Exosome system with high glycosidase expression, and preparation method therefor and use thereof

Abstract

Provided are a biomimetic exosome system with high glycosidase expression for treating glucose metabolism-based fatty liver-associated liver cancer, and a preparation method therefor. The method comprises transfecting a glycosidase sequence into a mesenchymal stem cell by means of lentiviral transfection and then performing an ultracentrifugation to obtain an exosome for treating fatty liver-associated liver cancer. The biomimetic exosome system has a targeted migration capability, low immunogenicity, and enhanced ability to deliver biologically active substances, restores disrupted glucose metabolism, reduces endoplasmic reticulum stress, and inhibits epithelial-mesenchymal transition signaling. It is verified by means of both in-vivo and in-vitro experiments that in vitro, the biomimetic exosome system can effectively target cancer cells, reduce aberrant O-GlcNAc modification, and inhibit malignant tumor behaviors; and in vivo, after being delivered to the fatty liver-associated liver cancer, the biomimetic exosome system can reduce O-GlcNAc modification levels, and restore disrupted metabolism, thereby inhibiting cancer progression.

Description

An exosome system with high expression of glycosidase, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a biomimetic exosome system for treating fatty liver-related liver cancer based on glucose metabolism, its preparation method, and its application. Background Technology

[0002] With the rising prevalence of obesity, the proportion of hepatocellular carcinoma (HCC) associated with fatty liver is also increasing. Surgical resection is the primary clinical treatment for these tumors, while drug therapy is also used as an intervention strategy to prevent tumor recurrence and improve patient survival. Drug therapy typically targets multiple signaling pathways to promote tumor cell death. Specifically, interventions through metabolic pathways play a crucial role in inducing tumor cell apoptosis and inhibiting tumor metastasis. Among these pathways, the regulation of glucose metabolism, particularly in cases of fatty liver and nutritional excess, is essential for suppressing tumor growth. Therefore, many anti-tumor therapies targeting glucose metabolism have been developed. However, despite significant progress, a thorough understanding of the mechanisms of glucose metabolism in fatty liver-associated HCC remains lacking. Therefore, the development of effective therapeutic strategies based on glucose metabolism remains a promising prospect.

[0003] Sequencing analysis of clinically resected samples from fatty liver-associated hepatocellular carcinoma (HCC) revealed a significant decrease in the O-GlcNAcase (OGA) enzyme in these tumor tissues. Previous studies have shown that the dramatic increase in O-linked N-acetylglucosamine (O-GlcNAc) modification plays a crucial role in abnormal tumor glucose metabolism. However, the underlying mechanisms of O-GlcNAc modification remain largely unexplored, leading to unsatisfactory results when targeting O-GlcNAc with drugs alone. We found that OGA, a key enzyme that dynamically and transiently regulates O-GlcNAc, is downregulated in fatty liver-associated HCC. By increasing OGA levels, abnormal O-GlcNAc modification can be reduced, thereby restoring disrupted energy metabolism and alleviating endoplasmic reticulum (ER) stress. We believe that regulating OGA simultaneously modulates metabolic reprogramming and the function of energy-related organelles, providing a promising pathway for targeted therapy of HCC.

[0004] Compared to conventional mesenchymal stem cells, exosomes possess targeted migration capabilities, low immunogenicity, and highly efficient bioactive substance transport capabilities, thus offering adaptability and controllability in therapeutic applications. Therefore, to verify this hypothesis, we developed a mesenchymal stem cell (MSC) with high OGA expression. OGA Exosome system (MSC) OGA -EXOs) to treat fatty liver-related liver cancer. SUMMARY

[0005] The present application aims to solve the above technical problems, and provides an exosome system (MSC OGA -EXOs) for treating fatty liver related liver cancer based on glucose metabolism, which is prepared by lentiviral transfection and ultracentrifugation, and has the advantages of simple method, strong universality and convenient mass production.

