CN111778212B - Preparation method and application of mobilized hematopoietic stem cell plasma exosome - Google Patents
Preparation method and application of mobilized hematopoietic stem cell plasma exosome Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an mobilized hematopoietic stem cell plasma exosome. The invention firstly discloses an exosome extraction method, which comprises the step of mobilizing peripheral blood hematopoietic stem cells by using a mobilizing agent. The invention further provides an exosome preparation containing the exosome obtained by the extraction method and application thereof. The method for extracting the mobilized hematopoietic stem cell plasma exosomes is simple and easy to operate, and the obtained exosomes are high in concentration and good in quality. In addition, in vitro experiments prove that the peripheral blood hematopoietic stem cell collection plasma exosome obtained by the invention has the effect of promoting the proliferation of peripheral blood mononuclear cells.
Description
Technical Field
The invention relates to a preparation method of an mobilized hematopoietic stem cell plasma exosome and application thereof.
Background
Currently, the promotion of hematopoietic injury repair is mainly performed by two ways: 1) hematopoietic growth factors: granulocyte colony stimulating factor, megakaryocyte growth factor, erythropoietin, thrombopoietin and the like, wherein the hematopoietic growth factor mainly promotes the proliferation of hematopoietic cells in bone marrow and restores normal hematopoiesis, but is difficult to exert the effect of promoting the hematopoietic recovery for hematopoietic suppression caused by severe radiation damage; 2) hematopoietic stem cell transplantation: hematopoietic stem cells can be differentiated downwards into various mature blood cells and immune cells, such as red blood cells, platelets, lymphocytes and the like, and the whole hematopoietic system is completely reconstructed in a recipient, so that hematopoietic stem cell transplantation provides a possible treatment method for malignant diseases and immune and gene abnormal diseases of various blood systems. At present, allogeneic hematopoietic stem cell transplantation is one of the most effective means recognized for treating malignant hematological diseases, and the cell source of the allogeneic hematopoietic stem cell transplantation mainly has three ways, namely bone marrow, peripheral blood and cord blood. The method releases stem cells in bone marrow into peripheral blood by drug mobilization, and then enriches and extracts hematopoietic stem cells in the peripheral blood by a method of apheresis and separation, is a method which is convenient for collecting, has small invasiveness and can obtain cells for multiple times in a short period. Currently, mobilized peripheral blood has gradually replaced bone marrow hematopoietic stem cells and become the primary source of cells for allogeneic hematopoietic stem cell transplantation, but a significant proportion of patients requiring transplantation still lack Human Leukocyte Antigen (HLA) matched donors, and HLA matching is the biggest barrier limiting the development of allogeneic hematopoietic stem cell transplantation. Although semi-compatible transplantation protocols are relatively mature, the incidence and severity of graft-versus-host disease after transplantation and various infections after immunosuppression are important factors that limit the development of transplantation techniques.
Microvesicles (MVs) are submicron bilayer membrane structures secreted by cells, can be secreted by various cells and widely distributed in various body fluids such as plasma, saliva, milk, sweat and the like, contain components such as proteins, lipids, mRNA and microRNA of maternal cells, are fused with other cells, release the contents thereof to target cells, influence the functions thereof, and play an important role in the information transfer process between different cells. Microvesicles vary with the origin, number, size and antigenic composition of the cells. Most of the circulating microvesicles are from blood platelets, and a small part of the circulating microvesicles are from other blood cells and endothelial cells, etc., the secreted microvesicles can be stored in tissue fluid near the mother cells and can also reach far along with the circulation of body fluid, and the microvesicles secreted by the cells have heterogeneity along with the differences of origin, number, size, cell state and stimulation conditions of the cells, and the microvesicles are divided into two groups according to the formation mechanism and physical characteristics: exosomes and microparticles. The exosome is a microbubble which is most clearly defined at present, has the diameter of between 20 and 100nm and the density of between 1.13 and 1.19g/ml, and the secretion of the exosome depends on the activation of cytoskeleton and the exocytosis mode regulated by P53 protein, is independent of the calcium ion concentration in cells, originates from an endosome, is firstly stored in a multivesicular body consisting of a single-layer membrane, and is secreted when being fused with the cell membrane.
