Preparation method of umbilical cord mesenchymal stem cell exosome and application of umbilical cord mesenchymal stem cell exosome in treatment of diabetes

Abstract

The invention discloses a GLP1 fusion protein with pancreas islet targeting, an exosome modified by the GLP1 fusion protein as well as a preparation method and application of the GLP1 fusion protein. The structure of the fusion protein is TP-linker-Lamp2b-linker-GLP1, the amino acid sequence of the pancreas islet targeting polypeptide TP is CPKTRRVY, and the pancreas islet targeting polypeptide TP can be specifically combined with pancreas islet beta cells; the amino acid sequence of the Linker is GSGSGSGSGS, so that the immunogenicity can be reduced, and the structural shielding can be avoided; the amino acid sequence of Lamp2b is as shown in SEQ ID NO.1, and exosome membrane anchoring can be realized; the amino acid sequence of the GLP1 mutant is HGEGTFPSDVSYLEGQAAKEFI AWLVKGR, and the half-life period of the GLP1 mutant is remarkably prolonged through A2G / T7P mutation. The invention also provides a preparation method of the fusion protein modified exosome. The preparation method comprises the steps of lentiviral vector construction, hUC-MSCs transduction and exosome extraction. In-vitro and in-vivo experiments prove that the modified exosome can target pancreas islet beta cells, efficiently exert the hypoglycemic effect of the GLP1 and remarkably improve the pancreas islet function, and a novel strategy which is high in targeting property, long-acting and efficient in delivery is provided for diabetes treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for preparing umbilical cord mesenchymal stem cell exosomes and their application in the treatment of diabetes. Background Technology

[0002] Diabetes mellitus, the most prevalent metabolic disease globally, has now surpassed 530 million cases worldwide, according to the latest statistics from the International Diabetes Federation, with type 2 diabetes (T2DM) accounting for over 90%. Its pathological progression exhibits a typical vicious cycle of dual defects: the pancreatic β-cell population undergoes progressive decline (encompassing increased apoptosis, phenotypic dedifferentiation, and circadian rhythm disturbances in insulin secretion), accompanied by a continuous increase in insulin resistance (IR) in peripheral tissues (liver, skeletal muscle, and adipose tissue). A long-term high-glucose microenvironment, through excessive activation of the polyol pathway, abnormal activation of protein kinase C, and dysregulation of the hexosamine pathway, induces microvascular complications (such as glomerulosclerosis and retinal capillary occlusion) and macrovascular complications (such as atherosclerosis and peripheral vascular disease), leading to a 2.3 times higher risk of serious adverse events in patients over 5 years compared to healthy individuals, significantly increasing the disease burden.

[0003] Current clinical interventions have significant shortcomings: while insulin replacement therapy can quickly correct insulin resistance, it requires lifelong administration and is prone to causing hypoglycemia and abnormal fat deposition; first-line oral hypoglycemic agents such as metformin can only partially alleviate insulin resistance (IR), have no direct protective effect on β-cell function, and about 30% of patients discontinue treatment due to gastrointestinal adverse reactions such as nausea and diarrhea; GLP-1 receptor agonists, although having both blood sugar control and weight loss effects, require subcutaneous injection and carry the risk of pancreatic exocrine dysfunction, and cannot reverse the already damaged β-cell population. Therefore, developing a synergistic treatment system that combines β-cell regeneration promotion and targeted IR improvement has become a core direction for overcoming the clinical challenges of type 2 diabetes mellitus (T2DM).

[0004] Mesenchymal stem cell-derived exosomes (MSC-Exos), as nanoscale vesicles with a diameter of 30-150 nm, exhibit unique value in T2DM intervention due to their carrying of functional miRNAs (such as miR-21, which regulates apoptosis, and miR-124, which improves insulin resistance), cytokines, and lipid mediators. Among them, human umbilical cord mesenchymal stem cell (hUC-MSC) exosomes have become a highly promising candidate vector due to their convenient sourcing, low immunogenicity (HLA-DR expression negative), and minimal ethical controversy. However, natural exosomes have two major bottlenecks: ① lack of targeting; after intravenous infusion, only 3.2% ± 1.5% can be directionally enriched in the pancreatic islet region, while the rest are non-specifically cleared by the reticuloendothelial system (such as splenic macrophages and hepatic sinusoidal endothelial cells); ② limited function; although they can mildly reduce β-cell apoptosis through secretory TGF-β1, they cannot effectively activate the IRS-1 / PI3K / Akt signaling pathway to improve insulin resistance, thus limiting their clinical translational efficacy.

[0005] Existing exosome modification technologies have significant limitations: ① At the level of targeted modification, they mostly rely on vascular endothelial markers or integrin ligands, which suffer from insufficient target cell recognition specificity (such as cross-reactivity with the tumor microenvironment) and short ligand half-lives (in vivo clearance half-life < 6 hours); ② At the level of functional enhancement, traditional drug encapsulation strategies easily damage the integrity of exosome membranes, leading to high leakage rates (leakage rates often exceed 60% within 48 hours); ③ At the level of anchoring strategies, modification methods based on membrane protein fusion often result in abnormal target protein conformation due to steric hindrance, with approximately 30% of fusion proteins losing their biological activity. Therefore, constructing an exosome modification system that combines high targeting specificity, dual-function synergy, and structural stability has become a pressing technical challenge in this field. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] Therefore, this invention discloses a GLP1 fusion protein with pancreatic islet targeting, wherein the structure of the fusion protein is TP-linker-Lamp2b-linker-GLP1, and the functional domains are sequentially linked as follows:

