CN114591905A - Method for preparing apoptosis vesicle from human red blood cell and application - Google Patents
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Abstract
The invention provides a method for preparing apoptotic vesicles from human erythrocytes, which comprises the steps of separating erythrocytes after collecting human venous blood, adding an erythrocyte apoptosis inducing drug in vitro to induce the rapid apoptosis of the erythrocytes, generating a large amount of apoptotic vesicles, and separating the apoptotic vesicles by a gradient centrifugation method. The apoptosis vesicle prepared by the method can promote osteogenic differentiation of the human mesenchymal stem cells, thereby improving the in-vitro osteogenic capacity of the human mesenchymal stem cells, achieving the aim of promoting bone regeneration, and having wide development prospect in the aspects of preparation of medicines for promoting osteogenic differentiation of the human mesenchymal stem cells and treatment of bone defects.
Description
Technical Field
The invention relates to the technical field of biological tissue engineering, in particular to a method for preparing an apoptotic vesicle by using human red blood cells for promoting osteogenic differentiation of mesenchymal stem cells and application thereof.
Background
Bone defects caused by trauma, infection, surgery or congenital deformity are very common in clinic, and bone regeneration treatment by bone tissue engineering is a hot spot in the field of tissue engineering research. In recent years, the use of extracellular vesicles such as exosomes in bone regeneration has been gaining attention. The extracellular vesicles are vesicle-shaped bodies secreted by cells and having a double-layer membrane structure, have diameters of 30nm to 5000nm, and are mainly classified into exosomes, microvesicles and apoptotic vesicles according to particle sizes and secretion modes. Cells in physiological states secrete mainly exosomes and microvesicles; the cells in the apoptotic state mainly secrete apoptotic vesicles (apoVs), which can be divided into apoptotic microvesicles with the particle size of 50-1000nm, apoptotic bodies with the particle size of 1000-5000nm and apoptotic exosomes with the particle size of less than 150 nm. Apoptosis is an essential link of organism metabolism, and damaged apoptosis and other processes also exist in the process of repairing the damaged bone. In the prior art, cytotoxic drugs such as staurosporine and the like are adopted to be co-cultured with cells for 12-24 hours when the apoptotic vesicles are extracted, so that the cells slowly die and the apoptotic vesicles are generated at the same time, the time is longer, the efficiency is lower, and the yield is not high. Red Blood Cells (RBCs) are the highest blood cell content in human body, and have wide sources and simple extraction. At present, most of research aiming at red blood cells utilizes red blood cells to prepare vesicle membranes or red blood cell membrane-coated medicines, nanoparticles and the like, and the method aiming at the application of apoptosis vesicles generated by red blood cell induction to bone regeneration treatment is less, and the method also has the technical problems of long time, low efficiency and low yield, so that a large amount of apoptosis vesicles are rapidly prepared by utilizing human red blood cells, and the method has wide development prospect in the aspects of bone tissue defect treatment and application to the preparation of medicines for promoting osteogenesis of human mesenchymal stem cells to differentiation.
Disclosure of Invention
The invention separates the red blood cells after collecting the human venous blood and adds the medicine to induce the apoptosis of the red blood cells, thereby rapidly generating a large amount of apoptotic vesicles from the red blood cells.
The invention provides a method for preparing apoptotic vesicles from human erythrocytes, which comprises the following steps:
1) collecting blood by vein, centrifuging for 10 min, extracting lower layer red precipitate, washing with sterile PBS for 2 times to obtain erythrocyte;
2) and adding erythrocyte apoptosis inducing liquid into the separated erythrocytes, carrying out ice induction treatment for 15-30 minutes, and obtaining the apoptotic vesicles from the human erythrocytes by a gradient centrifugation method.
The erythrocyte apoptosis-inducing solution is prepared from
、
And EDTA-2Na 0.1 mM.
Specifically, the volume ratio of the erythrocyte to the erythrocyte apoptosis inducing liquid is 1:6-1: 10.
The induction treatment time is preferably 30 minutes.
The apoptosis inducing solution pH7.1-7.4, and is used after being filtered and sterilized by a 0.22 μm filter.
The gradient centrifugation method comprises the following steps:
(1) centrifuging the erythrocyte suspension subjected to apoptosis induction treatment at 800 g and 4 ℃ for 10 minutes to remove cell debris;
(2) collecting supernatant, 16000g, centrifuging for 30 minutes at 4 ℃;
(3) collecting the precipitate, washing with sterile PBS for 1 time, 16000g, centrifuging for 30 minutes at 4 ℃;
(4) the pellet was collected and resuspended in PBS to obtain apoptotic vesicles derived from human erythrocytes.
