An engineered human umbilical cord mesenchymal stem cell overexpressing FGF2, an exosome and a preparation method and application thereof

Abstract

The application discloses an FGF2-overexpressed engineered human umbilical cord mesenchymal stem cell, an exosome and a preparation method and application thereof. An FGF2 gene is introduced into human umbilical cord mesenchymal stem cells (UC-MSCs) through a lentiviral vector, so that FGF2 protein is efficiently expressed in the UC-MSCs. High-purity exosomes secreted by the cells are obtained through the combination of ultrafiltration and differential centrifugation technology. The FGF2 gene modified UC-MSCs significantly enhance the effect of the UC-MSCs on improving liver steatosis in the treatment of metabolic dysfunction-associated steatohepatitis (MASH), and the exosomes derived from the UC-MSCs also have independent potential for treating MASH.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biomedical engineering technology, specifically to an FGF2-overexpressing engineered human umbilical cord mesenchymal stem cells and exosomes, their preparation method and application. Background Technology

[0002] Metabolic dysfunction-associated steatohepatitis (MAS), formerly known as non-alcoholic steatohepatitis (NASH), is a common chronic liver disease. Statistics show a global prevalence of up to 31%, with approximately 20% progressing to cirrhosis or liver cancer, making it one of the fastest-growing causes of liver transplantation demand. The main pathological features of MASH are excessive accumulation of triglycerides in hepatocytes, ballooning degeneration of hepatocytes, intralobular inflammation, and liver fibrosis. As the metabolic regulatory center, the liver releases cytokines and pro-inflammatory mediators based on lipid accumulation and cell damage (lipotoxicity). These mediators not only directly affect the function of target organs such as the heart, skeletal muscle, and kidneys, but also exacerbate local pathological changes, leading to multi-system involvement in clinical manifestations. The etiology of MASH is complex and diverse, driven by multiple factors including genetics, environment, obesity, immune imbalance, and diabetes. Currently, lifestyle interventions are the cornerstone of improving MASH, but they rely on long-term self-discipline and have significant individual differences in effectiveness, making them insufficient for fundamental disease control and pathological reversal. Furthermore, Resmetirom and Semaglutide, as FDA-approved drugs for treating adult MASH patients, have several limitations, including a limited target population and significant side effects. Therefore, finding effective treatments to improve hepatic lipid accumulation, inflammation, and fibrosis symptoms in MASH patients is of significant clinical importance.

[0003] Human umbilical cord mesenchymal stem cells (UC-MSCs) are self-renewing, low-immunogenic, and multi-lineage differentiation potential stem cells. They can migrate to damaged tissues, promote tissue repair and regeneration, and exert anti-inflammatory and immunomodulatory effects by paracrine secretion of various bioactive factors to regulate the microenvironment. Although UC-MSCs have shown some potential in the treatment of malignant angiopathy (MASH), their overall efficacy remains limited, mainly due to complex mechanisms of action, significant individual variability in efficacy, and insufficient cell targeting. Studies have shown that the therapeutic effect of UC-MSCs on MASH largely depends on their paracrine function, namely, regulating immunity, inhibiting fibrosis, and promoting tissue repair by releasing various bioactive factors (such as FGF2). Fibroblast Growth Factor 2 (FGF2) belongs to the fibroblast growth factor (FGF) family and plays an important role in promoting cell proliferation, participating in tissue repair, angiogenesis, and regulating metabolism. Therefore, developing FGF2-overexpressing UC-MSCs holds promise as a novel strategy for treating MASH. Summary of the Invention

[0004] The purpose of this invention is to provide FGF2-overexpressing engineered human umbilical cord mesenchymal stem cells, exosomes, their preparation methods and applications, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing FGF2-overexpressing engineered human umbilical cord mesenchymal stem cells, comprising the following steps:

[0007] S1: Human umbilical cord mesenchymal stem cells are isolated and passaged in vitro to obtain human umbilical cord mesenchymal stem cells.

[0008] S2: Construct a lentiviral vector overexpressing FGF2;

[0009] S3: The FGF2 overexpression lentiviral vector solution and Polybrene solution were mixed and added to the complete mesenchymal stem cell culture medium containing human umbilical cord mesenchymal stem cells obtained in step S1 for viral infection. The cells were cultured in an incubator at 37 ℃ and 5% CO2 for 6 h. After that, the culture medium was replaced and the cells were cultured for another 36 h. The cells and culture supernatant were then harvested. Proteins were extracted from the infected UC-MSCs and the overexpression of the FGF2 gene was detected by Western Blot. The validated FGF2 gene overexpressing UC-MSCs were then amplified and cultured in large quantities in serum-free medium.

[0010] In one or more embodiments, step S1, the in vitro isolation and passage culture of the human umbilical cord mesenchymal stem cells, includes the following steps:

[0011] S11: Rinse the umbilical cord with sterile saline in a sterile operating table, cut the umbilical cord into small segments of about 2 cm in length, peel off the arteries, veins and epidermis in the umbilical cord, and retain the Wharton's jelly in the middle.

[0012] S12: Cut the detached Wharton's jelly tissue into small pieces with a side length of 5 mm. The cut tissue pieces are seeded in 100 mm culture dishes with a spacing of 5 mm between the pieces. Add an appropriate amount of mesenchymal stem cell complete culture medium to each dish and culture at 37 ℃ and 5% CO2. Change the medium every three days. After 7-10 days of culture, the cells will crawl out of the tissue pieces and form P0 generation cells.

[0013] S13: P0 generation cells, after reaching over 90% confluence, are digested and passaged. Specifically:

[0014] Discard the old culture medium in the culture dish and gently rinse twice with PBS. Add an appropriate amount of mild stem cell digestive enzyme (Yokang, catalog number: NC1004.1) to each culture dish and incubate at room temperature for about 3 minutes. Observe under a microscope. When most of the cells are discrete spherical, add 4 mL of complete culture medium to stop the digestion. Gently pipette the cell suspension to mix and transfer it to a centrifuge tube. Centrifuge at 1300 rpm for 5 minutes and discard the supernatant. The cells harvested in this step are P0 generation cells. Seed the P0 generation cells at a density of 5000 cells / cm² into a new T175 culture flask, add an appropriate amount of culture medium to 20 mL, gently shake to mix, and place in a constant temperature and humidity incubator. Continue to culture under 5% CO2 and 37 ℃. When the cell confluence reaches 90%, perform cell digestion and passage to P1 generation cells. The passage operation uses the same steps and digestive enzymes as the primary generation. When P1 cells reach 90% confluence, they are passaged to generate P2 generation cells. After passing quality control, the harvested P2 cells are aliquoted and cryopreserved in liquid nitrogen to establish a master cell bank. P2 cells resuscitated from the master cell bank are further expanded and cultured, passaged sequentially to the P5 generation. After each passage, rigorous quality control is performed to ensure the cells possess good proliferative capacity and multi-lineage differentiation potential. Once the P5 cells meet quality standards, they are aliquoted and cryopreserved in liquid nitrogen to establish a working cell bank. The criteria for determining the quality of P5 cryopreserved cells are as follows: the cells are adherent, uniformly shaped, long spindle-shaped cells; the positive expression rates of CD73, CD90, and CD105 are greater than or equal to 95%, and the positive expression rates of CD11b, CD19, CD34, CD45, or HLA-II molecules should not exceed 2%; under in vitro induced differentiation conditions, they have the ability to differentiate into osteoblasts, chondrocytes, and adipocytes; the results of infectious disease and pathogenic microorganism tests are all negative, and the endotoxin result is less than 0.25 EU / mL; the inhibition rate of pro-inflammatory lymphocytes Th1 is greater than 50%, the inhibition rate of Th17 is greater than 40%, the promotion rate of anti-inflammatory lymphocytes Treg is greater than 40%; and the PBMC inhibition rate is greater than 50%.

