CN116143934B - Stem cell exosome extraction kit and application thereof - Google Patents
Stem cell exosome extraction kit and application thereof Download PDFInfo
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Abstract
The invention provides a stem cell exosome extraction kit and application thereof, wherein the kit comprises nano magnetic beads, eluent and magnetic bead cleaning liquid; the nanometer magnetic beads are used for capturing target exosomes from a sample, and the surfaces of the nanometer magnetic beads are combined with multispecific antigen conjugates; the multispecific antigen-binding comprises anti-CD 9, CD81, and EpCAM antigen-binding fragments that are sequentially linked. By adopting the kit or the method, the stem cell exosomes with specific antigens on the surface can be identified and obtained, and the exosomes have stronger cell proliferation promoting capacity and oxidative damage resisting capacity; the differential centrifugation method is combined with the immunomagnetic bead method, so that dependence on expensive large-scale equipment is avoided, and quality and efficiency of screening stem cell exosomes are provided; can regulate the secretion level of inflammatory factors of cells, and avoid excessive immune injury to normal cells of the body.
Description
Technical field:
the invention belongs to the technical field of biology, and particularly provides a stem cell exosome extraction kit and application thereof.
The background technology is as follows:
exosomes were found in sheep reticulocytes by researchers in 1983, but they were originally considered cellular waste and have long been unattractive to the industry; until 2007, students found that the nanoscale vesicles contained proteins, lipids and RNAs (including mRNA and miRNA) and could be transferred to other cells as signal molecules that exert biological functions, exhibiting biological research and clinical application values. The exosomes are the extracellular vesicles with nano-scale bilayer membranes, the surfaces of the extracellular vesicles are phospholipid bilayers, the exosomes have good stability and permeability, and after the exosomes enter target cells, the exosomes can regulate the physiological functions and signal transduction of the cells; the exosomes are in cup-shaped structures under a microscope, the diameter ranges from 30 nm to 200nm, the density is about 1.13-1.19g/mL, the exosomes can be stored for a long time in an environment of-80 ℃, the exosomes stored for a long time under different conditions can have different degrees of diameter increase compared with the exosomes which are freshly separated, and the exosomes in the supernatant are observed to have different degrees of protein leakage; the distribution of exosomes is quite broad, being present in almost all body fluids, including blood, saliva, urine, cerebrospinal fluid and milk.
Mesenchymal stem cells (mesenchymal stem cells, MSCs) have the potential to self-renew and differentiate in multiple directions, common MSCs are derived from fat (AMSCs), bone Marrow (BMSCs), umbilical cord (UCMSCs) and gums, and MSCs have attracted extensive attention since their discovery in 1968 and have been used in preclinical studies for many years. Various types of mesenchymal stem cells can secrete exosomes under normal and pathological conditions, the exosomes are formed by the invagination of intracellular lysosome particles, are fused with endosomal membranes and cell membranes by endocytosis, and are released into the extracellular matrix by paracrine signals. Although MSC derived exosomes of different origins have different yields, for example bone marrow mesenchymal stem cells release more exosomes than adipose derived mesenchymal stem cells; the yield of human amniotic mesenchymal stem cell exosomes is significantly higher than that of human bone marrow mesenchymal stem cells, but their physiological functions are similar, as studies have shown that 60% of the proteins in exosomes derived from human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, HUMSCs) and exosomes derived from bone marrow (human bone marrow mesenchymal stem cells, HBMSCs) are identical, and these proteins are associated with cell growth and antioxidant stress, both of which inhibit tumor cell growth.
The methods currently available for the efficient isolation of exosomes are the following: differential centrifugation, density gradient centrifugation, precipitation, wash separation, ultrafiltration, antibody affinity capture, microfluidic separation, and mass spectrometry.