[0006] Technical scheme: The exosome system (MSC OGA -EXOs) with high expression of OGA is prepared by transfecting a glycosidase sequence into human mesenchymal stem cells (MSC) through lentiviral transfection, and then obtained by ultracentrifugation, wherein the exosome is spherical in shape and has an average diameter of 120 nm to 125 nm.

[0007] The specific steps are as follows:

[0008] 1) Preparation of human mesenchymal stem cells (MSC);

[0009] 2) Preparation of human mesenchymal stem cells (MSC OGA） with high expression of glycosidase, wherein the high expression glycosidase lentivirus particles are diluted with a culture medium, the MSC is added to the culture medium, and the culture medium is incubated in an incubator, and the transfected cells expressing the drug resistance gene are selected by adding puromycin until no cell death occurs.

[0010] 3) Preparation of exosomes (MSC OGA -EXOs) with high expression of glycosidase, wherein the MSC is cultured in an exosome-free culture medium, and the exosome system is obtained after one centrifugation and two centrifugations of the culture medium.

[0011] Preferably, the titer of the high expression glycosidase lentivirus particles in step 2) is 10 9 copies / mL.

[0012] Preferably, the culture medium in step 2) is DMEM / F12 culture medium, and the volume ratio of the high expression glycosidase lentivirus particles to the DMEM / F12 culture medium is 1:1000.

[0013] Preferably, the concentration of the MSC in the culture medium is 10 6 cells / mL.

[0014] Preferably, the concentration of the puromycin in the culture medium after being added to the culture medium in step 2) is 2 μg / mL

[0015] Preferably, the centrifugation step in step 3) is as follows: under room temperature conditions, the culture medium is centrifuged sequentially at a speed of 300× g for 10 minutes, 2000× g for 10 minutes, and 10000× g for 30 minutes, and the obtained precipitate particles are resuspended in PBS.

[0016] Preferably, the secondary centrifugation step in step 3) is as follows: the precipitate obtained from the first centrifugation is centrifuged at 4°C and 100,000 × g for 70 minutes, the supernatant is removed, and the precipitate obtained is the exosome.

[0017] This invention also provides the application of an exosome system that highly expresses glycosidases in the preparation of drugs for fatty liver-related liver cancer.

[0018] Preferably, the drug is a drug for fatty liver-related liver cancer based on glucose metabolism.

[0019] Beneficial effects:

[0020] (1) This invention designs an exosome system (MSC) for high expression of nucleoside enzyme (OGA) in hepatocellular carcinoma associated with fatty liver. OGA -EXOs), which have superior targeted migration capabilities.

[0021] (2) The exosome system (MSC) for high expression of nucleoside enzyme (OGA) in hepatocellular carcinoma associated with fatty liver provided by the present invention OGA EXOs were prepared by lentivirus transfection and ultracentrifugation. The method is simple, easy to operate, highly reproducible, has low technical requirements, is versatile, flexible, and easy to prepare on a large scale.

[0022] (3) The exosome system (MSC) for high expression of nucleoside enzyme (OGA) in hepatocellular carcinoma with hepatic fatty liver prepared in this invention OGA O-GlcNAc (O-GlcNAc) possesses targeted migration capabilities, low immunogenicity, and enhances the transport of bioactive substances, normalizing disrupted glucose metabolism, reducing endoplasmic reticulum stress (ERS), and inhibiting epithelial-mesenchymal transition (EMT) signaling. In vivo and in vitro experiments validated its effective targeting of cancer cells in vitro, reducing abnormal O-GlcNAc modification and inhibiting tumor malignancy. In vivo, upon reaching fatty liver-related hepatocellular carcinoma, it reduced O-GlcNAc modification levels, restored disrupted metabolism, and thus inhibited cancer progression. Attached Figure Description