The extraction methods of microvesicles are various: the ultracentrifugation method, the most widely used technical means for extracting microvesicles at present, and the linear sucrose gradient centrifugation method are also used for separating microvesicles, but both methods are time-consuming. The immunomagnetic bead separation method is simple and rapid, but analysis deviation can be caused due to incomplete function of the extracted microvesicles and application of antibodies, so that subsequent functional research experiments are limited. The filtration method and the ultracentrifugation method can obtain more microvesicles, are simple and short in time consumption, but still have the problem of insufficient extraction purity. The amount of microbubbles extracted by chromatography is further increased and purity is high, but the requirement on extraction equipment is high. Currently, the extraction method mainly uses microvesicles to extract mononuclear cells from collected hematopoietic stem cells, culture the mononuclear cells in vitro, and extract exosomes from culture supernatant, and has the following main disadvantages: the culture process is long, and the problems of cell death and content release exist; the culture conditions are not suitable for the simultaneous growth of stem cells and immune cells, thus creating a difference between the exosomes contained in the supernatant and the exosomes in the harvest; the culture cell amount is small, and the amount of the extracted exosome is not enough for application; the cells after culture cannot be reused, resulting in waste.
Disclosure of Invention
The invention aims to solve the technical problem of how to quickly, simply and conveniently extract high-concentration exosomes from post-patient peripheral blood hematopoietic stem cell collection and application value thereof, wherein the hematopoietic stem cell exosomes can promote the repair of hematopoietic damage and/or peripheral blood mononuclear cell proliferation after radiotherapy and chemotherapy, and the graft-versus-host disease risk caused by implantation of the peripheral blood hematopoietic stem cells after infusion is avoided.
In order to solve the technical problems, the invention firstly provides an exosome extraction method.
The method for extracting exosomes provided by the invention comprises the step of mobilizing peripheral blood hematopoietic stem cells by using an mobilizing agent.
In the above extraction method, the mobilizing agent may be recombinant human granulocyte colony stimulating factor (G-CSF).
In the above extraction method, the mobilization time can be 4-5 days, such as 4.5 days.
The extraction method may further comprise collecting mobilized peripheral blood hematopoietic stem cells to obtain a peripheral blood hematopoietic stem cell collection, and standing the peripheral blood hematopoietic stem cell collection at 22 ℃ for 3 hours to allow exosomes in the peripheral blood hematopoietic stem cell collection to be secreted into plasma.
The extraction method can also comprise the steps of centrifuging the peripheral blood hematopoietic stem cell collection after placement, collecting blood plasma, and collecting exosome from the blood plasma, wherein the exosome is the peripheral blood hematopoietic stem cell collection blood plasma exosome.
In the above extraction method, the step of centrifuging the peripheral blood hematopoietic stem cell collection after placement to collect plasma, and collecting exosomes from the plasma may be:
1) centrifuging the placed peripheral blood hematopoietic stem cell collection at 400g for 15min at 22 ℃ to obtain plasma, transferring the plasma into centrifuge tubes (first centrifuge tubes), horizontally centrifuging the plasma for 10min at 300g at 4 ℃, and taking the supernatant and discarding the precipitate;
2) slowly adding the supernatant obtained in the step 1) into a centrifuge tube, balancing with a PBS buffer solution, horizontally centrifuging at 4 ℃ for 10min at 2000g, and reserving the supernatant and removing precipitates;
3) transferring the supernatant obtained in the step 2) into another new centrifuge tube (a second batch of centrifuge tubes), balancing with PBS buffer solution, horizontally centrifuging at 4 ℃ for 10000g for 30min, taking the supernatant, and discarding the precipitate;
4) transferring the supernatant obtained in the step 3) into another new special centrifuge tube (a third batch of centrifuge tubes), balancing with PBS buffer solution, horizontally centrifuging at 4 ℃ for 100000g for 2h, and reserving precipitates and discarding the supernatant; the sediment is the plasma exosome of the peripheral blood hematopoietic stem cell collection.
