Mesenchymal stem cells overexpressing CD63 gene and preparation method and application thereof

Abstract

The present invention relates to the field of biotechnology, and in particular to mesenchymal stem cells overexpressing the CD63 gene, and a preparation method and application thereof. After constructing a lentiviral vector overexpressing the CD63 gene, the present invention transfects mesenchymal stem cells with the vector, and finally obtains a CD63-MSC cell line stably expressing CD63. After extraction and detection, the cell line significantly improves the exosome yield per unit cell and per unit culture medium volume, and the exosome yield is significantly improved. In addition, the present invention separates, purifies and traces exosomes through a GFP tag carried by a lentivirus, which solves the defects of traditional methods to a certain extent and promotes the scientific research and clinical application of mesenchymal stem cell exosomes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to mesenchymal stem cells overexpressing CD63 gene and a preparation method and application thereof. Background Art

[0002] Exosome (Exo): A tiny membrane vesicle that can be secreted by most cells, with a diameter of about 30-150nm, a lipid bilayer membrane structure, carrying RNA and protein, released by most cell types, and is part of the intercellular communication system. Almost all cells secrete extracellular vesicles, including mesenchymal stem cells. Extracellular vesicles derived from mesenchymal stem cells contain components similar to those of parent cells, such as cytokines, growth factors, lipids, mRNAs, and regulatory miRNAs. They are mainly involved in cell-to-cell communication and changes in the microenvironment of tissues, and play an invaluable role in immune regulation and tissue damage repair.

[0003] Mesenchymal stem cells, as a type of adult multipotent stem cells, have multiple tissue sources, can differentiate into multiple cell lineages and have a strong paracrine factor effect. Mesenchymal stem cells have the advantages of low immunogenicity and high tissue repair and regeneration. Studies have found that the therapeutic effect of mesenchymal stem cells is mainly through their paracrine effects. Mesenchymal stem cells can not only secrete some soluble bioactive factors, but also secrete extracellular microvesicles, namely exosomes, which can promote angiogenesis, inhibit cell apoptosis and fibrosis, and thus promote the repair and regeneration of damaged tissues.

[0004] More and more studies have shown that stem cell exosomes can reduce cell apoptosis, reduce inflammatory response, promote angiogenesis, inhibit fibrosis, and improve tissue repair potential and other important biological effects. These effects of exosomes make them have good application prospects in the treatment of tissue injuries such as myocardial infarction and skin trauma, and there have been many in-depth studies. Compared with stem cell transplantation therapy, stem cell exosomes can reduce the risk of cell transplantation tumorigenesis. Therefore, promoting angiogenesis and enhancing damaged cell repair through stem cell-derived exosomes is expected to become a new therapeutic strategy for tissue damage repair. The unique biological advantages of exosomes have also opened up a new idea for the development of drug carriers in the future. Scientists believe that compared with stem cells, the use of exosomes secreted by stem cells is safer and more operable. Because stem cell exosomes as therapeutic products have certain characteristics that stem cell preparations do not have, such as exosomes have a relatively certain composition compared to living cells, which is easy to control quality; and the stability of exosomes in vitro is more conducive to transportation and preservation. Therefore, these characteristics make exosomes have great application value.

[0005] However, the preclinical and clinical development of exosome technology as a delivery platform requires large quantities of exosomes. The isolation method of exosomes needs to be easily scalable to support large-scale production. Unlike immortalized tumor cell lines, the expansion capacity of mesenchymal stem cells (MSCs) is limited. Currently, the yield of exosomes obtained by traditional extraction methods is low and not scalable, which hinders the large-scale production of mesenchymal stem cells for exosomes, and thus affects the efficacy evaluation of exosomes in preclinical animals. Summary of the invention

[0006] In view of this, the present invention provides mesenchymal stem cells overexpressing the CD63 gene and a preparation method and application thereof. The present invention obtains a CD63-MSC cell line stably expressing CD63 by overexpressing the CD63 gene. After extraction and detection, the cell line significantly improves the exosome yield per unit cell and per unit culture medium volume, and the exosome yield is significantly improved.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides the use of CD63 gene in promoting mesenchymal stem cells to produce and / or release exosomes.