[0023] Figure 10. O-GlcNAc-related markers expression profile in tumor of patients with hepatocarcinoma related to fatty liver. Panel A: Schematic representation of RNA sequencing, immunohistochemistry and Western blotting using clinical samples of patients with hepatocarcinoma related to fatty liver. Panel B: Comparison of disease-free survival (DFS) at 3 years after surgery between patients with hepatocarcinoma related to fatty liver and other hepatocarcinoma patients. Panel C: Comparison of disease-free survival (DFS) at 3 years between patients with hepatocarcinoma with and without metabolic syndrome. Panel D: Cox proportional hazards regression analysis of disease-free survival (DFS) data of patients at baseline. Panel E: Differentially expressed genes in tumor and peritumoral tissues of patients with hepatocarcinoma related to fatty liver identified by RNA sequencing analysis. Panel F: Western blotting detection of OGA and OGT expression levels in tumor (C group) and peritumoral (P group) tissues of patients with hepatocarcinoma related to fatty liver. Panel G-H: Immunohistochemical staining of OGA and OGT expression in tumor and peritumoral tissues of patients with hepatocarcinoma related to fatty liver. Scale bar, 100 pm.

[0024] Figure 2. MSC OGA - Preparation and characterization of EXOs. Panel A: Schematic representation of lentiviral system transfection of mesenchymal stem cells and secretion of exosomes. Panel B: Morphology of Vector and MSCs transfected with OGA, shown by green fluorescence of EGFP. Scale bar, 50 pm. Panel C-D: Flow cytometry analysis of positive and negative surface markers of MSCs after transfection with OGA. Panel E: TEM image showing the morphology of the obtained exosomes. Scale bar, 50 pm. Panel F: Nanoparticle tracking analyzer (NTA) showing the size distribution of MSC OGA - EXOs. Panel G: Western blotting identification of the obtained exosomes.

[0025] Figure 3. Uptake and function of MSC OGA - EXOs in hepatoma cells. Panel A-D: Uptake of MSC VEC - EXOs and MSC OGA - EXOs by tumor cells (T group) and primary hepatocytes (P group). Scale bar, 10 pm. Panel E: Western blotting quantitative analysis of OGA and OGT expression in tumor cells and primary hepatocytes before and after uptake of MSC OGA - EXOs. Panel F-G: Scratch assay showing untreated tumor cells, tumor cells treated with MSC Vec - EXOs and different concentrations of MSC OGA - EXOs. Scale bar, 100 pm. Panel H: Migration of untreated tumor cells (Con), tumor cells treated with MSC Vec- EXOs (Vec) and MSCs at different concentrations OGA - Migration of EXOs to tumor cells, scale bar 100 μm; Figure I is a Western blotting analysis of untreated tumor cells (Con), MSC Vec - EXOs-treated tumor cells (Vec) and MSCs at different concentrations OGA - Endoplasmic reticulum stress level of EXOs-treated tumor cells; Figure J is a Western blotting analysis of untreated tumor cells (Con), MSC Vec - EXOs-treated tumor cells (Vec) and MSCs at different concentrations OGA - EMT level of EXOs-treated tumor cells.

[0026] Figure 4 is an in vivo evaluation of MSC OGA - Schematic diagram of in vivo evaluation of EXOs: Figure A is a schematic diagram of construction of STAM model mice simulating fatty liver-related hepatocarcinoma and treatment with EXOs; Figure B is small animal in vivo imaging after injection of mice in each group: NC: normal mice injected with PBS; NC-Vec: normal mice injected with MSC Vec - EXOs; NC-OGA: normal mice injected with MSC OGA - EXOs; STAM: STAM mice injected with PBS; STAM - Vec: STAM mice injected with MSC Vec - EXOs; STAM-OGA: STAM mice injected with MSC OGA - EXOs; Figure C-E are the body weight, liver weight and liver / body ratio of the six groups of mice; Figure F is HE staining of tumor and paracancerous tissue of STAM, STAM-Vec and STAM-OGA mice, scale bar 400 μm.

[0027] Figure 5 is an in vivo evaluation of MSC OGA - Schematic diagram of in vivo evaluation of therapeutic effect of EXOs on fatty liver-related hepatocarcinoma mice: Figures A-C are the expression levels of ALT, AST and AFP indicators of the 6 groups of mice; Figures D-E are immunohistochemical staining showing the expression levels of OGA and OGT in cancer tissue and paracancerous tissue, scale bar 50 μm; Figures F-G are Western blotting analysis of the expression levels of endoplasmic reticulum stress and EMT of the six groups of mice. DETAILED DESCRIPTION

[0028] In order to deepen the understanding of the present application, the present application will be further described in detail below in combination with examples and drawings, which examples are only used to explain the present application and do not constitute a limitation on the protection scope of the present application.