The extraction method also comprises the step of gently resuspending the precipitate in the step 4) by using PBS buffer solution, and placing the precipitate in a refrigerator at 4 ℃ for standby.
The extraction method further comprises centrifuging the peripheral blood hematopoietic stem cell collection at 22 deg.C at 400g for 15min, collecting part of blood plasma, and returning the rest peripheral blood hematopoietic stem cell collection to patients or freezing according to clinical requirement.
The exosome obtained by the extraction method is also within the protection scope of the invention.
In order to solve the technical problems, the invention further provides an exosome preparation.
The exosome preparation provided by the invention comprises: peripheral blood hematopoietic stem cell collection plasma exosomes and a medically acceptable solvent; the peripheral blood hematopoietic stem cell collection plasma exosome is obtained according to the extraction method.
In the present invention, the solvent may be PBS buffer. The exosome preparation consists of the exosomes and PBS buffer.
In the present invention, the exosome formulation may be used for autologous, allogeneic subjects.
In order to solve the technical problems, the invention further provides an exosome obtained by the extraction method or an application of the exosome preparation:
the use of the exosome obtained by the extraction method or the exosome preparation in the preparation of a pharmaceutical composition for promoting the recovery of hematopoietic damage;
and/or the exosome obtained by the extraction method or the exosome preparation is used for preparing the pharmaceutical composition for promoting the proliferation of the peripheral blood mononuclear cells.
In the above uses, the hematopoietic damage is damage of hematopoietic function caused by various causes, such as radiation, radiotherapy or chemotherapy (bone marrow transplantation).
In the above uses, the exosome recipient is a human who has not undergone prevention and/or treatment of graft-versus-host disease.
The invention collects peripheral blood hematopoietic stem cells from human peripheral blood treated by the peripheral blood hematopoietic stem cell mobilizing agent by a blood cell separator to obtain a peripheral blood hematopoietic stem cell collection, collects plasma from the peripheral blood hematopoietic stem cell collection, and extracts exosomes from the plasma. Plasma is a waste product from which the extraction of exosomes can increase its availability without affecting the use of other components of the peripheral blood hematopoietic stem cell collection. The peripheral blood hematopoietic stem cell collection plasma exosome obtained by the invention is infused into an organism with hematopoietic injury, so that the recovery of the hematopoietic injury can be promoted, the risk of complications caused by implantation of the peripheral blood hematopoietic stem cell after infusion is avoided, the exosome can play a role of maternal cells, and the adverse reaction of the maternal cells is avoided.
Drawings
In FIG. 1, A and B are the results of transmission electron microscopy of plasma exosomes of human peripheral blood hematopoietic stem cell collection.
FIG. 2 is a Micro-BCA protein assay standard curve.
FIG. 3 is a flow chart of a test for promoting rapid recovery of hematopoietic injury after radiotherapy by using plasma exosomes of a peripheral blood hematopoietic stem cell collection.
FIG. 4 shows the result of transmission electron microscopy of plasma exosomes of mouse peripheral blood hematopoietic stem cell collection.
FIG. 5 shows the white blood cell, hemoglobin and platelet changes after infusion of hematopoietic stem cell exosomes in irradiated mice.
FIG. 6 is a growth curve of hematopoietic stem cells.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are all conventional ones unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The preparation method of the PBS buffer solution used in the following examples of the invention is as follows:
weighing 4g of sodium chloride, 0.71g of sodium dihydrogen phosphate, 0.1g of potassium chloride and 0.135g of potassium dihydrogen phosphate, adding 400 ml of distilled water for dissolving, dropwise adding hydrogen chloride to adjust the pH value to 7.4, fixing the volume to 500 ml, sterilizing under high pressure, and storing in a refrigerator at 4 ℃.