[0015] In one or more embodiments, step S2, the construction of the FGF2 overexpression lentiviral vector, specifically includes:

[0016] The full-length sequence of the FGF2 gene (Gene ID: 2247) was obtained from the NCBI database and cloned into the lentiviral expression vector pLV3. The FGF2 gene was inserted using the multiple cloning site to construct the pLV3-CMV-FGF2-EF1a-CopGFP-Puro plasmid. Subsequently, using EL transfection reagent, the constructed pLV3-CMV-FGF2-EF1a-CopGFP-Puro plasmid was mixed with the helper plasmids pMD2G (encoding VSV-G envelope protein) and psPAX2 (encoding viral packaging protein) in a 4:2:1 ratio and co-transfected into 293T cells for viral packaging. After 6 h, the culture medium was replaced and the cells and culture supernatant were harvested after 36 h of further culture.

[0017] In one or more embodiments, in step S3, the final concentration of the Polybrene is 5 μg / mL.

[0018] In a second aspect, the present invention provides engineered human umbilical cord mesenchymal stem cells overexpressing FGF2 obtained according to the preparation method of the first aspect.

[0019] Thirdly, the present invention provides a method for preparing FGF2-overexpressing engineered human umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

[0020] S1: Expand and culture the FGF2-overexpressing engineered human umbilical cord mesenchymal stem cells described in the second aspect of the present invention, and collect the cell culture supernatant;

[0021] S2: The cell culture supernatant collected in step S1 is purified by a combination of ultrafiltration and differential centrifugation to obtain FGF2-overexpressing engineered human umbilical cord mesenchymal stem cell exosomes.

[0022] In one or more embodiments, step S2, the purification step combining ultrafiltration and differential centrifugation, is as follows:

[0023] S21: Remove cell debris and suspended particles from the supernatant collected in S1: Place the supernatant collected in S1 into a 50 mL centrifuge tube and centrifuge at 300 g at 4 ℃ for 10 min to remove cell debris and suspended particles. After centrifugation, transfer the supernatant to a new centrifuge tube.

[0024] S22: Ultrafiltration concentration: Transfer the supernatant into a 100 kDa MWCO ultrafiltration tube and centrifuge at 5,000 g for 30 min at 4 ℃. The concentrate after centrifugation, i.e. the liquid above the filter membrane, is the part rich in exosomes.

[0025] S23: Differential centrifugation for exosome separation: Aliquot the concentrated supernatant into ultracentrifuge tubes with a difference not exceeding 0.02 g to ensure weight balance. Level the tubes with PBS buffer and then ultracentrifuge at 100,000 g for 70 min at 4 °C. Collect the exosome precipitate at the bottom of the tubes and gently pour off the supernatant, avoiding disturbing the exosome precipitate. Gently resuspend the exosome precipitate with 2 mL of sterile PBS buffer. To ensure the purity of the exosomes, centrifuge again at 100,000 g for 70 min, remove the supernatant, and retain the exosome precipitate at the bottom.

[0026] Fourthly, the present invention provides an engineered human umbilical cord mesenchymal stem cell exosome overexpressing FGF2, obtained by the preparation method of the second aspect.

[0027] Fifthly, the present invention provides the use of FGF2, engineered human umbilical cord mesenchymal stem cells overexpressing FGF2 as described in the second aspect, or engineered human umbilical cord mesenchymal stem cell exosomes overexpressing FGF2 as described in the fourth aspect in the treatment of metabolic dysfunction-related steatohepatitis.

[0028] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0029] 1. FGF2 Gene-Modified Human Umbilical Cord Mesenchymal Stem Cells: This invention introduces the FGF2 gene into UC-MSCs via a lentiviral vector, enabling efficient expression of the FGF2 protein in the cells. The gene-modified UC-MSCs provided by this invention exhibit stronger therapeutic effects in improving liver injury compared to traditional unmodified cells. Histological analysis shows that FGF2 gene-overexpressing UC-MSCs significantly improve liver appearance, weight, lipid accumulation, inflammation, and fibrosis compared to traditional UC-MSCs. Section staining and serological testing revealed that FGF2 gene-overexpressing UC-MSCs more significantly reduce liver TG / TC and serum AST levels compared to traditional UC-MSCs, and also show a more significant decrease in liver inflammation, lipid deposition, and fibrosis levels.

[0030] 2. Cell-Derived Exosomes: Another key innovation of this invention is the use of exosomes secreted by these cells and modified exosomes to verify therapeutic effects in in vitro experiments. Exosomes, as lipid bilayer nanoparticles secreted by mesenchymal stem cells, have become an important means of treating various diseases and delivering drugs due to their biocompatibility, stability, and safety. Because exosomes naturally have a small particle size and are rich in CD47 on their surface, they carry FGF2 protein and other important bioactive molecules, allowing them to evade macrophage phagocytosis and be directly delivered to the liver, showing significant effects in improving liver damage. Attached Figure Description

[0031] Figure 1Microscopic observation results of UC-MSCs P0 generation cells

[0032] Figure 2 Microscopic observation results of UC-MSCs P5 generation cells

[0033] Figure 3 Identification results of surface markers in UC-MSCs.

[0034] Figure 4 Figure 1 shows the results of exosome particle size, morphology, and surface markers.

[0035] Figure 5 Figure showing the effect of FGF2 recombinant protein reducing lipid deposition in AML12 cells.

[0036] Figure 6 Figure showing the results of the exosome uptake experiment.

[0037] Figure 7 Figure showing the effect of MSC-CM in reducing lipid deposition through exosomes.

[0038] Figure 8 Figure showing the results of knocking down FGF2 protein expression reversing the lipid deposition reduction effect of MSC-CM.

[0039] Figure 9 . Validation diagram of FGF2 protein overexpression

[0040] Figure 10 Figure showing the effect of FGF2 protein overexpression in reducing lipid deposition in MSC-CM.

[0041] Figure 11 Figure 1. Results of treating liver injury in MASH mice with UC-MSCs overexpressing FGF2 protein.

