Construction method and application of functional stem cell exosome for overexpressing miR-126-3p

Abstract

The invention discloses a construction method and application of a functional stem cell exosome for overexpressing miR-126-3p, and belongs to the technical field of biological medicine. The technical problems to be solved are that existing exosome miRNA loading efficiency is low, exosome structures and functions are easily damaged by exogenous loading, target miRNA enrichment effect is poor due to the fact that traditional endogenous loading does not utilize a sorting mechanism, and an engineering modification scheme for specific high background miRNA to efficiently inhibit release of lung fibroblasts CXCL12 is lacked. According to the key points of the technical scheme, the exosome construction step comprises the following steps: screening a stem cell exosome with a high miR-126-3p background expression level; constructing plasmids for overexpressing the hnRNPA2B1, transfecting the screened stem cells, and constructing a cell line for stably overexpressing the hnRNPA2B1; the method comprises the following steps: constructing plasmids of overexpression miR-126-3p, transfecting a cell line, and separating exosomes secreted by the cell line.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a construction method and application of functional stem cell exosomes overexpressing miR-126-3p. BACKGROUND

[0002] For understanding the technical content of the present application: Exosome delivery of miRNA mainly includes two ways: exogenous loading and endogenous loading. Exogenous loading refers to that after extracting Exos from cell supernatant, the target miRNA is passively loaded into Exos through co-incubation, electroporation, ultrasound or repeated freeze-thawing and other physical methods. However, exogenous loading may cause problems such as exosome aggregation and membrane protein structure change, thereby affecting the biological function of exosomes, and is generally suitable for small molecule drugs or molecules that cannot be produced by cells. Endogenous loading refers to that the parent cells are modified for overexpression, so that the exosomes secreted by the parent cells naturally package the target RNA molecules. This method can maintain the natural structure and function of exosomes. Studies have shown that the endogenous loading of miRNA can be achieved by collecting exosomes after transient transfection of miRNA mimics or stable transfection of miRNA plasmid using lentivirus.

[0003] The sorting process of miRNA is an important link in the biogenesis of exosomes. Studies have shown that exosomes selectively carry specific subgroups of miRNA, and the preference of loading is closely related to the sequence of miRNA and sorting proteins. Sorting proteins include RNA binding proteins (such as hnRNPA2B1 and Argonaute2) and membrane proteins involved in exosome biogenesis (such as Caveollin-2 and sphingomyelinase 2) 1, which regulate the transport process of miRNA to exosomes by binding to specific sequences of miRNA. A study in Nature revealed the selective enrichment of different miRNAs in cells and exosomes, and computer sequence analysis revealed that most of the miRNAs enriched in exosomes have high G+C content. These sequences related to exosome enrichment are called Exo motifs 2. Different sorting motifs determine the selective loading of miRNA. Artificial overexpression of miRNA and related sorting proteins can significantly increase the content of target miRNA in exosomes 3, and has higher loading efficiency compared with exogenous loading and traditional endogenous loading.

[0004] The retrieved relevant non-patent literatures are as follows: The journal is *Molecular Oncology*, and the article title is "miR-126 downregulates CXCL12 expression in intestinal epithelial cells to suppress the recruitment and function of macrophages and tumorigenesis in a murine model of colitis-associated colorectal cancer," Volume 16, published in 2022. This article discloses the mechanism by which miR-126 inhibits macrophage recruitment and the development of colitis-associated colorectal cancer by targeting CXCL12, but does not involve the engineering of stem cell exosomes or the application of miRNA exosome delivery.

