ISG15 overexpression cell line, SRM-enriched exosome as well as preparation method and application of ISG15 overexpression cell line and SRM-enriched exosome

Abstract

The invention discloses an ISG15 overexpression cell line, an SRM-enriched exosome as well as a preparation method and application of the ISG15 overexpression cell line and the SRM-enriched exosome, and ISGylation modification of spermidine synthase SRM is induced by constructing an ISG15 overexpression stably transfected cell line, so that packaging and sorting of the spermidine synthase SRM into the exosome are promoted. The technical scheme disclosed by the invention shows that the spermidine synthase transmitted by the exosome has biological activity and can catalyze to generate spermidine after being absorbed by target tissues / target cells, so that the spermidine synthase has wide application prospects in multiple aspects of disease treatment, medical care, beauty treatment, young keeping and the like. In addition, other protein or polypeptide drugs can be modified through a genetic engineering technology to be subjected to ISGylation modification so as to be wrapped in the exosome, and simultaneous administration of multiple proteins is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to an ISG15 overexpressing cell line, SRM-enriched exosomes, and a preparation method and application thereof. Background Art

[0002] Exosomes are small extracellular vesicles secreted by cells. The diameter of the vesicles is between 30-150nm. Under physiological conditions, they can mediate intercellular communication by transmitting active ingredients such as nucleic acids, proteins, and lipids. Since exosomes are produced by cells and have a typical phospholipid bilayer membrane structure, they have extremely low immunogenicity. Therefore, the development and utilization of exosomes for the diagnosis and treatment of diseases has become increasingly popular. Among them, using exosomes as delivery vehicles for a variety of drugs such as small molecules, proteins, and nucleic acids is an important application direction.

[0003] There are two main ways to load drugs into exosomes. One is exogenous cargo loading, that is, firstly separating and purifying exosomes, and then loading drugs into exosomes by ultrasound, electroporation, co-culture, etc. The other is endogenous cargo loading, that is, firstly loading drugs into donor cells, and then using the exosome biogenesis process to obtain exosomes loaded with target drugs. Protein and peptide drugs generally adopt endogenous packaging methods, but because the specific mechanism of intracellular protein packaging and sorting into exosomes has not been fully elucidated, at this stage, it mainly relies on cells to randomly select molecules in the cytoplasm for packaging. Therefore, the loading process of protein drugs is difficult to be artificially regulated, and the loading efficiency needs to be improved urgently.

[0004] Spermidine synthase is a catalytic enzyme for the endogenous synthesis of spermidine. Its product, spermidine, is a natural polyamine that participates in biological processes such as cell DNA replication, mRNA transcription, and protein translation. It has pharmacological effects such as anti-aging, anti-tumor, and prevention and treatment of cardiovascular diseases. It has broad application prospects in disease prevention and treatment, health product development, and beauty product development.

[0005] Therefore, it is necessary to develop a drug that can exert spermidine synthase activity and promote spermidine accumulation. Summary of the invention

[0006] The purpose of the present invention is to provide an ISG15 overexpressing cell line, SRM-enriched exosomes, and a preparation method and application thereof. The present invention proposes for the first time to use SRM encapsulated in the natural medium of exosomes to achieve the effect of promoting spermidine accumulation, and the encapsulated SRM still has biological activity.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, an ISG15 overexpressing cell line is provided, wherein the ISG15 overexpressing cell line is prepared by the following method:

[0009] The nucleic acid molecule encoding ISG15 is cloned into a eukaryotic expression vector to form a recombinant eukaryotic expression vector, which is then transferred into a host cell to form a stable cell line, namely the ISG15 overexpression cell line.

[0010] As a specific embodiment, the method for preparing the ISG15 overexpressing cell line comprises:

[0011] The ISG15 gene was cloned into the pLVX-IRES-ZsGreen1 vector to obtain the recombinant plasmid

[0012] pLVX-IRES-ZsGreenl-ISG15;

[0013] HEK293T cells were transfected with a mixture of three plasmids, pMD2.G, psPAX2 and pLVX-IRES-ZsGreen1-ISG15, and then cultured. The culture fluid was collected and processed to obtain a lentivirus packaged with the ISG15 gene.

[0014] The lentivirus packaged with the ISG15 gene was added to a mixture of polybrene to infect MCF-10A cells for culture, and then the ISG15 overexpression cell line was obtained by cell sorting.

[0015] Furthermore, the mass ratio of the three plasmids, pMD2.G, psPAX2 and pLVX-IRES-ZsGreen1-ISG15, is 1.8-2.2: 2.8-3.2: 3.8-4.2.

[0016] Furthermore, the concentration of the polybrene is 4.5-5.5 μg / ml.

[0017] Furthermore, the eukaryotic expression vector is pLVX-IRES-ZsGreen1.