The invention provides application of the method in promoting osteogenic differentiation of the human mesenchymal stem cells.
In particular, the application is to apply the erythrocyte apoptosis vesicle obtained by the method toThe preparation of the medicine for promoting the osteogenic differentiation of the human bone marrow mesenchymal stem cells. Human mesenchymal stem cells after addition of apoVsRUNX2、ALP、OCNThe gene expression level and the RUNX2 protein expression level are obviously improved, and the in vitro osteogenic differentiation capacity of the human bone marrow mesenchymal stem cells is increased.
Has the advantages that:
the invention separates the red blood cells after collecting the human venous blood and adds the red blood cell apoptosis inducing drug in vitro to induce the apoptosis of the red blood cells, thereby generating the apoptosis vesicle. The preparation method of the erythrocyte apoptotic vesicle (RBC-apoVs) is simple, short in time consumption (the apoptosis induction time is only 15-30 minutes), low in cost and large in yield (the concentration of the obtained RBC-apoVs can reach 5.9 multiplied by 10)11Particles/mL), high efficiency and can carry out rapid detection.
The preparation method of the invention adopts the erythrocyte in the blood of the human body to obtain the apoptotic vesicle, thereby avoiding the ethical problem and the immune problem.
The apoptosis vesicle obtained by the method can promote osteogenic differentiation of the mesenchymal stem cells, thereby improving the in-vitro osteogenic capacity of the mesenchymal stem cells, achieving the purpose of promoting bone regeneration and having wider development and application prospects in treatment of bone tissue defects.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic flow diagram of the in vitro induction of apoptosis and RBC-apoVs extraction of human erythrocytes;
FIG. 2 is a graph showing the result of RBC-apoVs extraction, wherein A is the observation result of the red blood cells separated from human blood by scanning electron microscopy; b is the transmission electron microscope observation result of RBC-apoVs; c is a nanoparticle tracking analysis detection result of RBC-apoVs; d is the flow cytometry detection result of RBC-apoVs high expression red blood cell characteristic marker protein CD235a and apoptosis vesicle surface marker phosphatidylserine;
FIG. 3 is a graph of RBC-apoVs promoting osteogenic differentiation of human mesenchymal stem cells in vitro; wherein A is alizarin red staining diagram; b is ALP staining pattern; c is ALP enzyme activity quantification result;
FIG. 4 shows mesenchymal stem cells of human bone marrow after addition of RBC-apoVsRUNX2、ALP、OCNThe results of the gene Real-time qPCR experiment and the RUNX2 protein imprinting experiment, wherein A isRUNX2The amount of gene expression; b isALPThe amount of gene expression; c isOCNThe amount of gene expression; d is the result of Western blot of RUNX2 protein expression level.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The percentages used in the following examples are by mass unless otherwise specified.
Example 1 efficient extraction and characterization of apoVs from human erythrocytes
1. The preparation process of the human erythrocyte apoptotic vesicle is concretely as follows (as shown in figure 1):
(1) collecting blood of healthy people intravenously, centrifuging at room temperature of 300g for 10 minutes, and extracting erythrocyte sediment at the lower layer; the pellet obtained was Red Blood Cells (RBCs) by washing with sterile PBS, centrifugation (300 g, room temperature, 10 min) and 2 repetitions.
The morphology of the red blood cells was observed using a field emission scanning electron microscope.
(2) And adding erythrocyte apoptosis inducing liquid into the separated erythrocytes in vitro, and inducing the erythrocytes to rapidly apoptosis on ice. The volume ratio of the red blood cells to the red blood cell apoptosis inducing liquid is about 1:6-1: 10.
The preparation method of the erythrocyte apoptosis inducing liquid comprises the following steps: ammonium chloride
8.3 g of potassium bicarbonate
1.0 g and 37.2 mg of ethylene diamine tetraacetic acid (EDTA-2 Na), adjusting the pH to 7.1-7.4, adding distilled water to a constant volume of 1L, and filtering and sterilizing by a 0.22 mu m filter.