[0029] The human mesenchymal stem cells used in the following examples were purchased from the American Type Culture Collection.

[0030] Example 1 Preparation of exosome system (MSC-EXOs) with high expression of nucleosidase (OGA) OGA -EXOs)

[0031] (1) Preparation of human mesenchymal stem cells (MSCs)

[0032] The cells were cultured in 75T culture flasks. They were incubated in a thermostatic cell incubator at a temperature of 37°C with 5% CO2. When the cells grew to 80-90% of the area of the bottom of the culture flask, they were passaged. First, the old culture medium in the culture dish was aspirated, washed twice with 3 ml of neutral phosphate buffered saline (PBS), then 3 ml of 0.25% trypsin was added and digested in a 37°C incubator for 2-3 minutes, then 3 ml of the above culture medium was added to stop the digestion. The cell suspension was aspirated into a 15 ml centrifuge tube with a pipette, centrifuged at 1000 rpm for 5 min, and the cells were resuspended with 3 ml of the above culture medium and passaged into new culture flasks at a ratio of 1:3.

[0033] (2) Preparation of lentivirus transfection of OGA

[0034] MSCs were prepared by stable transduction with lentivirus carrying human OGA gene (10 9 copies / mL) OGA OGA lentivirus particles were diluted with DMEM / F12 culture medium at a volume ratio of 1:1000, and the MSCs prepared in step 1 were added to the cell culture medium, so that the concentration of MSCs in the culture medium was 10 6 cells / mL. After incubation for 24 hours, the culture medium containing lentivirus particles was replaced with fresh culture medium and incubated for another 48 hours. Transfected cells expressing the drug resistance gene were selected by adding puromycin, and the concentration of puromycin in the culture medium was 2 μg / mL until no cells died. The expression of OGA was detected by qPCR and Western blot. After transfection, the purity of the cells was detected by flow cytometry, and positive and negative markers were detected.

[0035] (3) Preparation of exosome system (MSC OGA -EXOs) with high expression of nucleosidase (OGA)

[0036] To extract exosomes, MSCs were cultured in exosome-depleted medium. The medium was subjected to a series of centrifugation steps: 300 x g for 10 min at room temperature, followed by 2000 x g for 10 min, and then 10000 x g for 30 min. The supernatant was collected, and the pellet was resuspended in PBS. The resuspended pellet was then centrifuged at 100000 x g for 70 min at 4°C. The supernatant was removed, and the pellet was obtained as the exosomes.

[0037] Example 2. Expression profiling of O-GlcNAc-associated markers in tumors of patients with fatty liver-related hepatocarcinoma

[0038] To confirm the effect of nutritional excess on the malignant transformation of fatty liver-related hepatocarcinoma, we performed a retrospective analysis of the prognosis of patients with fatty liver-related hepatocarcinoma who underwent surgery (Fig. 1A). On one hand, we compared the 3-year disease-free survival (DFS) and overall survival of patients with fatty liver-related hepatocarcinoma with those of other patients with hepatocarcinoma. The results showed that the 3-year DFS of patients with hepatocarcinoma complicated with fatty liver was significantly shorter than that of other patients with hepatocarcinoma (Fig. 1B). On the other hand, we studied the 3-year DFS and overall survival of patients with and without metabolic syndrome. The results showed that the 3-year DFS of patients with metabolic syndrome was significantly shorter than that of patients without metabolic syndrome (Fig. 1C). To further explore the factors affecting the 3-year DFS and overall survival of patients, we performed Cox proportional hazards regression analysis of the baseline patient data. The analysis showed 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) were independent risk factors affecting the 3-year DFS of patients with hepatocarcinoma (Fig. 1D). These findings indicate that metabolic reprogramming, one of the ten hallmarks of malignant tumors, plays an indispensable role in the development of fatty liver-related hepatocarcinoma.