[0042] Figure 12 PCR results of MASH mice treated with UC-MSCs overexpressing FGF2 protein.

[0043] Figure 13 Image of a section of a MASH mouse treated with UC-MSCs overexpressing FGF2 protein.

[0044] Figure 14 Figure showing the reduction of lipid deposition in AML12 cells by exosomes extracted from UC-MSCs overexpressing FGF2 protein. Detailed Implementation

[0045] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0046] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0047] Example 1: Isolation and Identification of Human Umbilical Cord Mesenchymal Stem Cells (UC-MSCs)

[0048] This embodiment describes the methods for isolating, culturing, passage, and identifying human umbilical cord mesenchymal stem cells (UC-MSCs), aiming to obtain high-purity, high-activity UC-MSCs for subsequent gene modification and clinical applications.

[0049] 1.1 Acquisition and processing of human umbilical cord tissue

[0050] Fresh human umbilical cord tissue was collected from a top-tier hospital (ethics-approved), ensuring that all microbial tests (including bacteria, fungi, mycoplasma, and viruses) were negative before the samples entered the laboratory. The umbilical cord was rinsed with sterile saline and cut into approximately 2 cm segments. Next, the arteries, veins, and epidermis were carefully dissected from the umbilical cord, preserving the central Wharton's jelly portion. The dissected Wharton's jelly tissue was cut into 5 mm pieces to facilitate subsequent cell isolation and culture. The cut tissue pieces were seeded into 100 mm culture dishes, with uniform spacing of 5 mm between each piece. 10 mL of complete mesenchymal stem cell culture medium was added to each dish, and the dishes were cultured at 37 ℃ and 5% CO2 until cells emerged. The medium was changed every three days. After 7-10 days of culture, cells emerged from the tissue pieces, forming P0 generation cells.

[0051] 1.2 Culture and passage of UC-MSCs

[0052] Slowly add an appropriate amount of complete mesenchymal stem cell culture medium to each culture dish and incubate the dishes in a 5% CO2, 37 ℃ incubator for 24 h. Figure 1 The results of microscopic observation of UC-MSCs P0 generation cells are presented.

[0053] Before passage, multiple large clonal clusters were observed to form in the culture dish in the P0 generation cells. The distribution of these clonal clusters and the morphology of the cells could be clearly seen under a microscope. The cells showed good adherent growth ability and high activity.

[0054] When the cell density of the P0 generation reaches over 90%, cell digestion and harvesting can proceed. Discard the old culture medium from the culture dish and gently rinse twice with PBS to remove residual culture medium and airborne cells. Add an appropriate amount of mild stem cell digestive enzyme (Youkang, NC1004.1) to each culture dish and incubate at room temperature for approximately 3 minutes. Observe under a microscope until most cells are discrete spherical, then add 4 mL of complete mesenchymal stem cell culture medium to stop digestion. Gently pipette the cell suspension to mix and transfer it to a 50 mL centrifuge tube. Rinse the bottom of the culture dish again with PBS, collect all cell suspension, and transfer them all to a centrifuge tube. Centrifuge at 1300 rpm for 5 minutes and discard the supernatant. Then, resuspend the precipitated cells in an appropriate amount of complete mesenchymal stem cell culture medium and count them. The cells harvested in this step are P0 generation cells, with a total cell count potentially reaching several million.

[0055] The harvested P0 generation cells were seeded at a density of 5000 cells / cm² into new T175 culture flasks, with an appropriate amount of culture medium added to 20 mL. After gentle mixing, the flasks were placed in a constant temperature and humidity incubator and cultured at 37 °C with 5% CO₂. Cell growth was observed daily until cell confluence reached 90%. At this point, the cells were digested and passaged to generation 1 (P1) cells. The passage procedure used the same steps and digestive enzymes as the primary generation to ensure good cell viability and proliferation. When P1 cells reached 90% confluence, they were passaged to generate generation 2 (P2) cells. After passing quality control, the harvested P2 cells were aliquoted and cryopreserved in liquid nitrogen to establish a master cell bank. The criteria for determining the quality of P2 cryopreserved cells are as follows: the cells are adherent, uniformly shaped, and elongated spindle-shaped; the positive expression rates of CD73, CD90, and CD105 are greater than or equal to 95%, and the positive expression rates of CD11b, CD19, CD34, CD45, or HLA-II molecules should not exceed 2%; under in vitro induced differentiation conditions, they have the ability to differentiate into osteoblasts, chondrocytes, and adipocytes; the results of infectious disease and pathogenic microorganism tests are all negative, and the endotoxin result is less than 0.25 EU / mL. Generally, one umbilical cord tissue can yield approximately (0.5-1.5) × 10^9 master cell bank cells. P2 cells resuscitated from the master cell bank are further expanded and cultured, passaged through five generations to the fifth generation (P5). After each passage, rigorous quality testing is performed to ensure that the cells possess good proliferative capacity and multi-lineage differentiation potential. Once the P5 cells meet the quality standards, they are aliquoted and cryopreserved in liquid nitrogen to establish a working cell bank. The criteria for determining the quality of P5 cryopreserved cells are as follows: the cells are adherent, uniformly shaped, long spindle-shaped cells; the positive expression rates of CD73, CD90, and CD105 are greater than or equal to 95%, and the positive expression rates of CD11b, CD19, CD34, CD45, or HLA-II molecules should not exceed 2%; under in vitro induced differentiation conditions, they have the ability to differentiate into osteoblasts, chondrocytes, and adipocytes; the results of infectious disease and pathogenic microorganism tests are all negative, and the endotoxin result is less than 0.25 EU / mL; the inhibition rate of pro-inflammatory lymphocytes Th1 is greater than 50%, the inhibition rate of Th17 is greater than 40%, and the promotion rate of anti-inflammatory lymphocytes Treg is greater than 40%; the PBMC inhibition rate is greater than 50%. Generally, approximately (0.5-1.5) × 10^12 working cell bank cells can be cultured from one umbilical cord tissue. Figure 2 The results of microscopic observation of UC-MSCs P5 generation cells are presented.

[0056] 1.3 Identification of UC-MSCs

[0057] To confirm the characteristics of the isolated UC-MSCs, flow cytometry was used to identify their surface markers. Cells were cultured to passage P5, and when the confluence reached 90%, they were digested with 0.125% trypsin. After digestion was terminated, the cell suspension was collected. The cell concentration was adjusted to 5 × 10^5 cells / mL, and the cells were stained with antibodies (such as CD105, CD73, CD90, etc.) before flow cytometry analysis.

[0058] like Figure 3 Identification of surface markers in UC-MSCs showed that UC-MSCs exhibited high positive rates (>99%) for CD90, CD105, and CD73 markers, while expression of negative markers such as CD45, CD19, CD34, and HLA-DR was almost zero (<1%). These results confirm that the isolated UC-MSCs possess typical mesenchymal stem cell characteristics.

