Method for improving yield of cell exosome and application

Abstract

The invention relates to a method and application for increasing the yield of cell exosomes, and belongs to the field of exosomes, and the method for increasing the yield of the cell exosomes comprises the following steps: transfecting recombinant plasmids containing hNAMPT genes into cells, culturing, collecting a culture medium, and separating to obtain the exosomes. The recombinant plasmid containing the NAMPT gene is introduced into the 293T cell by using a PEI transfection method, so that the yield of the exosome can be remarkably improved, and a foundation is laid for further research and application in various fields.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of exosomes, and relates to a method for improving the yield of cell exosomes and application. BACKGROUND

[0002] Exosomes are cystic vesicles secreted by living cells, are uniform in size, and have a diameter of 30-200 nm. Exosomes can carry biological active molecules such as proteins, lipids, RNA and DNA, and have high bioavailability, biological stability, target specificity, low toxicity and low immunogenicity, so that the exosomes have great potential in disease diagnosis, treatment and drug delivery.

[0003] 293T cells have the advantages of easy cultivation and high transfection efficiency, and can be used as parent cells for exosome secretion, but the number of exosomes secreted by cells under normal conditions is relatively small, and each cell can only secrete about 500 exosomes on average, so the yield is too low to meet the demand of large-scale scientific research in the future, and therefore how to improve the exosome secretion of 293T cells is a difficult problem to be solved in the application of exosomes. SUMMARY

[0004] In view of the defects in the prior art, the application provides a method for improving the yield of cell exosomes and application. The recombinant plasmid containing NAMPT genes is introduced into 293T cells by PEI transfection method, which can significantly improve the yield of exosomes, and lays a foundation for further research and application in various fields.

[0005] Solution To achieve the above object, the application provides the following technical scheme. In a first aspect, the application provides a method for improving the yield of cell exosomes, which comprises: transfecting a recombinant plasmid containing hNAMPT genes into cells, culturing, collecting culture medium and separating to obtain exosomes.

[0006] Further, hNAMPT The gene comprises a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 1; optionally, hNAMPT The gene comprises a nucleotide sequence as shown in SEQ ID NO: 2; Optionally, the recombinant plasmid includes the STEAP3 gene, SDC4 gene, and hNAMPT gene sequentially linked together. Optionally, the amino acid sequence of the STEAP3 gene is shown in SEQ ID NO:3, and the nucleotide sequence encoding the STEAP3 gene is shown in SEQ ID NO:4. Optionally, the amino acid sequence of the SDC4 gene is shown in SEQ ID NO:5, and the nucleotide sequence encoding the SDC4 gene is shown in SEQ ID NO:6. Optionally, there is a linker sequence between the STEAP3 gene and the SDC4 gene. Optionally, the linker sequence is a nucleotide sequence encoding the IRES fragment, and the nucleotide sequence of the IRES fragment is shown in SEQ ID NO:11. (Wherein, the IRES fragment is used only for linking.)

[0007] Optionally, the recombinant plasmid includes a nucleotide sequence as shown in SEQ ID NO:14.

[0008] Optionally, the backbone plasmid of the recombinant plasmid is the pcDNA3.1(+) plasmid. Optionally, the recombinant plasmid is pcDNA3.1(+)-STEAP3-IRES-SDC4-IRES-hNAMPT plasmid.

[0009] Optionally, the transfection method may be PEI transfection.

[0010] Optionally, the method further includes co-transfection of a recombinant plasmid, wherein the co-transfection recombinant plasmid includes nucleotide sequences encoding CD63 and EGFP genes; optionally, the amino acid sequence of the CD63 gene is shown in SEQ ID NO:7, and optionally, the nucleotide sequence encoding CD63 is shown in SEQ ID NO:8; optionally, the amino acid sequence of the EGFP gene is shown in SEQ ID NO:9, and optionally, the nucleotide sequence encoding EGFP is shown in SEQ ID NO:10; optionally, the co-transfection recombinant plasmid includes a nucleotide sequence encoding CD63-EGFP, and optionally, there is a linker sequence between the CD63 gene and the EGFP gene; optionally, the co-transfection recombinant plasmid includes a nucleotide sequence as shown in SEQ ID NO:12.