Differential centrifugation, which is a method in which exosomes are separated from other substances present in a sample according to their volume and physical characteristics, the collected sample is centrifuged at different speeds to remove cells, cell debris and macromolecular proteins, and then ultracentrifuged at 100000×g for 70 minutes to obtain exosomes in the supernatant. Differential centrifugation is the gold standard in exosome separation methods, the most common method of extracting exosomes from cellular biological fluids and culture supernatants, but while differential centrifugation (including ultracentrifugation) is effective for exosome separation, this technique is time consuming, labor intensive and severely instrument dependent for research laboratories and clinical settings, and the ultracentrifugation process can lead to loss of large amounts of exosomes with a risk of reduced yields. Furthermore, this method is questioned in terms of separation effect, which on the one hand may lead to insufficient purity, which results in some studies indicating that the final centrifugation product is microvesicle instead of exosomes, since the identification of microvesicles and exosomes is not a unified standard, and is therefore considered unsuitable for extracting exosomes from a small number of serum samples; on the other hand, protein and RNA components existing on exosomes are greatly affected in the ultracentrifugation process, and are unfavorable for later use, so that the method is mainly applicable to large samples such as cell supernatant, urine and the like and protein research.
In the density gradient centrifugation method, a sample is added into an inert gradient medium to carry out centrifugal sedimentation or sedimentation balance in the center of a density gradient region, and different components of the sample can be sedimented into an equal density region of the sample under the action of a certain centrifugal force, so that the separation of an exosome and other components in the sample is realized. The most common of these methods is sucrose density gradient centrifugation, which builds a linear sucrose gradient (0.25-2.0M sucrose) into an ultracentrifuge tube, then deposits the sample on top of the linear sucrose gradient, and then ultracentrifuges the gradient solution at 210000g at 4 ℃ to obtain the exosomes. The advantage of this method is that, first of all, it has a higher separation efficiency, thus producing higher purity exosomes, compared to conventional ultracentrifugation methods; and secondly, the exosome particles are not easy to crush or deform in the separation process, so that the separated components are prevented from being mixed again. The method has the disadvantages of long centrifugation time and low yield of the final product. Furthermore, the need to prepare an inert gradient media solution prior to running the process, as well as the preparation work and the process itself are complex and time consuming, the instrumentation required for density gradient centrifugation is also expensive, and a large amount of space is taken up in the laboratory, which makes many laboratories unable to practice the process.
Precipitation methods which add solvents to a solution to alter the polarity and solubility of certain components, thereby precipitating them into solution. Compared with differential centrifugation, precipitation is effective in improving the separation efficiency of biological fluids, and has been developed TM Exo-Q and Exo-Spin TM Blood cell purification kit, mi-RCURY Exosome separation kit and Exo Quick-TC Exosome TM Various extraction kits such as a precipitation solution kit, a methanol precipitation and total exosome separation kit and the like. However, this method has an influence on the purity of exosomes due to the use of precipitation reagents.
The washing separation method, which uses the principle of chromatographic separation, adds a sample to one end of a chromatographic column, makes it adsorbed and dissolved on a stationary phase, and then uses a washing agent to separate the sample from the stationary phase. The method has the advantages of time and cost effectiveness, and has good large-scale clinical application prospect. However, the method is difficult to completely adsorb and wash the exosome, is easy to waste exosome samples, and is not applicable to separation and detection of small or trace samples.
The ultrafiltration method uses the pressure difference at two sides of the ultrafiltration membrane as driving force, under a certain pressure, the stock solution flows through the surface of the membrane, large-volume particles are trapped, and small-volume particles can pass through the ultrafiltration membrane, so that the purification, separation and concentration of the stock solution are achieved. The method can remove certain impurities which are difficult to remove by density gradient centrifugation, such as nonspecific AGO protein, but the ultrafiltration membrane is separated Kong Yifa to generate blockage, so that the treatment efficiency is gradually reduced, the separation time is prolonged, and the method is unfavorable for the treatment of a large number of samples.
An immunomagnetic bead method in which specific antibodies are immobilized on magnetic beads, and exosomes are selected and isolated by utilizing the antibody-antigen specific binding ability. The method can specifically screen exosomes expressing one or more specific antigens, and researches show that the antibody affinity capture method is more effective in separating exosomes than the centrifugation method or the density gradient method (Popovic M, mazzega E, toffoletto B, de Marco A. Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions.Microb Cell industries.2018; 17 (1): 6), thereby providing a solution for diagnosis or treatment of specific diseases. However, the traditional antibody has a complex structure and high production cost, and limits the application range.
