Exosome system for high expression of glycosidase, method for producing the same, and its application

Abstract

This invention discloses a biomimetic exosome system and method for producing a glucose metabolism-based glycosidase for the treatment of fatty liver-associated hepatocellular carcinoma, which involves transfecting mesenchymal stem cells with a glycosidase sequence by lentiviral transfection, followed by obtaining exosomes by ultracentrifugation. The biomimetic exosome system of this invention possesses target migration ability, low immunogenicity, improved ability to transport bioactive substances, normalizes disrupted glucose metabolism, reduces endoplasmic reticulum stress, and suppresses epithelial-mesenchymal transition signaling. Both in vivo and in vitro experiments have verified that it can effectively target cancer cells in vivo, reduce abnormal O-GlcNAc modifications, and inhibit tumor malignancy, and after reaching fatty liver-associated hepatocellular carcinoma in vivo, it can reduce O-GlcNAc modification levels, restore disrupted metabolism, and suppress cancer progression.

Description

[Technical Field]

[0001] This invention belongs to the field of biomedical materials, and more specifically, relates to a biomimetic exosome system that highly expresses glycosidase for treating fatty liver-related liver cancer based on glucose metabolism, as well as a method for producing the same and its applications. [Background technology]

[0002] With the rise in obesity rates, the proportion of fatty liver-associated liver cancers within hepatocellular carcinomas is also increasing. Surgical resection is the primary clinical treatment for such tumors, and drug therapy is also used as an intervention strategy to prevent tumor recurrence and improve patient survival. Drug therapy generally targets various signaling pathways that promote tumor cell death. Specifically, interventions via metabolic pathways play a crucial role in inducing apoptosis in tumor cells and suppressing tumor metastasis. Among these pathways, the regulation of glucose metabolism is particularly important for inhibiting tumor growth in cases of fatty liver and overnutrition. Therefore, many antitumor therapies targeting glucose metabolism have already been developed. However, despite significant progress, the mechanisms of glucose metabolism in fatty liver-associated liver cancer are still not fully understood. Therefore, the development of effective therapeutic strategies based on glucose metabolism remains highly anticipated.

[0003] Sequencing analysis of clinically resected fatty liver-associated hepatocellular carcinoma specimens revealed a marked decrease in the O-GlcNAcase (OGA) enzyme in these tumor tissues. Previous studies have shown that a rapid increase in O-linked N-acetylglucosamine (O-GlcNAc) modification plays a crucial role in glucose metabolism abnormalities in tumors. However, the potential mechanisms of O-GlcNAc modification are largely unexplored, and targeting O-GlcNAc with drugs alone yields unfavorable results. Furthermore, it was discovered that OGA, a key enzyme that dynamically and transiently regulates O-GlcNAc, is downregulated in fatty liver-associated hepatocellular carcinoma. Increasing OGA levels can reduce abnormal O-GlcNAc modification, restore disrupted energy metabolism, and alleviate endoplasmic reticulum (ER) stress. Additionally, regulating metabolic reprogramming and the function of energy-related organelles while regulating OGA is thought to offer a promising pathway for targeted therapy of hepatocellular carcinoma.

[0004] Compared to conventional mesenchymal stem cells, exosomes possess target migration ability, low immunogenicity, and efficient bioactive substance transport capabilities, making them adaptable and controllable for therapeutic applications. Therefore, to demonstrate this hypothesis, mesenchymal stem cells (MSCs) that highly express OGA are used to treat fatty liver-associated liver cancer. OGA ) Exosome system (MSC) OGA -EXOs) are under development. [Overview of the project]

[0005] Objective of the present invention: The present invention aims to solve the above technical problems and provides a biomimetic exosome system (MSC) that highly expresses glycosidase (OGA) and is used in the treatment of fatty liver-related liver cancer based on glucose metabolism. OGA We propose exosomes (EXOs), which are produced by lentiviral transfection and ultracentrifugation, and are simple, versatile, and easy to produce on a large scale.

[0006] Technical solution: Exosome system (MSC) that highly expresses OGA as described in the present invention OGA -EXOs) are obtained by introducing a glycosidase gene sequence into human mesenchymal stem cells (MSCs) via lentiviral transformation and then using ultracentrifugation. The exosomes are spherical in shape and have an average diameter of 120 nm to 125 nm.