[0009] The present invention has found that overexpression of the CD63 gene can promote the production and / or release of exosomes by mesenchymal stem cells.

[0010] In some embodiments, the CD63 gene is the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence obtained by deleting, replacing, or adding one or more bases of the sequence shown in SEQ ID NO: 1.

[0011] The present invention also provides mesenchymal stem cells overexpressing the CD63 gene.

[0012] In some embodiments, the CD63 gene is the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence obtained by deleting, replacing, or adding one or more bases of the sequence shown in SEQ ID NO: 1.

[0013] In some embodiments, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic membrane mesenchymal stem cells or dental pulp mesenchymal stem cells.

[0014] In some specific embodiments, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0015] In some embodiments, the mesenchymal stem cells overexpressing the CD63 gene are prepared by the following method:

[0016] Recombinant lentivirus overexpressing CD63 gene was constructed and transfected into mesenchymal stem cells.

[0017] Specifically, the mesenchymal stem cells overexpressing the CD63 gene of the present invention are prepared by the following method:

[0018] Using pLv122 vector as the base vector, a recombinant plasmid pLV122-CD63 overexpressing the CD63 gene was constructed, and the recombinant lentivirus was packaged by transfecting 293T cell lines, named LV122-CD63. The recombinant lentivirus LV122-CD63 was transfected into mesenchymal stem cells to obtain a cell line stably expressing CD63, named MSC-LV122-CD63.

[0019] The present invention also provides a method for preparing the above-mentioned mesenchymal stem cells overexpressing the CD63 gene, which are prepared by the following method:

[0020] Recombinant lentivirus overexpressing CD63 gene was constructed and transfected into mesenchymal stem cells.

[0021] In some embodiments, the method for preparing the mesenchymal stem cells overexpressing the CD63 gene is prepared by the following method:

[0022] Using pLv122 vector as the base vector, a recombinant plasmid pLV122-CD63 overexpressing the CD63 gene was constructed, and the recombinant lentivirus was packaged by transfecting 293T cell lines and named LV122-CD63; the recombinant lentivirus LV122-CD63 was transfected into mesenchymal stem cells to obtain a cell line stably expressing CD63, named MSC-LV122-CD63.

[0023] In some embodiments, the CD63 gene is the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence obtained by deleting, replacing, or adding one or more bases of the sequence shown in SEQ ID NO: 1.

[0024] In some embodiments, the lentivirus is Lv122.

[0025] In the present invention, a recombinant lentivirus overexpressing the CD63 gene was constructed and named LV122-CD63 lentivirus, which was transfected into MSCs and then screened to obtain MSC cell lines highly expressing CD63.

[0026] The present invention also provides the use of the mesenchymal stem cells prepared by the above-mentioned preparation method of the mesenchymal stem cells in the preparation of drugs for promoting tissue damage repair, regulating immune response or improving ischemia-reperfusion injury.

[0027] The present invention uses pLv122 vector as the basic vector, constructs a recombinant plasmid pLV122-CD63 that overexpresses the CD63 gene, transfects the 293T cell line to package and obtain a recombinant lentivirus, named LV122-CD63, and transfects the recombinant lentivirus LV122-CD63 into mesenchymal stem cells to obtain a cell line that stably expresses CD63, named MSC-LV122-CD63. After testing, the cell line significantly improves the exosome yield per unit cell and unit culture medium volume, and the exosome yield is significantly improved.

[0028] The present invention also provides a method for promoting mesenchymal stem cells to produce and / or release exosomes, wherein the mesenchymal stem cells overexpressing the CD63 gene are cultured and the exosomes are extracted.