Example 1 extraction of plasma exosomes from peripheral blood hematopoietic Stem cell Collection
1. Obtaining a Collection of donor peripheral blood hematopoietic Stem cells
A collection of peripheral blood hematopoietic stem cells from human healthy donors was used as the subject. rhG-CSF is given to a healthy donor for 5ug/(kg.d) q 12h, is injected subcutaneously for 4.5 days (namely, rhG-CSF is injected subcutaneously for 1 time every 12 hours, each dose is rhG-CSF 5ug/kg body weight, and administration is continuously carried out for 4.5 days), peripheral blood hematopoietic stem cells are mobilized on the 5 th day, peripheral blood is collected by a blood cell separator (purchased from Amicus, France Hewley) for peripheral blood hematopoietic stem cell collection (collection parameters are set according to a blood cell collection program), the general circulation total blood volume is 10000mL, and the peripheral blood hematopoietic stem cell collection of 200 mL and 300mL is obtained.
2. Exosome-rich plasma fractions from peripheral blood hematopoietic stem cell collection
1) The peripheral blood hematopoietic stem cell collection collected in step 1 was left at 22 ℃ for 3 hours to secrete exosomes in the peripheral blood hematopoietic stem cell collection into plasma.
2) And (3) centrifuging the placed peripheral blood hematopoietic stem cell collection in a low-speed centrifuge at 22 ℃ for 15min at 400g, separating out plasma, extracting 5-10 ml of plasma by using an injector for later use, and returning the residual peripheral blood hematopoietic stem cell collection to a patient or freezing and storing according to clinical requirements.
3. Separation of plasma exosomes of peripheral blood hematopoietic stem cell collection
1) Transferring the plasma obtained in the step 2 into a 15ml centrifuge tube, horizontally centrifuging the plasma at the temperature of 4 ℃ for 10min at 300g, and taking the supernatant and discarding the precipitate.
2) Slowly adding the supernatant obtained in the step 1) into a special centrifuge tube (a first batch of centrifuge tubes), balancing PBS buffer solution (the error is within 0.05 g), placing the special centrifuge tube into an ultracentrifuge (Saimeishei company, model number is Thermofisiher WX100+) for horizontal centrifugation at 4 ℃ for 2000g for 10min, taking the supernatant, and discarding the precipitate.
3) Transferring the supernatant obtained in the step 2) into another new special centrifuge tube (a second batch of centrifuge tubes), balancing with PBS buffer solution, horizontally centrifuging at 4 ℃ for 10000g for 30min in an ultracentrifuge, and taking the supernatant and discarding the precipitate.
4) Transferring the supernatant obtained in the step 3) into another new special centrifuge tube (a third batch of centrifuge tubes), balancing with PBS buffer solution, horizontally centrifuging at 4 ℃ for 100000g in an ultracentrifuge for 2h, and reserving the precipitate and discarding the supernatant.
5) And (3) lightly resuspending the precipitate obtained in the step (4) by using 100ul of PBS buffer solution, and placing the precipitate into a refrigerator at 4 ℃ for later use to obtain the plasma exosome of the peripheral blood hematopoietic stem cell collection.
4. Identification of plasma exosomes of peripheral blood hematopoietic stem cell collection by transmission electron microscope
1) And (4) sucking 10ul of the plasma exosomes of the peripheral blood hematopoietic stem cell collection obtained in the step (3) by a micropipette into the copper mesh grid, and standing for 5 min.
2) The water was gently blotted dry with filter paper along the edge of the copper mesh.
3) 20ul of 2% sodium phosphotungstate was added to the copper mesh grid for negative dyeing, and the mixture was left at room temperature for 5 min.
4) The water was gently blotted with filter paper along the edges of the copper mesh.
5) The incandescent lamp gently bakes the copper mesh dry.