Microfluidic separation, a way to precisely control and manipulate fluids at the microscale level, is capable of manipulating fluids in the micro/nano-scale space, extending reactions, separations and assays to chips of several square centimeters in size, with the greatest advantage of achieving flexible combination and large-scale integration of multi-unit technologies on an integrally controllable micro-platform. Microfluidic technology utilizes the physical and biochemical properties of the stereocomplex for microscale separation, detection and analysis, and in addition to utilizing mature separation methods/separation influencing factors, the technology uses new sorting mechanisms such as acoustic, electrophoretic and electromagnetic operations. The technology is fast, only needs a small amount of sample and a small amount of reagent, but the micro-fluidic device is complex and tiny in structure, and is difficult to produce exosomes in a large scale, and is mainly used in a laboratory research stage at present.
Mass spectrometry, using ionization of components in a test sample to produce ions of different charge-to-mass ratios, the ion beam being formed under the influence of an accelerating electric field and entering a mass return analyser, through which slower ions deflect more and faster ions deflect less; in the magnetic field, slower ions deflect more, faster ions deflect less, and mass spectrograms are respectively focused and separated according to different movement characteristics of different ions in the electromagnetic field. The mass spectrum data quality is high, and the rapid separation of exosomes can be realized, but although the mass spectrum retains the protein mass spectrum, the mass spectrum standard is not established yet, and the method is still in the research stage.
In order to overcome the above-mentioned difficulty, the present invention provides a stem cell exosome extraction kit, which comprises nano magnetic beads, eluent and a magnetic bead cleaning solution; the surface of the nano magnetic beads is combined with a multispecific antigen conjugate, and can be combined with CD9, CD81 and EpCAM antigens. The stem cell exosome extracted by the kit has stronger cell proliferation promoting capability and oxidation injury resisting capability, and can reduce the secretion level of inflammatory factors and prevent excessive injury to cells.
Disclosure of Invention
In order to solve the technical problems, the invention provides a stem cell exosome extraction kit which is characterized by comprising nano magnetic beads, eluent and a magnetic bead cleaning solution; the nanometer magnetic beads are used for capturing target exosomes from a sample, and the surfaces of the nanometer magnetic beads are combined with multispecific antigen conjugates; the multispecific antigen-binding substance comprises an anti-CD 9, CD81 and EpCAM antigen-binding fragment connected in sequence; wherein the amino acid sequence of the anti-CD 9 antigen binding fragment is shown as SEQ ID NO. 1, the amino acid sequence of the anti-CD 81 antigen binding fragment is shown as SEQ ID NO. 2, and the amino acid sequence of the anti-EpCAM antigen binding fragment is shown as SEQ ID NO. 3; the eluent is used for separating the specific binding bodies to form purified exosomes; the magnetic bead cleaning liquid is used for cleaning the exosome.
The nanometer magnetic beads are spherical magnetic particles, and are coated with monoclonal antibodies which are specifically bound with target substances, and the directional movement of the complexes can be induced under the attraction of an external magnetic field, so that exosomes are separated from a sample. This method has target specificity and ensures the integrity of the extracted exosomes, is also relatively easy to implement and does not require expensive instrumentation. In the present invention, the antigen binding fragment adopts a single domain antibody structure, so-called single domain antibody (Single Domain Antibody, sdAb), also called Nanobody (Nanobody) or VHH (Variable Domain ofHeavy Chain) antibody, which is an antibody with a natural deletion of light chain in the peripheral blood of camelid animals such as alpaca, camel, etc., and only comprises a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but is not easy to adhere to each other or even aggregate like a single chain antibody fragment (scFv) which is artificially modified, and the VHH structure has structural stability equivalent to that of the original heavy chain antibody and binding activity to the antigen, which is a known minimum unit capable of binding to the target antigen. The antibody has stable structure, easy design, can mark specific residues, has low mass production cost and stable clone, can directly attack soluble antigens and cells (Crepin R, genetien D, duche A, et al, nanobodies against surface biomarkers enable the analysis of tumor genetic heterogeneity in uveal Melanoma patient-modified xenografts, pigment Cell Melanoma Res.2017; 30:317-327), and therefore, the antibody is selected to construct the capture magnetic beads, so that the preparation is convenient, the target antigens can be effectively combined, and the high-efficiency separation is realized.