[0007] The specific steps are as follows: 1) The process of producing human mesenchymal stem cells (MSCs), 2) Lentiviral transfection is performed to dilute lentiviral particles that express high glycosidase in a culture medium, add MSCs to the medium, co-culture in an incubator, and add puromycin to select transfected cells that express drug resistance genes until cell death ceases. OGA The manufacturing process of ) and 3) Culturing MSCs in a medium that does not contain exosomes, and then performing primary and secondary centrifugation of the medium to obtain an exosome system, resulting in exosomes (MSCs) that highly express glycosidase. OGA -EXOs) manufacturing process.

[0008] Preferably, the titer of the glycosidase-highly expressing lentivirus particle described in step 2) is 10 9 The copy rate is per mL.

[0009] Preferably, the culture medium described in step 2) is DMEM / F12 medium, and the volume ratio of glycosidase-highly expressing lentivirus particles to DMEM / F12 medium is 1:1000.

[0010] Preferably, the concentration of the MSC in the culture medium described in step 2) is 10 6 It is the number of cells / mL.

[0011] Preferably, after adding the puromycin described in step 2) to the culture medium, the concentration in the culture medium becomes 2 μg / mL.

[0012] Preferably, as the primary centrifugation step described in step 3), at room temperature, the medium is sequentially treated at a centrifugation speed of 300 × g for 10 minutes, at a centrifugation speed of 2000 × g for 10 minutes, and at a centrifugation speed of 10000 × g for 30 minutes, and the obtained precipitate particles are resuspended in PBS.

[0013] Preferably, as the secondary centrifugation step described in step 3), the precipitate obtained by primary centrifugation is centrifuged at 4 °C at a centrifugation speed of 100000 × g for 70 minutes, the supernatant is removed, and the obtained precipitate becomes exosomes.

[0014] The present invention also provides an application of an exosome system that highly expresses glycosidase in the manufacture of drugs for fatty liver-related liver cancer.

[0015] Preferably, the drug is a drug for fatty liver-related liver cancer based on glucose metabolism.

[0016] It has the following beneficial effects.

[0017] (1) In the present invention, an exosome system (MSC OGA -EXOs) that highly expresses nucleosidase (OGA) for fatty liver-related liver cancer and has excellent target migration ability is designed.

[0018] (2) The exosome system (MSC OGA ] -EXOs) that highly expresses nucleosidase (OGA) for fatty liver-related liver cancer provided by the present invention is prepared by the methods of lentivirus transfection and ultracentrifugation. The method is simple, easy to operate, highly reproducible, has low technical requirements, high versatility, high flexibility, and is easy to prepare on a large scale. <##0000105##>

[0019] (3) The exosome system (MSC OGAEXOs (-EXOs) possess target migration capabilities, low immunogenicity, enhance the ability to transport bioactive substances, normalize disrupted glucose metabolism, reduce endoplasmic reticulum stress (ERS), and suppress epithelial-mesenchymal transition (EMT) signaling. Both in vivo and in vitro experiments have shown that it can effectively target cancer cells in vitro, reduce abnormal O-GlcNAc modifications, and inhibit tumor malignancy. Furthermore, in vivo, after reaching fatty liver-associated hepatocellular carcinoma, it can reduce O-GlcNAc modification levels, restore disrupted metabolism, and suppress cancer progression. [Brief explanation of the drawing]