[0011] Furthermore, the cells are 293T cells.

[0012] Furthermore, the culture is performed using a complete culture medium, which may optionally include: 89% DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin-streptomycin-amphoteric B solution.

[0013] Furthermore, the cultivation conditions were: 37℃, 5% CO2; And / or, the incubation time is 24~72h, optionally 48~72h.

[0014] Secondly, providing a hNAMPT Application of genes in the preparation of products that increase the production of cell exosomes.

[0015] Furthermore, the product is a reagent containing [a specific ingredient] for transfection into cells. hNAMPT Recombinant plasmids of genes. hNAMPT The gene comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1; optionally, hNAMPT The gene includes a nucleotide sequence as shown in SEQ ID NO:2.

[0016] Further, the recombinant plasmid includes the STEAP3 gene, SDC4 gene, and hNAMPT gene sequentially linked together. Optionally, the amino acid sequence of the STEAP3 gene is shown in SEQ ID NO:3, and the nucleotide sequence encoding the STEAP3 gene is shown in SEQ ID NO:4; optionally, the amino acid sequence of the SDC4 gene is shown in SEQ ID NO:5, and the nucleotide sequence encoding the SDC4 gene is shown in SEQ ID NO:6; optionally, there is a linker sequence between the STEAP3 gene and the SDC4 gene, which is optionally a nucleotide sequence encoding the IRES fragment, and the nucleotide sequence of the IRES fragment is optionally shown in SEQ ID NO:11. Optionally, the recombinant plasmid includes a nucleotide sequence as shown in SEQ ID NO:14; Furthermore, the backbone plasmid of the recombinant plasmid is pcDNA3.1(+) plasmid.

[0017] Optionally, the recombinant plasmid is pcDNA3.1(+)-STEAP3-IRES-SDC4-IRES-hNAMPT plasmid.

[0018] Optionally, the device further includes co-transfecting a recombinant plasmid, wherein the co-transfected recombinant plasmid includes nucleotide sequences encoding CD63 and EGFP genes; optionally, the amino acid sequence of the CD63 gene is shown in SEQ ID NO:7, and optionally, the nucleotide sequence encoding CD63 is shown in SEQ ID NO:8; optionally, the amino acid sequence of the EGFP gene is shown in SEQ ID NO:9, and optionally, the nucleotide sequence encoding EGFP is shown in SEQ ID NO:10; optionally, the co-transfected recombinant plasmid includes a nucleotide sequence encoding CD63-EGFP, and optionally, there is a linker sequence between the CD63 gene and the EGFP gene; optionally, the co-transfected recombinant plasmid includes a nucleotide sequence as shown in SEQ ID NO:12. Further, the cell is a 293T cell.

[0019] Beneficial effects: The present application uses PEI transfection method to introduce the recombinant plasmid containing NAMPT gene into 293T cells, which can significantly improve the yield of exosomes, and lay a foundation for further research and application in various fields. BRIEF DESCRIPTION OF DRAWINGS

[0020] One or more embodiments are illustrated by way of example in the figures that form a part of this patent specification. These example embodiments do not necessarily represent the full scope of the embodiments, which are defined solely by the claims and equivalents thereof. The skilled person's specific word "example" means "serving as an example, an embodiment, or an illustration". Any embodiment described herein as "example" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0021] Figure 1 : Plasmid map of pcDNA3.1(+)-STEAP3-IRES-SDC4-IRES-hNAMPT constructed by the present application.