[0018] Furthermore, the host cell is an MCF-10A cell.

[0019] Furthermore, the method for preparing the recombinant eukaryotic expression vector comprises: inserting the ISG15 nucleic acid molecule with a sequence as shown in SEQ ID NO.3 into the EcoRI and BamHI restriction sites in the eukaryotic expression vector pLVX-IRES-ZsGreen1.

[0020] A second aspect of the present invention provides an SRM-enriched exosome, wherein the SRM-enriched exosome is an SRM-containing exosome generated by the ISG15-overexpressing cell line.

[0021] In a third aspect of the present invention, a method for preparing the SRM-enriched exosomes is provided, the method comprising:

[0022] culturing the ISG15 overexpressing cell line to obtain a culture fluid;

[0023] The culture fluid is separated and purified to obtain exosomes.

[0024] Furthermore, the cell culture comprises:

[0025] The ISG15 overexpressing cell line is cultured to a density of 70% to 90%, washed with PBS and replaced with serum-free DMEM / F12 medium. Preferably, 20 ng / ml of EGF growth factor, 10 ug / ml of insulin, 0.5 mg / ml of hydrocortisone, 100 ng / ml of cholera toxin and other cell proliferation promoting components are simultaneously administered for culture.

[0026] Furthermore, the separation and purification includes: centrifuging the culture solution at 10,000 to 25,000 rpm, discarding the supernatant, and collecting the precipitate, which is the exosome.

[0027] In a fourth aspect of the present invention, provided is the use of the SRM-enriched exosomes in promoting collagen synthesis.

[0028] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0029] The SRM exosomes provided by the present invention are genetically engineered human mammary epithelial cell exosomes, which have a low immune response to humans. At the same time, such overexpressed SRM exosomes can be industrially mass-produced, and are simple and easy to operate in clinical treatment. In addition to intravenous injection, they can also be locally injected according to actual conditions. According to the efficacy of its active product spermidine, it can be applied to disease treatment, medical care, beauty and skin care, and other scenarios. The present invention has the following advantages:

[0030] (1) For the first time, it was proposed that SRM encapsulated by exosome natural medium can promote the accumulation of spermidine, and the encapsulated SRM still has biological activity.

[0031] (2) Exosome administration can achieve targeting and avoid immune rejection.

[0032] (3) Exosomes are stable in nature. The lipid bilayer of exosomes can protect active regulatory factors from degradation when transporting them, thereby preventing the active ingredients from being rapidly degraded during transfer within the body.

[0033] (4) There are more diverse ways of administration. Exosomes can be used as drug treatment carriers through multiple routes such as intravenous injection, local subcutaneous injection, and nebulized inhalation.

[0034] (5) The exosomes required for use can be enriched in large quantities through large-scale cell culture and collection of culture medium.

[0035] (6) Genetic engineering technology can be used to modify other proteins or peptide drugs to make them undergo ISGylation modification and thus be encapsulated into exosomes, thereby achieving the simultaneous administration of multiple proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0037] Figure 1 The western blot experiment results of MCF-10A cells overexpressing ISG15 are shown in Figure 2. Vec is the control of MCF-10A cells transfected with no expression vector, and ISG15 OE (over expression) is the MCF-10A cells stably transfected with ISG15. The results show that ISG15 is successfully overexpressed in MCF-10A cells.

[0038] Figure 2 The results of a western blot experiment show the SRM protein content in the exosomes of MCF-10A cells overexpressing ISG15. 10A / Vec EVs are exosomes (EVs) derived from untransfected MCF-10A cells, and 10A / ISG15 OE EVs are exosomes derived from MCF-10A cells overexpressing ISG15. The results show that the content of spermidine synthase SRM in the EVs secreted by MCF-10A cells overexpressing ISG15 increases significantly.

[0039] Figure 3 The immunocolloidal gold electron microscopy results show the SRM content in the exosomes of MCF-10A cells overexpressing ISG15.

[0040] 10A / Vec EVs are exosomes from MCF-10A cells without transfection, and 10A / SRM EVs are exosomes from ISG15-overexpressing MCF-10A cells.

[0041] MCF-10A cell-derived exosomes (i.e. Figure 210A / ISG15 OE EV). The result figure shows that the content of spermidine synthase SRM in the secreted EVs increased significantly after MCF-10A cells overexpressed ISG15.

[0042] Figure 4 The figure shows the quantitative results of spermidine in the gastrocnemius muscle of mice after being treated with SRM-rich EVs. The results show that SRM can promote the accumulation of the downstream product spermidine after being transmitted to mouse tissues via EVs.

[0043] Figure 5 The western blot results of the collagen content in the gastrocnemius muscle of mice treated with SRM-rich EVs are shown in Figure 1. As can be seen from the results, SRM can promote the accumulation of collagen after being transmitted to mouse tissues via EVs.

