An exosome system with high expression of glycosidase and a preparation method and application thereof

Abstract

The application discloses a kind of for treating based on glucose metabolism's lipaemia related hepatoma's biomimetic high expression glycosidase exosome system and preparation method, by lentivirus transfection glycosidase sequence is transfected to mesenchymal stem cell, then by ultracentrifugation method obtains exosome for the treatment of lipaemia related hepatoma.The exosome biomimetic system of the application has targeted migration ability, low immunogenicity, improves the ability of transporting bioactive substance, makes interrupted glucose metabolism normal, reduces endoplasmic reticulum stress, inhibits epithelial-mesenchymal transition signal transduction.It is verified by in-vivo and in-vitro experiment two aspects that it can effectively target cancer cells in vitro, reduce the abnormal modification of O-GlcNAc, inhibit the malignant behavior of tumor;After reaching lipaemia related hepatoma in vivo, it can reduce the modification level of O-GlcNAc, restore the damaged metabolism, so as to inhibit the progress of cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a kind of bionic high expression glycosidase exosome system for treating fat liver related liver cancer based on glucose metabolism and its preparation method and application. BACKGROUND

[0002] With the increase in the prevalence of obesity, the proportion of fat liver related liver cancer in hepatocellular carcinoma is also increasing. Surgical resection is the main clinical treatment for this type of tumor, and drug therapy is also used as an intervention strategy to prevent tumor recurrence and improve patient survival. Drug therapy usually targets multiple signaling pathways to promote tumor cell death. Specifically, intervention in metabolic pathways plays an important role in inducing tumor cell apoptosis and inhibiting tumor metastasis. Among these pathways, the regulation of glucose metabolism, especially in the context of fatty liver and overnutrition, is crucial for inhibiting tumor growth. Therefore, many anti-tumor therapies targeting glucose metabolism have been developed. However, despite significant progress, the mechanisms of glucose metabolism in fat liver related liver cancer are still not well understood. Therefore, the development of effective treatment strategies based on glucose metabolism is still promising.

[0003] Through sequencing analysis of clinical resection samples of fat liver related liver cancer, we found that O-GlcNAcase (OGA) enzyme was significantly reduced in these tumor tissues, and previous studies have shown that a sharp increase in O-linked N-acetylglucosamine (O-GlcNAc) modification plays a key role in abnormal tumor glucose metabolism. However, the underlying mechanisms of O-GlcNAc modification are still largely unexplored, resulting in suboptimal results when targeting O-GlcNAc with drugs alone. We found that in fat liver related liver cancer, the key enzyme OGA that dynamically and transiently regulates O-GlcNAc is downregulated. By increasing OGA levels, abnormal O-GlcNAc modification can be reduced, restoring disrupted energy metabolism and alleviating endoplasmic reticulum (ER) stress. We believe that regulating OGA simultaneously regulates metabolic reprogramming and the function of energy-related organelles, providing a promising approach for liver cancer targeted therapy.

[0004] Compared to conventional mesenchymal stem cells, exosomes have the ability to target migration, low immunogenicity, and efficient transport of bioactive substances, making them adaptable and controllable in therapeutic applications. Therefore, to test this hypothesis, we developed an OGA high expression mesenchymal stem cell (MSC OGA ) exosome system (MSC OGA -EXOs) to treat fat liver related liver cancer. SUMMARY

[0005] Invention purposes: In order to solve the above technical problems, we propose an exosome system (MSC OGA -EXOs) for the treatment of fatty liver related liver cancer based on glucose metabolism, which is prepared by lentiviral transfection and ultracentrifugation method, which is simple, universal and easy to mass production.

[0006] Technical scheme: The exosome system (MSC OGA -EXOs) with high expression of OGA is prepared by transfecting glycosidase sequence into human mesenchymal stem cells (MSC) by lentiviral transfection, and then obtained by ultracentrifugation method. The exosome is spherical in appearance, with 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, dilute the high expression glycosidase lentivirus particles with culture medium, add MSC to the culture medium, and incubate in the incubator. Select the transfected cells expressing the drug resistance gene by adding puromycin until no cell death occurs.