[0039] In different metabolic reprogramming, glucose metabolism disorder is considered as a key link between fatty liver and overnutrition and liver cancer development. However, the role of O-GlcNAc modification in fatty liver related hepatocarcinoma is still limited. To elucidate the O-GlcNAc modification mechanism and its potential targets related to fatty liver related hepatocarcinoma, we analyzed the samples of clinical surgery patients by RNA sequencing, immunohistochemistry and Western blotting. By RNA sequencing analysis of cancer tissues and adjacent tissues of fatty liver related hepatocarcinoma patients, we found that the genes responsible for glycosylation were up-regulated in cancer tissues compared with adjacent tissues, among which the genes related to O-GlcNAc transferase (OGT) were particularly significant (Figure 1E). On the contrary, the expression of genes involved in deglycosylation, mainly OGA, was found to be decreased. Immunohistochemistry and Western blot analysis results also showed that OGT expressed at a higher level in cancer tissues related to fatty liver, while OGA expressed at a lower level (Figure 1F-H). These observations suggest that the decrease of OGA expression in liver cancer tissues related to fatty liver disease may be associated with poor patient prognosis. Therefore, considering the key role of OGA in removing OGl cNAc modification, the decrease of OGA expression in these liver cancer tissues may lead to poor prognosis.

[0040] Example 3 MSC OGA - Preparation and characterization of EXOs

[0041] We developed an OGA high-expressing MSC OGA Exosome system (MSC OGA -EXOs) for the treatment of fatty liver related hepatocarcinoma (Figure 2A). For this purpose, we infected MSCs with lentiviral vectors carrying the OGA gene and selected them using puromycin. Under the microscope, we observed that MSCs maintained a spindle shape after infection (Figure 2B). To verify that lentiviral transduction did not induce MSCs differentiation, we also detected the expression of positive and negative surface markers specific to MSCs. Flow cytometry analysis confirmed that OGA-modified MSCs continued to express CD73, CD90 and CD105, but not CD34, CD45 and HLA-DR (Figure 2C, D). To obtain the desired exosomes, we centrifuged the supernatant and further isolated the exosomes from MSCs. Transmission electron microscopy (TEM) showed that MSC OGA -EXOs had a spherical morphology with an average diameter of about 123 nm (Figure 2E-F). In addition, exosomes were also confirmed using membrane proteins such as D63, CD81 and membrane-associated protein TSG101 (Figure 2G). These data indicate that we successfully obtained OGA overexpressing MSC-derived exosomes (MSC OGA -EXOs).

[0042] Example 4 MSCs OGA - Uptake and functional evaluation of EXOs in hepatoma cells

[0043] After generating MSCs OGA -EXOs, we next evaluated their function with hepatoma cells and primary hepatocytes to check their uptake efficiency. We labeled MSCs OGA -EXOs with 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine, 4-chlorobenzenesulfonate salt (DiD) and co-cultured with cells for 24 h, and observed the results using confocal laser scanning microscopy. The resulting images showed red fluorescence within the cytoplasm, confirming the intracellular internalization of exosomes (Fig. 3A-D). Interestingly, we also observed a peculiar phenomenon that the uptake of DiD by tumor cells was higher than that by hepatocytes, which indicated that tumor cells had more active phagocytic capacity. This phenomenon was beneficial for the efficient absorption of exosomes we prepared by tumor cells, allowing them to exert the expected effects.