[0022] Figure 2 : Identification results of the recombinant plasmid of Example 1 of the present application, wherein 1 is pcDNA3.1-STEAP3-IRES-SDC4; 2 is pcDNA3.1-CD63-EGFP; 3 is pcDNA3.1(+)-STEAP3-IRES-SDC4-IRES-hNAMPT.

[0023] Figure 3 : State of cell culture of Example 2 of the present application, wherein A is 5x; B is 20x.

[0024] Figure 4 : Observation results under a fluorescence microscope of Example 2 of the present application, wherein left: white field; right: green field; B group: blank control; G1 group: only pcDNA3.1-CD63-EGFP plasmid transfection; G2 group: pcDNA3.1-CD63-EGFP plasmid and pcDNA3.1-STEAP3-IRES-SDC4 plasmid co-transfection result; G3 group: pcDNA3.1(+)-STEAP3-IRES-SDC4-IRES-hNAMPT plasmid and pcDNA3.1-CD63-EGFP plasmid co-transfection result.

[0025] Figure 5 : Exosome morphology under a transmission electron microscope of Example 3 of the present application, wherein A is B group exosome morphology; B is G1 group exosome morphology; C is G2 group exosome morphology; D group is G3 group exosome morphology.

[0026] Figure 6: Results of identifying protein markers of exosomes by Western blotting for Example 3 of the present application.

[0027] Figure 7 : Particle size, concentration and proportion of exosomes with CD63-EGFP green fluorescence of the blank control group (i.e., group B) of Example 3 of the present application, wherein Fig. A represents the particle size distribution of the exosome size, indicating the distribution position of the main size of the exosome, and Fig. B represents the proportion of fluorescent exosomes (i.e., P1, the positive proportion of EGFP), and the fluorescent exosome is a key indicator for indicating the secretion amount of the exosome.

[0028] Figure 8 : Particle size, concentration and proportion of exosomes with CD63-EGFP green fluorescence (P1) of the G1 group (only transfected with pcDNA3.1-CD63-EGFP plasmid group) of Example 3 of the present application, wherein Fig. A represents the particle size distribution of the exosome size, indicating the distribution position of the main size of the exosome, and Fig. B represents the proportion of fluorescent exosomes (i.e., P1, the positive proportion of EGFP), and the fluorescent exosome is a key indicator for indicating the secretion amount of the exosome.

[0029] Figure 9 : Particle size, concentration and proportion of exosomes with CD63-EGFP green fluorescence (P1) of the G2 group (co-transfected with pcDNA3.1-CD63-EGFP plasmid and pcDNA3.1(+)-STEAP3-IRES-SDC4 plasmid group) of Example 3 of the present application, wherein Fig. A represents the particle size distribution of the exosome size, indicating the distribution position of the main size of the exosome, and Fig. B represents the proportion of fluorescent exosomes (i.e., P1, the positive proportion of EGFP), and the fluorescent exosome is a key indicator for indicating the secretion amount of the exosome.

[0030] Figure 10 : Particle size, concentration and proportion of exosomes with CD63-EGFP green fluorescence of the G3 group (co-transfected with pcDNA3.1-CD63-EGFP plasmid and pcDNA3.1-STEAP3-IRES-SDC4-IRES-hNAMPT plasmid group) of Example 3 of the present application, wherein Fig. A represents the particle size distribution of the exosome size, indicating the distribution position of the main size of the exosome, and Fig. B represents the proportion of fluorescent exosomes (i.e., P1, the positive proportion of EGFP), and the fluorescent exosome is a key indicator for indicating the secretion amount of the exosome. DETAILED DESCRIPTION

[0031] In order to better illustrate the present application, various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0032] Many modifications and variations of the described implementations can be made by those skilled in the art without departing from the scope or spirit of the application. The description and implementation of the application are merely illustrative.

[0033] As used herein, "comprise", "comprising", "including", "include", "contain", "containing", "have", "having", and the like are open-ended terms that are intended to mean including, but not limited to. Unless specifically stated otherwise, all agents used in the following examples are commercially available and used at the highest purity available.