[0020] [Figure 1] This is a schematic diagram of the expression profile analysis of O-GlcNAc-related markers in tumors of patients with fatty liver-associated hepatocellular carcinoma (LIC). Figure A is a schematic diagram of RNA sequencing, immunohistochemistry, and Western blotting performed using clinical specimens from patients with fatty liver-associated LIC. Figure B is a comparison of the 3-year disease-free survival (DFS) of patients with fatty liver-associated LIC with that of other patients with LIC. Figure C is a comparison of the 3-year disease-free survival (DFS) of patients with LIC with metabolic syndrome and those without metabolic syndrome. Figure D is a Cox proportional hazards regression analysis of DFS data from baseline patients. Figure E is the identification of differentially expressed genes in cancer and paracancerous tissues of patients with fatty liver-associated LIC by RNA sequencing analysis. Figure F is the measurement of OGA and OGT expression levels in cancer (group C) and paracancerous (group P) tissues of patients with fatty liver-associated LIC by Western blotting. Figures G-H show immunohistochemical staining of OGA and OGT expression in cancer and para-cancerous tissues of patients with fatty liver-associated liver cancer, scaled to 100 μm. [Figure 2]This is a schematic diagram illustrating the preparation and characterization of MSCOGA-EXOs. Figure A is a schematic diagram showing the lentiviral system transfecting mesenchymal stem cells and secreting exosomes. Figure B shows the morphology of MSCs transfected with Vector and OGA, as indicated by the green fluorescence from EGFP, at a scale of 50 μm. Figures C-D show the analysis of positive and negative surface markers of OGA-transfected MSCs by flow cytometry. Figure E is a TEM image of the morphology of the obtained exosomes, at a scale of 50 μm. Figure F shows the size distribution of MSCOGA-EXOs as measured by nanoparticle tracking analyzer (NTA). Figure G shows the identification of the obtained exosomes by Western blotting. [Figure 3]This is a schematic diagram of the uptake and function of MSCOGA-EXOs in hepatoma cells. Figures A-D show the uptake of MSCVEC-EXOs and MSCOGA-EXOs by tumor cells and primary hepatocytes, respectively, at a scale of 10 μm. Figure E shows quantitative analysis of OGA and OGT expression in tumor cells (group T) and primary hepatocytes (group P) before and after MSCOGA-EXOs uptake by Western blotting. Figures F-G show the migration of untreated tumor cells, tumor cells treated with MSCVec-EXOs, and tumor cells treated with different concentrations of MSCOGA-EXOs at 0, 12, and 24 hours, as shown by scratch experiments, at a scale of 100 μm. Figure H shows the migration of untreated tumor cells (Con), MSCVec-EXOs (Vec), and different concentrations of MSCOGA-EXOs to tumor cells at 0, 12, and 24 hours, at a scale of 100 μm. Figure I shows the analysis of endoplasmic reticulum stress levels in untreated tumor cells (Con), tumor cells treated with MSCVec-EXOs (Vec), and tumor cells treated with different concentrations of MSCOGA-EXOs, using Western blotting. Figure J shows the analysis of EMT levels in untreated tumor cells (Con), tumor cells treated with MSCVec-EXOs (Vec), and tumor cells treated with different concentrations of MSCOGA-EXOs, using Western blotting. [Figure 4]This is a schematic diagram of the in vivo evaluation of MSCOGA-EXOs in mice with orthotopic fatty liver-associated hepatocarcinoma. Figure A is a schematic diagram of the construction of a STAM model mouse simulating fatty liver-associated hepatocarcinoma and exosome therapy. Figure B shows in vivo development of small animals after injection into mice of each group: normal mice injected with NC:PBS, normal mice injected with NC-Vec:MSCVec-EXOs, normal mice injected with NC-OGA:MSCOGA-EXOs, STAM mice injected with STAM:PBS, STAM mice injected with STAM-Vec:MSCVec-EXOs, and STAM mice injected with STAM-OGA:MSCOGA-EXOs. Figures C-E show body weight, liver weight, and liver weight index of the six groups of mice. Figure F shows HE staining of tumors and paracancerous tissues in STAM, STAM-Vec, and STAM-OGA mice, scale 400 μm. [Figure 5] This is a schematic diagram of the in vivo evaluation of the therapeutic effect of MSCOGA-EXOs on fatty liver-associated liver cancer mice. Figures A-C show the expression levels of ALT, AST, and AFP in six groups of mice. Figures D-E show the expression levels of OGA and OGT in cancer tissue and para-cancerous tissue by immunohistochemistry, scaled to 50 μm. Figures F-G show the analysis of endoplasmic reticulum stress and EMT expression levels in six groups of mice by Western blotting. [Modes for carrying out the invention]

[0021] To further enhance the understanding of the present invention, the present invention will be described in more detail below with reference to examples and drawings. These examples are merely for interpretation purposes and do not limit the scope of protection of the present invention.