[0044] To verify the effect of exosome uptake on the overall O-GlcNAc modification level of tumor cells, we detected the expression of OGA and OGT proteins in these cells by Western blotting experiments. The results showed that, compared with the primary hepatocyte group (P group), the O-GlcNAc modification level of tumor cells (T group) was elevated, OGT production was enhanced, and OGA expression was reduced. This effect was reversed after the introduction of MSCs OGA -EXOs. In addition, compared with primary hepatocytes that absorbed MSCs OGA -EXOs (P + E group), tumor cells that absorbed MSCs OGA -EXOs (T + E group) exhibited significantly higher OGA expression and reduced OGT production (Fig. 3E). To determine the effect of OGA on the progression of hepatoma cells, we performed migration and invasion experiments to evaluate how the changes in OGA expression levels induced by exosome treatment affected the migration and invasion properties of hepatoma cells. These experimental data showed that the migration and invasion potential of hepatoma cells treated with OGA was significantly reduced (Fig. 3F-H).

[0045] To explore the effect of MSCs OGA- The effect of EXOs uptake on endoplasmic reticulum, we evaluated the level of endoplasmic reticulum stress by Western blotting analysis, in our results, we observed that overexpression of OGA can inhibit endoplasmic reticulum stress, and the level of endoplasmic reticulum stress can be further inhibited with the increase of EXOs concentration (Figure 31). In addition, we also explored the mechanism of OGA regulating the migration and invasion of hepatoma cells. By Western blotting analysis, we confirmed that overexpression of OGA can up-regulate the expression level of EMT related proteins such as ZEB1, Snail, N-cadherin and Vimentin, indicating that MSC OGA - EXOs treatment can inhibit the occurrence of EMT in hepatoma cells (Figure 3J). Therefore, from all the cellular levels of Figure 4, we found that tumor cells can internalize EXOs, overexpress OGA, and with the uptake of OGA, tumor cells show a decrease in migration and invasion ability. In addition, these results indicate that our MSC OGA - EXOs can effectively reduce the malignant behavior of tumor cells, and are expected to play a role in tumor treatment.

[0046] Example 5 In vivo evaluation of MSC OGA - EXOs in steatosis-related hepatocarcinoma mice

[0047] To study the actual role of OGA gene in steatosis-related hepatocarcinoma in vivo, we used Stelic animal model (STAM) and normal diet model to evaluate MSC OGA - EXOs test was modeled (Figure 4A). For the STAM model, male mice were injected intraperitoneally with 200g streptozotocin (STZ) within 5 days after birth, and then given a 60% high-fat diet (HFD) from 3 weeks of age. Normal diet mice and STAM model mice were divided into three groups, and injected with normal saline (NC, STAM) at 4 weeks; MSC Vec - EXOs (NC-Vec, STAM-vec); MSC OGA - EXOs (NC-OGA, STAM-OGA) at 4 weeks, and then samples were collected when the mice grew to 16 weeks of age. In order to confirm the targeting ability in vivo, bioluminescence imaging was used to track the entry of EXOs into the body. From the imaging results, we observed that most of the EXOs were located in the liver, which indicated their targeting ability and potential to subsequently play an anti-tumor role in the liver (Figure 4B). By detecting the liver and body weight of mice, we further analyzed the effect of MSC OGA- Effect of EXOs on tumorigenesis (Fig. 4C-E). Although the body weight of fatty liver mice was higher than that of control mice, their body weight at 16 weeks was actually lower due to the progression of tumors. In addition, the liver weight of STAM mice was also higher than that of control mice due to tumor burden, resulting in a larger liver weight ratio. Furthermore, the body weight of STAM-OGA group mice was higher than that of STAM-Vec group, and the liver weight and liver weight ratio were significantly lower than those of STAM-Vec group. It is worth mentioning that the liver of STAM mice had obvious carcinogenic effect, and there were single or multiple tumors. In contrast, the tumor size of the STAM-OGA group was significantly reduced compared with other groups (Fig. 4F). These data suggest that our liver-targeted MSC OGA - EXOs can significantly inhibit the occurrence and progression of liver cancer associated with fatty liver.