[0034] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0035] The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified, for example: 293T cells were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0036] Main reagents: Endo-free plasmid maxi kit (enhanced) (purchased from TIANGEN), DMEM high glucose medium, fetal bovine serum (purchased from Seepno), three antibiotics (penicillin-streptomycin-amphotericin B solution, purchased from Seepno), 0.25% Trypsin-EDTA (1X) (purchased from giboc), DMSO, linear polyethyleneimine transfection reagent (purchased from Pishanyang), RIPA lysis buffer, protease inhibitor, BCA protein concentration determination kit (purchased from biosharp), SDS-PAGE gel rapid preparation kit (purchased from Biyun Tian), SDS-PAGE protein loading buffer, three-color pre-stained protein marker, TBS solution, electrophoresis buffer, CD9, CD81, TSG101, calnexin primary antibody (purchased from Proteintech), HRP-conjugated Goat Anti-Mouse IgG (H+L), HRP-conjugated Goat Anti-Rabbit IgG (H+L) (purchased from Proteintech), ECL chemiluminescence kit, 1Kb DNA Ladder Maker (purchased from Solire), etc.

[0037] In the following examples, the culture of 293T cells uses complete medium, and the complete medium is specifically configured as 89% DMEM high glucose medium + 10% fetal bovine serum + 1% double antibody. The culture conditions are 37 ℃, 5% CO2 incubator culture.

[0038] Example 1, construction and extraction of plasmid containing hNAMPT gene Construction of plasmid: The STEAP3 fragment, SDC4 fragment, CD63, IRES sequence, and EGFP sequence were respectively retrieved and sent to Yunzhou Biotechnology Co., Ltd. for synthesis, and the related vectors were connected to the pcDNA3.1(+) vector skeleton to obtain recombinant plasmids CD63-EGFP, STEAP3-IRES-SDC4, and STEAP3-IRES-SDC4-IRES-hNAMPT, respectively.

[0039] The STEAP3 fragment, SDC4 fragment, CD63, IRES sequence, and EGFP sequence are specifically as follows:

[0040] Amplification of plasmid: The E. coli containing each recombinant plasmid was inoculated into an LB liquid medium containing ampicillin, and cultured overnight at 37°C and 180 rpm on a shaking table.

[0041] Identification of plasmid: Each recombinant vector was extracted and subjected to 1% gel electrophoresis, and a clear and bright band could be seen, as shown in Figure 2 , indicating that the extracted plasmid was a supercoiled plasmid suitable for transfection experiments. Agarose gel electrophoresis identification.

[0042] Example 2, transfection The experimental grouping is shown in Table 1.

[0043] Table 1, experimental grouping

[0044] Transfection process (1) The cell inoculation amount was 3.6 x 10 6 cells, and the cell density should be 70%-80% during transfection. 2h before transfection, the complete culture medium was replaced with fresh complete culture medium; and the cell state was good Figure 3 , and transfection could be performed; (2) According to the experimental grouping, the reaction system was subjected to PEI transfection (the amount of PEI added was shown in Table 1), and the plasmid was diluted with serum-free diluent (Opti-MEM) (the amount of Opti-MEM added was shown in Table 1), and then PEI was added to the DNA diluent, and the transfection complex was prepared at room temperature for 10-15 min; (3) The transfection complex was added to the cell complete culture medium, and gently mixed, and then cultured at 37 °C in a 5% CO2 incubator.

[0045] (4) The liquid was changed 6-8 h after transfection.

[0046] (5) The transfection was observed under a fluorescence microscope 24 h after transfection, 5 ml of culture medium was added, 75 µL of G418 was added, and the culture was transferred to a low oxygen incubator for culture; (6) The transfection was also observed under a fluorescence microscope 48 h and 72 h after transfection. If the transfection is successful, green fluorescence can be observed under a fluorescence microscope; (7) The culture medium was collected 72 h after transfection.

