Method for promoting stem cells to express long-acting fgf21 at high level and application thereof

Abstract

The application discloses a method for promoting stem cells to express long-acting FGF21 at a high level and application thereof, and belongs to the field of biotechnology, genes and cell therapy. The application realizes the purpose of high-level secretion of stem cells to express long-acting FGF21 by fusing and expressing insulin-like growth factor binding protein IGFBP2. The FGF21 expression amount of the gene modified mesenchymal stem cells is still stable to reach 6454 ng / mL after subculture to the 7th generation, is at least 67.69 times higher than that of a control group, and is up to 961.27 times. The gene modified stem cells obtained by the application can effectively reduce the blood sugar increase of type 2 diabetes mice caused by high-fat diet, the fasting blood sugar decrease value is 30.0% higher than that of the control group, and the muscle movement ability is improved, the mouse grip strength value is 21.9% higher than that of the control group, which indicates that the application has great application value.

Description

Technical Field

[0001] This invention relates to a method for promoting high-level expression of long-acting FGF21 in stem cells and its application, belonging to the fields of biotechnology, gene and cell therapy. Background Technology

[0002] FGF21 has significant drug development value in the treatment of metabolic diseases and addiction. It is a cytokine encoded by 209 amino acids. Its carboxyl terminus is easily degraded by FAP (Fibroblast activation protein) enzymes present in the bloodstream. Therefore, it is usually fused with human serum albumin (HSA) or antibody Fc fragments to form long-acting FGF21-HSA or FGF21-Fc. However, how to efficiently express active cytokines such as FGF21 has always been an important limiting bottleneck in this field (Zhao S, et al. Screening and validation of rabbit monoclonal antibody pair against human FGF21 for human plasma FGF21 high sensitivity bioanalysis. iScience. 2026 Jun 4;29(6):116260.). As an emerging living cell drug, stem cells have advantages that other drugs do not have in fundamentally improving the cause of diseases such as organ function repair. Using stem cells as a base to efficiently express functional proteins such as long-acting FGF21 is an important problem to be solved in this field. There is a huge demand and application value in developing a technology and method for efficiently expressing functional proteins such as FGF21 in living cell drugs such as stem cells.

[0003] Existing technologies for efficient protein expression mainly focus on strategies such as promoter optimization, codon optimization of the gene itself, or amino acid mutation of the protein itself (Liu, L., et al. Double promoter and tandem gene strategy for efficiently expressing recombinant FGF21. Microbial Cell Factories, 2024, 23(1), 171; Cui, W., et al. High-efficiency expression and secretion of human FGF21 in Bacillus subtilis by intercalation of a mini-cistron cassette and combinatorial optimization of cell regulatory components. Microbial Cell Factories, 2019, 18, 17.). However, these optimizations also have their technical limitations. Exploring new efficient expression methods, especially high-level expression in stem cells, the foundational cells of the emerging therapeutic field, is an important and highly valuable technical problem in the fields of biotechnology, gene therapy, and cell therapy. FGF21-related drugs are not yet on the market. In particular, gene and cell therapies based on stem cell secreted functional proteins still face limitations in drug development, such as improving expression levels and efficacy. Therefore, exploring related expression strategies, including FGF21, and developing cell drugs is of great practical significance in the field of drug development for metabolic and addiction-related diseases. Summary of the Invention

[0004] This invention unexpectedly achieved high-level expression of FGF21 in stem cells (the highest level known to date) by fusing expression of insulin-like growth factor binding protein 2 (IGFBP2). The secretion of FGF21 protein in stem cells was at least 67.69 times, and up to 961.27 times, higher than that of the control group fused with preproglucagon protein FGF21-HSA-Preproglucagon. Safety and efficacy were demonstrated in a mouse model of high-fat induced diabetes. This invention also reveals, for the first time, two indicators that improve FGF21-expressing stem cells compared to the control group: activity in reducing fasting blood glucose and activity in improving muscle grip strength. This provides a new and powerful method and tool for the development of in vivo drugs, including stem cells, based on long-acting FGF21 protein.

[0005] In summary, the purpose of this invention is to construct a novel stem cell that expresses high levels of long-acting FGF21, thereby achieving more effective blood glucose control for type 2 diabetes and more effective treatment for reduced exercise capacity caused by complications.

[0006] This invention provides a gene modification strategy to promote high-level expression of the functional protein FGF21 in mesenchymal stem cells, wherein the mesenchymal stem cells simultaneously express the FGF21 protein shown in SEQ ID NO.2 and the IGFBP2 protein shown in SEQ ID NO.3.

[0007] In one embodiment, the FGF21 protein and the IGFBP2 protein are further linked by an HSA protein or an Fc fragment.

[0008] In one embodiment, the signal peptide shown in SEQ ID NO.1, the FGF21 protein shown in SEQ ID NO.2, the linker shown in SEQ ID NO.5, the HSA protein shown in SEQ ID NO.4, the linker shown in SEQ ID NO.5, and the IGFBP2 protein shown in SEQ ID NO.3 are sequentially connected from the amino terminus to the carboxyl terminus.

[0009] In one embodiment, the nucleotide sequence encoding the signal peptide is shown in SEQ ID NO. 6; the nucleotide sequence encoding the FGF21 protein is shown in SEQ ID NO. 7; the nucleotide sequence encoding the linker is shown in SEQ ID NO. 10; the nucleotide sequence encoding the HSA protein is shown in SEQ ID NO. 9; and the nucleotide sequence encoding the IGFBP2 protein is shown in SEQ ID NO. 8.

[0010] In one embodiment, the mesenchymal stem cells are derived from the umbilical cord, adipose tissue, or bone marrow.