[0044] Figure 6 The figure shows the results of immunohistochemistry (IHC) experiments on the collagen content in the gastrocnemius muscle of mice after treatment with SRM-enriched EVs.

[0045] Figure 7 for Figure 6 The quantitative statistical results show that SRM can promote the accumulation of collagen after being transmitted to mouse tissues via EV. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0047] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0048] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0049] The embodiment of the present invention provides an exosome enriched with SRM, and the overall idea is as follows:

[0050] ISG15 is a 15kDa protein induced by type I interferon and belongs to the ubiquitin-like protein superfamily. It can be covalently bound to the target protein by ISG15-specific ligase to cause post-translational modification. This process is called ISGylation. Proteins can have a variety of biological effects after ISGylation modification. Among them, a small number of studies have reported on the function of protein ISGylation modification in exosome secretion, but whether it is involved in the sorting of exosome proteins has not been reported.

[0051] The inventors of this application have found through in-depth research that spermidine synthase SRM can be packaged into exosomes and released after ISGylation modification. Exosomes enriched with SRM can be absorbed after reaching skeletal muscle tissue through blood circulation, and exert spermidine synthase activity to promote spermidine accumulation. Specifically, by overexpressing ISG15 protein in cells, SRM can be promoted to undergo ISGylation, so that more exosomes containing SRM can be harvested. After being absorbed by target cells / target tissues, the synthesis of downstream product spermidine spermidine is increased, and further promotes the synthesis of collagen, especially type I collagen COL1A1, in an eIF5A-dependent manner.

[0052] The following is a detailed description of an SRM-enriched exosome, a preparation method, and an application thereof in combination with examples and experimental data.

[0053] Example 1: A method for preparing exosomes rich in spermidine synthase SRM.

[0054] 1. Construction of ISG15 overexpression cell line

[0055] Step S1, clone the ISG15 gene into the pLVX-IRES-ZsGreen1 vector (Takara, Cat#632187) to obtain the recombinant plasmid pLVX-IRES-ZsGreen1-ISG15; specifically, ISG15 was amplified by PCR using the following primers:

[0056] Forward primer(5'-3'):ATGGGCTGGGACCTGAC(SEQ ID NO.1);

[0057] Reverse primer (5'-3'): TTAGCTCCGCCCGCCA (SEQ ID NO.2);

[0058] The PCR product (SEQ ID NO.3) and the pLVX-IRES-ZsGreen1 plasmid were digested, ligated, transformed, screened, and sequenced to obtain the recombinant plasmid pLVX-IRES-ZsGreen1-ISG15.

[0059] Step S2, transfecting HEK293T cells with a mixture of three plasmids, pMD2.G (Takara, Cat#12260), psPAX2 (Takara, Cat#12259) and pLVX-IRES-ZsGreen1-ISG15 obtained in the above step, and culturing the cells, and then collecting the culture fluid to obtain a lentivirus packaged with the ISG15 gene;

[0060] Step S3, the lentivirus containing the ISG15 gene was added to the mixture of polybrene to infect MCF-10A cells for culture, and then the MCF-10A / ISG15 cell line was obtained by cell sorting. Figure 1 shown.

[0061] 2. Cell Culture

[0062] The ISG15 overexpressing cell line is cultured to obtain a culture fluid; the culture method comprises:

[0063] (1) Preheat the cell culture medium, autoclaved PBS solution, etc. in a 37°C water bath, turn on the UV light in the biosafety cabinet, and irradiate for 30 minutes.

[0064] (2) Wipe the biosafety counter and incubator handles with alcohol and remove the cell culture dish from the incubator.

[0065] (3) Aspirate the culture medium, wash the cells with 1× PBS, discard the PBS, add an appropriate amount of trypsin, and spread it evenly on the bottom of the culture flask. Place the cells in a cell culture incubator for digestion.

[0066] (4) Adjust the digestion time according to the cell type (15 min for MCF-10A cells and MCF-10A / ISG15 OE).

[0067] ①After digestion, add fresh culture medium with an equal volume of trypsin, gently shake the culture dish to mix to stop digestion, and collect the cell suspension in a centrifuge tube.

[0068] ② Centrifuge at 1400 rpm for 5 minutes to precipitate the cells and discard the supernatant in a biosafety cabinet.

[0069] ③ Add 1 mL of fresh culture medium and gently pipette to disperse and mix the cells. For subculture, add the cells to a cell culture dish containing fresh culture medium and shake the dish horizontally using the cross method to disperse the cells evenly.

[0070] ④ Observe the cell density after subculturing under an inverted microscope and place the cells in a cell culture incubator (37°C, 5% CO 2 ) were cultivated.