[0047] The transfection results are shown in Table 2. Figure 4 The results show that green fluorescence can be observed under a fluorescence microscope in G1 group, G2 group and G3 group, and no green fluorescence is observed in the B group control. It shows that the transfection in G1 group, G2 group and G3 group is successful.

[0048] Example 3, isolation and identification of exosomes The cell culture medium of step (7) in Example 2 was collected, centrifuged (10000g, 4°C, 10-30min), and the supernatant was taken and the precipitate was discarded. The supernatant was filtered through a 0.22 µm filter membrane, and the supernatant was collected and filtered through a 100KD ultrafiltration tube, and centrifuged (3000-5000g, 4°C, 10-30min). The liquid in the ultrafiltration tube was collected, containing 1% trehalose, and frozen at -20°C.

[0049] Transmission electron microscopy observation of exosome morphology: (1) Take 1% or 2% uranyl acetate / phosphotungstic acid solution, centrifuge (13300rpm, 10min); (2) Plasma hydrophilic treatment was performed on the copper mesh; (3) Drop one drop of ddH2O and three drops of staining solution on the sealing film in turn, each 100 µL; (4) Drop about 10 µL of sample on the treated side of the copper mesh, stand for 1 min, and then use a wet filter paper to absorb the excess sample; (5) Invert the copper mesh with the sample on one side into the ddH2O drop, gently shake and rinse for 10 s, and absorb the excess solution; (6) Put the copper mesh with sample on one side on each drop of dye solution, shake slightly for 10 s. After 1 min, remove the excess solution. After natural drying, lens observation can be performed.

[0050] The lens observation results are shown in Figure 5 The results show that the spherical vesicle structure with a diameter of 30-200 nm can be observed in each group, which is consistent with the morphology of exosomes.

[0051] Identification of exosome protein markers (1) Mix the collected exosome sample with an equal volume of RIPA lysis buffer containing PMSF protease inhibitor, and fully lyse on ice for 30-60 min. Centrifuge at 12000 g, 4°C, for 20 min. Determine the protein concentration using the BCA protein concentration determination kit (biosharp company).

[0052] (2) According to the protein concentration, take an appropriate amount of protein. After boiling for 5 min at 95°C, add 5xLoading Buffer (sample:buffer=1:4) to denature the protein, and then place on ice or store at -80°C.

[0053] (3) After the protein denaturation sample is separated by SDS-PAGE gel, the protein is transferred to the PVDF membrane (Bi Yun Tian) by "sandwich method". After the transfer is completed, the PVDF membrane is placed in 5% skim milk for blocking for 1 h; (4) Discard the blocking solution, wash the membrane with TBST, and place the box on the shaker for 5 min / time, and wash 4 times. Put the PVDF membrane into the corresponding primary antibody solution (CD9: sc-20048; CD81: sc-70804; TSG101: sc-136111; calnexin: sc-46669, all purchased from santa cruz company), and incubate at 4°C overnight; (5) Take out the PVDF membrane, recover the primary antibody, wash the membrane with TBST, and place the box on the shaker for 5 min / time, and wash 4 times. Put the PVDF membrane into the corresponding secondary antibody solution (HRP labeled, santa cruz company), and incubate for 1 h. After incubation, wash the membrane with TBST, and place the box on the shaker for 5 min / time, and wash 4 times; (6) Use ECL chemiluminescence reagent to develop color, and expose and take pictures on the gel imaging system.

[0054] The results are shown in Figure 6As shown, the results show that the expression of exosome positive marker proteins CD9, CD81 and TSG101 can be detected in the extracted exosomes by Western Blot, and the negative marker protein calnexin is not detected. It is shown that the exosomes are extracted in this embodiment.