[0022] The human mesenchymal stem cells used in the following examples are purchased from the American Type Culture Collection (ATCC) cell bank in the United States.

[0023] Example 1: Exosome system (MSC) that highly expresses nucleosidase (OGA). OGA -EXOs) Production Method (1) Generation of human mesenchymal stem cells (MSCs): Cells were cultured using 75T culture flasks. They were cultured in a constant-temperature cell incubator containing 5% CO2 at 37°C. Subculturing was performed when the cells had grown to cover 80% to 90% of the bottom surface area of ​​the culture flask. First, the old culture medium in the culture dish was absorbed and removed, then washed twice with 3 ml of neutral phosphate buffer solution (PBS), followed by 3 ml of 0.25% trypsinase and digestion in a 37°C incubator for 2-3 minutes, followed by 3 ml of the above culture medium to terminate digestion. The cell suspension was pipetteed into a 15 ml centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and 3 ml of the above culture medium was added again to resuspend the cells. Subculturing was then performed in a 1:3 ratio into a new culture flask.

[0024] (2) Preparation of lentiviral transfection OGA Human OGA gene (10 9 Stable transduction of MSCs using lentiviruses carrying copies / mL OGA The following was prepared: OGA lentivirus particles were diluted in DMEM / F12 medium at a volume ratio of 1:1000, and then the MSCs prepared in step 1) were added to the cell nutrient solution, and the concentration of MSCs in the medium was increased to 10 6 The cell volume was adjusted to 10 cells / mL. After incubation for 24 hours, the medium containing lentiviral particles was replaced with fresh medium and incubated for another 48 hours. Transfection cells expressing drug resistance genes were selected until the puromycin concentration in the medium reached 2 μg / mL and cell death ceased. OGA expression was measured by qPCR and Western blot. After transfection, cell purity was measured by flow cytometry, and positive and negative surface markers were detected.

[0025] (3) Exosome system (MSC) that highly expresses nucleosidase (OGA) OGA - EXOs) creation To extract exosomes, MSCs were cultured in an exosome-free medium. A series of centrifugation steps were performed on the medium. Specifically, the medium was first centrifuged at 300 × g for 10 minutes at room temperature, then at 2000 × g for 10 minutes, and then at 10000 × g for 30 minutes. The supernatant was collected, the precipitate was resuspended in PBS, and the precipitate resuspended in PBS was then centrifuged again at 4°C at a rate of 100000 × g for 70 minutes. The supernatant was then removed, and the resulting precipitate was identified as exosomes.

[0026] Example 2: Analysis of the expression profile of O-GlcNAc-related markers in tumors of patients with fatty liver-associated liver cancer. To demonstrate the impact of overnutrition on the malignant progression of fatty liver-associated liver cancer, a retrospective analysis was conducted on the prognosis of patients who underwent surgery for fatty liver-associated liver cancer (Figure 1A). Furthermore, the 3-year disease-free survival (DFS) and overall survival were compared between patients with fatty liver-associated liver cancer and patients with other liver cancers. The results showed that the 3-year DFS was significantly shorter in patients with fatty liver cancer than in patients with other liver cancers (Figure 1B). On the other hand, the 3-year DFS and overall survival rates of patients with and without metabolic syndrome were also studied. The results showed that the 3-year DFS was significantly shorter in patients with metabolic syndrome compared to patients without metabolic syndrome (Figure 1C). To further investigate factors influencing patients' 3-year DFS and overall survival, Cox proportional hazards regression analysis was performed on baseline patient data. Analysis revealed that fatty liver (univariate analysis, hazard ratio [HR], 2.188; 95% confidence interval [CI], 1.097–4.365) and metabolic syndrome (univariate analysis, HR, 2.564; 95% CI, 1.275–5.157) are independent risk factors affecting 3-year disease-free survival (DFS) in liver cancer patients (Figure 1D). These findings indicate that metabolic reprogramming is one of the ten key features of malignant tumors and plays an essential role in the progression of fatty liver-associated liver cancer.