[0048] Example 6 MSC OGA - In vivo evaluation of therapeutic effect of EXOs on fatty liver-related liver cancer mice

[0049] To further evaluate the therapeutic effect of MSC OGA To evaluate the effect of EXOs on tumor progression, we also evaluated the liver function indicators and alpha-fetoprotein (AFP) levels of liver cancer mice. Compared with normal diet mice, the levels of alanine transaminase (ALT), aspartate transaminase (AST) and alpha-fetoprotein (AFP) in the serum of three STAM groups of mice were increased. However, in the MSC OGA - After EXOs treatment, the levels of ALT, AST and AFP in the STAM-OGA group were significantly lower than those in the STAM and STAM-Vec groups (5A-C). This indicates the improvement of liver function in the STAM-OGA group and the effectiveness of anti-tumor therapy. These positive results prompted us to further study the expression levels of OGA and OGT in these mice. The results also showed that in the STAM-OGA group, the expression of OGA was effectively enhanced, and the level of OGT was reduced (Fig. 5D, E), indicating that MSC OGA - EXOs can effectively regulate the recovery of dysregulated O-GlcNAc modification to control the progression of tumors.

[0050] To elucidate the inhibition mechanism of MSC OGA To elucidate the inhibition mechanism of MSC OGA- EXOs can significantly inhibit endoplasmic reticulum stress, thereby inhibiting tumor progression (Figure 5F). In addition, we studied the proteins related to EMT. The results showed that the cell phenotype changed, E-cadherin expression decreased, leading to decreased cell adhesion, and acquired invasive and migratory characteristics. We also found that the loss of E-cadherin expression was the most significant feature of EMT in fatty liver-related hepatocarcinoma mice, and exosome treatment could prevent the progression of EMT (Figure 5G). These results show that our exosomes can be a viable strategy for treating EMT in mice with liver-related hepatocarcinoma, paving the way for a new treatment for cancer therapy.

[0051] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A high glycosidase expressing exosome system, characterized in that, The exosome system is obtained by transfecting glycosidase sequences into human mesenchymal stem cells through lentivirus transfection, and then through ultracentrifugation method, the exosome is spherical in appearance, and the average diameter is 120 nm-125 nm.

2. The method of claim 1, wherein the high glycosidase-expressing exosome system is prepared by the steps of: (a) culturing the cell expressing the glycosidase in a culture medium; (b) collecting the exosome from the cell; and (c) purifying the exosome. The specific steps are as follows: 1) Preparation of human mesenchymal stem cells; 2) Preparation of human mesenchymal stem cells with high expression of glycosidase, high expression of glycosidase lentivirus particles are diluted with culture medium, human mesenchymal stem cells are added to the culture medium, and the culture medium is incubated in an incubator, and the transfected cells expressing the drug resistance gene are selected by adding puromycin until no cell death occurs; 3) Preparation of exosomes with high expression of glycosidase, human mesenchymal stem cells are cultured in exosome-free medium, and the exosome system is obtained after centrifugation of the medium once and twice.

3. The production method according to claim 2, characterized by, Step 2) the titer of the high expression glycosidase lentivirus particles is 10 9 copies / mL.

4. The preparation method according to claim 2, characterized in that, Step 2) The culture medium is DMEM / F12 medium, and the volume ratio of high expression of glycosidase lentivirus particles to DMEM / F12 medium is 1:1000.

5. The preparation method according to claim 2, characterized in that, Step 2) the concentration of the human mesenchymal stem cells in the culture medium is 10 6 cells / mL.

6. The preparation method according to claim 2, characterized in that, Step 2) The concentration of puromycin in the culture medium after adding to the culture medium is 2 μg / mL.

7. The preparation method according to claim 2, characterized in that, Step 3) The first centrifugation step is: under room temperature conditions, the medium is centrifuged at 300xg for 10 minutes, 2000xg for 10 minutes, and 10000xg for 30 minutes, and the obtained precipitated particles are resuspended in PBS.

8. The preparation method according to claim 2, characterized in that, Step 3) The second centrifugation step is: the precipitate obtained by the first centrifugation is centrifuged at 100000xg at 4°C for 70 minutes, the supernatant is removed, and the obtained precipitate is the exosome.

9. Use of the exosome system with high expression of glycosidase according to any one of claims 1-8 in the preparation of a drug for fatty liver-related liver cancer.

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

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