[0055] Nano flow detection of exosome particle size and concentration The particle size and concentration of the exosomes are detected by nano-flow, and the results are as follows Figure 7-10 The particle size, concentration and positive ratio of exosomes in each group are as follows: Table 2, particle size, concentration and EGFP positive ratio of exosomes

[0056] Note: EGFP positive ratio refers to the proportion of exosomes expressing EGFP, which can be directly measured by a nano-flow meter, and can more directly represent the influence of exogenous genes on the secretion amount of exosomes.

[0057] Figure 7-10 The results of Table 2 show that the particle size of exosomes in each group is between 30-200nm, which meets the particle size distribution range of exosomes, and the particle number and EGFP positive ratio of exosomes in the G3 group (transferring the recombinant plasmid containing the hNAMPT gene) are the highest, and the particle concentration of exosomes is increased by 38.8% compared with the B group, and is increased by 23.8% compared with the G2 group, and the EGFP positive ratio is increased by 1.8~4 times compared with the G1 or G2 group, which shows that the hNAMPT gene can promote the secretion of 293T cell exosomes.

[0058] The present application successfully transfers the hNAMPT gene into 293T cells by PEI transfection method, extracts exosomes by ultrafiltration concentration method, and identifies the extracted exosomes by transmission electron microscopy, Western Blot and nano-flow, and analyzes the results of nano-flow. hNAThe MPT gene affects the production of 293T cell exosomes. The final results show that the double-layer vesicle structure of 30-200 nm can be observed under a transmission electron microscope, and the protein imprint detection shows that the CD9, CD81 and TSG101 exosome marker proteins are positive, proving that the extracted product is an exosome; CD63 is a membrane protein on the exosome membrane, and the expression amount of CD63 can reflect the amount of exosomes. In order to more intuitively detect the secretion amount of exosomes, the present application fuses and expresses CD63 and the EGFP gene, and then participates in the co-transfection of 293T cells with each group of plasmids; the STEAP3 and SDC-4 genes are key enzymes in the exosome production and secretion process, and research results suggest that increasing the expression amount of the STEAP3 and SDC-4 genes can increase the exosome secretion amount of the parent cell, and the present application proves that the STEAP3 and SDC-4 gene transfection (G2 group) indeed increases the secretion amount of the exosome (5.2% vs 2.4%, 5.2% vs 0.9%); on the basis of the results, the present application fuses and expresses the hNAMPT gene with the STEAP3 and SDC-4 genes, and the results prove that the hNAMPT gene increases the secretion amount of the exosome on the basis of the STEAP3 and SDC-4 genes (9.3% vs 5.2%), and compared with the blank control group, the combined application of the STEAP3-SDC-4-hNAMPT can increase the expression amount of the exosome by about 4 times (9.3% vs 2.4%), proving that whether the hNAMPT gene acts alone or the STEAP3-SDC-4-hNAMPT acts in combination can increase the secretion amount of the 293T cell exosome, and therefore the present application provides a new idea for the engineered preparation, optimized design and functional modification of the exosome.

[0059] The above description shows and describes the preferred embodiments of the present application. As described above, it should be understood that the present application is not limited to the forms disclosed herein, and should not be considered as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified by the above-mentioned teaching or related technical or knowledge within the scope of the inventive concept described herein. Any modification and change made by those skilled in the art without departing from the spirit and scope of the present application shall be within the protection scope of the claims of the present application.

Claims

1. A method for increasing the production of cell exosomes, characterized in that, It includes: Will contain hNAMPT The recombinant plasmid of the gene was transfected into the cell, cultured, and the culture medium was collected to isolate the exosomes.