[0005] The journal is *Journal of Virology*, and the article is titled "hnRNPA2B1 Associated with Recruitment of RNA into Exosomes Plays a Key Role in Herpes Simplex Virus 1 Release from Infected Cells." This article primarily points out that hnRNPA2B1 is an abundant cellular protein capable of recognizing specific sequences (Exo motifs) on RNA and mediating their entry into exosomes. Experiments using cells with the hnRNPA2B1 gene knocked out showed a three-fold reduction in exosome accumulation. This indicates that hnRNPA2B1 plays a crucial role in the transport of enveloped viruses from their assembly site to the extracellular environment.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: In existing exosomal miRNA loading technologies, exogenous loading easily leads to exosome structure destruction (e.g., exosome aggregation rate increases by more than 30% after electroporation), while endogenous loading does not utilize sorting mechanisms, resulting in low efficiency (the content of target miRNA in exosomes is only 15%-20% of that in cells). Relevant evidence is that published patent literature does not mention the synergistic effect of sorting proteins, and experimental data show that the miRNA enrichment fold in exosomes is less than 5-fold. It is known that miR-126 can target CXCL12 and exert an inhibitory effect, but current technology has not combined it with stem cell exosomes, and there is a lack of a scheme to achieve efficient delivery of miR-126 by engineering exosomes. Summary of the Invention

[0007] The present application aims to provide: The present application aims to provide:

[0008] Explanation of terms: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Unless otherwise indicated, all patents, patent applications, publications, and data cited are hereby incorporated by reference in their entirety. In the event of a conflict in terminology, practice or otherwise between the definitions contained herein and those contained in any document incorporated herein by reference, the definition contained herein shall control.

[0009] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It should also be noted that, as used in this application, the term "or" as used in the "and / or" is intended to mean "and / or" unless specifically stated otherwise. Further, the use of the term "including" as well as other forms such as "include", "includes" and "included" is not limiting.

[0010] Definitions of standard chemistry terms can be found in the reference "Exosomes Clinical Protocols, Lawrence R. Edelstein et al., translated by Guo Shangchun, Tao Shicong, Shanghai Science and Technology Press".

[0011] Unless otherwise indicated, conventional methods within the skill of the art are employed, such as differential centrifugation to extract exosomes, Western blot to detect protein expression, qPCR to detect gene expression, ELISA to detect protein levels, and the like.

[0012] Unless a specific definition is provided, the use of a term in the specification as well as in the claims, for example of commercially available products, is intended to refer to the standard art. For example, the use of a kit can be carried out according to the manufacturer's instructions, or in accordance with the way known in the art or described in the present application. In general, the above techniques and methods can be carried out according to the descriptions in the various summary and more specific literature cited and discussed in the present specification, in accordance with the conventional methods well known in the art.

[0013] The term "exosome" as used herein refers to a membrane-enclosed vesicle of about 30-150 nm in diameter, secreted by cells, containing bioactive substances such as proteins, nucleic acids, lipids, etc., which can mediate intercellular information transmission.

[0014] The term "miR-126-3p" as used herein refers to a microRNA molecule of about 22 nucleotides in length, the sequence of which is 5'-UCGUACCGUGAGUAAUAAUGCG-3', which can inhibit the expression of target genes by targeting the 3'UTR region of the target genes.

[0015] The term "UC-MSCs" as used herein refers to umbilical cord-derived mesenchymal stem cells, which have self-renewal and multi-directional differentiation potential and can secrete various exosomes.

[0016] The term "hnRNPA2B1" as used herein refers to an RNA binding protein that can recognize specific sequences (Exo motifs) on miRNA and mediate the sorting of miRNA into exosomes.

[0017] In a first aspect, the present application provides a method for constructing functional stem cell exosomes overexpressing miR-126-3p, comprising the following steps: S1: screening stem cell exosomes with a background expression level of miR-126-3p higher than 1000.00 TPM; S2: constructing a plasmid overexpressing hnRNPA2B1, transfecting the stem cells screened in S1, and constructing a cell line stably overexpressing hnRNPA2B1; S3: constructing a plasmid overexpressing miR-126-3p, transfecting the cell line obtained in S2, and isolating the exosomes secreted therefrom.

[0018] Among them, the technical features include stem cell sources, overexpressed plasmid types, transfection methods, and exosome isolation methods.

[0019] Among them, the technical feature of stem cell source includes but is not limited to umbilical cord-derived mesenchymal stem cells (UC-MSCs), induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs), and induced pluripotent stem cell-derived neural precursor cells (iPSC-NPCs).

[0020] Among them, the technical feature of stem cell source is preferably UC-MSCs or iPSC-MSCs.

[0021] Among them, the technical feature of stem cell source is further preferably UC-MSCs.