(3) RBC-apoVs were obtained by gradient centrifugation 15-30 minutes after apoptosis induction treatment. Specifically, the method comprises the following steps:
centrifuging 800 g of erythrocyte suspension subjected to apoptosis induction at 4 ℃ for 10 minutes to remove cell debris;
collecting supernatant, 16000g, centrifuging for 30 minutes at 4 ℃;
collecting precipitate, washing with sterile PBS, 16000g, centrifuging at 4 deg.C for 30 min;
the pellet was collected and resuspended in 200. mu.l sterile PBS to obtain RBC-apoVs.
Morphology of RBC-apoVs was examined by cryo-transmission electron microscopy, particle size and concentration were examined by ZetaView nanoparticle tracking analysis, and RBC-apoVs were analyzed by flow cytometry for the red blood cell signature protein CD235a and the apoptotic vesicle surface signature marker phosphatidylserine.
2. Field emission scanning electron microscope:
1) taking freshly separated red blood cells, adding freshly prepared 2.5% glutaraldehyde, and fixing at 4 ℃ for more than 30 minutes;
2) washing with PBS for 2 times, adding appropriate amount of Milli-Q water for resuspension, taking 2-3 drops, and dripping onto silicon wafer;
3) putting the mixture into a glass drier containing a silica gel drying agent, and naturally drying for 24 hours;
4) and (3) sticking the dried silicon wafer on a sample table by using a double-sided adhesive tape, spraying gold by using an ion sputtering instrument, and observing by using a Hitachi S4800 scanning electron microscope.
As shown in FIG. 2A, the surface of the red blood cells was regular and had a biconcave disk shape. The method is proved to be capable of effectively separating the red blood cells.
3. Freezing transmission electron microscope:
1) 5 mul of the apoptosis micro-vesicle suspension is absorbed and dropped on a copper net, and the mixture is kept stand for 1min at room temperature;
2) sucking more liquid along the outer side of the copper mesh by using filter paper, sucking 5 mu l of 2% uranyl acetate, dripping the uranyl acetate on the copper mesh, and standing the mixture for 30 seconds at room temperature;
3) absorbing the redundant liquid along the outer side of the copper mesh by using filter paper, and standing and drying at room temperature;
4) images were taken under a transmission electron microscope with the voltage set at 120 kV.
The result is shown in fig. 2B, where the apoptotic vesicle of human erythrocytes is in the shape of a disk with a concave center, conforming to the characteristics of apoptotic vesicles.
4. ZetaView nanoparticle tracking analysis:
1) diluting a sample by PBS (phosphate buffer solution) in a proper amount, and analyzing by using a ZetaView nanoparticle tracking analyzer;
2) and observing the Brownian motion track of the vesicle to be detected.
The results are shown in FIG. 2C, which shows that the average particle size is 180.4nm and the average concentration can be reached
It is demonstrated that RBC-apoVs extracted by the method of example 1 of the present application have high yield, and the apoptotic vesicles obtained by the method are mainly apoptotic microvesicles.
5. Flow cytometry detection:
1) identifying the human erythrocyte surface specific marker protein CD235a on the surface of RBC-apoVs: the apoptotic vesicle sample liquid obtained in this example was diluted with a flow buffer to
PE-anti-human CD235a antibody was added as a test group, and blank control groups without the addition of antibody were established and incubated at 4 ℃ for 30 minutes in the absence of light.
2) Identification of RBC-apoVs surface phosphatidylserine: sample is diluted to
Annexin V-FITC staining was performed according to the Annexin V-FITC kit (Biyuntian, C1062L) instructions, and Annexin-V could specifically bind phosphatidylserine. A blank control group was set without Annexin V-FITC.
The obtained result is shown in fig. 2D, the left light peak represents a blank control group, the right dark peak represents an experimental group, and the experimental result shows that the surface of the RBC-apoVs highly expresses erythrocyte characteristic protein CD235a, and the positive rate is 99.9%; meanwhile, the apoptosis vesicle marker phosphatidylserine is highly expressed, and the positive rate is 92.4%. The above results demonstrate that apoptotic vesicles derived from erythrocytes obtained by the method have the surface characteristics of erythrocytes and apoptotic vesicles.
Example 2 in vitro staining and semi-quantitative experiments to examine the Effect of apoVs derived from human erythrocytes on in vitro osteogenic differentiation of human mesenchymal stem cells
Human mesenchymal stem cells were subjected to conventional proliferation culture (PM) or osteogenic induction culture (OM) or human erythrocyte-derived apoVs (OM + RBC-apoVs: 0.50. mu.g/ml) were added under osteogenic induction culture conditions. Alizarin red staining, ALP staining and ALP enzyme activity quantitative detection are carried out after 7 days of osteogenic induction culture.