6) And (4) placing the treated copper mesh grid into an electron microscope, adjusting the electron microscope, and observing.
The transmission electron microscope results of plasma exosomes of peripheral blood hematopoietic stem cell collection are shown as A and B in figure 1: the plasma exosome form of the peripheral blood hematopoietic stem cell collection is in a similar circle shape, the size is not uniform, and the diameter is about 100 nm.
5. Quantitative detection of plasma exosomes of peripheral blood hematopoietic stem cell collection
Detection was carried out using the Micro-BCA protein quantification kit (Thermo Fisher, USA, cat # 23235) as follows
1) And opening the water bath tank, setting the temperature to be 37 ℃, taking a foam tank, and adding a certain amount of liquid nitrogen.
2) Taking out 30ul of exosome, putting the exosome into an EP tube, clamping the upper end of the EP tube by hemostatic clips, immersing the liquid containing part into liquid nitrogen to rapidly freeze the liquid, rapidly taking out the exosome, putting the exosome into a water bath tank, and slightly shaking the exosome to melt the liquid. And repeating the steps, freezing and thawing for 5-7 times repeatedly, and fully cracking the exosomes to prepare the exosome sample to be detected.
3) According to the formula of solution A: and B, liquid B: and preparing the working solution (MR)/a standard product by using the solution C in a ratio of 25:24: 1.
4) The 96-well plate is taken and the holes are made one circle at the edge to prevent errors in reading. Two rows are selected, with 1 row labeled with holes No. 1-7 and 2 rows labeled with holes No. 1-2. 150. mu.L of the prepared standard was added to well 1, line 1, at a concentration of 40. mu.g/mL, and 150. mu.L of PBS buffer was added to well 2-7.
5) 150ul of the sample was added to row 1 and row 2 wells as above and the exosome sample to be detected was added by fold dilution from well 3 to well 6. 150 μ L of PBS buffer was added to the middle of the remaining one well.
6) The lysed exosomes were diluted 100-fold with PBS buffer to a final volume of 300 ul. 150 μ L of the exosome sample to be detected was added to each of the 2 nd row, 1-2 wells.
7) 150 mu L of the prepared working solution is added into each well, and the mixture is placed into an incubator at 37 ℃ and incubated for 2 hours in a dark place.
8) And (3) after 2h, putting the 96-well plate into an enzyme-labeling instrument, adjusting the laser wavelength to 562nm, and reading the absorbance value.
Drawing a standard curve of concentration-OD value according to the concentration and the absorbance OD value of the standard substance to obtain a linear formula, and then obtaining the standard curve according to R 2 The fitting degree and reliability of the standard curve are judged according to the value, and R is generally considered to be 2 Values above 0.99 provide good fit and reliability. The results are shown in FIG. 2, and the protein concentration in exosomes was calculated by substituting the OD values measured in the sample wells into the formula. The plasma exosome concentration was about 1mg/ml per hematopoietic stem cell harvest. The resulting mean was calculated to obtain 1000. mu.g exosomes per ml plasma.