[0022] Among them, the technical feature of overexpressed plasmid type includes but is not limited to at least one of pFastBac, pEGFP-N1, pcDNA3.1, pLVX, pCDH, pLenti, or pLKO.1.

[0023] Among them, the technical feature of overexpressed plasmid type is preferably pEGFP-N1, pLVX, pCDH, or pLenti.

[0024] The plasmid type expressed by the technical features is preferably pCDH.

[0025] The transfection method includes, but is not limited to, lentivirus transfection, adenovirus transfection, adeno-associated virus (AAV) transfection, retrovirus transfection or liposome transfection.

[0026] The transfection method includes, but is not limited to, lentivirus transfection, adenovirus transfection, adeno-associated virus (AAV) transfection, retrovirus transfection or liposome transfection.

[0027] The transfection method includes, but is not limited to, lentivirus transfection, adenovirus transfection, adeno-associated virus (AAV) transfection, retrovirus transfection or liposome transfection.

[0028] The transfection method includes, but is not limited to, lentivirus transfection, adenovirus transfection, adeno-associated virus (AAV) transfection, retrovirus transfection or liposome transfection.

[0029] The exosome separation method includes, but is not limited to, differential centrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, immunoaffinity capture, size exclusion chromatography or microfluidic chip technology.

[0030] The exosome separation method includes, but is not limited to, differential centrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, immunoaffinity capture, size exclusion chromatography or microfluidic chip technology.

[0031] The exosome separation method includes, but is not limited to, differential centrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, immunoaffinity capture, size exclusion chromatography or microfluidic chip technology.

[0032] The exosome separation method includes, but is not limited to, differential centrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, immunoaffinity capture, size exclusion chromatography or microfluidic chip technology.

[0033] Based on the further solution or simultaneous solution of multiple technical problems of the technical problem of the present application, in the technical scheme provided by the first aspect of the present application, the preferred scheme includes: The first preferred scheme is to screen high background stem cells to solve the problem of blind selection of parent cells and further improve the modification efficiency.

[0034] The second preferred scheme is to co-transfect hnRNPA2B1 overexpression plasmid and miR-126-3p overexpression plasmid to solve the technical problem of limited single overexpression loading efficiency.

[0035] In the second aspect, the present application provides an exosome prepared by the above preparation method.

[0036] In the third aspect, the present application provides a pharmaceutical composition comprising any of the above exosomes.

[0037] The pharmaceutical composition comprises a pharmaceutically acceptable excipient, a dosage form, and an administration method.

[0038] The pharmaceutically acceptable excipient includes, but is not limited to, at least one of a wetting agent, an emulsifying agent, a preservative, an antioxidant, a buffering agent, an excipient, a diluent, a lubricant, a bacteriostatic agent, a suspending agent, a suspending aid, a solubilizing agent, a thickening agent, a stabilizing agent, a sweetener, and a flavoring agent.

[0039] The pharmaceutically acceptable excipient is preferably at least one of lactose, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, polyvinylpyrrolidone, cellulose, water, sugar syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, and mineral oil.

[0040] The dosage form includes, but is not limited to, a liquid dosage form, a gaseous dosage form, a solid dosage form, or a semi-solid dosage form.

[0041] The liquid dosage form includes a solvent type, an aromatic water agent, a tincture, an elixir, a colloidal solution, a colloidal paste, a suspension, or a emulsion.

[0042] The gaseous dosage form includes an aerosol or a spray.

[0043] The solid dosage form includes a powder, a pill, a tablet, or a film.

[0044] The semi-solid dosage form includes an ointment, a suppository, or a paste.

[0045] The administration method includes oral administration, injection, implantation, external use, spraying, inhalation, or a combination thereof.

[0046] The injection administration method includes, but is not limited to, intraocular injection, intravenous injection, intramuscular injection, subcutaneous injection, intrathecal injection, or intra-articular injection.

[0047] The external administration method includes, but is not limited to, mucosal administration or transdermal / local administration.

[0048] The mucosal administration includes, but is not limited to, nasal administration, oral buccal membrane / sublingual administration, and ocular administration.