Further, the amino acid sequence of the multispecific antigen conjugate is shown as SEQ ID NO. 4.
Further, the preparation method of the nano magnetic beads comprises the following steps: washing the carboxyl magnetic beads by using PBS solution, then adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide solution, and incubating for 30 minutes at room temperature; adding the multispecific antigen conjugate into the magnetic bead solution, slowly stirring to uniformly mix the multispecific antigen conjugate, and reacting for 3-4h at room temperature; and (3) washing for 3 times by using a PBS solution, and re-suspending in the PBS solution after magnetic attraction separation to obtain the nano magnetic beads.
The nanometer magnetic beads are coated with the multi-specific antigen conjugate, so that the screening specificity of exosomes can be improved, the exosomes can be rapidly separated through the reciprocating motion of an externally applied magnetic field, the dependence on large-scale equipment is reduced, and the quality and the efficiency of exosome separation are improved.
Further, the eluate was an aqueous solution containing 2% trypsin, 20mM EDTA-Na2 and 10mM Tris HCl.
Further, the bead washing solution is PBS buffer solution with pH 7.4.
There is provided a method for extracting exosomes using the stem cell exosome extraction kit, comprising the steps of: primary culturing of mesenchymal stem cells; performing primary separation by using a differential centrifugation method, collecting the supernatant of the mesenchymal stem cells, and centrifuging at 300g for 10min at 4 ℃; centrifuging at 3000g for 15min; centrifuging for 45min at 12000 g; filtering the obtained supernatant by a 0.22 mu m filter membrane, and concentrating to obtain a crude exosome; immunomagnetic bead separation, mixing the crude exosomes with nano magnetic beads according to the ratio of 1:3, incubating for 2-3h at room temperature, and separating by using a magnetic field; washing 3-5 times with cleaning solution after separation, and eluting with eluent to obtain exosome.
The method combines the differential centrifugation method and the immunomagnetic bead method, avoids the centrifugal operation of more than one hundred thousand revolutions, reduces the requirement on centrifugal equipment, reduces the production cost, is easy to operate, and can be carried out in a common biological laboratory; meanwhile, the stem cell exosomes with specific functions are screened and obtained by utilizing the specific reaction of antigen and antibody, so that the biological activity of the subsequent exosomes is improved.
Further, the mesenchymal stem cells are derived from umbilical cord, bone marrow or adipose tissue.
Provides an application of the stem cell exosome extraction kit in extracting stem cell exosomes.
Further, the stem cells are umbilical cord mesenchymal stem cells.
Advantageous effects
The invention provides a stem cell exosome extraction kit and application thereof, and has the following advantages:
(1) Provided is a multi-specific antigen conjugate targeting anti-CD 9, CD81 and EpCAM, capable of recognizing and acquiring stem cell exosomes having specific antigens on the surface, the exosomes having a stronger cell proliferation promoting ability and an anti-oxidative damage ability;
(2) The method combines the differential centrifugation method and the immunomagnetic bead method, avoids the dependence on expensive large-scale equipment, and provides the quality and efficiency of screening the stem cell exosomes;
(3) The stem cell exosome obtained by the kit and the method can regulate the secretion level of inflammatory factors of cells and avoid excessive immune injury to normal cells of organisms.
Drawings
Fig. 1: schematic of the structure of the multispecific antigen-binding substance;
fig. 2: exosome cell proliferation promoting ability;
fig. 3: IL-2 expression level;
fig. 4: IL-1 beta expression levels.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. All techniques implemented based on the above description of the invention should be within the scope of the protection claimed in this application.
The experimental methods described in the following examples, unless otherwise specified, are all conventional; the reagent biological material and the detection kit can be obtained from commercial sources unless otherwise specified.