[0027] In different metabolic reprogramming pathways, glucose metabolism disorders are considered a crucial component in fatty liver, overnutrition, and liver cancer progression. However, the role of O-GlcNAc modification in fatty liver-associated liver cancer remains limited. To elucidate the O-GlcNAc modification mechanism and its potential targets in fatty liver-associated liver cancer, we analyzed specimens from patients who underwent clinical surgery using RNA sequencing, immunohistochemistry, and Western blotting. RNA sequencing analysis of cancerous and paracial tissues from fatty liver-associated liver cancer patients revealed upregulation of glycosylation-related genes in cancerous tissue compared to paracial tissue, with O-GlcNAc transferase (OGT)-related genes being particularly prominent (Figure 1E). In contrast, gene expression, mainly OGA, was found to be decreased in genes involved in deglycosylation. Immunohistochemistry and Western blot analysis also showed high OGT expression levels and decreased OGA expression levels in fatty liver-associated cancer tissue (Figures 1F-H). These observations suggest that decreased OGA expression in liver cancer tissue associated with fatty liver disease may be linked to a poor prognosis in patients. Therefore, considering the important role of OGA in the removal of OGlcNAc modifications, decreased OGA expression in these liver cancer tissues may lead to a poor prognosis.

[0028] Example 3 MSC OGA - EXO creation and characterization MSCs that highly express OGA for the treatment of fatty liver-associated liver cancer (Figure 2A) OGA Exosome System (MSC) OGA-EXOs) have been developed. Therefore, MSCs were infected with a lentiviral vector carrying the OGA gene and selected with puromycin. Under a microscope, it was observed that the MSCs remained spindle-shaped after infection (Figure 2B). To verify that lentiviral transduction does not induce MSC differentiation, the expression of MSC-specific positive and negative surface markers was further measured. Flow cytometry analysis demonstrated that OGA-modified MSCs continued to express CD73, CD90, and CD105 without expressing CD34, CD45, and HLA-DR (Figure 2C, D). To obtain the desired exosomes, the supernatant was centrifuged and exosomes were further separated from the MSCs. Transmission electron microscopy (TEM) was used to analyze the MSCs. OGA -EXOs have a spherical morphology and an average diameter of approximately 123 nm (Figures 2E-F). Furthermore, exosomes were identified using membrane proteins such as D63, CD81, and the membrane-related protein TSG101 (Figure 2G). These data were obtained from exosomes derived from MSCs overexpressing OGA (MSCs). OGA This indicates that the EXOs were successfully retrieved.

[0029] Example 4: MSCs in liver tumor cells OGA - EXOs import and functional evaluation MSC OGA - After EXOs were generated, their function was subsequently evaluated by hepatoma cells and primary hepatocytes, and their uptake efficiency was detected. 1,1-dioctadecyl-3,3,3,3-tetramethyldicarbocyanine, 4-chlorobenzenesulfonic acid (DiD) was used to analyze MSCs. OGA-EXOs were labeled, co-cultured with cells for 24 hours, and the results were observed using a confocal laser scanning microscope. The resulting images showed red fluorescence in the cytoplasm, demonstrating the internalization of exosomes into cells (Figures 3A-D). Interestingly, a unique phenomenon was further observed in which tumor cells took up DiD more than hepatocytes, indicating that tumor cells have a more active phagocytic capacity. This phenomenon contributes to the effective absorption of the fabricated exosomes by tumor cells, allowing them to exert the expected effects.

[0030] To investigate the effect of exosome uptake on the overall O-GlcNAc modification level of tumor cells, OGA and OGT protein expression in these cells was measured using Western blotting experiments. The results showed that, compared to primary hepatocytes (P group), tumor cells (T group) exhibited increased O-GlcNAc modification levels, increased OGT production, and decreased OGA expression. Such effects were observed in MSCs. OGA -The situation reversed after the introduction of EXOs. Also, MSC OGA -Compared to primary hepatocytes (P+E group) that absorbed EXOs, MSC OGA Tumor cells that absorbed EXOs (T+E group) showed significantly higher OGA expression and decreased OGT production (Figure 3E). To determine the effect of OGA on hepatoma cell progression, migration and invasiveness studies were conducted to evaluate how changes in OGA expression levels induced by exosome therapy affect the migration and invasiveness characteristics of hepatoma cells. These experimental data show that the migration and invasiveness of hepatoma cells treated with OGA were significantly reduced (Figure 3F-H).