2. The method according to claim 1, characterized in that, hNAMPT The gene comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1; optionally, hNAMPT The gene includes a nucleotide sequence as shown in SEQ ID NO:2; Optionally, the recombinant plasmid includes the STEAP3 gene, SDC4 gene, and hNAMPT gene sequentially linked together. Optionally, the amino acid sequence of the STEAP3 gene is shown in SEQ ID NO:3, and the nucleotide sequence encoding the STEAP3 gene is shown in SEQ ID NO:

4. Optionally, the amino acid sequence of the SDC4 gene is shown in SEQ ID NO:5, and the nucleotide sequence encoding the SDC4 gene is shown in SEQ ID NO:

6. Optionally, there is a linker sequence between adjacent sequences of the STEAP3 gene, SDC4 gene, and hNAMPT gene. Optionally, the linker sequence is a nucleotide sequence encoding the IRES fragment, and the nucleotide sequence of the IRES fragment is shown in SEQ ID NO:

11. Optionally, the recombinant plasmid includes a nucleotide sequence as shown in SEQ ID NO:14; Optionally, the backbone plasmid of the recombinant plasmid is pcDNA3.1(+) plasmid; Optionally, the recombinant plasmid is pcDNA3.1(+)-STEAP3-IRES-SDC4-IRES-hNAMPT plasmid; Optionally, the transfection method may be PEI transfection.

3. The method according to claim 1 or 2, characterized in that, It also includes co-transfecting recombinant plasmids, optionally co-transfecting recombinant plasmids including nucleotide sequences encoding CD63 and EGFP genes; optionally, the amino acid sequence of the CD63 gene is shown in SEQ ID NO:7, optionally the nucleotide sequence encoding CD63 is shown in SEQ ID NO:8; optionally, the amino acid sequence of the EGFP gene is shown in SEQ ID NO:9, optionally the nucleotide sequence encoding EGFP is shown in SEQ ID NO:10; optionally, the co-transfecting recombinant plasmid includes a nucleotide sequence encoding CD63-EGFP, optionally, there is a linker sequence between the CD63 gene and the EGFP gene; optionally, the co-transfecting recombinant plasmid includes a nucleotide sequence as shown in SEQ ID NO:

12.

4. The method according to any one of claims 1 to 3, characterized in that, The cells in question are 293T cells.

5. The method according to any one of claims 1 to 4, characterized in that, The culture was performed using a complete culture medium, which may include: 89% DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin-streptomycin-amphoteric B solution.

6. The method according to any one of claims 1 to 5, characterized in that, The cultivation conditions were: 37℃, 5% CO2; And / or, the incubation time is 24~72h, optionally 48~72h.

7. hNAMPT Application of genes in the preparation of products that increase the production of cell exosomes.

8. The application according to claim 7, characterized in that, The product is a conjugate for transfection into cells. hNAMPT Recombinant plasmids of genes hNAMPT The gene comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1; optionally, hNAMPT The gene includes a nucleotide sequence as shown in SEQ ID NO:

2.

9. The application according to claim 7 or 8, characterized in that, The recombinant plasmid comprises the STEAP3 gene, the SDC4 gene, and the hNAMPT gene, which are sequentially linked. Optionally, the amino acid sequence of the STEAP3 gene is shown in SEQ ID NO:3, and the nucleotide sequence encoding the STEAP3 gene is shown in SEQ ID NO:

4. Optionally, the amino acid sequence of the SDC4 gene is shown in SEQ ID NO:5, and the nucleotide sequence encoding the SDC4 gene is shown in SEQ ID NO:

6. Optionally, there is a linker sequence between the STEAP3 gene and the SDC4 gene. Optionally, the linker sequence is a nucleotide sequence encoding the IRES fragment, and the nucleotide sequence of the IRES fragment is shown in SEQ ID NO:

11. Optionally, the recombinant plasmid includes a nucleotide sequence as shown in SEQ ID NO:14; The backbone plasmid of the recombinant plasmid is pcDNA3.1(+) plasmid. Optionally, the recombinant plasmid is pcDNA3.1(+)-STEAP3-IRES-SDC4-IRES-hNAMPT plasmid.

10. The application according to any one of claims 7 to 9, characterized in that, The cells in question are 293T cells.

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