[0011] This invention provides a method for preparing gene-modified mesenchymal stem cells with high FGF21 expression, comprising the following steps: S1. Insert the target gene into the lentiviral expression vector plvx-IRES-Neo to construct the plvx-IRES-Neo-FGF21-HSA-IGFBP2 recombinant plasmid; S2. The recombinant plasmid plvx-IRES-Neo-FGF21-HSA-IGFBP2 obtained in step S1 is co-transfected with the packaging plasmid into host cells to prepare lentiviral particles. S3. Infect mesenchymal stem cells with the lentiviral particles obtained in step S2 to obtain gene-modified mesenchymal stem cells with high expression of FGF21.

[0012] In one embodiment, the target gene, from its 5' end to its 3' end, comprises, in sequence, the nucleotide sequence encoding the signal peptide shown in SEQ ID NO.6, the nucleotide sequence encoding the FGF21 protein shown in SEQ ID NO.7, the nucleotide sequence encoding the linker shown in SEQ ID NO.10, the nucleotide sequence encoding the HSA protein shown in SEQ ID NO.9, the nucleotide sequence encoding the linker shown in SEQ ID NO.10, and the nucleotide sequence encoding the IGFBP2 protein shown in SEQ ID NO.8.

[0013] In one embodiment, the host cells comprise 293T cells.

[0014] This invention provides gene-modified mesenchymal stem cells prepared by the method described above.

[0015] The present invention provides a pharmaceutical composition of the genetically modified mesenchymal stem cells, the pharmaceutical composition comprising the genetically modified mesenchymal stem cells and / or their cultures.

[0016] This invention provides the use of the genetically modified mesenchymal stem cells or the pharmaceutical composition in the preparation of a medicament for treating metabolic syndrome.

[0017] In one embodiment, the metabolic syndrome includes type 2 diabetes; the drug further comprises a pharmaceutically acceptable sterile isotonic or non-aqueous solution, dispersion, suspension, or emulsion.

[0018] Beneficial effects: This invention unexpectedly achieved highly efficient expression of FGF21 by fusing the expression of IGFBP2 protein, achieving the following results: 1. This invention provides FGF21-HSA-IGFBP2 gene-modified stem cells, wherein the secretion of FGF21 protein in the stem cells is at least 67.69 times and up to 961.27 times higher than that in the control group (expressing FGF21-HSA-Preproglucagon), and the secretion of active protein is more stable during passage culture compared with the control group. 2. This invention is the first to verify the safety and efficacy of FGF21-HSA-IGFBP2 gene-modified stem cells in regulating glucose metabolism in a high-fat-fed mouse model that more closely resembles human type 2 diabetes. Compared with the control group, it showed better control of fasting blood glucose, with a 30.0% increase in the rate of decrease. 3. This invention is the first to discover that FGF21-HSA-IGFBP2 gene-modified stem cells have a long-lasting effect on the treatment of type 2 diabetes. Fasting blood glucose and glucose tolerance in mouse models remained normal 48 hours after administration, indicating that this invention maintains normal blood glucose metabolism in mouse models for at least 48 hours. 4. Compared with the control group FGF21-HSA-Preproglucagon stem cells, FGF21-HSA-IGFBP2 gene-modified stem cells have a stronger expansion capacity, which is 1.45 times that of the latter. This reduces cell culture costs by 45%, significantly lowering the cost of this technology compared to the control group and significantly increasing the development value of this approach. 5. The FGF21-HSA-IGFBP2 gene-modified stem cells described in this invention can improve aging symptoms caused by abnormal blood sugar, especially the decline in muscle movement ability. Behavioral experiments in mouse models have verified the improvement in muscle movement ability. After two injections, the grip strength index of mice treated with this method increased by 21.9% compared with the control group. This method fills the gap in the treatment of muscle atrophy, an aging complication related to diabetic metabolic problems, and provides a novel cell therapy that simultaneously improves blood sugar and muscle atrophy. Attached Figure Description

[0019] Figure 1 This is a plasmid map containing the FGF21-HSA-IGFBP2 nucleotide sequence; Figure 2 Identification of surface antigens of umbilical cord mesenchymal stem cells; Figure 3 Comparison of cell morphology and growth of gene-modified stem cells; Figure 4 Comparison of FGF21 protein secretion levels in gene-modified stem cells; Figure 5 Comparison of the average decrease in fasting blood glucose between the two cell types after two injections; Figure 6This study compares the grip strength of mice with two different cell types after two injections of gene-modified stem cells. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0021] The experimental methods involved in the following examples are as follows.

[0022] (1) Preparation of recombinant lentivirus with FGF21-HSA and IGFBP2 fusion gene 1.1 Construction of the lentiviral expression plasmid plvx-IRES-Neo-FGF21-HSA-IGFBP2 fusion gene The target gene fragment synthesized by Suzhou Silicon-based Biosynthesis Co., Ltd. consists of the following sequences from the amino terminus: FGF21 (SEQ ID No. 7) containing its own signal peptide (SEQ ID No. 6), a linker (SEQ ID No. 10), plasma half-life extension protein HSA (gene sequence with signal peptide removed, SEQ ID No. 9), a linker (SEQ ID No. 10), and IGFBP2 (gene sequence with signal peptide removed, SEQ ID No. 8). This fragment is then loaded into the lentiviral expression vector plvx-IRES-Neo (Clontech, catalog number 632181), with the upstream restriction enzyme site being [insert site here]. Eco RⅠ, downstream restriction site Bam H Ⅰ. Carrier design diagram as follows Figure 1 As shown. Primers for the target vector backbone and / or insert sequence were designed, and the nucleic acid sequences were identified by Sanger sequencing, with complete alignment. The HSA gene in the above vector can be replaced by commonly used antibody Fc fragment genes that extend plasma half-life.