1. Cell culture: human mesenchymal stem cells were purchased from ScienCell Research Laboratories (Carlsbad, Calif., USA).
Growth medium (PM): alpha-MEM medium containing 10% fetal bovine serum, 1% penicillin/streptomycin.
Osteogenic differentiation medium (OM): alpha-MEM medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 0.2 mM vitamin C, 10 mM sodium beta-phosphate and 100 nM dexamethasone.
0.90. mu.g/ml human red blood cell-derived apoVs (OM + RBC-apoVs) were added under osteogenic induction culture conditions.
Cells and method for producing the same
Inoculating to 12-well plate at 37 deg.C and 5% CO2Culturing under the condition, and changing the culture solution 1 time every 3 days.
2. Alizarin red staining:
1) after 7 days of osteogenic induction of the cells, removing the culture medium, washing with PBS for 3 times, adding 95% ethanol into each hole, and fixing for 30 minutes at room temperature;
2) discarding 95% ethanol, washing with Milli-Q water for 3 times;
3) adding a proper amount of alizarin red dye solution into each hole, and dyeing at room temperature until difference exists, and stopping;
4) the samples were washed 3 times with Milli-Q water and scanned.
The staining results are shown in fig. 3A, compared with the PM group, the OM group is darker, and the OM + RBC-apoVs group is darker than the OM group, which indicates that the mineralization capability of the cells is significantly enhanced after the RBC-apoVs is added, and thus, the addition of RBC-apoVs is proved to promote the osteogenic differentiation of the human mesenchymal stem cells.
3. ALP staining:
1) after 7 days of osteogenic induction of the cells, removing the culture medium, washing with PBS for 3 times, adding 95% ethanol into each hole, and fixing at room temperature for 30 min;
2) discarding 95% ethanol, washing 3 times with PBS;
3) adding alkaline phosphatase dye solution into each hole, and dyeing at room temperature in a dark place until difference exists;
4) the samples were washed 3 times with PBS and scanned.
The staining results are shown in fig. 3B, compared with the PM group, the OM group is darker, and the OM + RBC-apoVs group is darker than the OM group, which indicates that the alkaline phosphatase activity of the cells is significantly improved after RBC-apoVs is added, the alkaline phosphatase is one of the activity markers of osteoblasts, and the results indicate that the addition of RBC-apoVs promotes osteogenic differentiation of human mesenchymal stem cells.
4. Quantitative determination of ALP enzymatic Activity:
alkaline phosphatase test kit (microplate reader method) was purchased from Nanjing institute of bioengineering.
1) After 7 days of osteogenic induction of the cells, the culture medium was discarded, and the cells were washed 3 times with PBS, 100. mu.l of 1% TritonX-100 was added to each well, and treated for 10 minutes on ice;
2) transferring the lysed cells into a centrifuge tube, centrifuging for 15min at 4 ℃ and 14000g, and collecting a supernatant:
3) the BCA method measures the total protein concentration per sample;
4) adding 30 mul of supernatant and 100 mul of detection working solution into each well of a 96-well plate;
5) mixing, and incubating at 37 deg.C for 15 min;
6) mu.l of developing solution was added to each well, and the absorbance at 520nm was measured.
The results are shown in fig. 3C, in which the OM group after osteogenic induction culture showed a significant increase in ALP expression compared to the normal proliferation culture group (PM), and in which the OM + RBC apoVs group showed a significant increase in ALP expression compared to the OM group (×)P<0.001). The above results show that the addition of RBC-apoVs is effective in promoting the in vitro osteogenic capacity of stem cells.
Example 3 in vitro molecular biological assay to examine the Effect of apoVs derived from human erythrocytes on osteogenic differentiation of human mesenchymal stem cells
Human bone marrow mesenchymal stem cells are cultured with
The density of each well was inoculated into 6-well plates and cultured under the following three culture conditions: 1) proliferation Medium (PM); 2) osteogenic induction (OM); 3) human erythrocyte-derived apoVs (OM + RBC-apoVs: 0.50. mu.g/ml). Collecting cells after 14 days, and detecting osteogenic key genes by qRT-PCRRUNX2、 ALP、OCNExpression of (2).