[0059] Example 2: Isolation and Identification of Exosomes

[0060] This embodiment details how to isolate and identify exosomes from human umbilical cord mesenchymal stem cells (UC-MSCs). The process includes specific operational steps, required reagents, and equipment used to ensure precise operation and the acquisition of high-purity exosomes.

[0061] 1. Isolation of exosomes

[0062] a. Cell culture and supernatant collection

[0063] Reagents: UC-MSCs, complete culture medium for mesenchymal stem cells.

[0064] b. Extraction of exosomes

[0065] Equipment and reagents: ultracentrifuge, 100 kDa MWCO ultrafiltration tubes, PBS buffer.

[0066] Procedure: Preliminary centrifugation: Place the collected supernatant in a 50 mL centrifuge tube and centrifuge at 300 g at 4 °C for 10 min to remove cell debris and suspended particles. After centrifugation, transfer the supernatant to a new centrifuge tube. Ultrafiltration concentration: Transfer the supernatant to a 100 kDa MWCO ultrafiltration tube and centrifuge at 5,000 g for 30 min at 4 °C. The concentrate after centrifugation (the liquid above the filter membrane) is the exosome-rich fraction. Differential centrifugation to separate exosomes: Aliquot the concentrated supernatant into equal volumes in ultracentrifuge tubes, ensuring weight balance (difference not exceeding 0.02 g), and level with PBS buffer. Then, ultracentrifuge at 100,000 g at 4 °C for 70 min and collect the exosome precipitate at the bottom of the centrifuge tube. Gently pour off the supernatant, avoiding disturbance of the exosome precipitate. Gently resuspend the exosome precipitate with 2 mL of sterile PBS buffer. To ensure the purity of exosomes, centrifuge again at 100,000 g for 70 min, remove the supernatant, and retain the exosome precipitate at the bottom.

[0067] c. Storage of exosomes

[0068] The exosome pellet was resuspended in 100-200 μL of sterile PBS. The isolated exosomes were aliquoted into sterile EP tubes and can be stored at 4 °C for short-term storage and at -80 °C for long-term storage.

[0069] 2. Identification of exosomes

[0070] a. Particle size analysis

[0071] Equipment and reagents: Nanoparticle tracking analyzer (NTA, ZetaView PMX 110, ParticleMetrix GmbH, Germany), PBS buffer.

[0072] Procedure: Calibrate the NTA instrument using 110 nm polystyrene microspheres to ensure accurate measurements. Dilute the separated exosome sample with PBS to an appropriate concentration, avoiding overly dense particles or excessive dilution that could affect detection. Add the sample to the NTA sample cell and run the program to analyze the exosome particle size distribution. Typical exosome particle sizes range from 30 to 150 nm; record the concentration based on the data displayed by the instrument.

[0073] like Figure 4 A shows the particle size and distribution of exosomes extracted by NTA detection, with a peak particle size of approximately 100 nm.

[0074] b. Observation of exosome morphology using transmission electron microscopy (TEM)

[0075] Equipment and reagents: Transmission electron microscope (HT7700, Hitachi), 200-mesh carbon film copper mesh, 2% phosphotungstic acid or 1% uranic acid.

[0076] Procedure: Add 10 μL of exosome sample to a 200-mesh carbon copper grid and let stand for 5 min to allow the liquid to spread evenly. Carefully blot away excess liquid with filter paper to ensure even sample spread. Add 10 μL of phosphotungstic acid or uranic acid to the surface of the exosomes on the copper grid and let stand for 2 min to negatively stain the exosomes. Gently blot away excess staining solution with filter paper. After the sample is completely dry, place the copper grid under a transmission electron microscope for observation.

[0077] like Figure 4 B-mode transmission electron microscopy revealed that the exosomes all exhibited a typical circular, double-membrane vesicle morphology, with diameters ranging from 50 to 150 nm.

[0078] c. Western Blot detection of surface markers in exosomes

[0079] Equipment and reagents: SDS-PAGE electrophoresis equipment (Bio-Rad), PVDF membrane, CD9 antibody, Alix antibody, Calnexin antibody.

[0080] Procedure: Sample Preparation: Mix exosome protein samples with SDS-PAGE Loading Buffer at a ratio of 4:1 and heat at 95 °C for 5 min to denature the proteins. Electrophoresis: Perform SDS-PAGE protein electrophoresis. Load the exosome samples and pre-stained protein markers into the electrophoresis tank. Set the voltage to 60 V and electrophoresis for 30 min. After the samples enter the separating gel, adjust the voltage to 120 V and continue electrophoresis until complete separation. Transfer: Transfer the electrophoresed proteins to a PVDF membrane using a current of 150 mA for 90 min. Blocking and Antibody Incubation: Block the PVDF membrane for 1 h in 5% skim milk powder or BSA solution. Then, dilute the antibodies (CD9, Alix, Calnexin) proportionally and incubate the membrane at 4 °C overnight. Secondary Antibody Incubation and Development: The next day, recover the primary antibody and wash the membrane three times with TBST for 10 min each time. Then add HRP-labeled goat anti-rabbit secondary antibody and incubate at room temperature for 1 h, followed by three washes with TBST. Finally, the protein bands were developed using an ECL luminescent reagent and recorded using a chemiluminescence imaging system.

[0081] Western blotting results are as follows: Figure 4 As shown in Figure C, the extracted exosomes showed positive expression for Alix and CD9 molecular markers, but negative expression for Calnexin.

[0082] Example 3: Recombinant FGF2 protein reduces lipid deposition in hepatocytes

[0083] FGF2 is a multifunctional and pleiotropic growth factor, a core member of the FGF family. FGF2 is present in various tissues and cell types, especially mesenchymal-derived cells, fibroblasts, endothelial cells, and nerve cells. Under the regulation of factors such as cellular stress and injury, FGF2 is directly transported through cell membranes or released from cellular vesicles such as exosomes. It is a core regulator of tissue homeostasis, repair, and regeneration, playing important roles in promoting cell proliferation and survival, tissue repair and regeneration, and regulating lipolysis and glucose metabolism. To investigate the role of FGF2 protein in metabolic dysfunction-related steatohepatitis, this study used recombinant FGF2 protein purchased from MedChemExpress, USA, and also established an oleic acid / palmitic acid (OA / PA)-induced in vitro steatosis model.

[0084] In the experiment, AML12 cells were cultured for 24 h in DMEM medium (Gibco, catalog number: 8123324) containing 10% fetal bovine serum (FBS) and 1% insulin-transferrin-selenium supplement (ITS) to ensure cell viability and proliferation. Subsequently, OA stock solution was diluted to 1.0 mM and PA stock solution was diluted to 0.5 mM with DMEM complete medium, and the cells were treated for 24 h. By adding OA / PA (the most commonly used and representative fatty acids) to the cell culture system, cells were placed under conditions of excessive fatty acid growth, mimicking the metabolic environment of the human body under nutrient excess, thus successfully inducing lipotoxicity in the cells.