[0023] 1.2 Plasmid amplification and endotoxin removal extraction: 1. Three glycerol bacteria DH5α containing the lentiviral expression plasmid (plvx-IRES-Neo-FGF21-HSA-IGFBP2) constructed in step 1.1 and the packaging plasmids pSPAX2 (Addgene, catalog number 12260) and pMD2.G (Addgene, catalog number 12259) were used. Administer 50 doses at a 1% vaccination rate. 1. Add 1 ml of LB liquid medium containing 100 μg / mL ampicillin; 2. Incubate at 37 ℃ and 220 rpm for 15 h with constant temperature shaking; 3. After incubation, strictly follow the instructions of the endotoxin-free plasmid extraction kit (Tiangen Biotech, catalog number DP108) to obtain endotoxin-free plasmid DNA; 4. Measure the plasmid concentration and A using a full-wavelength microplate reader (ThermoFisher, Multiskan SkyHigh). 260 / A 280 Ratio, purity meets standards (A) 260 / A 280 Plasmids with a density of 1.8-1.9 were aliquoted and stored at -20 °C.

[0024] 1.3 293T cell resuscitation and passage culture 1. Cell resuscitation: 30 min in advance, preheat the complete culture medium (DMEM + 10% FBS + 1×Penicillin-Streptomycin solution, final penicillin concentration: 100 U / mL, final streptomycin concentration: 100 μg / mL) in a 37 ℃ incubator. Thaw the cell cryopreservation tubes in a 37 ℃ water bath, centrifuge at 1000 rpm for 1 min, discard the cryopreservation solution, resuspend the cells in the preheated complete culture medium, and seed them into T25 culture flasks. Add culture medium to 5 mL and incubate at 37 ℃ with 5% CO2. 2. Cell medium change: 24 h after resuscitation, discard the old culture medium containing dead cells and replace with 5 mL of fresh complete culture medium. 3. Cell passage: When cell confluence reaches 80%-90%, wash twice with PBS, add 1 mL of trypsin and digest at room temperature until the intercellular spaces widen. Add 3 mL of complete culture medium to stop digestion, pipette to prepare a single-cell suspension, and centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant and passage the cells in a 1:3 ratio; 4. Cell cryopreservation: After digestion and centrifugation of cells in the logarithmic growth phase, resuspend them in cryopreservation solution (92% complete culture medium + 8% DMSO) to a final volume of 2-3 × 10⁻⁶ cells / mL. 6 Aliquots were dispensed into cryovials and placed in a programmable cooling box (corning, catalog number 432001) at -80°C overnight, then transferred to liquid nitrogen for long-term storage.

[0025] 1.4 Cell plating before transfection 1. 24 h before transfection, digest and count 293T cells in the logarithmic growth phase; 2. Spread 293T cells evenly in a 100 mm culture dish, using 10 mL of culture medium; 3. Incubate overnight at 37 ℃ with 5% CO2. The cell confluence must be strictly controlled at 80%-90% during transfection.

[0026] 1.5 Polyethylenimine 40000 (PEI 40000, Yisheng, catalog number 40816ES02, batch number WP5430010) mediated triplasmic co-transfection: 1. Replace the culture medium with fresh medium 1 hour before transfection; 2. Prepare a plasmid mixture according to the target plasmid:pSPAX2:pMD2.G = 5:2.5:1 mass ratio, with a total plasmid amount of 25.5 μg per 100 mm dish; 3. Prepare two 1.5 mL EP tubes: Tube A (plasmid dilution): 250 μL of antibiotic-free and serum-free DMEM + the calculated volume of each plasmid, gently mix and incubate at room temperature for 5 min. Tube B (transfection reagent dilution): 250 μL antibiotic-free and serum-free DMEM + 76.5 μL PEI (PEI volume: total plasmid mass ratio = 3:1), mix gently and let stand at room temperature for 5 min; 4. Add the liquid from tube B dropwise to tube A, mixing by pipetting while adding, and let stand at room temperature for 15 min to form the transfection complex; 5. Add the complex dropwise evenly to the culture dish, shake gently and return to the incubator; 6. 16 h after transfection, discard the old culture medium containing the complex and replace it with 10 mL of fresh complete culture medium.

[0027] 1.6 Lentiviral supernatant collection and pretreatment 1. 48 h post-transfection: Collect the supernatant into a 50 mL centrifuge tube, store temporarily at 4 ℃, and add 10 mL of fresh complete culture medium for continued culture; 2. 72 h post-transfection: Collect the supernatant a second time and combine it with the supernatant collected at 48 h; 3. Centrifuge at 3500 rpm and 4 ℃ for 5 min to remove cell debris, collect the supernatant into a new 50 mL centrifuge tube, and immediately concentrate or store temporarily at 4 ℃ (not exceeding 24 h).

[0028] 1.7 Lentiviral Concentration and Purification All operations were performed on an icebox to maintain low temperatures and preserve virus activity.

[0029] 1. Initial purification: Filter the supernatant using a 0.22μm PES syringe filter (Merck, catalog number SLGPR33RB) to remove residual cell debris and bacteria; 2. Concentration and precipitation: Add EZ lentivirus concentrate (Liji Biotechnology, catalog number AC04L441) at a virus supernatant:concentrate ratio of 4:1, mix by inversion, and incubate overnight (16 h) at 4℃; 3. Centrifugation and collection: Centrifuge at 3500 g for 15 min at 4℃. A white virus precipitate will be visible at the bottom of the tube. Carefully aspirate the supernatant, briefly centrifuge at 1500 g for 30 s, and then aspirate any remaining liquid; 4. Resuspension and aliquoting: Gently resuspend the virus precipitate in 100μL of pre-chilled PBS, aliquot into 20-30μL / tube, and store at -80℃ (avoid repeated freeze-thaw cycles).

