, accurate and universal disease diagnosis marker is provided.
Drawings
FIG. 1 shows the affinity comparison of CD3 aptamer and A1 with different cells (Jurakt, hepG2, WRL 68).
FIG. 2 shows gel electrophoresis to verify formation of dual aptamer structure and release of complementary strand.
FIG. 3 is a schematic diagram of formation of a dual aptamer structure and complementary strand release.
FIG. 4 shows DLS detection of dual aptamer modified nanogold.
FIG. 5 is an identification of model EVs; wherein fig. 5a: TEM images of EVs; fig. 5b: particle size distribution of EVs; fig. 5c: protein expression status of EVs and Jurkat cell lysates.
FIG. 6 is a SEM characterization of the morphology of the nanogold and Au@Apts@EVs complex. Fig. 6a: auNPs; fig. 6b: au@apts@evs.
FIG. 7 is a fluorescent nanoflow of Au@Apts@EVs complexes; wherein i: au@apts; ii: au@apts@evs complex; iii: au@apts@evs complex+c1+c2.
FIG. 8 is a view of the mice on days 4 and 8 after skin grafting; NR: c57 BL/6- > C57BL/6 allogeneic genome skin graft group; ACR: BALB/C- > C57BL/6 allogeneic genome skin graft group.
FIG. 9 is a schematic diagram of Au@Apts capture and isolation of a subset of TCR-CD3 dimeric EVs.
Fig. 10 is a comparison of CD3 fluorescence of isolated mcd3+ EVs and dcd3+ EVs subpopulations of ACR group plasma.
Fig. 11 is a comparison of particle size of mcd3+ EVs and dcd3+ EVs subpopulations isolated from NR and ACR plasma (n=5).
FIG. 12 is an analysis of the number of CD3+ EVs isolated in the NR and ACR groups. Fig. 12a: the number of total cd3+ EVs is compared, n=5; fig. 12b: the ratio of the number of dcd3+evs to the number of total cd3+evs in its samples, n=5.
Fig. 13 is the difference in expression of miRNA contained in dcd3+evs compared to mcd3+evs (n=4).
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1 preparation and characterization of Au@Apts
The Au@Apts is prepared by taking nano gold as a carrier, taking nucleic acid aptamer (A1, A2) as a targeting reagent, taking a mercapto-modified DNA chain (SH-T1) as a medium for connecting the aptamer and the nano gold and regulating the distance between the double aptamers.
SH-T1 sequence:
c5 sequence:
aptamer chain:
a1 sequence:
a2 sequence:
CD3 aptamer sequence:
complementary strand:
c1 sequence:
c2 sequence:
wherein the role of C5: and the protein is combined with a spacer part sequence of an aptamer on SH-T1, so that the rigidity of a chain is enhanced, and the interval between two aptamers is ensured. Design principle of connecting chain: the distance between two aptamer chains is controlled to be just the size of dimeric TCR-CD3, the distance is more suitable between 15 and 18nm, and the design interval is 50 bases and about 16nm.
The double underlined sequence is a CD3 aptamer, and the underlined sequence is a part of SH-T1 combined with A1 and A2; italics and bolded sequences are the parts that bind to C1, C2; the italics and bold sequences are the portions that bind to C5.
(1) Affinity validation of aptamer to CD3
Jurkat, hepG2, WRL68 cells in logarithmic growth phase were taken in sterilized 1.5mL EP tubes, each cell number was 2X 10 5 . Three cells were incubated with 2. Mu.M FAM fluorescence-modified A1 and CD3 aptamers at 37℃for 2h, respectively, with replacement of fresh serum-free medium. During this period, cells were gently blown every 30min. After incubation, unbound nucleic acid strands were washed with PBS, cells were resuspended in 400. Mu.L of PBS, and fluorescence detection was performed by flow cytometry. The results are shown in figure 1, where the CD3 aptamer has the strongest affinity for Jurkat cells compared to HepG2 and WRL68 cells that do not express CD 3; the fluorescence intensity of binding of A1 to Jurkat cells was similar to that of CD3 aptamer, indicating that the extension sequence of A1 except for the aptamer had little effect on binding to CD 3.
