Exosome system for highly expressing glycosidase as well as preparation method and application of exosome system

Abstract

The invention discloses a bionic high-expression glycosidase exosome system for treating fatty liver-related liver cancer based on glucose metabolism and a preparation method thereof, a glycosidase sequence is transfected to mesenchymal stem cells through lentivirus transfection, and then exosomes are obtained through an ultracentrifugation method for treating the fatty liver-related liver cancer. The exosome bionic system disclosed by the invention has targeted migration capability and low immunogenicity, improves the capability of transporting bioactive substances, normalizes interrupted glucose metabolism, reduces endoplasmic reticulum stress and inhibits epithelial-mesenchymal transition signal transduction. In-vivo and in-vitro experiments prove that the compound can effectively target cancer cells in vitro, reduce abnormal modification of O-GlcNAc and inhibit malignant behaviors of tumors; after reaching the fatty liver related liver cancer in vivo, the compound can reduce the O-GlcNAc modification level and recover damaged metabolism, thereby inhibiting the progress of cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a biomimetic exosome system with high expression of glycosidase for treating fatty liver-related liver cancer based on glucose metabolism, and a preparation method and application thereof. Background Art

[0002] As the prevalence of obesity increases, the proportion of fatty 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 has also been 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 through 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, there is still a lack of in-depth understanding of the mechanism of glucose metabolism in fatty liver-related liver cancer. Therefore, the development of effective therapeutic strategies based on glucose metabolism is still worth looking forward to.

[0003] Through sequencing analysis of clinical resection samples of fatty liver-related HCC, we found that the O-GlcNAcase (OGA) enzyme was significantly reduced in these tumor tissues. Previous studies have shown that a dramatic 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 remain largely unexplored, resulting in suboptimal results when O-GlcNAc is targeted with drugs alone. We found that OGA, a key enzyme that dynamically and temporally regulates O-GlcNAc, is downregulated in fatty liver-related 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 to simultaneously regulate metabolic reprogramming and the function of energy-related organelles provides a promising avenue for targeted therapy of liver cancer.

[0004] Compared with conventional mesenchymal stem cells, exosomes have targeted migration ability, low immunogenicity, and efficient bioactive substance transport capacity, so they are adaptable and controllable in therapeutic applications. Therefore, to confirm 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 of the invention

[0005] Purpose of the invention: The purpose of the present invention is to solve the above technical problems. We propose a biomimetic exosome system (MSC OGA -EXOs), used for the treatment of fatty liver-related liver cancer based on glucose metabolism, the exosome system was prepared by lentiviral transfection and ultracentrifugation, the method is simple, versatile and easy to large-scale production.

[0006] Technical solution: The exosome system (MSC) highly expressing OGA described in the present invention OGA -EXOs), which are obtained by transfecting the glycosidase sequence into human mesenchymal stem cells (MSCs) through lentiviral transfection and then obtaining them through ultracentrifugation. The exosomes are spherical in shape with an average diameter of 120 nm~125 nm.

[0007] The specific steps are as follows: 1) Preparation of human mesenchymal stem cells (MSC); 2) Human mesenchymal stem cells (MSC) with high expression of glycosidase OGA） Preparation, by lentiviral transfection, diluting the highly expressed glycosidase lentiviral particles with culture medium, adding MSCs to the culture medium, incubating them together in an incubator, and selecting the transfected cells expressing the drug-resistant gene by adding puromycin until there is no cell death; 3) Exosomes with high expression of glycosidase (MSC OGA -EXOs) were prepared by culturing MSCs in a medium lacking exosomes, and the medium was centrifuged once and twice to obtain the exosome system.

[0008] Preferably, the titer of the highly expressed glycosidase lentiviral particles in step 2) is 10 9 copies / mL.

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

[0010] Preferably, the concentration of MSC in the culture medium is 10 6 cells / mL.