[0030] 1.8 Lentiviral titer determination (ELISA method) Follow the instructions for the Lentiviral Titer ELISA Kit (Shanghai Sangon Biotech, catalog number B605114-0096).

[0031] (2) Lentiviral infection of human umbilical cord mesenchymal stem cells (hUC-MSCs) 2.1 Pre-infection cell plating 1. One day prior to infection, hUC-MSCs in the logarithmic growth phase were digested and counted, and the density was adjusted to 5 × 10⁻⁶. 4 Cells / mL were cultured in serum-free MSC-T4 human mesenchymal stem cell medium (Zhuhai Beso, catalog number 04304P05), supplemented with UltraGROTM-Advanced cell nutrition additive (HELIOS, catalog number HPCFDCGL50) at a ratio of 20:1; 2 mL of cell suspension (approximately 1 × 10⁶ cells / mL) was seeded in each well of a 2.6-well plate. 5 (1 cell); 3. Incubate overnight at 37 ℃ with 5% CO2, and control cell confluence at 70%-80% during infection.

[0032] 2.2 Steps for lentiviral gradient infection of umbilical cord mesenchymal stem cells: 1. The known titer of the FGF21-HSA-IGFBP2 recombinant virus is 2.33 × 10⁻⁶. 8 TU / mL, Experimental groups: Set 5 L, 10 L, 15 L, 20 L, 30 L (corresponding to the virus-to-cell ratio—multiples of infection of 12, 23, 35, 47, and 70, respectively) and blank control group (NC); 2. Prepare 2 mL EP tubes, add the corresponding volume of lentivirus, dilute to 2 mL with complete culture medium, and add 2 L-polybrene (Beyotime, product number C0531) final concentration 10 g / mL; 3. Discard the old culture medium in the 6-well plate and add virus-polybrene mixture; 4. Incubate at 37 ℃ and 5% CO2; observe cell status 6 h after infection, and change the medium immediately if obvious virulence is observed; 5. Strictly control the total infection time to ≤16 h, then replace with fresh complete culture medium and continue culturing until the cell confluence reaches 80%-90%. Collect the cell supernatant and perform ELISA to detect FGF21 secretion to confirm protein expression. Digest each type of cell, plate it, and expand it into cell culture flasks. Continue to expand the culture of cells with the best cell status and the best protein expression.

[0033] 2.3 Detection of target protein and gene expression 2.3.1 Detection of FGF21 and IGFBP2 mRNA expression levels in gene-modified stem cells by Q-PCR After expanding the cell culture, 1 million cells were collected. Cells were lysed using Trizol lysis buffer (Takara, catalog number RNAisoPlus 9108) and total RNA was extracted. The expression levels of the target genes FGF21 and IGFBP2 mRNA relative to the internal reference gene GAPDH mRNA in the infected and control groups were then detected using a fluorescent probe method. Primers and probes are shown in Table 1 below. Table 1 Primer and probe sequences for FGF21 and IGFBP2

[0034] According to the instructions of the Takara Reverse Transcription Kit (Takara, catalog number 2641A), the extracted RNA was reverse transcribed into cDNA, and then the reaction system and instructions of the Hieff Unicon Universal TaqMan multiplex qPCR master mix (11211ES03) were followed to detect the expression of related genes. (1) The cDNA stock solution obtained by reverse transcription was used for qPCR experiments. 2 µL of cDNA stock solution was used as a template for qPCR in each reaction well. (2) Three technical replicates were set up for each sample. All components of the qPCR reaction system except cDNA were separated into a master tube, and the corresponding cDNA samples were added to each tube to minimize pipetting errors. (3) Three NTC control wells were set up. (4) The following mixture was prepared in the qPCR tube to make the final concentration of primers 0.5 µM and the final concentration of probes 0.1 µM. The Bio-Rad CFX 96 real-time fluorescence quantitative PCR instrument was used for detection. The reaction system and procedure are shown in Table 2.

[0035] Table 2. qPCR reaction procedures for FGF21 and IGFBP2

[0036] (5) Confidential data standards: (5-1) The amplification curve is a standard S-shape, without waveform jitter or other abnormal phenomena, and there is no NTC amplification or the NTC amplification differs from the target gene Cq value by more than 10; (5-2) The Cq value of each target gene is subtracted from the corresponding internal control Cq value to obtain ΔCq. For each sample, the Cq difference between replicates is ≤1, and the ΔCq between replicates is ≤0.5 as reliable data; (6) Data processing: The Cq values ​​of each target gene and internal control in the sample are obtained by qPCR detection. The Cq value of each target gene is subtracted from the corresponding internal control Cq value to obtain ΔCq. The average ΔCq value and ΔCq SD value are calculated. The expression value of the target gene is calculated according to 2 -ΔCq Algorithm calculation.