[0031] MSCs in the endoplasmic reticulum OGA-To investigate the effects of EXOs uptake, the level of endoplasmic reticulum stress was evaluated by Western blotting analysis. The results showed that overexpression of OGA could suppress endoplasmic reticulum stress, and that the level of endoplasmic reticulum stress was further suppressed with increasing exosome concentration (Figure 3I). Furthermore, the mechanism by which OGA controls the migration and invasion of hepatoma cells was investigated. Western blotting analysis demonstrated that overexpression of OGA could upregulate the expression levels of EMT-related proteins such as ZEB1, Snail, N-cadherin, and Vimentin, and that MSCs... OGA -EXOs therapy has been shown to suppress the development of EMT in liver cancer cells (Figure 3J). Therefore, from all cellular levels in Figure 4, it was discovered that tumor cells can internalize exosomes and overexpress OGA, and that with OGA uptake, tumor cells show a decrease in migration and invasiveness. Furthermore, these results suggest that MSC OGA EXOs have been shown to effectively mitigate the malignant behavior of tumor cells and are expected to play a role in tumor treatment.

[0032] Example 5: MSCs in mice with orthotopic fatty liver-associated liver cancer OGA - In vivo evaluation of EXOs To study the actual role of the OGA gene in fatty liver-associated liver cancer in vivo, we are using stelic animal models (STAM) and normally feeding models to study MSCs. OGA -A modeling study was conducted for the EXOs test (Figure 4A). In the STAM model, male mice were intraperitoneally injected with 200g of streptozotocin (STZ) within 5 days of birth, followed by a 60% high-fat diet (HFD) from 3 weeks of age. Normal feeding mice and STAM model mice were further divided into three groups, and at 4 weeks, they were divided into phosphate-buffered saline (NC, STAM) and MSC, respectively. Vec -EXOs (NC-Vec, STAM-vec), MSC OGA-EXOs (NC-OGA, STAM-OGA) were injected into mice, and samples were collected when the mice reached 16 weeks of age. To confirm the targeting ability in vivo, bioluminescence imaging was used to track the exosomes in vivo. Imaging results showed that most of the exosomes localized to the liver, indicating their targeting ability and the potential to exert subsequent antitumor effects in the liver (Figure 4B). By measuring the liver and body weight of the mice, the MSCs were identified as being responsible for tumor development after targeting the liver. OGA -The effects of EXOs were further analyzed (Figure 4C-E). Despite fatty liver mice having a heavier body weight than control mice, their body weight at 16 weeks was actually lower due to tumor progression. Also, compared to control mice, STAM mice had a higher liver weight due to tumor load, resulting in a larger liver-to-body weight ratio. Furthermore, while the body weight of STAM-OGA mice was higher than that of STAM-Vec mice, both liver weight and liver-to-body weight ratio were significantly lower than in the STAM-Vec group. Notably, the livers of STAM mice exhibited clear carcinogenicity and contained single or multiple tumors. In contrast, the tumor size in the STAM-OGA group was significantly smaller compared to the other groups (Figure 4F). These data are from liver-targeted MSCs. OGA -EXOs have been shown to significantly suppress the development and progression of liver cancer associated with fatty liver disease.

[0033] Example 6: MSCs in mice with fatty liver-associated liver cancer OGA - In vivo evaluation of the therapeutic effects of EXOs MSCs in tumor progression OGA -To further evaluate the effects of EXOs, liver function indicators and alpha-fetoprotein (AFP) levels were also further assessed in liver cancer mice. Compared to mice with normal feeding, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alpha-fetoprotein (AFP) were elevated in all three STAM groups of mice. However, MSC OGA- After EXOs treatment, ALT, AST, and AFP levels were significantly lower in the STAM-OGA group compared to the STAM and STAM-Vec groups (5A-C). This indicates improved liver function and the effectiveness of antitumor therapy in the STAM-OGA group. Due to these positive results, the expression levels of OGA and OGT in these mice were further investigated. As a result, OGA expression was effectively enhanced and OGT levels decreased in the STAM-OGA group (Figure 5D, E), thus MSC OGA -EXOs have been shown to effectively regulate the restoration of atrophied O-GlcNAc modification, thereby controlling tumor progression.