[0011] Preferably, the concentration of puromycin in the culture medium after being added to the culture medium in step 2) is 2 μg / mL Preferably, the one centrifugation step in step 3) is: at room temperature, the culture medium is centrifuged at 300× g for 10 minutes, 2000× g for 10 minutes, and 10000× g for 30 minutes, and the obtained precipitated particles are resuspended in PBS.

[0012] Preferably, the secondary centrifugation step in step 3) is: centrifuging the precipitate obtained by the primary centrifugation at 4° C. and 100,000×g for 70 minutes, removing the supernatant, and the obtained precipitate is the exosomes.

[0013] The present invention also provides the use of an exosome system that highly expresses glycosidase in the preparation of fatty liver-related liver cancer drugs.

[0014] Preferably, the drug is a fatty liver-related liver cancer drug based on glucose metabolism.

[0015] Beneficial effects:

[0016] (1) The present invention designs an exosome system (MSC) for the treatment of fatty liver-related liver cancer with high expression of nucleosidase (OGA) OGA -EXOs), with superior targeted migration ability.

[0017] (2) The exosome system (MSC) for the treatment of fatty liver-related liver cancer with high expression of nucleosidase (OGA) provided by the present invention OGA -EXOs) are prepared by lentiviral transfection and ultracentrifugation, which is simple, easy to operate, highly reproducible, low in technical requirements, highly versatile, highly flexible, and easy to prepare on a large scale.

[0018] (3) The exosome system (MSC) prepared by the present invention for the treatment of fatty liver-related liver cancer with high expression of nucleosidase (OGA) OGA -EXOs) have targeted migration ability, low immunogenicity, improved the ability to transport bioactive substances, normalized interrupted glucose metabolism, reduced endoplasmic reticulum stress (ERS), and inhibited epithelial-mesenchymal transition (EMT) signal transduction. Through in vivo and in vitro experiments, it was verified that it can effectively target cancer cells in vitro, reduce abnormal O-GlcNAc modification, and inhibit tumor malignant behavior; after reaching fatty liver-related liver cancer in vivo, it can reduce the level of O-GlcNAc modification and restore the damaged metabolism, thereby inhibiting the progression of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Schematic diagram of expression profile analysis of O-GlcNAc-related markers in tumors of patients with fatty liver-related liver cancer: Figure A is a schematic diagram of RNA sequencing, immunohistochemistry and Western blotting using clinical samples of patients with fatty liver-related liver cancer; Figure B is a comparison of the 3-year disease-free survival (DFS) of patients with fatty liver-related liver cancer after surgery with that of other liver cancer patients; Figure C is a comparison of the 3-year disease-free survival (DFS) of liver cancer patients with and without metabolic syndrome; Figure D is a Cox proportional hazard regression analysis of baseline patient disease-free survival (DFS) data; Figure E is the identification of differentially expressed genes in cancer and adjacent tissues of patients with fatty liver-related liver cancer by RNA sequencing analysis; Figure F is the detection of the expression levels of OGA and OGT in cancer (group C) and adjacent tissues (group P) of patients with fatty liver-related liver cancer by Western blotting; Figure GH is the immunohistochemical staining of OGA and OGT expression in cancer and adjacent tissues of patients with fatty liver-related liver cancer, and the scale bar is 100 μm.

[0020] Figure 2 MSC OGA Schematic diagram of the preparation and characterization of -EXOs: Figure A is a schematic diagram of the lentiviral system transfecting mesenchymal stem cells and secreting exosomes; Figure B is the morphology of Vector and OGA-transfected MSCs, as shown by the green fluorescence of EGFP, with a scale bar of 50μm; Figures CD are flow cytometric 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, with a scale bar of 50μm; Figure F is a nanoparticle tracking analyzer (NTA) showing the morphology of MSCs OGA -Size distribution of EXOs; Figure G shows the exosomes obtained by Western blotting.