[0037] 2.3.2 Sandwich ELISA method for detecting FGF21 expression level in supernatant of gene-modified stem cell culture To confirm the successful overexpression of FGF21 secreted by stem cells after gene modification, the FGF21 content in the culture supernatant of the uninfected and infected groups was detected by ELISA (the amount of FGF21 secreted can reflect the expression level of FGF21-HSA and its fusion protein, because the three are expressed in a coupled manner to form a fusion protein). The specific method is as follows (the FGF21 20C3 capture antibody and FGF21 detection antibody 5F6-Biotin used are self-developed reagents, patent authorization announcement number CN118909111B): (1) Dilute the coated antibody 20C3 with CBS (50 mM carbonate buffer) solution to 0.2 μg / mL, add 100 μL / well to the high affinity microplate, and incubate overnight at 4 ℃; (2) Wash the plate 3 times with PBST at 300 μL / well; (3) Add 300 μL / well of 3% BSA-PBST to the microplate, and incubate at 37 ℃ for 90 minutes; (4) Wash the plate 3 times with PBST at 300 μL / well; (5) Dilute the FGF21 standard with 0.5% BSA-PBST serially to 781.3 pg / mL-12.21 pg / mL, add 100 μL / well to the microplate, and incubate at 0.5% BSA-PBST serially to the microplate. BSA-PBST served as a blank control, and the samples were culture supernatants from both the uninfected and infected groups. (6) Wash the plate 3 times with PBST at 300 μL / well; (7) Dilute the biotin-labeled antibody (5F6-Biotin) with PBS to 0.1 μg / mL, add 100 μL / well to the microplate, and incubate at 37℃ for 1 hour; (8) Wash the plate 3 times with PBST at 300 μL / well; (9) Dilute the horseradish peroxidase-labeled streptavidin with PBS at 1:32250, add 100 μL / well to the microplate, and incubate at 37℃ for 30 minutes; (10) Wash the plate 3 times with PBST at 300 μL / well; (11) Add 100 μL / well TMB solution and incubate at room temperature in the dark for 4 minutes; (12) Add 50 μL / well ELISA stop solution; (13) Read the absorbance values ​​at 450 nm and 620 nm wavelengths using a microplate reader. The expression level of FGF21 in the culture supernatant was calculated based on the ELISA standard curve.

[0038] (3) Lentiviral gene-modified FGF21-HSA-IGFBP2 high-expression stem cells were injected via tail vein into a type 2 diabetic mouse model. Fasting blood glucose levels in mice were measured before and after injection using a Sinocare blood glucose meter, and a 2-hour oral glucose tolerance test (OGTT 2 h) was performed to determine the glucose tolerance response in animals on a long-term high-fat diet. After 12 h of fasting with free access to water, blood glucose levels in all animals were measured (time, 0 min). Subsequently, animals were administered 20% glucose (2 g / kg) by gavage. Blood was collected using the tail puncture method to measure blood glucose concentration. Blood glucose concentrations were measured using a Sinocare blood glucose meter at 30, 60, 90, and 120 min after the glucose load (OGTT 2 h).

[0039] The tail vein injection dose in mice was 2.5 × 10⁻⁶. 5 Inject 25 g cells / 200 μL, with the control group receiving the same number of control cells. Blood was collected from the tail vein (tail tip) for fasting blood glucose and OGTT 2-hour blood glucose testing. In the examples below, the regulatory effect of gene-modified stem cell injection on blood glucose stability in mice was maintained for at least 48 hours.

[0040] Example 1: Comparison of FGF21 secretion expression levels in umbilical cord mesenchymal stem cells transduced by three recombinant lentiviruses 1.1 Preparation of FGF21-HSA-IGFBP2, FGF21-HSA-Preproglucagon, and FGF21-HSA recombinant lentiviruses Lentiviral expression plasmids (plvx-IRES-Neo-FGF21-HSA-IGFBP2) and packaging plasmids (pSPAX2, pMD2.G) were amplified and endotoxin-free extracted according to the aforementioned methods to obtain endotoxin-free plasmid DNA. Recombinant lentivirus was then packaged in HEK-293T cells using the aforementioned method, and the viral titer was determined. The target gene fragment FGF21-HSA-Preproglucagon was synthesized by Suzhou Silicon Biotechnology. The target gene fragment, starting from the amino terminus, consists of: signal peptide (SEQ ID No. 6), FGF21 (SEQ ID No. 7), linker (SEQ ID No. 10), plasma half-life extension protein HSA (gene sequence with signal peptide removed, SEQ ID No. 9), linker (SEQ ID No. 10), and Preproglucagon (SEQ ID No. 11), and was loaded upstream of the lentiviral expression vector plvx-IRES-Neo. Xba I. Enzyme cleavage site, downstream BamHⅠ restriction site. The target gene fragment FGF21-HSA was synthesized by Suzhou Silicon-based Biotechnology. The target gene fragment, starting from the amino terminus, consists of: signal peptide (SEQ ID No. 6), FGF21 (SEQ ID No. 7), linker (SEQ ID No. 10), and plasma half-life extension protein HSA (gene sequence with signal peptide removed, SEQ ID No. 9). It was loaded into the upstream of the lentiviral expression vector plvx-IRES-Neo. Eco RⅠ restriction site, downstream Bam HⅠ restriction site. Recombinant lentivirus containing FGF21-HSA-Preproglucagon and FGF21-HSA genes was prepared according to the method described above, and the viral titer was determined.

[0041] Umbilical cord mesenchymal stem cells were isolated and cultured from perinatal umbilical cord tissue of ethically approved healthy women using standard techniques. The obtained mesenchymal stem cells were cultured and expanded in a 37 ℃, 5% CO2 incubator. Flow cytometry identified positive expression of four mesenchymal stem cell surface antigens: CD44, CD73, CD90, and CD105; and negative expression of four antigens: CD11b, CD19, CD34, and CD45. The results are as follows: Figure 2 As shown.