[0029] In some embodiments, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0030] In some embodiments, the culture medium used for the culture is a serum-free low-glucose DMEM culture medium, and the culture medium also contains gelatin microcarriers.

[0031] The present invention has no special requirements for the preparation of gelatin microcarriers, and methods well known in the art can be used. In a specific embodiment of the present invention, gelatin microcarriers are prepared with reference to patent application number 201811445712.9 "A method for large-scale expansion of mesenchymal stem cells".

[0032] In some specific embodiments, the method for producing and / or releasing exosomes is: selecting umbilical cord mesenchymal stem cells transfected with a recombinant lentiviral vector in the P3 growth logarithmic phase, culturing them in a serum-free, low-glucose DMEM medium containing gelatin microcarriers until the cell confluence reaches 70% to 80%, and extracting them by ultracentrifugation to obtain exosomes.

[0033] The present invention also provides the use of exosomes produced and / or released by the above method in promoting the proliferation of fibroblasts.

[0034] The present invention also provides the use of the exosomes produced and / or released by the above method in the preparation of drugs for promoting tissue damage repair, regulating immune response or improving ischemia-reperfusion injury.

[0035] The present invention uses Lv122 as the basic vector to construct a lentiviral vector that overexpresses the CD63 gene, which is transfected into mesenchymal stem cells, and finally obtains a CD63-MSC cell line that stably expresses CD63. Through exosome extraction and detection, the cell line significantly improves the exosome yield per unit cell and per unit culture medium volume, and the exosome yield is significantly improved. In addition, the present invention purifies, separates and traces exosomes through the GFP tag carried by the lentivirus, which solves the defects of traditional methods to a certain extent and promotes the scientific research and clinical application of mesenchymal stem cell exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0037] Figure 1 This is a schematic diagram of the structure of the basic carrier Lv122 in the present invention;

[0038] Figure 2 This is a schematic diagram of the structure of the recombinant lentiviral vector in the present invention;

[0039] Figure 3 This is a fluorescence image of the LV122 lentivirus 293 cell line in the present invention, wherein: Figure 3 A is the fluorescence image of MSC-LV122-CD63 cultured using GE's Cytodex. Figure 3 B is the fluorescence analysis of 293-LV122 and 293-LV122-CD63 mesenchymal stem cells;

[0040] Figure 4 Fluorescence analysis diagram of MSC-LV122-CD63 mesenchymal stem cells constructed by the present invention and control MSC-LV122 mesenchymal stem cells;

[0041] Figure 5 This is an electron micrograph of the Exo-LV122-CD63 exosomes in the present invention;

[0042] Figure 6 The protein content of exosomes extracted from different mesenchymal stem cells;

[0043] Figure 7 The number of exosome nanoparticles extracted for different mesenchymal stem cells;

[0044] Figure 8 The results of western blot detection of CD63 protein expression in different mesenchymal stem cells, where 1 is the control group Exo-MSC; 2 is the experimental group Exo-MSC-Lv122; 3 is the experimental group Exo-MSC-Lv122-CD63;

[0045] Fig. 9 This is a fluorescence image of skin fibroblasts taking up two types of exosomes, where the green fluorescence is the exosome signal absorbed by skin fibroblasts;

[0046] Fig.10 Effects of exosomes extracted from different mesenchymal stem cells in promoting the proliferation of skin fibroblasts. DETAILED DESCRIPTION

[0047] The present invention discloses stem cells overexpressing the CD63 gene and methods for preparing and using the stem cells. A person skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0048] The stem cells overexpressing CD63 gene provided by the present invention, and the preparation method thereof, raw materials and reagents used in the application thereof can all be purchased from the market.

[0049] The present invention will be further described below in conjunction with embodiments:

[0050] Example 1 Construction of CD63 Lentivirus

[0051] According to the CD63 gene sequence information (Gene ID: 967), the CD63 gene shown in SEQ ID NO: 1 was cloned and the pLv122-based vector (such as Figure 1 As shown), a recombinant vector pLV122-CD63 containing the CD63 gene was constructed and packaged to generate LV122-CD63 lentivirus by transfecting 293T cell lines. (See Figure 2 ).