Inoculating human bone marrow mesenchymal stem cells into a 60mm culture dish, respectively carrying out common proliferation culture (PM) or osteogenesis induction culture (OM) or adding apoVs derived from human red blood cells under osteogenesis induction culture conditions for total culture for 14 days, and detecting the expression of an osteogenesis key protein RUNX 2.
qRT-PCR experiment for detecting osteogenic key geneRUNX2、ALP、OCNThe expression steps are as follows:
1. total RNA extraction from cells
Human mesenchymal stem cells are cultured with
The density of each well was inoculated into 6-well plates, and total RNA was extracted 14 days after induction in 3 different conditions. The method comprises the following specific steps:
1) the medium was aspirated and washed with PBS.
2) Trizol reagent (1 ml/well) was added and transferred to a 1.5ml centrifuge tube.
3) 200. mu.l of chloroform was added, shaken for 30 seconds, and left on ice for 3 minutes.
4) Centrifuge at 12000g for 15min at 4 ℃.
5) Standing, sucking the upper aqueous phase and transferring to another centrifuge tube.
6) Add 400. mu.l of isopropanol, mix well and let stand on ice for 5 minutes.
7) Centrifuge at 12000g for 10 min at 4 ℃.
8) The supernatant was discarded, 1ml of 75% ethanol prepared from precooled absolute ethanol and DEPC water was added, and the precipitate was washed.
9) Centrifuge at 7500g for 5min at 4 ℃.
10) Discarding the supernatant, sucking off the liquid, drying the precipitate at room temperature, adding an appropriate amount of DEPC water, and subpackaging the extracted total RNA at-80 ℃ for storage or carrying out the next experiment.
2. Reverse transcription to synthesize cDNA
1) The reverse transcription reaction system is 20. mu.l, and the total RNA consumption is about 1000 ng.
2) 1000ng of the extracted total RNA was taken and a reverse transcription reaction solution was prepared on ice according to the kit instructions.
3) Reverse transcription reaction conditions: carrying out reverse transcription reaction at 37 ℃ for 15 minutes; carrying out reverse transcriptase inactivation reaction at 85 ℃ for 5 seconds; keeping at 4 deg.C, and storing at-20 deg.C or performing the next experiment.
3. Real-time quantitative PCR reaction
1) Three auxiliary holes are detected for each gene of each sample, 20 mu l of reaction system is configured in each hole of the eight-tube, the reagents and the dosage are as follows, wherein the primer sequences are shown in the table 1:
2) the PCR reaction conditions are as follows: hot starting at 95 ℃ for 10 minutes, and denaturing at 95 ℃ for 30 seconds; annealing and extending for 1 minute at 60 ℃ for 40 cycles;
3) to be provided withGAPDHFor internal reference, data were analyzed using the Δ Δ Ct method, experimental data in triplicateMean ± sd of experiments.
The primer sequences are shown in table 1:
TABLE 1 qRT-PCR primer sequences
The results are shown in FIGS. 4A-C: osteogenesis-related genes after osteogenic induction culture compared to proliferation Medium group (PM)RUNX2、ALP、OCNThe expression level is obviously increased. After addition of human red blood cell-derived apoVs under osteogenic induction culture conditions as compared to control (OM),RUNX2、ALP、OCNthe gene expression level is obviously improved, and the RBC-apoVs can promote the expression of the osteogenesis related gene of the human bone marrow mesenchymal stem cellsP<0.005）。
4. Western blot experiment
(1) Collecting cells in 60mm culture dishes, adding a proper amount of RIPA lysate and protease inhibitor into each culture dish, and collecting the cells into a centrifuge tube by using a cell scraper;
(2) standing on ice for 30min to completely lyse cells;
(3) centrifuging at 12000g for 20 min at 4 ℃, sucking supernatant, detecting protein concentration by using a BCA method, and denaturing the protein at 99 ℃ for 5min to obtain a protein sample;
(4) a 10% SDS-PAGE gel was prepared and equal amounts of protein sample were added to each well:
(5) performing constant-voltage 80V electrophoresis, and adjusting the voltage to 120V when the electrophoresis strip completely runs out of the concentrated gel until bromophenol blue completely runs out of the separation gel;
(6) placing a membrane rotating clamp according to a model of sponge, filter paper, separation gel, membrane, filter paper and sponge, and electrically rotating for 1.5 hours at 110V and 300 mA;
(7) after the electrotransformation is finished, taking out the PVDF membrane, placing the PVDF membrane in 5% skimmed milk powder, sealing the shaking table for 1 hour:
(8) cutting off RUNX2 and GAPDH protein bands according to the molecular weight of the protein, and incubating corresponding primary antibodies at 4 ℃ overnight;
(9) washing the membrane for 3 times by TBST, incubating the secondary antibody for 10 minutes each time, and incubating for 60 minutes by a shaking table at room temperature;
(10) the membrane was washed 3 times for 10 minutes each with TBST, and then exposed.