[0034] MSC OGA -To elucidate the inhibition mechanism of EXOs, we further measured in vivo endoplasmic reticulum stress (ERS) and EMT levels, which are tumor-associated malignancy indicators. Results from the STAM-OGA group showed a significant decrease in endoplasmic reticulum stress levels, thus indicating that MSC OGA -EXOs were found to significantly inhibit endoplasmic reticulum stress and suppress tumor progression (Figure 5F). Furthermore, EMT-related proteins were studied. The results showed that changes in cell phenotype and decreased E-cadherin expression led to reduced cell adhesion and increased invasiveness and migratory characteristics. Loss of E-cadherin expression was also the most prominent feature of EMT in fatty liver-associated hepatocarcinoma mice, and it was further discovered that exosome therapy can suppress the progression of EMT (Figure 5G). These results suggest that exosomes represent a viable strategy for treating EMT in mouse liver-associated hepatocarcinoma, opening the way for new therapeutic methods in cancer treatment.

[0035] While preferred embodiments of the present invention have been described above, the present invention is not limited thereto. Within the spirit and scope of the present invention, any modifications, substitutions with equivalents, improvements, etc., made by those skilled in the art are included within the scope of protection of the present invention.

Claims

1. An exosome system for high-expression of glycosidase, comprising transfecting human mesenchymal stem cells with a glycosidase sequence by lentiviral transfection, and subsequently obtaining glycosidase by ultracentrifugation, wherein the exosomes are spherical in shape and have an average diameter of 120 nm to 125 nm.

2. 1) The process of producing human mesenchymal stem cells, 2) A process for producing human mesenchymal stem cells that highly express glycosidase, comprising: diluting lentiviral particles that highly express glycosidase by lentiviral transfection in a culture medium; adding human mesenchymal stem cells to the culture medium; co-culturing in an incubator; and selecting transfected cells that express drug resistance genes by adding puromycin until cell death ceases; 3) A process for producing exosomes that highly express glycosidase, in which human mesenchymal stem cells are cultured in a medium that does not contain exosomes, and an exosome system is obtained after primary and secondary centrifugation of the medium, A method for producing an exosome system that highly expresses the glycosidase described in claim 1, characterized by including the following:

3. The titer of the glycosidase-highly expressing lentivirus particle described in step 2) is 10 ９ The method for producing according to claim 2, characterized in that the amount is copies / mL.

4. The method for producing according to claim 2), characterized in that the culture medium described in step 2) is DMEM / F12 medium, and the volume ratio of glycosidase-highly expressing lentivirus particles to DMEM / F12 medium is 1:1000.

5. The concentration of human mesenchymal stem cells in the culture medium described in step 2) is 10 ６ The method for producing according to claim 2, characterized in that the amount is 1 cell / mL.

6. The method for producing according to claim 2), characterized in that after adding the puromycin described in step 2) to the culture medium, the concentration in the culture medium becomes 2 μg / mL.

7. The method for producing according to claim 2, characterized in that, as the primary centrifugation step described in step 3), the culture medium is sequentially processed at room temperature under the following conditions: at a centrifugation speed of 300 × g for 10 minutes, at a centrifugation speed of 2000 × g for 10 minutes, and at a centrifugation speed of 10000 × g for 30 minutes, and the resulting precipitate particles are resuspended in PBS.

8. The method for producing exosomes according to claim 2, characterized in that the secondary centrifugation step described in step 3) involves centrifugation of the precipitate obtained by primary centrifugation at 4°C and a centrifugation speed of 100,000 × g for 70 minutes, removing the supernatant, and the resulting precipitate becoming exosomes.

9. Application of an exosome system that highly expresses the glycosidase described in any one of claims 1 to 8 in the manufacture of a drug for fatty liver-related liver cancer.

10. The application according to claim 9, characterized in that the drug is a drug for fatty liver-related liver cancer based on glucose metabolism.

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