(2) Hybridization of thiol strand with double-aptamer and complementary strand
mu.L of 10. Mu.M SH-T1, A1, A2, C5 were each aspirated and mixed in an enzyme-free PCR tube, and 10mM MgSO was added 4 The chain hybridization reaction was promoted, and the solution was replaced with a calcium ion or magnesium ion solution, and the total volume was made up to 20. Mu.L with PBS, and the mixed solution was placed in a constant temperature bath at 37℃and reacted for 2 hours. Then, 2. Mu.L of 10. Mu. M C1 and C2 were added to the mixture after the reaction, and the reaction was continued in a constant temperature bath at 37℃for 2 hours. The raw material chain and the product of the above reaction were each prepared to a final concentration of 0.5. Mu.M, and the hybridization reaction was verified by polypropylene gel electrophoresis. The results are shown in FIG. 2. SH-T1, A1, A2 and C5 successfully hybridize to form a double-aptamer structure (Apts), and if complementary strands C1 and C2 are added, the Apts hybridizes to the complementary strands C1 and C2, and then the A1 and A2 are released, and the schematic diagram is shown in FIG. 3.
(3) Double-aptamer modified nano-gold
After forming the Apts according to the above procedure, 100 μl of 2 μM Apts was mixed with 100 μl of 5mg/mL nanogold suspension, and 0.2M NaCl solution was added, wherein the purpose of the salt addition (sodium chloride) was to stabilize the structure of DNA strands, reduce the repulsive force between strands, reduce repulsive force with nanoparticles, make DNA more easily attach to nanogold, and place in a mixing shaker, and react overnight at 4 ℃ in a refrigerator. The free aptamer is removed by centrifugation at 1000rpm for 15min, and the gold surface which is not bound with the proper ligand is blocked by 1mM mercaptohexanol and 1% bovine serum albumin respectively, so as to prevent nonspecific adsorption in the process of capturing EVs by the nano gold. The thiol-hexanol and bovine serum albumin were removed by centrifugation at 1000rpm for 15min, the remaining free components were washed off with PBS, and finally the pellet was resuspended in 200. Mu.LPBS and sonicated to make Au@Apts suspension. To verify the change in particle size of the nanogold, the particle size of the nanogold before and after the Apts modification was detected using a Dynamic Light Scattering (DLS). As shown in FIG. 4, after Apts modification, the average particle size of the nano-gold increased by about 55nm, which is similar to the theoretical calculated length of SH-T1, indicating that the nano-gold was successfully modified with the double aptamer.
Example 2 Capture and separation verification of EVs
(1) Extraction and characterization of model EVs
At 37℃and 5% CO 2 Jurkat cells were cultured under conditions of 5X 10 cell density 5 And (3) replacing the complete culture medium with a culture medium containing 1% of serum without external vesicles at a concentration of 1% per mL, continuously culturing at 37 ℃ for 48 hours, collecting cell supernatant, centrifuging at 300 Xg and 3000 Xg for 15 minutes, centrifuging at 10000 Xg for 30 minutes, removing cell fragments and large vesicles, centrifuging at 137000 Xg for 2 hours by using a refrigerated ultracentrifuge, discarding the supernatant, and re-suspending the precipitate with 200 mu L of PBS to obtain the model EVs. Diluting 1 mu L of model EVs with ultrapure water for 10 times, dripping the diluted EVs on an ultrathin carbon film supporting copper net, dripping 1% phosphotungstic acid after drying, standing and dyeing for 15s, immediately washing the residual phosphotungstic acid with ultrapure water, drying the copper net in vacuum for 12h, and shooting the morphology of the EVs with a TEM; diluting 1 mu L of model EVs with PBS for 500 times, and detecting the particle size of the EVs with NTA; taking 10 mu L of model EVs and Jurkat cell lysate, and detecting two samples by using a Western blot methodProtein expression of CD63, GAPDH, CD 3. As shown in FIG. 5, the morphology of the model EVs is in a tea tray shape, and the diameter is about 100nm (FIG. 5 a); the average particle size of the NTA-detected EVs was about 155nm (FIG. 5 b); and the EVs express CD63 and CD3 proteins, but not negative protein GAPDH (figure 5 c), which shows that the appearance, particle size and protein expression condition of the model EVs meet the identification requirement of the EVs, and the next experiment can be carried out.