[0010] 3) Preparation of exosome (MSC OGA -EXOs) with high expression of glycosidase, culture MSC in exosome-free medium, and obtain exosome system after primary centrifugation and secondary centrifugation 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 high expression glycosidase lentivirus particles to DMEM / F12 culture medium is 1:1000.

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

[0014] Preferably, the concentration of puromycin in the culture medium after adding puromycin 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 1Figure A is a schematic diagram of RNA sequencing, immunohistochemistry and Western blotting using clinical samples of patients with liver cancer related to fatty liver; Figure B is a comparison chart of disease-free survival (DFS) of patients with liver cancer related to fatty liver and other liver cancer patients for 3 years after surgery; Figure C is a comparison of 3-year disease-free survival (DFS) of liver cancer patients with and without metabolic syndrome; Figure D is a Cox proportional hazards regression analysis of baseline patient disease-free survival (DFS) data; Figure E is the identification of differentially expressed genes in cancer and paracancer tissues of patients with liver cancer related to fatty liver by RNA sequencing analysis; Figure F is the expression level of OGA and OGT in cancer (C group) and paracancer (P group) tissues of patients with liver cancer related to fatty liver detected by Western blotting; Figures G-H are immunohistochemical staining of OGA and OGT expression in cancer and paracancer tissues of patients with liver cancer related to fatty liver, the scale bar is 100 μm.

[0024] Figure 2 MSC OGA Figure A is a schematic diagram of lentivirus system transfection of mesenchymal stem cells and secretion of exosomes; Figure B is the morphology of Vector and MSCs transfected with OGA, shown by green fluorescence of EGFP, the scale bar is 50 μm; Figures C-D are flow cytometry analysis of positive and negative surface markers of MSCs after OGA transfection; Figure E is a TEM image showing the morphology of the obtained exosomes, the scale bar is 50 μm; Figure F is a nanoparticle tracking analyzer (NTA) showing the size distribution of MSC OGA EXOs; Figure G is the identification of the obtained exosomes by Western blotting.

[0025] Figure 3 MSC OGA Figure A-D are the uptake of MSC VEC EXOs and MSC OGA EXOs by tumor cells (T group) and primary hepatocytes (P group), the scale bar is 10 μm; Figure E is the quantitative analysis of the uptake of MSC OGA EXOs before and after by Western blotting; Figures F-G are scratch experiments showing untreated tumor cells, MSC Vec EXOs-treated tumor cells and different concentrations of MSC OGA EXOs-treated tumor cells at 0, 12 and 24 hours, the scale bar is 100 μm; Figure H is the migration of untreated tumor cells (Con), MSCVec -EXOs (Vec) and different concentrations of MSCs OGA - EXOs migration to tumor cells, scale bar 100 μm; Figure I shows Western blotting analysis of untreated tumor cells (Con) and MSCs. Vec -EXOs-treated tumor cells (Vec) and different concentrations of MSCs OGA -Endoplasmic reticulum stress level in EXOs-treated tumor cells; Figure J shows Western blotting analysis of untreated tumor cells (Con) and MSCs. Vec -EXOs-treated tumor cells (Vec) and different concentrations of MSCs OGA - EMT levels in tumor cells treated with EXOs.

[0026] Figure 4 MSCs in mice with in situ fatty liver-associated hepatocellular carcinoma OGA - Schematic diagram of in vivo evaluation of EXOs: Figure A is a schematic diagram of the construction of STAM model mice simulating fatty liver-related hepatocellular carcinoma and exosome treatment; Figure B is a small animal in vivo imaging after injection in each group of mice: NC: normal mice injected with PBS; NC-Vec: normal mice injected with MSCs Vec -EXOs; NC-OGA: Normal mice injected with MSCs OGA -EXOs; STAM: STAM mice injected with PBS; STAM-Vec: mice injected with MSCs Vec STAM mice with EXOs; STAM-OGA: injected with MSCs OGA STAM mice with EXOs; Figure CE shows the body weight, liver weight, and liver-to-body ratio of the six groups of mice; Figure F shows HE staining of tumors and adjacent tissues in STAM, STAM-Vec, and STAM-OGA mice, with a scale bar of 400 μm.