[0085] To evaluate the effects of FGF2 protein on promoting lipolysis and reducing lipid deposition, two concentrations of exogenous recombinant FGF2 protein (25 ng / mL and 50 ng / mL) were added to AML12 cells treated with OA / PA. The experiment was divided into four groups: blank control (NC group), OA / PA control group (OA / PA treatment only), OA / PA + FGF2-25 ng / mL group, and OA / PA + FGF2-50 ng / mL group. Specifically, AML12 cells were cultured in DMEM complete medium at 37 ℃ in a 5% CO2 incubator for 24 h. Subsequently, each well was replaced with the corresponding group's medium (1 mL). Except for the NC group, OA / PA mixture was added to all other groups during the medium replacement. Cells were then cultured for another 24 h at 37 ℃ in a 5% CO2 incubator before harvesting.

[0086] The following groups and corresponding culture media were used:

[0087] (1) NC group: DMEM medium (containing 10% FBS + 1% ITS).

[0088] (2) OA / PA group: DMEM medium (containing 10% FBS + 1% ITS) + 1.0 mM OA / 0.5 mM PA

[0089] (3) OA / PA+25ng FGF2 group: DMEM medium (containing 10% FBS+1% ITS) + 1.0 mM OA / 0.5mM PA+FGF2 recombinant protein (25 ng / mL).

[0090] (4) OA / PA+50ng FGF2 group: DMEM medium (containing 10% FBS+1% ITS) + 1.0 mM OA / 0.5mM PA+FGF2 recombinant protein (50 ng / mL).

[0091] After treatment, Oil Red O staining was used to detect cellular lipid deposition, and PCR was used to detect the expression of lipid metabolism-related genes. For example... Figure 5 Results showed that, compared with the control group treated with OA / PA only, lipid deposition was significantly reduced in both groups treated with recombinant FGF2 protein, especially in the 50 ng FGF2 treatment group (9.151% vs. 29.331%), indicating that FGF2 protein has a significant ameliorative effect on OA / PA-induced cellular lipid deposition. Furthermore, to further verify the function of FGF2 protein in promoting fatty acid oxidation, PCR was used to detect genes related to lipid synthesis and metabolism in cells. Figure 5 Results B showed that the expression levels of lipid metabolism-related genes (including Acox1, Cpt1a, and Ppara) in AML12 cells treated with 25 ng FGF2 and 50 ng FGF2 were significantly higher than those in the control group. These data indicate that FGF2 protein can effectively promote intracellular lipid metabolism and reduce lipid deposition, thereby improving cytotoxicity.

[0092] In conclusion, the results of this experiment demonstrate that FGF2 protein can effectively reduce intracellular lipid deposition in an OA / PA-induced lipotoxicity model. This regulatory role of FGF2 protein suggests its potential application in the treatment of metabolic dysfunction-related steatohepatitis.

[0093] Example 4: MSC-CM exerts its effects of reducing cellular lipid deposition and promoting lipid oxidation and decomposition through exosomes (EVs).

[0094] To verify whether exosomes can enter AML12 cells, we labeled the exosomes with Dio dye, performed exosome tracking experiments, and observed them using a laser confocal microscope. Simultaneously, to clarify whether MSC-CM (conditioned medium containing UC-MSCs-derived exosomes) exerts its biological effects through exosomes, we removed exosomes from the collected MSC-CM by ultracentrifugation, and used the resulting ultrafiltration supernatant (MSC-UF) for subsequent experiments.

[0095] The specific procedures for the exosome uptake experiment are as follows: AML12 cells were seeded in 12-well plates, and cell slides were placed inside the plates. 1 mg of Dio dye was dissolved in 453.67 μL of DMSO to a final concentration of 2.5 mM. Exosomes (300 μg / 60 μL) were resuspended in 2 mL of PBS containing 4 μL, mixed well, and incubated in the dark for 30 min. Then, the cells were centrifuged at 15000 g for 40 min at 4 ℃ to remove excess dye. AML12 cells cultured on coverslips were co-cultured with Dio-labeled exosomes for 24 h. Finally, the coverslip samples were observed using a confocal laser scanning microscope. At the beginning of the experiment, UC-MSCs were revived and cultured in a 37 ℃, 5% CO2 incubator for 72 h to obtain their culture supernatant MSC-CM. Subsequently, the cell culture supernatant collected in step one was subjected to ultrafiltration centrifugation to remove exosomes, obtaining exosome-free ultrafiltration supernatant MSC-UF. The specific operating steps are as follows: The collected cell culture supernatant was placed in a 50 mL centrifuge tube and centrifuged at 300 g at 4 ℃ for 10 min to remove cell debris and suspended particles. The supernatant was then transferred to a 100 kDa MWCO ultrafiltration tube and centrifuged at 5,000 g at 4 ℃ for 30 min. The liquid above the filter membrane is the exosome-rich portion. The liquid below the filter membrane was collected as the exosome-free ultrafiltration supernatant (MSC-UF) for subsequent experiments. AML12 cells treated with OA / PA were divided into three groups: control group (OA / PA treatment only), MSC-CM group (OA / PA + MSC-CM), and MSC-UF group (OA / PA + MSC-UF). Oil Red O staining and PCR were used to detect lipid deposition in each group.

[0096] like Figure 6 The results showed that exosome tracing experiments revealed that exosomes labeled with Dio dye exhibited green fluorescence in AML12 cells (without OA / PA treatment) after 24 h of co-culture, indicating that exosomes could enter AML12 cells in large quantities. Figure 7Results A showed that, according to Oil Red O staining, MSC-CM significantly reduced OA / PA-induced lipid deposition compared to the control group (17.086% vs. 27.336%). However, after ultrafiltration to remove exosomes, intracellular lipid deposition in the MSC-UF-treated group was similar to that in the control group (27.793% vs. 27.336%), indicating that the lipid metabolism-promoting effect of MSC-CM on AML12 cells is dependent on the presence of exosomes. Furthermore, Figure 7 PCR results in group B showed that, compared with the control group, the expression levels of lipid metabolism-related genes Acox1 and Cpt1a were significantly increased in the MSC-CM treatment group, while the expression levels of lipid synthesis-related genes Srebp-1c and Fasn were significantly decreased. Conversely, the ultrafiltrate MSC-UF treatment group showed the opposite trend. This indicates that MSC-CM mainly exerts its lipid-promoting effect by entering target cells via exosomes.

[0097] In summary, the experimental results indicate that exosomes derived from UC-MSCs play a crucial role in promoting lipidolysis in MSC-CM. The MSC-UF supernatant after exosome removal via ultrafiltration centrifugation had no significant effect on OA / PA-induced lipidolysis in AML12 cells. These findings confirm that exosomes are the main active component of MSC-CM in promoting lipidolysis in a cellular lipotoxicity model, providing important evidence regarding the intercellular signaling and therapeutic applications of exosomes.