[0021] Figure 3 For MSC OGA -Schematic diagram of the uptake and function of EXOs in liver tumor cells: Figures AD show the uptake of MSCs by tumor cells and primary hepatocytes VEC -EXOs and MSC OGA -EXOs uptake, scale bar is 10μm; Figure E is Western blotting quantitative analysis of MSC uptake OGA -EXOs before and after the expression of OGA and OGT in tumor cells (T group) and primary hepatocytes (P group); Figures FG are scratch experiments showing the expression of OGA and OGT in untreated tumor cells and MSCs Vec -EXOs-treated tumor cells and MSCs at different concentrations OGA -EXOs treated tumor cells at 0, 12 and 24 hours, scale bar is 100μm; Figure H shows the migration of untreated tumor cells (Con), MSCs at 0, 12 and 24 hours.Vec -EXOs (Vec) and different concentrations of MSC OGA -EXOs migration experiment to tumor cells, scale bar is 100μm; Figure I is Western blotting analysis of untreated tumor cells (Con), MSC Vec -EXOs-treated tumor cells (Vec) and different concentrations of MSC OGA -EXOs-treated tumor cells ER stress level; Figure J is Western blotting analysis of untreated tumor cells (Con), MSC Vec -EXOs-treated tumor cells (Vec) and different concentrations of MSC OGA -EMT level of tumor cells treated with EXOs.

[0022] Figure 4 MSCs in orthotopic fatty liver-related hepatocellular carcinoma mice 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 liver cancer and exosome treatment; Figure B is the in vivo imaging of small animals after injection of each group of mice: NC: normal mice injected with PBS; NC-Vec: normal mice injected with MSC Vec -EXOs; NC-OGA: normal mice injected with MSCs OGA -EXOs; STAM: STAM mice injected with PBS; STAM - Vec: injected with MSC Vec -STAM mice with EXOs; STAM-OGA: injected with MSC OGA -EXOs STAM mice; Figures CE are the body weight, liver weight and liver-to-body ratio of the six groups of mice; Figure F is HE staining of tumor and adjacent cancer tissues of STAM, STAM-Vec and STAM-OGA mice, scale bar is 400μm.

[0023] Figure 5 For MSC OGA Schematic diagram of in vivo evaluation of the therapeutic effect of -EXOs on fatty liver-related liver cancer in mice: Figures AC show the expression levels of ALT, AST, and AFP in the six groups of mice; Figures DE show the expression levels of OGA and OGT in cancer tissues and adjacent tissues by immunohistochemistry, with a scale 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 DESCRIPTION

[0024] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments and drawings. The embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0025] The human mesenchymal stem cells used in the following examples were purchased from the American Type Culture Collection (ATCC).

[0026] Example 1 Exosome system (MSC) highly expressing nucleosidase (OGA) OGA -EXOs) preparation method (1) Preparation of human mesenchymal stem cells (MSC): Use 75T culture flasks to culture cells. Place them in a constant temperature cell culture incubator at 37°C and 5% CO2. When the cells grow to cover 80%-90% of the bottom area of ​​the culture flask, subculture them. First, remove the old culture medium in the culture dish, wash twice with 3ml of neutral phosphate buffered saline (PBS), then add 3ml of 0.25% trypsin and digest in a 37°C incubator for 2-3 minutes, then add 3ml of the above culture medium to terminate digestion. Use a pipette to draw the cell suspension into a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, add 3ml of the above culture medium again to resuspend the cells, and subculture them in a new culture flask at a ratio of 1:3.

[0027] (2) Preparation of lentiviral transfected OGA Carrying human OGA gene (10 9 Preparation of MSCs by stable transduction with lentivirus containing 500 copies / mL OGA After diluting the OGA lentiviral particles with DMEM / F12 medium at a volume ratio of 1:1000, 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 24 hours of incubation, the medium containing the lentiviral particles was replaced with fresh medium and incubated for another 48 hours. Transfected cells expressing the drug resistance gene were selected by adding puromycin at a concentration of 2 μg / mL in the medium until there was no cell death. The expression of OGA was detected by qPCR and Western blot. After transfection, cell purity was detected by flow cytometry and positive and negative markers were detected.