[0027] Figure 5 For MSC OGA - Schematic diagram of the in vivo evaluation of the therapeutic effect of EXOs on fatty liver-related hepatocellular carcinoma in mice: Figures AC and D show the expression levels of ALT, AST, and AFP in the six groups of mice; Figures DE and E show the expression levels of OGA and OGT in cancerous and adjacent tissues as shown by immunohistochemistry, with a scale bar of 50 μm; Figure FG shows the expression levels of endoplasmic reticulum stress and EMT in the six groups of mice analyzed by Western blotting. Detailed Implementation

[0028] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

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

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

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

[0032] The cells were cultured in 75T culture flasks. They were placed in a constant temperature cell incubator at a temperature of 37°C containing 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] Preparation of MSCs stably transduced 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 to make the concentration of MSCs in the culture medium 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-resistant 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-EXOs) with high expression of nucleosidase (OGA) 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 obtained was exosomes.

[0037] Example 2. Expression profiling of O-GlcNAc-related 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. Figure 1 A). On the 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 hepatocarcinoma patients. The results showed that the 3-year DFS of patients with hepatocarcinoma complicated with fatty liver was significantly shorter than that of other hepatocarcinoma patients ( Figure 1 B). 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 ( Figure 1 C). 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 for the 3-year DFS of hepatocarcinoma patients ( Figure 1 D). These findings suggest that metabolic reprogramming, one of the ten hallmarks of malignancy, plays an indispensable role in the development of fatty liver-related hepatocarcinoma.

[0039] In various metabolic reprogramming processes, glucose metabolism disorders are considered a key factor in the development of fatty liver and overnutrition, and ultimately, hepatocellular carcinoma (HCC). However, our understanding of the role of O-GlcNAc modification in fatty liver-related HCC remains limited. To elucidate the mechanisms of O-GlcNAc modification and its potential targets in fatty liver-related HCC, we analyzed samples from clinical surgical patients using RNA sequencing, immunohistochemistry, and Western blotting. RNA sequencing analysis of cancerous and adjacent normal tissues from patients with fatty liver-related HCC revealed that, compared to adjacent normal tissues, genes responsible for glycosylation were upregulated in cancerous tissues, with genes associated with O-GlcNAc transferases (OGTs) being particularly prominent. Figure 1 E). Conversely, the expression of genes involved in deglycosylation, primarily OGA, was found to be reduced. Immunohistochemical and Western blot analyses also indicated that OGT expression was high in fatty liver-associated cancer tissues, while OGA expression was decreased. Figure 1 These observations suggest that reduced OGA expression in hepatocellular carcinoma tissues associated with fatty liver disease may be associated with poorer patient prognosis. Therefore, given the crucial role of OGA in the removal of OGlcNAc modification, reduced OGA expression in these hepatocellular carcinoma tissues may lead to poor prognosis.

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

[0041] We developed a method for high expression of OGA-MSCs OGA Exosome system (MSC) OGA -EXOs), used for the treatment of fatty liver-related liver cancer ( Figure 2 A). Therefore, we infected MSCs with a lentiviral vector carrying the OGA gene and selected them using puromycin. Under a microscope, we observed that MSCs maintained a spindle-shaped morphology after infection. Figure 2 B). To verify that lentiviral transduction does not induce MSC differentiation, we also examined the expression of MSC-specific positive and negative surface markers. Flow cytometry analysis confirmed that OGA-modified MSCs continued to express CD73, CD90, and CD105, but not CD34, CD45, or HLA-DR (B). Figure 2 (C, D). To obtain the desired exosomes, we centrifuged the supernatant to further isolate exosomes from MSCs. Transmission electron microscopy (TEM) showed that MSCs... OGA -EXOs have a spherical morphology with an average diameter of approximately 123 nm. Figure 2 EF). Furthermore, exosomes were confirmed using membrane proteins such as D63, CD81, and the membrane-associated protein TSG101.Figure 2 G). These data indicate that we have successfully obtained MSC-derived exosomes overexpressing OGA (MSCs). OGA -EXOs).