Example 2 exosomes of peripheral blood hematopoietic stem cell collections promote rapid recovery of hematopoietic damage following radiation therapy
A method for promoting rapid recovery of hematopoietic injury after radiotherapy by peripheral blood hematopoietic stem cell collection exosomes is shown in figure 3, and specifically comprises the following steps:
1. healthy male C57 mice (6-8W old, SPF grade, purchased from Beijing Wilton technology Ltd.) were administered with subcutaneous injection of G-CSF (purchased from Beijing Shunlu pharmaceutical Co., Ltd., product number 87189190000087272782)5ug/(kg.d) q 12h subcutaneous injection for 4.5 days (i.e., subcutaneous injection of G-CSF 1 time per 12 hours, each dose of G-CSF 5ug/kg body weight, continuous administration for 4.5 days) to mobilize, and spleen cell suspensions (i.e., peripheral blood of mice) were preparedBlood stem cell collection) to simulate the method of example 1 for obtaining a human peripheral blood hematopoietic stem cell collection after human mobilization, the specific method is as follows: mice were sacrificed 5 days after mobilization and soaked in 75% ethanol for 3 minutes. Splitting the left lower abdomen of the mouse by using sterilized scissors and tweezers, and taking out the spleen; gently grinding spleen in RPMI-1640 culture solution (Gibico, cat # C11875500BT) with a 2ml syringe core and a 400 mesh screen, washing the screen with RPMI-1640 culture solution after grinding, and filtering again with the screen to obtain a cell suspension; 15ml of sigma cell separation solution (purchased from Histopaque, with the product number of 1083) is added into a 50 ml corning pointed-bottom centrifuge tube, and 15ml of the cell suspension is carefully added onto the separation liquid surface by a liquid transfer device, so that the interface is ensured to be clear; centrifuging at 400g for 20 minutes at 20 ℃ in a horizontal centrifuge (slow acceleration, slow deceleration); after centrifugation was completed, the cell layer was gently removed and the middle white cell layer was carefully aspirated off with a pipette. Adding 10 times volume of PBS buffer solution for washing once, centrifuging 400g for 5 minutes, removing supernatant after centrifugation, adding 3 times volume of erythrocyte lysate (purchased from BD company, with the product number of 349202), and carefully mixing; standing for 5 minutes, adding PBS buffer solution to 40 ml, centrifuging for 5 minutes at 400g, and removing supernatant; washing twice with 30 ml PBS buffer solution, centrifuging for 5min at 400g, removing supernatant to obtain cell suspension, counting viable cell number with trypan blue, filtering the cell suspension with 50um cell sieve, and adjusting cell concentration to 1 × 10 8 And (c) obtaining a spleen cell suspension (namely, a mouse peripheral blood hematopoietic stem cell collection) which is equivalent to the human peripheral blood hematopoietic stem cell collection in the example 1). The mouse peripheral blood hematopoietic stem cell collection material is placed in PBS buffer solution to be incubated for 4 hours at 37 ℃, supernatant is obtained by centrifugation and separated according to the method of the step 3 in the embodiment 1 to obtain mouse peripheral blood hematopoietic stem cell collection exosomes, the morphology of the mouse peripheral blood hematopoietic stem cell collection exosomes observed by the method for identifying the mouse peripheral blood hematopoietic stem cell collection exosomes by using the transmission electron microscope in the step 4 in the embodiment 1 is shown in figure 4, and the result shows that the exosomes can be prepared by the method and have the diameter of about 50-100 nm.
2. 24 female CB6F1 mice (6-8W old, SPF grade, purchased from Beijing Wintonlifys technology Co., Ltd.) weighing 20g were divided into three groups, i.e., transplantedGroups, exosome groups and G-CSF groups, 8 per group. Three groups of mice are irradiated by 8Gy whole body (TBI) to establish a mouse radiation hematopoietic injury model (hereinafter referred to as radiation injury mice). After the model building is successful, the transplanted mice are treated by tail vein infusion to obtain mouse peripheral blood hematopoietic stem cell samples in the step 1, and the dose of each sample is 3 multiplied by 10 7 (ii) injecting exosome into peripheral blood hematopoietic stem cell collection exosome of mice obtained in step 1 via tail vein of mice of each splenocyte and exosome group in PBS buffer solution at a dose of 3 × 10 7 The total amount of exosomes extracted from each splenocyte, G-CSF group mice were injected with PBS buffer solution of the same volume through tail vein, G-CSF was injected subcutaneously, hematopoietic recovery of three groups of radiation-damaged mice, donor cell chimera detection (Y chromosome sry gene detection by PCR method) and graft-versus-host disease expression were observed.