[0042] 1.2 Lentiviral infection of umbilical cord mesenchymal stem cells and detection of FGF21 secretion expression level 1.2.1 Prepare FGF21-HSA, FGF21-HSA-IGFBP2, and FGF21-HSA-Preproglucagon gene-modified stem cells according to the aforementioned method: 1) Experimental grouping: 1 μL of recombinant lentivirus containing FGF21-HSA, 5 μL of recombinant lentivirus containing FGF21-HSA-IGFBP2 gene, and 10 μL of recombinant lentivirus containing FGF21-HSA-Preproglucagon gene (corresponding to virus-to-cell ratios—multiples of infection of the three were 2, 12, and 22, respectively. These three multiples of infection represent the optimal infection doses of the three recombinant viruses, which can achieve optimal cell growth and high expression of the target protein) were used to infect fourth-generation (P4) umbilical cord mesenchymal stem cells, respectively. 2) Prepare a 2 mL EP tube, add the corresponding volume of lentivirus, dilute with complete culture medium to 2 mL, and add 2 μL of lybrene to a final concentration of 10 μg / mL; 3) Discard the old culture medium from the 6-well plate and add the virus-Polybrene mixture; 4) Culture at 37 ℃ and 5% CO2; observe cell status 6 h after infection, and change the medium immediately if obvious toxicity is observed; 5) The total infection duration was strictly controlled to ≤16 h. After that, the culture medium was replaced with fresh complete medium and the cells were cultured until the confluence reached 80-90%. The cell supernatant was collected and the FGF21 secretion expression level was detected by ELISA. The result for FGF21-HSA gene-modified stem cells was 27.3 ng / mL, the result for FGF21-HSA-Preproglucagon gene-modified stem cells was 279.8 ng / mL, the result for FGF21-HSA-IGFBP2 gene-modified stem cells was 6366 ng / mL, and the result for wild-type stem cells was 23.60 pg / mL. After confirming the protein expression of the infected cells, the infected cells were digested, plated into cell culture flasks, and cultured for further expansion. 6) Because stem cells modified with the FGF21-HSA-Preproglucagon gene grow significantly slower than those modified with FGF21-HSA-IGFBP2, their cell morphology is as follows: Figure 3 As shown, 1-2 more passages are needed in the process of preparing the required number of cells for animal experiments. Both cell types were administered the drug within 10 passages when the cells were in relatively good condition. The expression of the target protein FGF21 in the supernatant of the gene-modified cells used in the two animal experiments was detected using the aforementioned ELISA method. The ELISA results of the target protein expression detection are shown in Table 3 below. Figure 4 As shown: Table 3 Comparison of FGF21 supernatant secretion in three gene-modified cells

[0043] Results analysis: The expression level of FGF21-HSA-IGFBP2 fusion protein was significantly higher than that of FGF21-HSA-Preproglucagon, with the former being at least 67.69 times and up to 961.27 times higher. Compared with FGF21-HSA-Preproglucagon and FGF21-HSA gene modification, FGF21-HSA-IGFBP2 gene modification significantly increased the expression level of FGF21 fusion protein, and the secretion capacity of the fusion protein was more stable during cell passage (the secretion expression level of FGF21 fusion protein in FGF21-HSA-Preproglucagon gene-modified stem cells decreased from 279.8 ng / mL in the initial passage to 6.714 ng / mL and 21.60 ng / mL in the subsequent passages, while the secretion expression level of FGF21-HSA-IGFBP2 gene-modified stem cells remained above 1000 ng / mL during passage and showed an increasing trend with subsequent passages).

[0044] 1.2.2 Q-PCR detection showed increased expression levels of FGF21 and IGFBP2 mRNA in gene-modified stem cells. Total RNA was extracted from wild-type stem cells and two different gene-modified stem cells (FGF21-HSA-Preproglucagon fusion expression and FGF21-HSA-IGFBP2 fusion expression) as described above. The expression levels of FGF21 mRNA and IGFBP2 mRNA in the cells relative to the internal reference gene GAPDH mRNA were detected by fluorescent probe Q-PCR. The results are shown in Table 4 below. The FGF21 mRNA in the FGF21-HSA-IGFBP2 fusion expression stem cells was 5.390 times higher than that in the FGF21-HSA-Preproglucagon fusion expression stem cells, which is consistent with the result of higher FGF21 protein expression. Furthermore, the IGFBP2 mRNA in the FGF21-HSA-IGFBP2 fusion expression stem cells also showed a high expression level.

[0045] Table 4 Comparison of FGF21 and IGFBP2 mRNA expression levels in gene-modified stem cells

[0046] Example 2: Comparison of cell expansion capacity between FGF21-HSA-IGFBP2 modified stem cells and FGF21-HSA-Preproglucagon modified stem cells 2.1 Comparison of passage expansion capacity: Compared with FGF21-HSA-IGFBP2 gene-modified stem cells, FGF21-HSA-Preproglucagon gene-modified stem cells have a stronger expansion capacity. The comparison results of the 8th generation of the former and the 7th generation of the latter are shown in Table 5. After 6 days, the former expanded 2.79 times and the latter expanded 1.93 times, which means that the former has a 1.45 times expansion capacity of the latter. That is, the FGF21-HSA-IGFBP2 gene-modified stem cells of the higher generation still have a stronger expansion capacity than the FGF21-HSA-Preproglucagon gene-modified stem cells of the lower generation, which fully demonstrates that the expansion capacity of FGF21-HSA-IGFBP2 gene-modified stem cells is significantly higher than that of FGF21-HSA-Preproglucagon gene-modified stem cells.

[0047] Table 5 Comparison of the Proliferative Capacity of Genetically Modified Stem Cells

[0048] 2.2 Comparison of doubling time of stem cells from two gene-modified types: (a) Constructing a cell number-OD standard curve: 1. Take P9 generation FGF21-HSA-IGFBP2 gene-modified stem cells and P8 generation FGF21-HSA-Preproglucagon gene-modified stem cells in the exponential growth phase and prepare single-cell suspensions respectively; 2. Seedling is performed in a gradient manner on 96-well plates: 0, 500, 1000, 2000, 4000, 6000, 8000, 12000, 16000, 32000 cells / well, with a volume of 100 μL per well and 3 replicates. 3. Incubate for 6 h (until cells are fully adhered and have not proliferated), then add 10 μL of CCK8 (Beyotime, catalog number C0048M) reagent to each well; 4. Incubate at 37℃ for 1 h, measure absorbance at 450 nm using a microplate reader, and plot a standard curve with cell number as the X-axis and OD value (minus blank well) as the Y-axis.