[0098] Example 5: Knockdown of FGF2 protein expression inhibits the lipidolysis-promoting effect of MSC-CM

[0099] To verify the crucial role of FGF2 protein in promoting lipidolysis in AML12 cells within MSCs, this invention knocked down FGF2 protein expression in UC-MSCs using siRNA technology. The specific steps are as follows: siRNAs were designed and synthesized: siNC5′-ACGUGACACGUUCGGAGAA-3′ and siRNA-FGF2 5′-GCUACAACUUCAAGCAGAATT-3′. Then, the FGF2-specific siRNA (or negative control siRNA, siNC) was transfected into…

[0100] In UC-MSCs, culture medium was collected 48 h after transfection to obtain FGF2 knockdown conditioned medium (siFGF2-CM) and control conditioned medium (siNC-CM). Subsequently, AML12 cells in an OA / PA-induced mouse hepatocyte lipotoxicity model were treated with siNC-CM and siFGF2-CM, respectively, to assess whether FGF2 protein is a key factor in the lipolysis-promoting effect of MSC-CM.

[0101] like Figure 8 Results A showed that, according to Oil Red O results, lipid deposition in AML12 cells was significantly reduced under control conditioned medium (siNC-CM) treatment (15.337% vs. 34.664%), indicating that MSC-CM promotes lipolysis. However, after FGF2 protein knockdown (siFGF2-CM treatment group), lipid deposition in AML12 cells significantly increased to 31.851% (p < 0.05), indicating that knockdown of FGF2 protein expression inhibited the lipolysis-promoting effect of MSC-CM. PCR experiments were used to further verify the expression levels of genes related to intracellular lipid synthesis and metabolism. The experimental results are as follows: Figure 8 B showed that after treatment with the control conditioned medium (siNC-CM), the expression levels of fatty acid degradation-related genes Acox1 and Cpt1a were significantly increased, while the expression level of lipid synthesis-related gene Srebp-1c was significantly decreased. In contrast, the expression levels of lipid synthesis-related genes were increased in the siFGF2-CM treatment group, indicating that knockdown of FGF2 protein expression inhibited the lipid-promoting effect of MSC-CM.

[0102] Example 6: Gene modification for FGF2 gene overexpression in human umbilical cord mesenchymal stem cells and preparation of injection solution

[0103] This embodiment describes the gene modification process of UC-MSCs overexpressing the FGF2 gene, including the construction of lentiviral vectors for the FGF2 gene, viral packaging and purification, FGF2 gene infection and screening of UC-MSCs, and the preparation of injection solution for UC-MSCs overexpressing the FGF2 gene.

[0104] 1. Construction and packaging of lentiviral vectors containing the FGF2 gene

[0105] a. Carrier construction

[0106] First, the full-length sequence of the FGF2 gene (Gene ID: 2247) was obtained from the NCBI database and cloned into the lentiviral expression vector pLV3, inserting the FGF2 gene using the multiple cloning site. As a control, the pLV3-GFP plasmid was also constructed. After constructing the pLV3-CMV-FGF2-EF1a-CopGFP-Puro plasmid, other reagents were prepared: pMD2G plasmid, psPAX2 plasmid, OPTI-MEM medium (Merco-Sigma, USA), and EL transfection reagent (TransGen Biotech, Beijing). Subsequently, using the EL transfection reagent, the constructed pLV3-CMV-FGF2-EF1a-CopGFP-Puro plasmid or pLV3-GFP plasmid was mixed with the helper plasmids pMD2G (encoding the VSV-G envelope protein) and psPAX2 (encoding the viral packaging protein) in a 4:2:1 ratio and co-transfected into 293T cells for viral packaging.

[0107]

[0108] The amino acid sequence of FGF2 protein is: MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS

[0109] b. Virus collection and purification

[0110] Thirty-six hours after transfection, the culture supernatant was collected and stored at 4 °C for use in the infection of UC-MSCs.

[0111] 2. Infection with FGF2 gene and screening of gene-modified UC-MSCs

[0112] a. Viral infection

[0113] Reagents: Polybrene (Wuhan Yisheng Biotechnology Co., Ltd.), UC-MSCs (umbilical cord mesenchymal stem cells).

[0114] UC-MSCs were infected with packaged pLV3-CMV-FGF2-EF1a-CopGFP-Puro plasmid and pLV3-GFP lentiviral particles, respectively. During infection, 5 μg / mL of polybrene was added to improve infection efficiency. The viral particles were mixed with polybrene and then added to complete mesenchymal stem cell culture medium for UC-MSCs. The cells were cultured at 37 °C and 5% CO2 for 6 h, then the medium was replaced, and the cells and culture supernatant were harvested after another 36 h.

[0115] b. Verification of FGF2 gene overexpression

[0116] Reagents: FGF2 antibody, Western blotting reagent; FGF2 primers, PCR reagent.

[0117] Overexpression of the FGF2 gene was detected in post-infected UC-MSCs using Western blotting. After protein extraction, FGF2 protein expression in cells was detected using an FGF2 antibody to verify whether FGF2 protein overexpression was successful. Figure 9 Results A showed that these cells significantly expressed FGF2 protein (P<0.001). On the other hand, RNA was extracted and reverse transcribed, and FGF2 primers were used to detect FGF2 gene expression in the cells to verify whether the FGF2 gene was successfully overexpressed. Figure 9Results B showed that these cells significantly expressed the FGF2 gene, proving that the FGF2 gene was successfully overexpressed.

[0118] 3. Preparation of FGF2 gene overexpressing UC-MSCs injection solution

[0119] a. Cell expansion and collection

[0120] Validated FGF2-overexpressing UC-MSCs were expanded and cultured in large quantities in complete mesenchymal stem cell culture medium. Once the cell count reached the required therapeutic level, the cells and culture supernatant (FGF2-OE) were collected. The collected cells were washed with PBS to remove residual culture medium and other impurities.

[0121] b. Preparation of injection solution

[0122] After washing, the cells were counted and suspended in PBS solution to prepare a sterile cell suspension. The cell concentration was adjusted to ensure that each milliliter of injection solution contained 4 × 10^6 UC-MSCs, thus preparing the final FGF2 gene-overexpressing UC-MSCs injection solution. This FGF2 gene-overexpressing UC-MSCs injection solution preparation process underwent strict aseptic operation and quality control, and was stored at 2-8 °C to ensure cell viability and safety. During the injection solution preparation process, trypan blue staining was used to detect cell viability, ensuring that the cell viability was above 90%.