Example 1 exosome capture antibody design
anti-CD 9, CD63 and CD81 antibodies are commonly used for exosome capture and screening, but early studies by the inventors showed that the combined effect of using CD9 and CD81 antibodies was better; also, the inventors creatively proposed in the present invention that anti-CD 9 and CD81 antibodies, further combined with anti-epithelial cell adhesion molecule (EpCAM) antibodies, were constructed as multi-specific antigen conjugates, capable of capturing and sorting exosomes having stronger epithelial cell modulating activity.
The heavy chain variable region of the single domain antibody is used as a specific capture fragment of a target antigen, and specific anti-CD 9, CD81 and EpCAM antigen binding fragments are obtained by screening phage libraries in early stage, wherein the amino acid sequences of the specific capture fragments are shown as SEQ ID NO. 1-3, and the amino acid sequences of related antigen binding fragments are shown in Table 1, and the antibody can bind with high affinity to the target antigen fragment.
TABLE 1 antigen binding fragments
Remarks: the amino acids are underlined as CDR regions.
In order to reduce the subsequent preparation and application difficulty, the trispecific antigen conjugate in the invention adopts a simplified structure, and different antigen binding fragments are connected by using flexible linkers, wherein the schematic diagram is shown in figure 1, and the amino acid sequence of the trispecific antigen conjugate is shown as SEQ ID NO. 4.
Example 2 preparation of mesenchymal Stem cell exosomes
2.1 Primary culture of human umbilical cord mesenchymal Stem cells
The umbilical cord of a healthy fetus is taken and preserved in PBS solution containing 2% penicillin and 2% streptomycin. Cleaning umbilical cord tissue in an ultra clean bench by using sterile PBS to remove blood clots and other impurities on the surface of the tissue; shearing umbilical cord with sterile surgical scissors, removing umbilical vein, umbilical artery and umbilical cord adventitia, separating out Wharton's jelly tissue, cleaning with sterile PBS for 5 times, shearing Wharton's jelly tissue into 2-3 mm tissue pieces, spreading in 60mm culture dish, adding DMEM culture medium containing 10% foetus calf serum and 1% penicillin+1% streptomycin, and concentrating at 37deg.C under 5% CO 2 Culturing in a saturated humidity incubator; observing the growth condition of cells every day, and changing fresh culture medium according to the condition; when the cell fusion degree reaches more than 80%, 0.25% trypsin is used for digestion and passage, and the human umbilical cord mesenchymal stem cells (rhUC-MSCs) are obtained after 3-5 passages of total passage
2.2 differential centrifugal extraction of exosomes
Collecting rhUC-MSCs cell culture supernatant, and centrifuging at 4 ℃ for 10min at 300 g; centrifuging at 3000g for 15min; centrifuging for 45min at 12000 g; filtering the obtained supernatant with 0.22 μm filter membrane, placing into clean centrifuge tube, centrifuging at 4deg.C for 20min at 3000g, concentrating cell supernatant, re-suspending with sterile PBS, and placing into refrigerator at 4deg.C for use.
2.3 sorting exosomes by immunomagnetic bead method
100. Mu.g of carboxyl magnetic beads (available from tin-free Bai Mei Biotech Co., ltd.) were washed 3-5 times with sterile PBS solution pH7.4, and then 500. Mu.L of 0.26M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution was added, and incubated at room temperature for 30 minutes to activate carboxyl groups on the surface of the magnetic beads; adding 200-500 mug of the multi-specific antigen conjugate provided by the invention into a magnetic bead solution, slowly stirring to uniformly mix the multi-specific antigen conjugate, reacting for 3-4 hours at room temperature, and modifying the magnetic bead by amino-carboxyl reaction. After the reaction was completed, the solution was washed 3 times with sterile PBS, unbound antigen conjugate was removed, and the solution was magnetically separated and resuspended in PBS for use.
Mixing the exosomes obtained in the step 2.2 with the magnetic beads according to the proportion of 1:3, incubating for 2-3 hours at room temperature, and separating by using a magnetic field; after separation, the purified exosomes were obtained by washing 3-5 times with PBS solution pH7.4 as washing liquid and eluting with aqueous solution containing 2% trypsin, 20mM EDTA-Na2 and 10mM Tris-HCl. And detecting under an electron microscope, wherein the diameter of the exosome is in the range of 50-150nm, and the particle size is uniform and stable.