[0049] (II) Calculation of doubling time in 96-hour cell culture: 1. Take two types of cells, FGF21-HSA-IGFBP2 gene-modified stem cells and FGF21-HSA-Preproglucagon gene-modified stem cells in the exponential growth phase, and prepare single-cell suspensions respectively; 2. Seed 300 cells / well into 96-well plates, with a volume of 100 μL per well, and set 4 replicates; 3. After culturing for 24, 48, 72, and 96 hours, remove the cells and add 10 μL of CCK8 reagent; 4. Incubate at 37 ℃ for 1 hour, and measure the absorbance at 450 nm using a microplate reader; calculate the cell count at each time point according to the standard curve, and then substitute the results into the formula to calculate the doubling time: ; Where N1 represents the number of viable cells at time point 1, N2 represents the number of viable cells at time point 2, and t represents the time interval. The calculation results are shown in Table 6 below: the doubling times of the two types of stem cells are 37.06 h and 43.64 h, respectively. The doubling time of the higher-generation FGF21-HSA-IGFBP2 stem cells is actually 6.58 h shorter than that of FGF21-HSA-Preproglucagon stem cells. This result reflects that the former has a stronger cell proliferation capacity, which is consistent with the previous statement that the former has a stronger passage expansion capacity.

[0050] Table 6 Comparison of doubling time of gene-modified stem cells

[0051] Example 3: Comparison of the therapeutic effects of FGF21-HSA-IGFBP2 modified stem cells and FGF21-HSA-Preproglucagon modified stem cells on type 2 diabetes induced by high-fat diet in 12.5-month-old mice. 3.1 Comparison of blood sugar lowering effects The diagnostic criteria for diabetes were based on the 2024 International Diabetes Federation (IDF) recommended diagnostic criteria: fasting blood glucose ≥ 7.0 mmol / L, and oral glucose tolerance test (OGTT) 1 h ≥ 11.6 mmol / L (Bergman M, et al. International Diabetes Federation Position Statement on the 1-hour post-load plasma glucose for the diagnosis of intermediate hyperglycaemia and type 2 diabetes. Diabetes ResClin Pract. 2024 Mar;209:111589.). C57 mice (12.5 months old; 6-month-old mice were fed a high-fat diet for 4 months, then switched to a normal diet for 2 months, and continued high-fat diet for half a month before injection) induced type 2 diabetes were injected twice with two types of genetically modified stem cells (4 male mice per group). The two injections were two weeks apart. The improvement in blood glucose metabolism and muscle atrophy (grip strength index) associated with type 2 diabetes was compared before and after the second injection. The specific procedure is as follows: According to 2.5×10 5 Animal models were injected with 25 g cells per cell (Table 7). The mouse models were randomly divided into two groups of four mice each. Two types of stem cells were injected twice, with a two-week interval between the injections. The specific groupings and the number of cells injected are shown in Table 7. Fasting blood glucose and OGTT 2-hour curves were measured one day before the second injection and 48 hours after the second injection.

[0052] Three days prior to the first injection, 8th generation (P8) FGF21-HSA-Preproglucagon gene-modified umbilical cord mesenchymal stem cells and 6th generation (P6) FGF21-HSA-IGFBP2 gene-modified umbilical cord mesenchymal stem cells were resuscitated and routinely cultured at a volume of 12 mL. The medium was changed after 16 h, and culture continued. Cells and culture supernatant were collected after 72 h. Harvested cells were centrifuged at 1800 rpm at room temperature for 5 min, resuspended in PBS, and counted for subsequent tail vein injection. Cell supernatant was centrifuged at 3000 rpm at 4°C for 10 min and stored at -20°C, avoiding repeated freeze-thaw cycles. Some cells were cryopreserved at -80°C at a density of 1×10⁻⁶ cells / cm². 6 Each vial is labeled as Generation 9 (P9) and Generation 7 (P7) for future injections.

[0053] Three days before the second injection, the frozen FGF21-HSA-Preproglucagon P9 cells and FGF21-HSA-IGFBP2 P7 cells were revived and transferred to T75 culture flasks with a culture volume of 12 mL. After 16 h, the medium was changed and cultured for another 72 h. Cells and culture supernatant were collected after 72 h using the method described above.

[0054] As shown in Table 8, after injection of both types of genetically modified stem cells, glucose metabolism in mice returned to normal (fasting blood glucose was below 7.0 mmol / L, and metabolic data within 2 hours of OGTT also returned to normal, including 1 hour of OGTT below 11.6 mmol / L), and the glucose control was long-lasting, remaining effective for at least 48 hours after injection. Fasting blood glucose, a more crucial indicator reflecting glucose metabolism, showed an average decrease of 2.925 mmol / L for FGF21-HSA-IGFBP2 and 2.250 mmol / L for FGF21-HSA-Preproglucagon genetically modified stem cells, as shown in Table 9. Figure 5 As shown, the former group showed a 30.0% greater decrease than the latter, and the decrease in fasting blood glucose between the two groups was significantly different (p < 0.05).

[0055] Table 7 Experimental Groups and Cell Injection Volumes

[0056] Table 8. Blood glucose detection in mice

[0057] Table 9 Comparison of fasting blood glucose levels after two injections of gene-modified stem cells.

[0058] 3.2 Comparison of cell costs for FGF21-HSA-IGFBP2 and FGF21-HSA-Preproglucagon gene-modified stem cells Compared to FGF21-HSA-IGFBP2-modified stem cells, FGF21-HSA-Preproglucagon stem cells have a stronger expansion capacity, with the former being 1.45 times that of the latter. This reduces cell culture costs by 45%, significantly lowering the cost of this technology compared to the control group and greatly enhancing the development value of this approach.