[0123] Example 7: Genetically modified MSC-CM enhances the lipid oxidative degradation of hepatocytes

[0124] This embodiment uses UC-MSCs modified with the FGF2 gene (see Example 6) to prepare an in vitro cytotoxicity model induced by OA / PA treatment with FGF2-OE supernatant (see Example 3), and evaluates its effect on promoting lipolysis in the in vitro cytotoxicity model. The experiment included a control group (NC), an OA / PA model group (OA / PA), an OA / PA+MSC (Vector)-CM group (Vector-CM containing unmodified UC-MSCs was added while inducing cytotoxicity), and an OA / PA+MSC (FGF2-OE)-CM group (FGF2-CM containing FGF2-modified UC-MSCs was added while inducing cytotoxicity) to compare their effects on cytotoxicity. After 24 h of treatment, the lipid synthesis and metabolism of AML12 cells were detected by Oil Red O staining and PCR experiments. Figure 10As shown in Figure A, compared with the natural MSC-CM treatment group, treatment with FGF2-modified MSC culture supernatant (MSC(FGF2-OE)-CM) significantly reduced intracellular lipid deposition (8.656% vs. 28.863%). PCR experiments were used to further verify the expression levels of genes related to intracellular lipid synthesis and metabolism. The experimental results are as follows... Figure 10 B showed that, compared with the natural MSC-CM group, the expression levels of fatty acid degradation-related genes Acox1, Ppara, and Cpt1a in the OA / PA+MSC(FGF2-OE)-CM group were significantly increased, while the expression levels of lipid synthesis-related genes Srebp-1c and Fasn were significantly decreased (P<0.05), indicating that MSCs-CM overexpressing FGF2 promoted lipid degradation.

[0125] Example 8: Establishment of the MASH mouse model

[0126] This invention utilizes a high-fat, high-cholesterol (HFHC) diet to induce the establishment of a MASH mouse model. The specific steps are as follows:

[0127] 1. Laboratory animals and grouping

[0128] Forty 8-week-old male C57BL / 6 mice were randomly divided into two groups: the NC group (normal control group) and the HFHC group (metabolic dysfunction-associated steatohepatitis model group), with 8 mice in the NC group and 32 mice in the HFHC model group.

[0129] 2. HFHC Dietary Management

[0130] HFHC model mice were fed a high-fat, high-cholesterol diet for 16 weeks to induce metabolic dysfunction-related steatosis, while NC mice were fed a normal diet as a control.

[0131] Example 9: Application of Genetically Modified Umbilical Cord Mesenchymal Stem Cells in the Treatment of MASH Mouse Models

[0132] This embodiment investigated the efficacy of FGF2-overexpressing human umbilical cord mesenchymal stem cells (UC-MSCs (FGF2-OE)) in the treatment of metabolic dysfunction-associated steatohepatitis (MASH), comparing the therapeutic effects of UC-MSCs (FGF2-OE) and ordinary UC-MSCs in a high-fat, high-cholesterol diet (HFHC)-induced MASH mouse model. Thirty-two male C57BL / 6 mice from the HFHC model group in Example 8 were randomly divided into four groups of eight mice each: the HFHC group (receiving PBS injection), the HFHC+UC-MSCs group (receiving ordinary UC-MSCs), the HFHC+UC-MSCs (Vector) group (receiving UC-MSCs infected with control GFP), and the HFHC+UC-MSCs (FGF2-OE) group (receiving UC-MSCs overexpressing FGF2). The NC group (normal control group) from Example 8 served as the control. Figure 10 Mice were fed a high-fat, high-cholesterol diet for 12 weeks. Each mouse was then injected via tail vein with 200 μL of PBS buffer containing the corresponding MSCs (8 × 10^5 cells / kg) once a week for a total of four weeks. After the experiment, the mice were sacrificed, and relevant indicators such as liver function, liver homogenate total cholesterol (TC), serum triglycerides (TG), AST levels, liver section staining, and expression levels of genes related to liver lipid synthesis and metabolism were observed and analyzed. Morphologically, the livers of the model group mice were significantly enlarged and had a pale yellow appearance. Figure 11 A). Compared with the model group, the liver appearance, color, and size of mice in the UC-MSCs treatment group, UC-MSCs (Vector) treatment group, and UC-MSCs (FGF2-OE) treatment group were significantly improved, and the levels of liver homogenate TC, serum TG, serum AST, and insulin were significantly reduced. Figure 11 The UC-MSCs (FGF2-OE) treatment group showed a more significant improvement in liver damage (P<0.05), indicating that UC-MSCs overexpressing the FGF2 gene have a better therapeutic effect in improving liver injury. The mRNA expression levels of inflammation and lipid metabolism-related genes were detected by RT-qPCR. The results showed that compared with the normal group, the expression levels of Fasn, Tnf-α, and Cxcl2 in the liver of the model group mice were significantly increased (P<0.05), while the expression levels of Acox1, Cpt1a, and Ppara were significantly decreased. Treatment with UC-MSCs reversed this trend, and the therapeutic effect of UC-MSCs (FGF2-OE) was more significant (P<0.05). Figure 12This indicates that overexpression of FGF2 can enhance the anti-inflammatory and lipid metabolism-promoting capabilities of UC-MSCs. Section staining and NAS statistical results suggest that the HFHC diet successfully induced the mouse MASH model, and the liver structure of mice in the UC-MSCs (FGF2-OE) treatment group was further improved compared to the model group and the UC-MSCs treatment group. Oil Red O staining results showed that, compared to the normal group, the model group mice exhibited large red lipid droplets, indicating significant lipid accumulation. Compared to the model group and the UC-MSCs treatment group, the number of lipid droplets in the liver of mice in the UC-MSCs (FGF2-OE) treatment group was significantly reduced (P<0.05). Similarly, Sirius red staining clearly showed a certain degree of fibrosis in the liver of the model group mice. After treatment with both the UC-MSCs and UC-MSCs (FGF2-OE) treatment groups, liver fibrosis was improved to some extent, with the improvement being more significant in the UC-MSCs (FGF2-OE) treatment group (P<0.05). Figure 13 This indicates that overexpression of FGF2 enhances the ability of UC-MSCs to improve steatosis and fibrosis in the liver of MASH mice. In conclusion, FGF2-overexpressing UC-MSCs showed superior therapeutic efficacy compared to unregistered UC-MSCs in the MASH mouse model. These findings provide strong experimental evidence for the application of FGF2-modified UC-MSCs in the treatment of metabolic dysfunction-related steatohepatitis, demonstrating that FGF2 overexpression significantly enhances the therapeutic effect of UC-MSCs.

[0133] Example 10: Application of Genetically Modified Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes in In Vitro Cell Lipid Toxicity Model Therapy

[0134] To investigate the efficacy of FGF2-overexpressing umbilical cord mesenchymal stem cell (UC-MSC)-derived exosomes (EVs) in an in vitro cytotoxicity model, two different culture supernatants were collected according to the method described in Example 6. Two types of exosomes, EVs (Vector) and EVs (FGF2-OE), were collected from the culture supernatants using the method described in Example 2. Western blot analysis confirmed that EVs (FGF2-OE) highly expressed FGF2 (… Figure 14A) The above-mentioned EVs (Vector) and EVs (FGF2-OE) were added to the cell lipotoxicity model described in Example 3. The experiment was divided into the following 4 groups: blank control NC group, OA / PA control group (OA / PA treatment only), OA / PA+EVs (Vector) group, and OA / PA+EVs (FGF2-OE) group. The specific operation was as follows: AML12 cells were cultured in DMEM complete medium at 37 ℃ and 5% CO2 in an incubator for 24 h. Subsequently, the medium in each well was replaced with the corresponding group medium (1 mL). Except for the NC group, the OA / PA mixture was added to the other groups at the same time as the medium was replaced. The cells were cultured for another 24 h at 37 ℃ and 5% CO2 in an incubator, and then the cells were harvested.