(2) Characterization of au@apts capture EVs
40. Mu.L of model EVs extracted as described above were added to 200. Mu.L of the suspension of Au@Apts@EVs, slowly spun at 37℃for 1h, centrifuged at 10000rpm for 15min, the supernatant was discarded, unbound free EVs were washed off with PBS, and then 200. Mu.L of PBS was added to resuspend the Au@Apts@EVs complex. And (3) dripping 10 mu L of nano gold suspension and Au@Apts@EVs complex suspension on a sample stage, and after vacuum drying for 12 hours, using a Scanning Electron Microscope (SEM) to characterize the morphology of the Au@Apts surface before and after EVs combination. As a result, as shown in FIG. 6, the pure nano gold was spherical with a smooth surface, and after EVs were captured, the nano gold surface was rough, and many small vesicle structures were present, the vesicle diameter was about 50nm. The au@apts successfully captures the model EVs in the solution, and the morphology and the particle size of the EVs are not obviously changed.
(3) Verification of complementary strand separation EVs
According to the capture method, the Au@Apts@EVs complex is formed, 1 mu L of PKH67 is added into the complex, the EVs combined on the surface of the nano gold is subjected to membrane dyeing, centrifugation is carried out at 10000rpm for 15min, free dye in supernatant is removed, and 200 mu L of LPBS is used for resuspension of the complex, so that the dyed Au@Apts@EVs complex suspension is obtained.
Thereto were added 20. Mu.L of 10. Mu. M C1, C2 and 10mM MgSO, respectively 4 The captured EVs were then separated from the nanogold by slow rotation at 37℃for 1h, centrifugation at 10000rpm for 15min, removal of EVs from the supernatant and resuspension with 200. Mu.L PBS. And respectively carrying out fluorescence analysis on the Au@Apts and the Au@Apts@EVs complexes before and after EVs separation by using a nanoflow detector (NanoFCM), and verifying the combination condition of the EVs on the surface of the complex. As a result, as shown in FIG. 7, au@Apts served as a negative control, the surface was not fluorescent (i), and the complex of Au@Apts@EVs showed strong fluorescence (ii) after capturing EVs, and after adding complementary strands C1, C2 and incubating, the sample was fluorescentThe light pattern was divided into two parts (iii), with the non-fluorescent part accounting for 69.8% of the total weight of the composition, au@apts after EVs release, and 30.2% of the fluorescent material was au@apts@evs composite with a small amount of EVs. It is demonstrated that complementary strands C1 and C2 completely separate at least 70% of the captured EVs from the Au@Apts surface.
Example 3 heterogeneity analysis and application of CD3+EVs subpopulations
(1) Establishment of mouse skin graft model
The skin transplantation of mice was performed using 8-10 weeks C57BL/6 male mice and BALB/C male mice, 10C 57BL/6 mice were randomly selected as the allogeneic genome skin transplantation group (C57 BL/6- > C57BL/6, NR group), 5 BALB/C mice and 5C 57BL/6 mice were randomly selected, BALB/C mice were used as the donor, and C57BL/6 mice were used as the recipient, as the allogeneic genome skin transplantation group (BALB/C- > C57BL/6, ACR group). The experimental mice were anesthetized with 2% chloral hydrate, the back hairs of the BALB/C mice and the C57BL/6 mice were removed with a hair pusher, the skin was leaked, the back skin of the mice was sterilized once with iodophor, sterilized twice with alcohol, the back skin was cut off with an autoclaved surgical instrument, and placed in PBS buffer containing penicillin and streptomycin. Subcutaneous tissue was carefully removed, then trimmed to a size of about 1X 1cm and placed in pre-chilled antibiotic-PBS buffer for use. Then the skin of the donor mouse is transplanted to the back of the receptor mouse, the skin suture needles are used for suturing at the four corners respectively, the aseptic dressing is covered, the skin is wrapped, and the donor mouse is put into an aseptic proper environment for breeding.