EXAMPLE 3 mesenchymal Stem cell exosomes promote cell proliferation
The exosomes have pro-proliferative effects on a variety of cells, and recent studies have shown that stem cell exosomes have significant promoting and modulating effects on the digestive system, including repairing gastrointestinal damage, promoting nutrient absorption, inhibiting excessive inflammatory reactions, etc. (see Yang Ke, zhao Jiang, etc., preclinical research progress of stem cells and exosomes for treatment of necrotizing enterocolitis, chinese journal of cell biology, 2022, 44 (09), 1847-1860). Therefore, in order to study the proliferation promoting effect of exosomes on cells, in this example, caco-2 cells are selected as the study object, and the Caco-2 cells are derived from a human colon cancer cell line, and exhibit many characteristics similar to those of small intestine epithelial cells, and have a microvilli structure, brush-like edges, intercellular tight junctions and the like, which are one of the most widely used and most classical models at present.
Inoculating Caco-2 cells into culture flask, culturing with DMEM medium containing 10% fetal calf serum, 1% penicillin+1% streptomycin, and culturing at 37deg.C and 5% CO 2 Culturing in a saturated humidity incubator, observing the growth condition of cells every day, and carrying out cell passage when the cell fusion degree reaches more than 80%, wherein the total cell passage is 3-5 generations.
Taking the crystal to be in logarithmic growthCaco-2 cells at stage 1X 10 4 The cells/wells were inoculated into 96-well plates and incubated overnight. The human umbilical cord mesenchymal stem cell exosomes extracted by the conventional differential centrifugation method (designated as EXO-1) were set up as EXO-1, 100 μg of EXO-1 was added to each well, EXO-2 (the human umbilical cord mesenchymal stem cell exosomes prepared by the method of the present invention, specific method was referred to as EXO-2 in example 2, 100 μg of EXO-2 was added to each well) and the control (equal volume of cell culture medium was added) were treated with the corresponding drugs for 24 hours. 10. Mu.L MTT was added to each well and incubated for 4h at 37 ℃; the culture medium is discarded, 150 mu of LDMSO is added for shaking for 10min, and an enzyme-labeled instrument is adopted to measure the absorbance (A) value at 490nm according to the formula: cell viability = a dosing/a control, cell viability was calculated.
As shown in figure 2, mesenchymal stem cell exosomes can promote proliferation of Caco-2 cells, and EXO-2 obtained by using the method disclosed by the invention has stronger proliferation promoting capability, which indicates that the exosomes are beneficial to repair of intestinal mucosa and improvement of physiological functions.
Example 4 mesenchymal Stem cell exosomes reduce oxidative stress to cell injury
Caco-2 cells were inoculated into 96-well plates, and after cell fusion reached 80%, H was added to each well at a final concentration of 100. Mu. Mol/L 2 O 2 As an oxidative damage molding agent, 5% CO at 37 DEG C 2 Culturing for 4h. The cells were then divided into three groups, EXO-1 (100. Mu.g EXO-1 per well), EXO-2 (100. Mu.g EXO-2 per well) and control (equal volume of cell culture medium) and treated with the corresponding drugs for 24h. Superoxide dismutase (superoxide dismutase, SOD) (purchased from the institute of biotechnology, south ky), reduced Glutathione (GSH) (purchased from the institute of bioengineering, south ky) in the cell supernatant was detected using a test kit.
The results are shown in Table 2, the addition of mesenchymal stem cell exosomes can resist the damage of oxidative stress to Caco-2 cells, and the SOD and GSH content in the treatment group are improved, but the effect of EXO-2 is more obvious in promoting SOD secretion, and the regulation capacity of GSH is not obviously different.