[0071] 3. Extraction of Exosomes

[0072] The culture fluid is separated and purified to obtain exosomes. The specific operation is as follows:

[0073] (1) Exosome extraction

[0074] ① Clean high-speed centrifuge bottles, ultracentrifuge tubes, etc., sterilize the centrifuge bottles by high pressure and dry them, and sterilize them together with the ultracentrifuge tubes by ultraviolet irradiation in a cell-internal biosafety cabinet for 30 minutes.

[0075] ② Collect the supernatant of the cell culture medium into a centrifuge bottle in a cell-free biosafety cabinet, centrifuge at 500g for 15min, and transfer the supernatant to a 500ml high-speed centrifuge bottle.

[0076] ③ Use Hitachi high-speed centrifuge at 10,000 rpm, 20 min, 4°C. After centrifugation, divide the supernatant into ultracentrifuge tubes, 35 ml per tube, and balance them in pairs.

[0077] ④ Centrifuge at 25,000 rpm in a Beckman ultracentrifuge for 70 min at 4°C. Discard the supernatant and the precipitate is the exosomes.

[0078] ⑤ Resuspend the exosomes in PBS (30ul PBS per 15cm cell culture dish of exosomes secreted by cells).

[0079] Ultracentrifugation was used to extract high-purity exosomes expressing SRM from the cell culture medium. The SRM content was significantly increased compared with the wild-type cells in the control group. The identification results were as follows: Figure 2 , Figure 3 shown.

[0080] Example 2: SRM exosomes can promote the accumulation of spermidine and collagen after being absorbed by tissues.

[0081] 1. Injection of exosomes into the tail vein of mice

[0082] (a) MCF-10A / Vec and MCF-10A / ISG15 cells were cultured in complete medium containing exosome-free serum. After 48 h of culture, the cell culture supernatant was collected and exosomes (10A / Vec EVs and 10A / SRM EVs) were isolated by ultracentrifugation.

[0083] (b) EV injection was performed via the tail vein (the experimental group was injected with SRM exosomes prepared in Example 1, and the control group was injected with exosomes that did not overexpress the ISG15 gene). Each mouse was injected with 10 μg EV each time, twice a week, for a total of 5 weeks.

[0084] (c) Mice were killed at week 6, and the gastrocnemius muscle tissues of the hind limbs were collected for ELISA, western blot, and IHC tests.

[0085] 2. If Figure 4 As shown, after 5 weeks of exosome treatment, ELISA detection found that the content of spermidine in the gastrocnemius tissue of mice in the SRM exosome injection group prepared in Example 1 was significantly higher than that in the control group, indicating that the SRM transported by exosomes has catalytic activity and can promote the accumulation of the downstream product spermidine.

[0086] 3. The results are as follows Figure 5-7 As shown in the figure, after 5 weeks of exosome treatment, western blot and IHC detection found that the collagen content in the gastrocnemius muscle tissue of mice injected with SRM exosomes was significantly higher than that in the control group, indicating that the downstream product of SRM, spermidine, has the function of promoting collagen synthesis.

[0087] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0088] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0089] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An ISG15 overexpressing cell line, characterized in that: The ISG15 overexpressing cell line was prepared by the following method: The nucleic acid molecule encoding ISG15 is cloned into a eukaryotic expression vector to form a recombinant eukaryotic expression vector, which is then transferred into a host cell to form a stable cell line, namely the ISG15 overexpression cell line.

2. The ISG15 overexpressing cell line according to claim 1, characterized in that: The eukaryotic expression vector is pLVX-IRES-ZsGreen1.

3. The ISG15 overexpressing cell line according to claim 1, characterized in that: The host cell is MCF-10A cell.

4. An exosome enriched with SRM, characterized in that: The SRM-enriched exosomes are SRM-containing exosomes generated by the ISG15-overexpressing cell line according to any one of claims 1 to 3.

5. A method for preparing SRM-enriched exosomes according to claim 4, characterized in that: The method comprises: Culturing the ISG15 overexpressing cell line according to any one of claims 1 to 3 to obtain a culture fluid; The culture fluid is separated and purified to obtain the exosomes.

6. The preparation method according to claim 5, characterized in that: The separation and purification comprises: centrifuging the culture solution at high speed, discarding the supernatant, and collecting the precipitate to obtain the exosomes.

7. Use of the ISG15 overexpressing cell line according to any one of claims 1 to 3 in the preparation of a collagen synthesis promoter.

8. Use of the SRM-enriched exosomes according to claim 4 in the preparation of a collagen synthesis promoter.

9. A method for promoting the secretion of spermidine synthase encapsulated by exosomes, characterized in that: The method comprises: preparing a stable cell line overexpressing ISG15, promoting endogenous loading of exosome proteins, and promoting ISGylation modification of encapsulated proteins in cells, thereby inducing their packaging and sorting into exosomes.

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