The graft survival was observed at day 4 post-surgery until the graft was completely rejected and necrotic, and the time and images were recorded. And (3) evaluation of the effect of transplanting the skin graft: by observing the color, hardness and fitting degree of the graft, the graft can be detached, necrotic, hair growth and the like. The grafting sheet certificate is attached, soft and ruddy, and the grafting is successful; rejection is determined to occur if the cortical sheet is whitened, crusted, sloughed or necrotic. The transplantation results are shown in fig. 8, and on the 4 th day after the transplantation, the transplanted skin of the mice in the NR group is well attached and ruddy; the transplanted skin pieces of ACR group are fallen off, and the skin is whitened; on the 8 th postoperative day, the transplanted mice in the NR group had intact skin patches attached and had hair growing; mice of ACR group were transplanted for skin necrosis and crusting. Indicating that the mice in ACR group have rejection reaction and immune cells are activated.
(2) Heterogeneity analysis of CD3+ EVs subpopulations
1. Whole blood from day 8 after the transplantation of mice of NR and ACR groups was collected, anticoagulated with heparin sodium, and centrifuged at 3000 Xg for 15 minutes to obtain plasma. The CD3+ EVs in the plasma were captured according to the EVs capture method in the above procedure, and centrifuged at 10000rpm for 15min to obtain Au@Apts@EVs complexes, which were resuspended in 200. Mu.L PBS.
First 20. Mu.L of 10. Mu. M C1 and 10mM MgSO were added 4 Slowly rotating at 37 ℃ for 1h, centrifuging at 10000rpm for 15min to obtain supernatant which is mCD3+EVs; the pellet was resuspended in 200. Mu.L PBS and 20. Mu.L 10. Mu. M C2 and 10mM MgSO were added 4 Slowly spin at 37℃for 1h and centrifuge at 10000rpm for 15min, the second supernatant was dCD3+EVs. To each of the two EVs samples, 5. Mu.L of FITC-labeled CD3 antibody was added and incubated at 37℃for 30min in the absence of light. A schematic diagram of the Au@Apts capture and isolation of a subset of TCR-CD3 dimeric EVs is shown in FIG. 9.
Subsequently, the two samples were subjected to 13700 Xg, 2h freeze ultracentrifugation, unbound fluorescent antibody was removed from the supernatant, EVs pellet was resuspended in 100. Mu.L PBS, and fluorescent nanofluidic analysis was performed. As a result, as shown in fig. 10, the fluorescence intensity pattern of mcd3+ EVs was concentrated and uniform single peak, the fluorescence intensity pattern of dcd3+ EVs was multimodal, and the ratio of dcd3+ EVs to dcd3+ EVs at a larger part of the fluorescence intensity was 63.1% at strong fluorescence, and 26.2% at mcd3+ EVs, indicating that the expression level of CD3 at the surface of dcd3+ EVs was greater than that of mcd3+ EVs, with the peak approximate to that of mcd3+ EVs being a limit; wherein d is a dimer; m is a monomer of a monomer.
2. To compare the particle sizes of mcd3+ EVs and dcd3+ EVs, NTA detection was performed on supernatants after C1 and C2 separation. Results as shown in fig. 11, two subpopulations of EVs were isolated from the same plasma sample, with dcd3+evs having an average particle size greater than mcd3+evs, and two subpopulations of NR and ACR groups exhibiting similar differences.
3. The number of mcd3+ EVs and dcd3+ EVs in each sample group was calculated by NTA testing the supernatant after C1 and C2 separation against the number difference between NR and ACR groups for the comparative mcd3+ EVs and dcd3+ EVs subpopulations. The results are shown in FIG. 12, where the total CD3+ EVs isolated from NR and ACR groups were not statistically different in number (FIG. 12 a), indicating that the difference in number was not statistically correlated with the occurrence or non-occurrence of rejection. The number of dcd3+ EVs per sample was divided by the total number of cd3+ EVs per sample to give the number of dcd3+ EVs per mouse (fig. 12 b). It can be found that in the ACR group, the dcd3+evs account for approximately 28.2%, whereas in the NR group, the dcd3+evs account for only 13.9%, with a significant statistical difference (p < 0.01), indicating that the number of dcd3+evs subgroups is associated with rejection in mice.