[0135] The following groups and corresponding culture media were used:

[0136] (1) NC group: DMEM medium (containing 10% FBS + 1% ITS).

[0137] (2) OA / PA group: DMEM medium (containing 10% FBS + 1% ITS) + 1.0 mM OA / 0.5 mM PA

[0138] (3) OA / PA+EVs(Vector) group: DMEM medium (containing 10% FBS+1% ITS) + 1.0 mM OA / 0.5 mMPA+EVs(Vector) (50 μg / mL).

[0139] (4) OA / PA+EVs(FGF2-OE) group: DMEM medium (containing 10% FBS+1% ITS) + 1.0 mM OA / 0.5 mMPA+EVs(FGF2-OE) (50 μg / mL).

[0140] After treatment, Oil Red O staining was used to detect cellular lipid deposition, and PCR was used to detect the expression of lipid metabolism-related genes. For example... Figure 14The results showed that lipid deposition was significantly reduced in both groups with added exosomes compared to the control group treated with OA / PA alone, especially in the EVs(FGF2-OE) treatment group (10.009% vs. 33.204%), indicating that UC-MSCs-derived exosomes overexpressing the FGF2 gene significantly improved OA / PA-induced cellular lipid deposition. Furthermore, to further verify that EVs(FGF2-OE) could enhance its function in promoting fatty acid oxidation, PCR experiments were used to detect genes related to lipid synthesis in cells. The results showed that compared to EVs(Vector), the expression levels of genes related to lipid metabolism (including Acox1, Cpt1a, and Ppara) in AML12 cells were significantly increased after EVs(FGF2-OE) treatment (P<0.05), while the expression level of the lipid synthesis-related gene Srebp-1c was significantly decreased (P<0.05). These data indicate that UC-MSCs-derived exosomes overexpressing the FGF2 gene can effectively promote intracellular lipid metabolism, reduce lipid deposition, and thus improve cytotoxicity.

[0141] In summary, our results indicate that exosomes derived from UC-MSCs overexpressing the FGF2 gene can effectively reduce intracellular lipid deposition in an OA / PA-induced lipotoxicity model. This regulatory effect of exosomes derived from UC-MSCs overexpressing the FGF2 gene suggests their potential application in the treatment of metabolic dysfunction-related steatohepatitis.

[0142] The present invention has been illustrated through the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing FGF2-overexpressing engineered mesenchymal stem cells, characterized in that, Includes the following steps: S1: Human umbilical cord mesenchymal stem cells are isolated and passaged in vitro to obtain human umbilical cord mesenchymal stem cells. S2: Construct a lentiviral vector overexpressing FGF2; S3: The FGF2 overexpression lentiviral vector solution and Polybrene solution were mixed and added to the complete culture medium containing human umbilical cord mesenchymal stem cells obtained in step S1 for viral infection. After culturing in an incubator at 37 ℃ and 5% CO2 for 12 h, the culture medium was replaced and cultured for another 48 h. Cells and culture supernatant were then harvested. Protein was extracted from the infected UC-MSCs, and the overexpression of FGF2 protein was detected by Western Blot. It was verified that the protein expression level increased by about 313.2% after FGF2 overexpression. The verified FGF2 overexpressing UC-MSCs were then expanded and cultured in large quantities in complete mesenchymal stem cell culture medium.

2. The preparation method according to claim 1, characterized in that, In step S2, the construction of the FGF2 overexpression lentiviral vector specifically involves: The full-length sequence of the FGF2 gene (Gene ID: 2247) was obtained from the NCBI database and cloned into the lentiviral expression vector pLV3. The FGF2 gene was inserted using the multiple cloning site to construct the pLV3-CMV-FGF2-EF1a-CopGFP-Puro plasmid. Subsequently, using EL transfection reagent, the constructed pLV3-CMV-FGF2-EF1a-CopGFP-Puro plasmid was mixed with the helper plasmids pMD2G (encoding VSV-G envelope protein) and psPAX2 (encoding viral packaging protein) in a 4:2:1 ratio and co-transfected into 293T cells for viral packaging. After 6 h, the culture medium was replaced and the cells and culture supernatant were harvested after 36 h of further culture.

3. An engineered human umbilical cord mesenchymal stem cell overexpressing FGF2 obtained by the preparation method according to claims 1-2.

4. A method for preparing FGF2-overexpressing engineered human umbilical cord mesenchymal stem cell exosomes, characterized in that, Includes the following steps: S1: Expand and culture the FGF2-overexpressing engineered human umbilical cord mesenchymal stem cells as described in claim 1 of this invention, and collect the cell culture supernatant; S2: The cell culture supernatant collected in step S1 is purified by a combination of ultrafiltration and differential centrifugation to obtain FGF2-overexpressing engineered human umbilical cord mesenchymal stem cell exosomes.

5. The method for preparing FGF2-overexpressing engineered human umbilical cord mesenchymal stem cell exosomes according to claim 4, characterized in that, In step S2, the purification step combining ultrafiltration and differential centrifugation is as follows: S21: Remove cell debris and suspended particles from the supernatant collected in S1: Place the supernatant collected in S1 into a 50 mL centrifuge tube and centrifuge at 300 g for 10 min at 4 ℃ to remove cell debris and suspended particles. After centrifugation, transfer the supernatant to a new 50 mL centrifuge tube. S22: Ultrafiltration concentration: Transfer the supernatant into a 100 kDa MWCO ultrafiltration tube and centrifuge at 5,000 g for 30 min at 4 ℃. The concentrate after centrifugation, i.e. the liquid above the filter membrane, is the part rich in exosomes. S23: Differential centrifugation to separate exosomes: The concentrated supernatant was aliquoted into ultracentrifuge tubes with a difference of no more than 0.02 g to ensure weight balance. The tubes were leveled with PBS buffer and centrifuged at 100,000 g for 70 min at 4 °C. The supernatant was removed and the exosome precipitate was gently resuspended with 2 mL of sterile PBS buffer. To ensure the purity of exosomes, centrifuge again at 100,000 g for 70 min, remove the supernatant and retain the exosome precipitate at the bottom.

6. An engineered human umbilical cord mesenchymal stem cell exosome overexpressing FGF2, obtained by the preparation method according to claim 4.

7. The use of engineered human umbilical cord mesenchymal stem cells overexpressing FGF2 as described in any one of claims 1-2 or exosomes of engineered human umbilical cord mesenchymal stem cells overexpressing FGF2 as described in claims 4-5 in a medicament for treating metabolic dysfunction-related steatohepatitis.

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