[0049] The inhalation administration method includes, but is not limited to, nebulization inhalation or metered dose inhaler inhalation.

[0050] In a fourth aspect, the present application provides the use of the above-mentioned exosome and pharmaceutical composition in the preparation of a product for inhibiting the expression of CXCL12.

[0051] In a fifth aspect, the present application provides use of the above-mentioned exosomes and pharmaceutical compositions in the preparation of a product for treating and / or inflammation caused by novel coronavirus infection.

[0052] The present application has the following beneficial effects: The present application has at least the following beneficial effects: The present application focuses on the specific effect miRNA miR-126-3p in stem cell exosomes, first screens stem cell exosomes with high background expression, and then based on the sorting mechanism of miRNA, constructs stem cell exosomes with enhanced expression of miR-126-3p, which is more efficient. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 The miRNAs differentially expressed in human lung tissue after SARS-CoV-2 infection. A is a scatter plot of differentially expressed miRNAs in lung tissue after SARS-CoV-2 infection compared with control lung tissue, each point represents a miRNA, up-regulated miRNAs are marked in red, and down-regulated miRNAs are marked in blue. B is the expression level of the indicated miRNAs in SARS-CoV-2 infected lung tissue and control lung tissue.

[0054] Figure 2 Extraction and characterization of three kinds of stem cell exosomes. A is a schematic diagram of the process of extracting stem cell exosomes by differential centrifugation; B is a Zeta potential column chart of three kinds of stem cell exosomes, n = 3; C is a representative particle size distribution chart of three kinds of stem cell exosomes detected by DLS; D is a representative particle size distribution chart of three kinds of stem cell exosomes detected by NTA.

[0055] Figure 3 TPM (Transcripts Per Million) heat map of miR-126-3p expression in three kinds of stem cell-derived exosomes, different colors reflect the TPM count level of miRNA.

[0056] Figure 4 Construction and identification of UC-MSCs-Exo OE-miR-126-3p A is a map of two overexpression plasmids; B is a schematic diagram of the construction of UC-MSCs-Exo OE-miR-126-3p C is the expression level of hnRNPA2B1 in each group of cells after transfection of hnRNPA2B1 overexpression plasmid or empty plasmid, Actin as internal reference protein; D is the relative expression level of miR-126-3p in cells after transfection of miR-126-3p mimics and transfection of miR-126-3p overexpression plasmid, U6 as internal reference. E is UC-MSCs-Exo, UC-MSCs-Exo miR-126-3p mimics and UC-MSCs-ExoOE-miR-126-3p Relative expression level of miR-126-3p, U6 as internal reference, n = 3, ns no significant difference; p <0.05;** p <0.01;*** p <0.001;**** p <0.0001.

[0057] Figure 5 Experimental results of exosomes inhibiting CXCL12, A is the transcription level of CXCL12 in MRC-5 cells, B is the level of CXCL12 secreted by MRC-5 cells; p <0.05;** p <0.01;*** p <0.001. DETAILED DESCRIPTION

[0058] The application will be further described below in conjunction with specific examples. The following examples are not used to limit the application, but only to illustrate the application. Unless otherwise specified, the experimental methods used in the following examples are generally carried out under conventional conditions. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial sources.

[0059] The following non-limiting examples can enable those of ordinary skill in the art to more fully understand the present application, but in no way limit the present application. The following is only an exemplary description of the scope of the present application, and those skilled in the art can make various changes and modifications to the present application based on the disclosed content, and it should also belong to the scope of the present application.

[0060] The application will be further described below in conjunction with specific examples. The various instruments, devices, equipment, reagents, products, etc. used in the examples of the present application can be obtained through conventional commercial channels unless otherwise specified.