[0052] Example 2 CD63 stably expressed mesenchymal stem cell line

[0053] The lentivirus LV122-CD63 constructed in Example 1 was used to transfect umbilical cord mesenchymal stem cells (virus infection multiplicity MOI value: 20-100) to obtain mesenchymal stem cells overexpressing the CD63 gene, namely MSC-LV122-CD63, which were cultured using gelatin microcarriers (the mainstream GE Cytodex product in the market was used as a control) and the growth of the mesenchymal stem cells was observed and recorded. The results are shown in FIG. Figures 3-4 Among them, the gelatin microcarrier is prepared according to the patent application number 201811445712.9 "A method for large-scale expansion of mesenchymal stem cells".

[0054] The results show that the cell viability of the gelatin microcarriers of the present invention can reach more than 90%, and the cell proliferation can reach more than 15 times, which is significantly higher than that of the same product (GE Cytodex product). The cell surface markers were detected by flow cytometry, and the results are shown in Table 1.

[0055] Table 1 Flow cytometry results of mesenchymal stem cell surface markers

[0056]

[0057] The results showed that the surface markers of mesenchymal stem cells overexpressing CD63 gene cultured by gelatin microcarriers in the present invention were expressed normally.

[0058] In summary, the present invention utilizes gelatin microcarriers to three-dimensionally culture MSC-LV122-CD63 mesenchymal stem cells, which can achieve cell adherent growth and more than 15 times cell proliferation, which is significantly better than the culture effect of the control sample (GE Cytodex product), and the obtained cells have good morphology, high vitality, normal expression of surface markers, and the cells still have good differentiation potential, laying a foundation for large-scale culture of mesenchymal stem cells.

[0059] Example 3 Extraction and identification of exosomes derived from mesenchymal stem cells

[0060] (1) Extraction of exosomes from mesenchymal stem cells

[0061] Three types of stem cells, namely MSCs in the logarithmic growth phase of P3, MSC-LV122 (umbilical cord mesenchymal stem cells transfected with the Lv122 empty vector) and MSC-LV122-CD63 (umbilical cord mesenchymal stem cells overexpressing the CD63 gene of the present invention), were selected for culture and exosome extraction.

[0062] The specific method is as follows: When the cell confluence reaches 70% to 80%, wash the cells with PBS, select serum-free low-glucose DMEM medium for 48 hours, and then use ultra-high-speed centrifugation to separate and extract exosomes. Take 200mL of cell culture medium and centrifuge at 300g for 10 minutes to remove cell debris, then centrifuge at 2000g for 10 minutes to further remove cell debris and impurities, and then centrifuge at 10,000g for 30 minutes. The supernatant is then collected through a 0.22μm disposable filter, and finally centrifuged at 100,000g for 70 minutes to discard the supernatant, resuspend the precipitate with 1mL PBS, and then centrifuge at 100,000g for 70 minutes to discard the supernatant, resuspend in PBS to obtain exosomes, and store at -80℃ after aliquoting.

[0063] (2) Electron microscopic examination of exosomes derived from mesenchymal stem cells

[0064] The extracted exosomes were resuspended in 20-30uL PBS, 10μL was added to the copper mesh of the transmission electron microscope for precipitation for 1min, and the floating liquid was removed with filter paper. 10μL of uranyl acetate was added to the copper mesh of the transmission electron microscope for precipitation for 1min, and the floating liquid was removed with filter paper. After drying at room temperature for several minutes, the results were observed on the microscope. Figure 5 .

[0065] The results showed that the size of exosomes in the electron microscope image was about 30 to 150 nm, and the exosome structure was round, with low refractive index in the middle and high refractive index around. This showed that there was no difference between the exosomes overexpressing CD63 and the conventional group, which was consistent with the classic structure of exosomes.