[0028] (3) Exosome system (MSC) with high expression of nucleosidase (OGA) OGA Preparation of -EXOs To extract exosomes, MSCs were cultured in a medium lacking exosomes. The medium was centrifuged for 10 minutes at 300 × g at room temperature, then at 2000 × g for 10 minutes, and then at 10,000 × g for 30 minutes. The supernatant was collected and the pellet was resuspended in PBS. The pellet resuspended in PBS was centrifuged again at 4°C and 100,000 × g for 70 minutes. The supernatant was removed and the pellet obtained was the exosome.

[0029] Example 2 Analysis of the expression profile of O-GlcNAc-related markers in tumors of patients with fatty liver-related liver cancer To confirm the effect of overnutrition on the malignancy of fatty liver-related HCC, we retrospectively analyzed the prognosis of patients with fatty liver-related HCC 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 liver cancer with those of other liver cancer patients. The results showed that the 3-year DFS of patients with liver cancer and fatty liver was significantly shorter than that of other liver cancer 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 patients' 3-year DFS and overall survival, we performed Cox proportional hazards regression analysis on 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 3-year DFS in patients with liver cancer ( Figure 1 D). These findings suggest that metabolic reprogramming, one of the ten hallmarks of malignancy, plays an integral role in the development of fatty liver-related HCC.

[0030] Among different metabolic reprogramming, glucose metabolism disorders are considered to be a key link in the development of fatty liver and overnutrition and liver cancer. However, the understanding of the role of O-GlcNAc modification in fatty liver-related liver cancer remains limited. In order to elucidate the mechanism of O-GlcNAc modification and its potential targets related to fatty liver-related liver cancer, we used RNA sequencing, immunohistochemistry and Western blotting to analyze samples from clinical surgical patients. Through RNA sequencing analysis of cancer tissues and paracancerous tissues of patients with fatty liver-related liver cancer, we found that compared with paracancerous tissues, genes responsible for glycosylation were upregulated in cancer tissues, among which genes related to O-GlcNAc transferase (OGT) were particularly significant ( Figure 1 E). In contrast, the expression of genes involved in deglycosylation, mainly OGA, was found to be reduced. Immunohistochemistry and Western blot analysis results also showed that OGT was expressed at a higher level in cancer tissues associated with fatty liver, while OGA expression level was reduced ( Figure 1 FH). These observations suggest that reduced OGA expression in HCC tissues associated with fatty liver disease may be associated with poor patient outcomes. Therefore, given the critical role of OGA in the removal of OGlcNAc modifications, reduced OGA expression in these HCC tissues may contribute to poor outcomes.

[0031] Example 3 MSC OGA Preparation and characterization of -EXOs We developed a type of OGA-high-expressing MSC OGA Exosome system (MSC OGA -EXOs), for the treatment of fatty liver-related liver cancer ( Figure 2 A). To this end, 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 detected the expression of positive and negative surface markers specific to MSCs. Flow cytometric analysis confirmed that OGA-modified MSCs continued to express CD73, CD90, and CD105, but did not express CD34, CD45, and HLA-DR ( Figure 2 C, D). In order to obtain the desired exosomes, we centrifuged the supernatant and further isolated exosomes from MSCs. Transmission electron microscopy (TEM) showed that MSC OGA -EXOs have a spherical morphology with an average diameter of approximately 123 nm ( Figure 2 EF). In addition, 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 OGA-overexpressing MSC-derived exosomes (MSC OGA -EXOs).