[0042] Example 4 MSC OGA Evaluation of EXOs uptake and function in liver tumor cells

[0043] In the generation of MSC OGA Following EXOs, we next assessed the function of hepatoma cells and primary hepatocytes to examine their uptake efficiency. We labeled MSCs with 1,1-octadecyl-3,3,3,3-tetramethyldicarbocyanine, 4-chlorobenzenesulfonate (DiD). OGA -EXOs were co-cultured with cells for 24 h, and the results were observed using confocal laser scanning microscopy. The images showed red fluorescence in the cytoplasm, confirming the internalization of exosomes into the cells. Figure 3 (AD). Interestingly, we also observed a peculiar phenomenon: tumor cells took up more DiD than hepatocytes, indicating that tumor cells have a more active phagocytic capacity. This phenomenon facilitates the effective absorption of our prepared exosomes by tumor cells, enabling them to exert their intended effects.

[0044] To verify the effect of exosome uptake on the overall O-GlcNAc modification level in tumor cells, we used Western blotting to detect the expression of OGA and OGT proteins in these cells. The results showed that compared with the primary hepatocyte group (P group), tumor cells (T group) had increased O-GlcNAc modification levels, enhanced OGT production, and decreased OGA expression. This effect was observed after introducing MSCs... OGA -EXOs were reversed afterward. Furthermore, with the absorption of MSCs... OGA Compared to primary hepatocytes (P+E group) of EXOs, MSCs were absorbed. OGA Tumor cells of -EXOs (T+E group) showed significantly higher OGA expression and reduced OGT production. Figure 3 E). To determine the impact of OGA on hepatocellular carcinoma cell progression, we conducted migration and invasion assays to assess how exosome-induced changes in OGA expression levels affected the migration and invasion characteristics of hepatocellular carcinoma cells. These experimental data showed that OGA-treated hepatocellular carcinoma cells had significantly reduced migration and invasion potential (E). Figure 3 FH).

[0045] To explore MSC OGA-The effect of EXOs uptake on the endoplasmic reticulum (ER): We assessed ER stress levels using Western blotting analysis. In our results, we observed that OGA overexpression inhibited ER stress, and that ER stress levels were further suppressed with increasing exosome concentration. Figure 3 I). Furthermore, we explored the mechanism by which OGA regulates the migration and invasion of hepatocellular carcinoma cells. Through Western blotting analysis, we confirmed that overexpression of OGA can upregulate the expression levels of EMT-related proteins, such as ZEB1, Snail, N-cadherin, and Vimentin, indicating that MSCs… OGA -EXOs treatment can inhibit the occurrence of EMT in liver cancer cells ( Figure 3 J). Therefore, from Figure 4 At all cellular levels, we found that tumor cells could internalize exosomes, overexpress OGA, and with OGA uptake, tumor cells exhibited reduced migration and invasion abilities. Furthermore, these results indicate that our MSCs... OGA EXOs can effectively reduce the malignant behavior of tumor cells and are expected to play a role in cancer treatment.

[0046] Example 5: MSCs in mice with orthotopic fatty liver-associated hepatocellular carcinoma OGA -EXOs's internal evaluation

[0047] To investigate the actual role of the OGA gene in fatty liver-related hepatocellular carcinoma in vivo, we used the Stelic animal model (STAM) and a normal diet model to study MSCs. OGA -Modeling research was conducted on EXOs testing ( Figure 4 A). For the STAM model, male mice were intraperitoneally injected with 200g streptozotocin (STZ) within 5 days of birth, and then fed a 60% high-fat diet (HFD) starting at 3 weeks of age. Normal-diet mice and STAM model mice were then divided into three groups, and at week 4, they were injected with phosphate-buffered saline (NC), STAM, and MSC, respectively. Vec -EXOs (NC-Vec, STAM-vec); MSC OGA -EXOs (NC-OGA, STAM-OGA) were collected from mice at 16 weeks of age. To confirm their in vivo targeting ability, bioluminescent imaging was used to track exosomes entering the body. Imaging results showed that most exosomes were localized to the liver, suggesting their targeting ability and potential for subsequent antitumor effects in the liver. Figure 4 B). By examining mouse liver and body weight, we further analyzed MSCs after targeting the liver. OGA -The effect of EXOs on tumorigenesis ( Figure 4C-E). Although the body weight of the fatty liver mice was higher than that of the control mice, their body weight at 16 weeks was actually lower due to the progression of the tumor. In addition, the liver weight of the STAM mice was also higher due to the tumor burden, resulting in a larger liver weight ratio compared to the control mice. Furthermore, the body weight of the STAM-OGA group mice was higher than that of the STAM-Vec group, and the liver weight and liver weight ratio were significantly lower than those of the STAM-Vec group. It is worth mentioning that the livers of the STAM mice had obvious carcinogenic effects, with single or multiple tumors. In contrast, the tumor size of the STAM-OGA group was significantly reduced compared to the other groups (F). Figure 4 F). 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 -EXOs for the treatment of fatty liver-related liver cancer in mice