TABLE 2 Effect of exosomes on Caco-2 cell SOD and GSH
EXAMPLE 5 mesenchymal Stem cell exosome promotes cytokine secretion
Cell modelling and administration were carried out in the same manner as in example 4, and cells were collected last time after the administration treatment, and the content of IL-6 and IL-1. Beta. In the cell supernatant was detected using ELISA kit (purchased from Beijing Soy Bao technology Co., ltd.) according to the kit instructions. As shown in fig. 3 and 4, the mesenchymal stem cell exosomes can significantly inhibit the expression of interleukin inflammatory factors, and the inhibition effect of EXO-2 on IL-6 is more significant, and although EXO-2 seems to have a stronger regulatory effect in IL-1β, no significant difference is seen.
IL-6 and IL-1 beta are main inflammatory factors in colonitis, the high-level expression of which is closely related to the damage of intestinal mucosa epithelium, and the stem cell exosome provided by the invention can effectively inhibit the expression of the factors, thereby providing a new treatment idea for treating intestinal diseases, promoting the absorption of nutrient components and maintaining normal digestion function.
Claims (10)
1. A multispecific antigen-binding article characterized by: the multispecific antigen-binding substance comprises an anti-CD 9, CD81 and EpCAM antigen-binding fragment connected in sequence; wherein the amino acid sequence of the anti-CD 9 antigen binding fragment is shown as SEQ ID NO. 1, the amino acid sequence of the anti-CD 81 antigen binding fragment is shown as SEQ ID NO. 2, and the amino acid sequence of the anti-EpCAM antigen binding fragment is shown as SEQ ID NO. 3.
2. The multi-specific antigen-binding article of claim 1, wherein: the amino acid sequence of the multispecific antigen conjugate is shown as SEQ ID NO. 4.
3. A stem cell exosome extraction kit, characterized in that: the kit comprises nano magnetic beads, eluent and a magnetic bead cleaning solution; the nano magnetic beads are used for capturing a target exosome from a sample, and the surface of the nano magnetic beads is combined with the multi-specific antigen conjugate as claimed in any one of claims 1-2; the eluent is used for separating the multi-specific antigen conjugate to form a purified exosome; the magnetic bead cleaning liquid is used for cleaning the exosome.
4. A stem cell exosome extraction kit according to claim 3, wherein: the preparation method of the nano magnetic beads comprises the following steps: washing the carboxyl magnetic beads by using PBS solution, then adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide/N-hydroxysuccinimide solution, and incubating for 30 minutes at room temperature; adding the multispecific antigen conjugate into the magnetic bead solution, slowly stirring to uniformly mix the multispecific antigen conjugate, and reacting for 3-4h at room temperature; and (3) washing for 3 times by using a PBS solution, and re-suspending in the PBS solution after magnetic attraction separation to obtain the nano magnetic beads.
5. The stem cell exosome extraction kit according to any one of claims 3-4, wherein: the eluate was an aqueous solution containing 2% trypsin, 20mM EDTA-Na2 and 10mM Tris-HCl.
6. The stem cell exosome extraction kit according to any one of claims 3-4, wherein: the magnetic bead cleaning solution is PBS buffer solution with pH of 7.4.
7. A method of extracting exosomes using the stem cell exosome extraction kit of any one of claims 3-6, comprising the steps of: primary culturing of mesenchymal stem cells; performing primary separation by using a differential centrifugation method, collecting the supernatant of the mesenchymal stem cells, and centrifuging at 300g for 10min at 4 ℃; centrifuging at 3000g for 15min; centrifuging for 45min at 12000 g; filtering the obtained supernatant by a 0.22 mu m filter membrane, and concentrating to obtain a crude exosome; immunomagnetic bead separation, mixing the crude exosomes with nano magnetic beads according to the ratio of 1:3, incubating for 2-3h at room temperature, and separating by using a magnetic field; washing 3-5 times with cleaning solution after separation, and eluting with eluent to obtain exosome.
8. The method of extracting exosomes of claim 7, wherein: the mesenchymal stem cells are derived from umbilical cord, bone marrow or adipose tissue.
9. Use of a stem cell exosome extraction kit according to any one of claims 3-6 for extracting stem cell exosomes.
10. The use according to claim 9, characterized in that: the stem cells are umbilical cord mesenchymal stem cells.
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