[0032] Example 4 MSC OGA -EXOs uptake and functional evaluation in liver tumor cells In the production of MSC OGA -EXOs, we next evaluated their functionality using liver tumor cells and primary hepatocytes to examine their uptake efficiency. We labeled MSCs with 1,1-dioctadecyl-3,3,3,3-tetramethyldicarbocyanine, 4-chlorobenzenesulfonate (DiD). OGA-EXOs were co-cultured with cells for 24 h and the results were observed using a confocal laser scanning microscope. The resulting images showed red fluorescence in the cytoplasm, confirming the cellular internalization of exosomes ( Figure 3 AD). Interestingly, we also observed a peculiar phenomenon that the uptake of DiD by tumor cells was higher than that by liver cells, indicating that tumor cells have more active phagocytic ability. This phenomenon is conducive to the effective absorption of our prepared exosomes by tumor cells, allowing them to play the expected role.

[0033] 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 increased, OGT production was enhanced, and OGA expression was reduced. This effect was not observed after the introduction of MSCs. OGA -EXOs were reversed. OGA -EXOs were absorbed by primary hepatocytes (P+E group) compared to MSCs OGA -EXOs tumor cells (T+E group) showed significantly higher OGA expression and reduced OGT production ( Figure 3 E). To determine the effect of OGA on the progression of liver cancer cells, we performed migration and invasion assays to evaluate how changes in OGA expression levels induced by exosome treatment affect the migration and invasion properties of liver cancer cells. These experimental data showed that the migration and invasion potential of liver cancer cells treated with OGA was significantly reduced ( Figure 3 FH).

[0034] To explore MSC OGA -The effect of exosome uptake on the ER, we evaluated the ER stress level by Western blotting analysis. In our results, we observed that overexpression of OGA could inhibit ER stress, and the ER stress level could be further inhibited as the exosome concentration increased ( Figure 3 I). In addition, we also explored the mechanism by which OGA regulates the migration and invasion of liver cancer 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 MSC OGA -EXOs treatment can inhibit the occurrence of EMT in liver cancer cells ( Figure 3 J). Therefore, from Figure 4At all cellular levels, we found that tumor cells could internalize exosomes, overexpress OGA, and that following OGA uptake, tumor cells showed reduced migration and invasion abilities. Furthermore, these results suggest that our MSCs OGA -EXOs can effectively reduce the malignant behavior of tumor cells and are expected to play a role in tumor treatment.

[0035] Example 5 MSCs in mice with in situ fatty liver-related liver cancer OGA -EXOs' in vivo evaluation To investigate the actual role of OGA gene in fatty liver-related HCC in vivo, we used the Stelic animal model (STAM) and the normal diet model to regulate MSCs. OGA -EXOs tests were conducted for modeling studies ( Figure 4 A). For the STAM model, male mice were intraperitoneally injected with 200 g of streptozotocin (STZ) within 5 days after birth, and then given a 60% high-fat diet (HFD) from 3 weeks of age. Mice on a normal diet and STAM model mice were further divided into three groups and injected with phosphate-buffered saline (NC, STAM); MSC Vec -EXOs (NC-Vec, STAM-vec); MSC OGA -EXOs (NC-OGA, STAM- OGA), and then samples were collected when the mice were 16 weeks old. To confirm the in vivo targeting ability, bioluminescence imaging was used to track the exosomes entering the body. From the imaging results, we observed that most of the exosomes were localized in the liver, indicating their targeting ability and subsequent potential to exert anti-tumor effects in the liver ( Figure 4 B). We further analyzed the effects of MSC targeting on the liver by measuring the liver and body weight of mice. OGA -The effect of EXOs on tumorigenesis ( Figure 4 CE). Although the weight of mice with fatty liver was higher than that of control mice, they actually weighed less at 16 weeks due to tumor progression. In addition, the liver weight of STAM mice was also higher due to tumor burden compared with control mice, resulting in a larger liver-to-body weight ratio. In addition, the weight of mice in the STAM-OGA group was higher than that of the STAM-Vec group, and the liver weight and liver-to-body weight ratio were significantly lower than those in the STAM-Vec group. It is worth mentioning that the liver of STAM mice has obvious carcinogenic effects, with single or multiple tumors. In contrast, the tumor size in the STAM-OGA group was significantly reduced compared with the other groups ( Figure 4 F). These data suggest that our liver-targeted MSCs OGA -EXOs can significantly inhibit the occurrence and progression of liver cancer associated with fatty liver.