[0059] 3.3 Comparison of muscle motor ability and grip strength in c57 mice before and after being fed a high-fat diet with FGF21-HSA-IGFBP2 and FGF21-HSA-Preproglucagon gene-modified stem cells. Literature reports that muscle atrophy in human type 2 diabetes model mice resulted in a significant decrease in grip strength of 22.4% compared to the normal group (Okamura T, et al. A multi-omics approach to overeating and inactivity-induced muscle atrophy in db / db mice, Journal of Cachexia, Sarcopenia and Muscle 2024; 15: 2030–2045). In the above experiment 3.1, grip strength tests were conducted before and after the second injection to compare the grip strength data of the two groups of animals. Experimental preparation: (1) Equipment and calibration: Ugo Basile brand Cat.47200 grip strength meter was used. Calibration was required before the experiment according to the instruction manual. Ensure that the sensor connection is stable and set to peak tensile mode, using the default unit g and a resolution of 0.1 g. (2) Animal preparation: The mice were brought into the testing room at least 1 hour in advance to adapt to the environment. The test should be conducted at the same time every day as much as possible to reduce the influence of circadian rhythm. Experimental measurement of limb grip strength: During the test, the movements were kept gentle. The mouse was grasped at the base of its tail and placed on a grid, with its forepaws and hind paws gripping the grid simultaneously, and its torso parallel to the grid. The tail was pulled horizontally backward, and the peak pulling force was recorded. After each mouse was tested, the grid was cleaned with 70% ethanol and allowed to dry completely before testing the next mouse to prevent odor interference. The grip strength data for each animal was measured three times, and the average value was taken. See Tables 10 and 11 below. Figure 6 As shown, between the first and second injections, the average grip strength / body weight (g / g) of mice treated with FGF21-HSA-IGFBP2 gene-modified stem cells was 3.4, which was 13.3% higher than the 3.0 grip strength of the FGF21-HSA-Preproglucagon gene-modified stem cell group. After the second injection, the average grip strength / body weight (g / g) of the two groups were 3.9 and 3.2, respectively. The grip strength of mice treated with FGF21-HSA-IGFBP2 gene-modified stem cells was 21.9% higher than that of the FGF21-HSA-Preproglucagon gene-modified stem cell group.

[0060] Table 10. Grip strength measurement after the first injection and before the second injection.

[0061] Table 11. Grip strength measurement after the second injection.

[0062] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for promoting high-level expression of FGF21 in mesenchymal stem cells, characterized in that, The target genes encoding FGF21 and IGFBP2 proteins are co-expressed in mesenchymal stem cells; the FGF21 and IGFBP2 proteins are linked by an HSA; the mesenchymal stem cells are derived from the umbilical cord; When HSA is linked between FGF21 protein and IGFBP2 protein, the target gene, from the 5' end to the 3' end, consists of the nucleotide sequence encoding the signal peptide shown in SEQ ID NO. 6, the nucleotide sequence encoding the FGF21 protein shown in SEQ ID NO. 7, the nucleotide sequence encoding the linker shown in SEQ ID NO. 10, the nucleotide sequence encoding the HSA protein shown in SEQ ID NO. 9, the nucleotide sequence encoding the linker shown in SEQ ID NO. 10, and the nucleotide sequence encoding the IGFBP2 protein shown in SEQ ID NO.

8.

2. The method as described in claim 1, characterized in that, The FGF21-HSA-IGFBP2 fusion protein was overexpressed in stem cells; the fusion protein, from the amino terminus to the carboxyl terminus, consisted of the signal peptide shown in SEQ ID NO.1, the FGF21 protein shown in SEQ ID NO.2, the Linker shown in SEQ ID NO.5, the HSA protein shown in SEQ ID NO.4, the Linker shown in SEQ ID NO.5, and the IGFBP2 protein shown in SEQ ID NO.

3.

3. A method for preparing gene-modified mesenchymal stem cells with high expression of FGF21, characterized in that, Includes the following steps: S1. Insert the target gene into a lentiviral expression vector to construct a recombinant plasmid; S2. Co-transfect host cells with the recombinant plasmid obtained in step S1 and the packaging plasmid to prepare lentiviral particles; S3. Infect mesenchymal stem cells with the lentiviral particles obtained in step S2 to obtain gene-modified mesenchymal stem cells with high expression of FGF21. The target gene consists of, from 5' to 3', the following nucleotide sequences: the nucleotide sequence encoding the signal peptide shown in SEQ ID NO. 6, the nucleotide sequence encoding the FGF21 protein shown in SEQ ID NO. 7, the nucleotide sequence encoding the linker shown in SEQ ID NO. 10, the nucleotide sequence encoding the HSA protein shown in SEQ ID NO. 9, the nucleotide sequence encoding the linker shown in SEQ ID NO. 10, and the nucleotide sequence encoding the IGFBP2 protein shown in SEQ ID NO.

8.

4. The method as described in claim 3, characterized in that, The host cells include 293T cells; the lentiviral expression vector includes plvx-IRES-Neo.

5. Genetically modified mesenchymal stem cells prepared by the method of claim 3 or 4.

6. A pharmaceutical composition comprising the gene-modified mesenchymal stem cells of claim 5.

7. The use of the gene-modified mesenchymal stem cells of claim 5 or the pharmaceutical composition of claim 6 in the preparation of a medicament for treating metabolic syndrome, characterized in that, The metabolic syndrome is type 2 diabetes.

8. The application as described in claim 7, characterized in that, The drug may also contain pharmaceutically acceptable sterile isotonic or non-aqueous solutions, suspensions or emulsions.

9. The application as described in claim 7, characterized in that, The drug also contains a dispersion.

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