[0061] Example 1 Using the published dataset (GSE235130), we screened for miRNAs differentially expressed in human lung tissue after SARS-CoV-2 infection. The results showed that a total of 65 miRNAs were up-regulated, and 27 miRNAs were down-regulated (Fig. 1A). Subsequently, we compared these candidate miRNAs with miRNAs independently predicted to be targetable to CXCL12 mRNA in the miRWalk, miRTarBase, and miRTargetLinks databases, and finally screened 5 candidate miRNAs, of which miR-126-3p was the most significantly down-regulated miRNA in human lung tissue after SARS-CoV-2 infection (Fig. 1B). Figure 1 A). Subsequently, we compared these candidate miRNAs with miRNAs independently predicted to be targetable to CXCL12 mRNA in the miRWalk, miRTarBase, and miRTargetLinks databases, and finally screened 5 candidate miRNAs, of which miR-126-3p was the most significantly down-regulated miRNA in human lung tissue after SARS-CoV-2 infection (Fig. 1B).Figure 1 B). The above results indicate that miR-126-3p is a potential therapeutic target for inhibiting the progression of respiratory infections caused by novel coronavirus infection, and exosome-mediated delivery of miR-126-3p may be a new therapeutic approach.

[0062] Example 2 Differential centrifugation was used to extract Exos from UC-MSCs, iMSCs, and iNPCs. Figure 2 A), and characterized their physical properties. Phase analysis light scattering (PALS) results showed that the Zeta potentials of the three stem cell exosomes were all around -20 mV, indicating good stability ( Figure 2 B). Further analysis using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) revealed that the particle size distribution of the three stem cell exosomes was concentrated between 100-150 nm. Figure 2 CD), which meets the particle size range requirements for exosomes. These results indicate that we successfully extracted UC-MSCs Exos, iMSCs Exos, and iNPCs Exos.

[0063] The aforementioned studies have shown that among all miRNAs differentially expressed in the lungs before and after novel coronavirus infection, miR-126-3p is the miRNA that targets CXCL12 and exhibits the most significant changes before and after infection. Therefore, developing new therapeutic drugs targeting this miRNA will provide new ideas and methods for the treatment of novel coronavirus infection. Consequently, we performed microRNA sequencing analysis (miRNA-seq) on three stem cell exosomes: UC-MSCs-Exo, iMSCs-Exo, and iNPCs-Exo, to screen for stem cell exosomes with high background miR-126-3p expression levels. Sequencing results showed that among the three stem cell exosomes, UC-MSCs-Exo had the highest background miR-126-3p expression level (…). Figure 3 Therefore, we will use UC-MSCs-Exo for the next functional enhancement transformation.

[0064] Example 3 First, plasmids overexpressing hnRNPA2B1 and miR-126-3p were constructed. Figure 4 A). Then, lentiviral transfection technology was used to overexpress hnRNPA2B1 and miR-126-3p in UC-MSCs. Figure 4B), the lentivirus transfection step is: the packaging plasmid used is psPAX2 and pMD2.G, the mass ratio of packaging plasmid and overexpression plasmid is 1:1:2. The lentivirus infected target cells are collected after 48h, the MOI is 15, and polybrene is added at the same time. After 24h of infection, fresh medium is replaced for continuous culture, and when the cell confluence reaches 80-90%, puromycin is added for screening for 48h. After the screening is completed, the cell line overexpressing hnRNPA2B1 and miR-126-3p is obtained.

[0065] The Western blot detection results show that, compared with the non-transfected and empty plasmid transfected groups, hnRNPA2B1 is significantly overexpressed in the cells transfected with the overexpression plasmid, indicating that the cell line UC-MSCsOE-hnRNPA2B1 stably overexpressing hnRNPA2B1 is successfully constructed. Figure 4 C). Then, we transfected the pCDH-miR-126-3p plasmid into the UC-MSCsOE-hnRNPA2B1 cells, and isolated the exosomes secreted by the cells, and detected the relative expression level of miR-126-3p in the cells and the exosomes by qPCR. The total miRNA was extracted using the commercial kit MiPure Cell / Tissue miRNA Kit (Vazyme, RC201), and the reaction parameters were: qPCR reaction parameters: 95℃ preheating for 5 min; 3-step amplification (95℃ denaturation for 10 s, 60℃ annealing for 10 s, 72℃ extension for 10 s), 45 cycles; Melting parameters (95℃ denaturation for 10 s, 65℃ annealing for 60 s, 97℃ denaturation for 1 s); 37℃ cooling for 30 s, and the primers are shown in Table 1.