[0036] Example 6 MSC OGA In vivo evaluation of the therapeutic effect of -EXOs on fatty liver-related hepatocellular carcinoma in mice To further evaluate the MSC OGA -EXOs on tumor progression, we also evaluated liver function indicators and alpha-fetoprotein (AFP) levels in mice with liver cancer. Compared with mice on a normal diet, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alpha-fetoprotein (AFP) were increased in the three STAM groups. OGA After treatment with -EXOs, the levels of ALT, AST, and AFP were significantly decreased in the STAM-OGA group compared with the STAM and STAM-Vec groups (5A-C). This indicates the improvement of liver function and the effectiveness of anti-tumor treatment in the STAM-OGA group. 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, OGA expression was effectively enhanced and OGT levels were decreased ( Figure 5 D, E), indicating that MSC OGA -EXOs can effectively regulate the restoration of dysregulated O-GlcNAc modification, thereby controlling tumor progression.

[0037] To clarify MSC OGA -EXOs inhibition mechanism, we also examined the endoplasmic reticulum stress (ERS) and EMT levels in vivo, both of which are indicators of tumor-related malignancies. Our results in the STAM-OGA group showed a significant decrease in ERS levels, indicating that MSC OGA -EXOs can significantly inhibit endoplasmic reticulum stress, thereby inhibiting tumor progression ( Figure 5 F). In addition, we investigated proteins associated with EMT. The results showed that the cell phenotype changed and E-cadherin expression decreased, resulting in decreased cell adhesion and acquisition of invasive and migratory characteristics. We also found that loss of E-cadherin expression was the most prominent feature of EMT in mice with fatty liver-related liver cancer, and exosome treatment could prevent the progression of EMT ( Figure 5 G). These results suggest that our exosomes can serve as a feasible strategy for EMT-induced liver-related HCC treatment in mice, paving the way for new therapeutic approaches for cancer treatment.

[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An exosome system for highly expressing glycosidase, characterized in that: The exosome system is obtained by transfecting the glycosidase sequence into human mesenchymal stem cells through lentiviral transfection and then ultracentrifugation. The exosomes are spherical in shape with an average diameter of 120 nm to 125 nm.

2. The method for preparing the exosome system with high expression of glycosidase according to claim 1, characterized in that: The specific steps are as follows: 1) Preparation of human mesenchymal stem cells; 2) Preparation of human mesenchymal stem cells that highly express glycosidases, diluting the highly expressed glycosidase lentiviral particles with culture medium through lentiviral transfection, adding human mesenchymal stem cells to the culture medium, incubating them together in an incubator, and selecting transfected cells that express drug-resistant genes by adding puromycin until no cells die; 3) Preparation of exosomes with high expression of glycosidase: Human mesenchymal stem cells were cultured in a medium lacking exosomes, and the exosome system was obtained after one and two centrifugations of the medium.

3. The preparation method according to claim 2, characterized in that: Step 2) The titer of the highly expressed glycosidase lentiviral particles is 10 9 copies / mL.

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

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

6. The preparation method according to claim 2, characterized in that: After the puromycin described in step 2) is added to the culture medium, the concentration in the culture medium is 2 μg / mL.

7. The preparation method according to claim 2, characterized in that: The one centrifugation step in step 3) is: at room temperature, the culture medium is centrifuged at 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.

8. The preparation method according to claim 2, characterized in that: The secondary centrifugation step in step 3) is as follows: the precipitate obtained by the primary centrifugation is centrifuged at 4°C and 100,000×g for 70 minutes, and the supernatant is removed to obtain the precipitate, i.e., the exosomes.

9. Use of the exosome system with high expression of glycosidase as described in any one of claims 1 to 8 in the preparation of fatty liver-related liver cancer drugs.

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

Images