The results of the effects on the hematopoietic recovery of the radiation-damaged mice are shown in fig. 5, and the changes of the white blood cells (indicated by "WBC"), hemoglobin (indicated by "Hb") and platelets (indicated by "PLT") of three groups of radiation-damaged mice are shown in fig. 5, respectively, and compared with the recovery of the hematopoietic G-CSF group (indicated by "G-CSF") by using only the stimulating factor, the exosome group (indicated by "G-CSF-MV + G-CSF") can increase the minimum of the white blood cells, hemoglobin and platelets of the radiation-damaged mice and increase the hematopoietic recovery rate. Although the transplanted group (indicated as "TBI" in the figure) can rapidly restore hematopoiesis, graft-versus-host disease occurs due to the complete engraftment of the donor murine cells. The exosome group can rapidly recover hematopoiesis and no adverse reactions such as graft-versus-host disease and the like are observed.
Example 3 peripheral blood hematopoietic Stem cell Collection plasma exosomes promote peripheral blood mononuclear cell proliferation
In vitro experiments verify whether the plasma exosome of the peripheral blood hematopoietic stem cell collection has the effect of promoting proliferation of peripheral blood mononuclear cells or not, and the specific steps are as follows:
human healthy donors are administered rhG-CSF 5ug/(kg.d) q 12h for 4.5 days (i.e. 1 time of rhG-CSF per 12 hours, each dose being rhG-CSF 5ug/kg body weight, and continuous administration for 4.5 days) to mobilize peripheral blood hematopoietic stem cells on day 5A blood cell separator is adopted to separate a peripheral blood hematopoietic stem cell collection, and the circulating total blood volume is 10L. Separating peripheral blood mononuclear cells from 1mL of separated hematopoietic stem cell collection with human lymphocyte separation medium (purchased from Ficoll, GE, Cat. No. 17-1440-02-1), washing, resuspending in 2mL of PBS to obtain mononuclear cell suspension, adjusting cell concentration to 1 × 10 6 Each ml of the cells are divided into two groups, namely an exosome group and a control group, and the two groups are inoculated into a 24-well culture plate, each group comprises 3 multiple wells, 1ml of mononuclear cell suspension and 10mg of exosomes are added into each well, the control group is added with PBS with the same volume, and the culture conditions are 37 ℃ and 5% CO 2 The cells were incubated at 95% humidity, and the number of mononuclear cells of peripheral blood hematopoietic stem cells in each group was measured every two hours.
As shown in FIG. 6, the number of cells in the exosome group (indicated as "MV" in the figure) increased with the time of co-culture, and the number of cells in the exosome group at 2d of co-culture was 1.5 times (P >0.05) that in the control group (indicated as "CC" in the figure); the number of the cells of the exosome group is 2.2 times of the number of the cells of the control group (P <0.05) in the co-culture period of 4d, and the number of the cells of the exosome group is 2.4 times of the number of the cells of the control group (P <0.05) in the co-culture period of 6d, which shows that the exosome of the peripheral blood hematopoietic stem cell collection plasma has obvious effect of promoting the expansion of the peripheral blood mononuclear cells.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.
Claims (3)
1. Use of an exosome or exosome preparation in the preparation of a pharmaceutical composition for promoting peripheral blood mononuclear cell proliferation;
the exosome is extracted according to an exosome extraction method, the extraction method comprises the steps of mobilizing peripheral blood hematopoietic stem cells by using a mobilizing agent, collecting the mobilized peripheral blood hematopoietic stem cells to obtain a peripheral blood hematopoietic stem cell collection, placing the peripheral blood hematopoietic stem cell collection at 22 ℃ for 3 hours, centrifuging the placed peripheral blood hematopoietic stem cell collection, collecting plasma, and collecting exosome from the plasma, wherein the exosome is a peripheral blood hematopoietic stem cell collection plasma exosome;
the exosome preparation comprises the exosome and a medically acceptable solvent.
2. The use of claim 1, wherein the mobilizing agent is recombinant human granulocyte colony-stimulating factor.
3. Use according to claim 1 or 2, characterized in that the mobilization period is 4-5 days.
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