4. The difference in the expression level of the miRNA of the contents of the comparative mcd3+ EVs and dcd3+ EVs was compared, the EVs subpopulation of ACR group plasma was separated by the above-described separation method, the supernatant after separation of C1 and C2 was centrifuged at 137000×g for 2 hours, the supernatant was discarded, and the pellet was resuspended in 100 μl PBS and quick frozen at-80 ℃. miRNA sequencing analysis was performed on mcd3+ EVs and dcddd3+ EVs subpopulations of samples, comparing the miRNA expression differences between the two groups of samples (4 parallel samples per group). As shown in fig. 13, the miRNA expression of dcd3+ EVs was both up-regulated and down-regulated compared to the miRNA expressed by mcd3+ EVs, with a total of 104 miRNA expression statistically different between the two subgroups (p < 0.1). This suggests that dcd3+ EVs and mcd3+ EVs have a certain heterogeneity in the expression amount of the content miRNA.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> university of Zhongshan, university of Zhongshan affiliated third Hospital
<120> a method for isolating extracellular vesicle subpopulations and uses thereof
<130>
<160> 7
<170> PatentIn version 3.5
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tagtagagct accgaagcca tcgagatgat gtagtagtta ccatcctacc attgatgatg 60
tgttgttaga tacttttcat agattgttgt 90
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catcatcaat ggtaggatgg 20
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Claims (6)
1. A method for isolating a subset of TCR-CD3 dimerization vesicles in plasma, comprising the steps of:
s1: capturing EVs in a plasma sample through the nano gold modified by the double aptamer; centrifugally separating to obtain a nano gold-EVs compound;
s2: the separated nano gold-EVs complex reacts with a complementary strand C1 and is centrifuged, the precipitate is resuspended, and then reacts with a complementary strand C2 and is centrifuged to obtain the TCR-CD3 dimeric vesicle subgroup; the preparation method of the double-aptamer modified nano gold comprises the following steps: forming a double-aptamer structure through a nucleic acid aptamer, a connecting chain and C5, and then reacting with the nano gold particles;
the sequence of the nucleic acid aptamer is as follows:
a1 sequence: 5'-GCCGCGGGGTGGGTCTAGTGTGGATGTTTAGGGGGCGGCCTACATCATCTCGATGGC-3';
a2 sequence: 5'-GCCGCGGGGTGGGTCTAGTGTGGATGTTTAGGGGGCGGCATCTATGAAAAGTATCTA-3';
the sequence of the connecting chain is as follows:
SH-T1：5’-SH-(CH 2 ) 6 -TAGTAGAGCTACCGAAGCCATCGAGATGATGTAGTAGTTACCATCCTACCATTGATGATGTGTTGTTAGATACTTTTCATAGATTGTTGT-3’；
the C5 sequence is as follows:
5’-CATCATCAATGGTAGGATGG-3’；
the sequence of the complementary strand is:
c1 sequence: 5'-ACAACAATCTATGAAAAGTATCTAACAACA-3';
c2 sequence: 5'-TAACTACTACATCATCTCGATGGCTTCGGT-3'.
2. The method of claim 1, wherein the method of preparing the dual aptamer comprises: SH-T1, A1, A2 and C5 are mixed and placed at the temperature of 35-38 ℃ to react for 1.5-2.5 h.
3. The method of claim 2, wherein the molar ratio of SH-T1, A1, A2, C5 is (0.5-1.5): 1.
4. The method of claim 1, wherein the concentration of the diadaptomic ligand is 1 to 3 μΜ; the concentration of the nano gold is 4-6 mg/mL.
5. The method according to claim 1, wherein the volume ratio of the dual aptamer to the nanogold is (0.5-1): 1.
6. The method according to claim 1, wherein the conditions for the reaction of the complementary strand C1 and the complementary strand C2 are each 35 to 38℃for 1.5 to 2.5 hours.
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| JP2017106915A (en) * | 2015-12-07 | 2017-06-15 | 株式会社ビー・エム・エル | Method of monitoring t-cell activation from bodily fluid |
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| JP2017106915A (en) * | 2015-12-07 | 2017-06-15 | 株式会社ビー・エム・エル | Method of monitoring t-cell activation from bodily fluid |
| CN111826353A (en) * | 2019-07-24 | 2020-10-27 | 上海斯丹赛生物技术有限公司 | Methods of modulating T cell function and response |
| CA3148937A1 (en) * | 2019-08-02 | 2021-02-11 | Immatics Biotechnologies Gmbh | Antigen binding proteins specifically binding mage-a |
| WO2021129559A1 (en) * | 2019-12-24 | 2021-07-01 | 南京北恒生物科技有限公司 | T cell receptor fusion protein and use thereof |
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