As shown in fig. 4D, under the osteogenic induction (OM) culture condition, the induction and the addition of apoVs significantly increased the expression level of the osteogenic marker protein RUNX2, demonstrating that apoVs derived from human red blood cells promotes osteogenic differentiation of human mesenchymal stem cells.
It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are given by way of illustration only, not limitation, and it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention; as long as the use requirements are met, the method is within the protection scope of the invention.
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Claims (8)
1. A method for preparing apoptotic vesicles from human erythrocytes is characterized by comprising the following steps: the method comprises the following steps:
1) collecting blood from vein, centrifuging, extracting lower layer red precipitate, washing with sterile PBS for 2 times to obtain erythrocyte;
2) adding erythrocyte apoptosis inducing liquid into the separated erythrocytes, carrying out ice induction treatment for 15-30 minutes, and obtaining apoptotic vesicles derived from human erythrocytes by a gradient centrifugation method;
the erythrocyte apoptosis-inducing solution is prepared from
、
And EDTA-2Na 0.1 mM.
2. The method according to claim 1, wherein the ratio of the volume of the erythrocyte and the apoptosis-inducing liquid is 1:6 to 1: 10.
3. The method of claim 1, wherein the induction treatment time is 30 minutes.
4. The method according to claim 1, wherein the apoptosis-inducing solution is at pH7.1 to 7.4.
5. The method according to claim 4, wherein the apoptosis-inducing solution is used after being sterilized by filtration through a 0.22 μm filter.
6. The method of claim 1, wherein said gradient centrifugation comprises the steps of:
(1) centrifuging the erythrocyte suspension subjected to apoptosis induction treatment at 800 g and 4 ℃ for 10 minutes to remove cell debris;
(2) collecting supernatant, 16000g, centrifuging for 30 minutes at 4 ℃;
(3) collecting the precipitate, washing with sterile PBS for 1 time, 16000g, centrifuging for 30 minutes at 4 ℃;
(4) the precipitate was collected and resuspended in PBS to obtain apoptotic vesicles derived from human erythrocytes.
7. Use of the method of any one of claims 1-6 to promote osteogenic differentiation of human mesenchymal stem cells.
8. The use according to claim 7, wherein the use is the use of the apoptotic vesicles obtained by the method of any one of claims 1 to 6 in the preparation of a medicament for promoting osteogenic differentiation of human mesenchymal stem cells.
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| CN202210338560.2A CN114591905B (en) | 2022-04-01 | 2022-04-01 | Method for preparing apoptotic vesicles from human erythrocytes and application of apoptotic vesicles |
| PCT/CN2022/136389 WO2023185066A1 (en) | 2022-04-01 | 2022-12-02 | Method for preparing apoptotic vesicles from human erythrocytes and use thereof |
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| CN114525244A (en) * | 2022-02-15 | 2022-05-24 | 中南大学 | Mechanism research method of pyroptosis product of mesenchymal stem cells in periodontal dental pulp combined diseased tissue in occurrence and development of diseases |
| CN115152742A (en) * | 2022-07-06 | 2022-10-11 | 中国人民解放军空军军医大学 | Extracellular vesicle preservation method |
| CN115820548A (en) * | 2022-12-12 | 2023-03-21 | 北京大学口腔医学院 | Preparation method and application of apoptotic vesicles derived from animal tissues |
| WO2023169594A1 (en) * | 2022-03-08 | 2023-09-14 | 中山大学 | Application of blood-derived sample in preparation of vesicles |
| WO2023185066A1 (en) * | 2022-04-01 | 2023-10-05 | 北京大学口腔医学院 | Method for preparing apoptotic vesicles from human erythrocytes and use thereof |
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| CN115820548A (en) * | 2022-12-12 | 2023-03-21 | 北京大学口腔医学院 | Preparation method and application of apoptotic vesicles derived from animal tissues |
| CN115820548B (en) * | 2022-12-12 | 2023-10-27 | 北京大学口腔医学院 | Preparation method and application of animal tissue-derived apoptosis vesicles |
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| CN114591905B (en) | 2022-09-02 |
| WO2023185066A1 (en) | 2023-10-05 |
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