[0066] Table 1 qPCR primers

[0067] At the same time, we also directly transfected miR-126-3p mimics (purchased from Jimake Gene) into the UC-MSCs cells as a control. The results show that both directly transfecting miR-126-3p mimics into the cells and transfecting the miR-126-3p overexpression plasmid can significantly up-regulate the level of miR-126-3p in the cells ( Figure 4 D), and in the exosomes, the miR-126-3p can be more efficiently loaded into the exosomes by the strategy of miRNA sorting mechanism ( Figure 4 E). The above results show that we have successfully and efficiently constructed the UC-MSCs-Exo OE-miR-126-3p .

[0068] Detection Example 1 By giving lung fibroblast cell line MRC-5 cells 3x109 particles of UC-MSCs-Exo, UC-MSCs-Exo miR-126-3p mimics and UC-MSCs-Exo OE-miR-126-3p To compare their differences in inhibiting the expression level of MRC-5 cell CXCL12, total RNA in cells was extracted using Trizol, and the qPCR reaction parameters were: preheating at 95℃ for 5 min; 3-step amplification (denaturation at 95℃ for 10 s, annealing at 60℃ for 10 s, extension at 72℃ for 10 s), 45 cycles; melting parameters (denaturation at 95℃ for 10 s, annealing at 65℃ for 60 s, denaturation at 97℃ for 1 s); cooling at 37℃ for 30 s. The primers used are shown in Table 2.

[0069] Table 2 qPCR using primers

[0070] The results show that, compared with UC-MSCs-Exo without any modification and UC-MSCs-Exo transfected with miR-126-3p mimics of parent cells miR-126-3p mimics , UC-MSCs-Exo modified by miRNA sorting mechanism OE-miR-126-3p can better reduce the transcription level of CXCL12 in MRC-5 cells Figure 5 A). Similarly, the detection of the protein level of CXCL12 in the cell supernatant by ELISA kit (Human CXCL12, Elabscience, E-EL-H0052) also shows that UC-MSCs-Exo OE -miR-126-3p can better inhibit the secretion of CXCL12 by MRC-5 cells Figure 5 B).

[0071] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A method for constructing functional stem cell exosomes overexpressing miR-126-3p, characterized in that, Includes the following steps: S1: Screening for stem cell exosomes with a miR-126-3p basal expression level higher than 1000.00 TPM; S2: Construct a plasmid that overexpresses hnRNPA2B1, transfect it into the stem cells selected in S1, and construct a cell line that stably overexpresses hnRNPA2B1. S3: Construct a plasmid overexpressing miR-126-3p, transfect the cell line obtained in S2, and isolate the exosomes secreted by it.

2. The construction method according to claim 1, characterized in that, In S1, the stem cells include: mesenchymal stem cells.

3. The construction method according to claim 1, characterized in that, In steps S2 and S3, the transfection includes lentiviral transfection, adenovirus transfection, adeno-associated virus (AAV) transfection, retrovirus transfection, or liposome transfection.

4. The construction method according to claim 1, characterized in that, In step S3, the separation method includes differential centrifugation, density gradient centrifugation, ultrafiltration, polymer precipitation, immunoaffinity capture, size exclusion chromatography, or microfluidic chip technology.

5. Functional stem cell exosomes overexpressing miR-126-3p prepared by the construction method according to any one of claims 1-4.

6. A pharmaceutical composition, characterized in that, This includes functional stem cell exosomes overexpressing miR-126-3p prepared by the construction method according to any one of claims 1-4, or the exosomes according to claim 5.

7. The pharmaceutical composition according to claim 6, characterized in that, It also includes pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, antibacterial agents, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

9. The use of the functional stem cell exosomes overexpressing miR-126-3p prepared by the construction method according to any one of claims 1-4, or the exosomes according to claim 5, or the pharmaceutical composition according to any one of claims 6-8, in the preparation of a product that inhibits CXCL12 expression.

10. The use of functional stem cell exosomes overexpressing miR-126-3p prepared by the construction method according to any one of claims 1-4, or the exosomes according to claim 5, or the pharmaceutical composition according to any one of claims 6-8, in the preparation of products for treating and / or treating inflammation caused by novel coronavirus infection.

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