[0049] To further evaluate the effect of MSC OGA -EXOs on tumor progression, we also evaluated the liver function indicators and alpha-fetoprotein (AFP) levels of the liver cancer mice. Compared with the normal diet mice, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alpha-fetoprotein (AFP) in the serum of the three STAM groups of mice were increased. However, after MSC OGA -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 investigate 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 (5D, E), indicating that MSC Figure 5 D, E), indicating that MSC OGA -EXOs can effectively regulate the recovery of dysregulated O-GlcNAc modification, thereby controlling tumor progression.

[0050] To elucidate the inhibition mechanism of MSC OGA -EXOs, we also detected the levels of endoplasmic reticulum stress (ERS) and EMT in vivo, both of which are indicators of tumor-related malignancy. Our results in the STAM-OGA group showed that the level of endoplasmic reticulum stress was significantly reduced, indicating that MSC OGA -EXOs can significantly inhibit endoplasmic reticulum stress, thereby inhibiting tumor progression (6A, B). Figure 5F). In addition, we investigated 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 5 G). These results show that our exosomes can be a viable strategy for treating EMT in mice with liver-related hepatocarcinoma, paving the way for new therapeutic approaches to cancer treatment.

[0051] The above only describes the 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. Use of an exosome system highly expressing glycosidase O-GlcNAcase (OGA) in the preparation of a drug for treating fatty liver related hepatocarcinoma, characterized in that, The exosome system is obtained by transfecting glycosidase sequences into human mesenchymal stem cells through lentiviral transfection, and then obtained by ultracentrifugation method, the exosome is spherical in appearance, and the average diameter is 120nm-125nm; the preparation steps of the high expression glycosidase exosome system are as follows: Step 1) preparation of human mesenchymal stem cells; Step 2) preparation of human mesenchymal stem cells with high expression of glycosidase O-GlcNAcase (OGA), the high expression glycosidase lentivirus particles are diluted with culture medium, and the human mesenchymal stem cells are added to the culture medium, and then incubated in the incubator, and the transfected cells expressing the drug resistance gene are selected by adding puromycin until no cell death occurs; Step 3) preparation of high expression glycosidase exosome, the human mesenchymal stem cells are cultured in exosome-free medium, and the exosome system is obtained after once centrifugation and twice centrifugation of the medium, the once centrifugation step is: under room temperature conditions, the medium is sequentially centrifuged at 300x g for 10 minutes, 2000x g for 10 minutes and 10000x g for 30 minutes, and the obtained precipitated particles are resuspended in PBS; The twice centrifugation step is: the precipitate obtained by once centrifugation is centrifuged at 100000x g for 70 minutes at 4°C, the supernatant is removed, and the obtained precipitate is the exosome.

2. Use according to claim 1, characterized in that, Step 2) the titer of the high expressing glycosidase lentivirus particles is 10 9 copies / mL.

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

4. Use according to claim 1, characterized in that, Step 2) the concentration of the human mesenchymal stem cells in the culture medium is 10 6 cells / mL.

5. The use according to claim 1, characterized in that, Step 2) the concentration of puromycin in the culture medium after adding to the culture medium is 2μg / mL.

6. Use according to claim 1, characterized in that, The drug is a fatty liver related hepatocellular carcinoma drug based on glucose metabolism.

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