[0066] (3) BCA detection of exosome proteins

[0067] For the exosome proteins prepared by lysis, the protein quantification was performed using the Bio-Tech BCA Protein Quantification Kit according to the operating instructions, and a standard curve was drawn to calculate the protein mass of exosomes of mesenchymal stem cells from different sources (MSCs, MSC-LV122, MSC-LV122-CD63). The results are shown in Figure 6 .

[0068] The results showed that the exosome protein content obtained by the MSC-LV122-CD63 umbilical cord mesenchymal stem cells overexpressing the CD63 gene of the present invention was significantly higher than that of the other two groups.

[0069] (4) NTA detection of exosome concentration and particle size:

[0070] Use nanoparticle tracking analyzer to analyze the size and concentration of exosomes. Use 1 mL of filtered PBS to resuspend the exosomes in the separated exosome precipitate, seal and place on ice; select a disposable clean sample pool, wipe the light with dust-free paper to ensure that there are no particles attached to the outer tube wall in the light path, slowly inject the exosome solution to avoid bubbles, tilt the sample pool appropriately, and seal the sample pool with a lid; place the sample pool in the instrument, operate the instrument according to standard operating procedures, save the data, and analyze the size range and concentration of the exosomes. The number of mesenchymal stem cell exosome nanoparticles from different sources (MSCs, MSC-LV122, MSC-LV122-CD63) was calculated, and the results are shown in Figure 7 .

[0071] The results showed that the number of exosome particles extracted from the MSC-LV122-CD63 umbilical cord mesenchymal stem cells overexpressing the CD63 gene of the present invention was significantly higher than that of the other two groups.

[0072] (5) Western blot detection of exosomes

[0073] Protein lysis buffer was added to the extracted exosome precipitate, and the mixture was placed on ice for 5-15 minutes, with 3-4 oscillations for 30 seconds each time. The mixture was then centrifuged at 12,000 rpm for 5 minutes at 4°C, and the supernatant was transferred to a new centrifuge tube for Western blot detection of CD9, CD63, and GFP protein expression. The results are shown in Figure 8 .

[0074] The results showed that the exosomes derived from the three mesenchymal stem cells obtained by ultracentrifugation were able to detect the expression of CD9 and CD63 proteins. Among them, the CD63 protein content in the MSC-LV122-CD63 mesenchymal stem cells of the present invention was significantly higher than that in the other two groups, indicating that the CD63 gene was highly expressed in the mesenchymal stem cells.

[0075] (6) Cellular exosome uptake tracking experiment

[0076] Two types of mesenchymal stem cells, MSC-LV122 and MSC-LV122-CD63, were used for uptake by skin fibroblasts, and the fluorescence expression of exosomes was observed under a fluorescence microscope. The results are shown in Fig. 9 The results showed that obvious exosome green fluorescence signals could be observed in both types of mesenchymal stem cells.

[0077] (7) Exosomes promote cell proliferation experiment

[0078] Exosomes were extracted from the above three mesenchymal stem cells, MSCs, MSC-LV122 and MSC-LV122-CD63, and used for skin fibroblast culture. The proliferation of skin fibroblasts was detected by CCK8. The results are shown in Fig.10 .

[0079] The results showed that all three types of exosomes had a significant effect in promoting cell proliferation, among which the proliferation effect of exosomes extracted from mesenchymal stem cells overexpressing the CD63 gene of the present invention was significantly higher than that of the other two groups (p<0.01).

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing exosomes, characterized in that: Umbilical cord mesenchymal stem cells that overexpressed the CD63 gene were cultured and exosomes were extracted; The sequence of the CD63 gene is shown in SEQ ID NO:

1.

2. Use of the exosomes produced and / or released by the preparation method according to claim 1 in the preparation of a product that promotes fibroblast proliferation.

Images