[0008] (1) Pancreatic islet-targeting polypeptide (TP): The sequence is CPKTRRVY, which can form a stable β-turn, specifically bind to the receptor on the surface of pancreatic β cells, and mediate targeted delivery;

[0009] (2) Linker: The sequence is GSGSGSGSGS, which reduces the immunogenicity of the fusion protein and avoids spatial masking of each functional domain;

[0010] (3) Lamp2b anchoring sequence: The sequence is shown in SEQ ID NO.1. It has 77.4% homology with the known Lamp2b sequence (B4DF49) and contains a linker binding site, which can anchor the fusion protein to the exosome membrane;

[0011] (4) GLP1 mutant: The sequence is HGEGTFPSDVSSYLEGQAAKEFIAWLVKGR. Through the A→G mutation at position 2 and the T→P mutation at position 7, it is significantly resistant to DPP-4 digestion and the half-life is extended to 45 minutes (the half-life of natural GLP1 is 2 minutes).

[0012] (5) The complete amino acid sequence of the fusion protein is shown in SEQ ID NO.2.

[0013] On the other hand, the present invention also discloses a method for preparing fusion protein-modified exosomes:

[0014] (1) Construction of recombinant lentiviral vector: The nucleic acid sequence encoding the fusion protein (SEQ ID NO.3) was artificially synthesized and inserted into the pLenti-CMV-Lamp2b-Puro vector through EcoRI and XhoI restriction sites. The sequence was verified by PCR, restriction enzyme digestion and sequencing.

[0015] (2) Lentiviral packaging: The recombinant vector, psPAX2 (packaging plasmid), and pMD2.G (enveloping plasmid) were co-transfected into HEK293T cells at a ratio of 4:3:1. After 48 hours, the viral supernatant was collected, filtered, and the titer was measured (approximately 1.2 × 10⁻⁶). 8 TU / mL);

[0016] (3) hUC-MSCs transduction and screening: hUC-MSCs were transduced with lentivirus (MOI=50), and 8 μg / mL Polybrene was added to enhance the infection efficiency. After 72 hours, cells were screened with 2 μg / mL puromycin to obtain cells that stably expressed the fusion protein.

[0017] (4) Exosome extraction and identification: The culture supernatant of transduced hUC-MSCs was collected and impurities were removed by gradient centrifugation at 300×g, 2000×g and 10000×g. After ultrafiltration and concentration, exosomes were obtained by ultracentrifugation at 100,000×g. The morphology (80-150nm cup-shaped vesicles) was observed by TEM, the particle size (average 113.2nm) was analyzed by NTA, and the fusion proteins on the membrane (CD63, CD9, Lamp2b, GLP1 positive) were verified by Western Blot.

[0018] The technical solution of this invention has significant advantages: First, it has strong targeting; the specific recognition mediated by TP peptides leads to local enrichment of GLP1 in the islets, reducing the side effects caused by systemic distribution. Second, it has outstanding long-lasting effect; the anti-enzymatic properties of GLP1 mutants significantly prolong the duration of action and reduce the frequency of administration. Third, it has high delivery efficiency; exosomes, as natural carriers, can protect GLP1 from degradation, while the anchoring effect of Lamp2b ensures the stable display of functional molecules. Fourth, it has good biocompatibility; exosomes derived from hUC-MSCs have low immunogenicity and higher safety. Fifth, the process is stable and controllable; the stable expression system mediated by lentiviruses and the gradient centrifugation-ultrafiltration extraction method can achieve large-scale preparation. In the future, this technology can be directly applied to the treatment of type 2 diabetes and diseases related to pancreatic islet function impairment, developing novel GLP1 drugs with strong targeting, long-lasting effects, and safety. Simultaneously, its targeted delivery system can be extended to the delivery of other islet-related factors, providing a new tool for the prevention and treatment of diabetic complications and islet regeneration research, with broad prospects for clinical translation and scientific research applications.

[0019] It should be noted that the schematic diagram of the preparation and function of the pancreatic islet-targeting GLP1 fusion protein-modified exosomes disclosed in this invention is as follows: Figure 1 As shown. Attached Figure Description

[0020] Figure 1 A schematic diagram illustrating the preparation and function of exosomes modified with GLP1 fusion protein that targets the pancreas.

[0021] Figure 2 Results of transmission electron microscopy (TEM) of exosomes.

[0022] Figure 3 Results of exosome nanoparticle tracking analysis (NTA).

[0023] Figure 4 Results of Western Blot validation of exosomes. Detailed Implementation

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0026] Example 1: Design of a GLP1 fusion protein with pancreatic islet targeting

[0027] The fusion protein sequence disclosed in this embodiment is abbreviated as TP-linker-Lamp2b-linker-GLP1, and the sequence and characteristics of each part are as follows:

[0028] 1. The amino acid sequence of the pancreatic islet-targeting polypeptide (TP) is CPKTRRVY. Secondary structure prediction shows that it can form a stable β-turn. This structural characteristic gives it a unique binding mode with β-cell surface receptors, enabling it to specifically target pancreatic β-cells.

[0029] 2. The linker sequence uses GSGSGSGSGS. The use of this sequence can effectively reduce the immunogenicity of the fusion protein, while avoiding the masking of the C-terminus and ensuring the normal function of each functional domain.

[0030] 3. The amino acid sequence of the anchoring sequence Lamp2b is shown in SEQ ID NO.1. This sequence has 77.4% homology with the known Lamp2b sequence (B4DF49), and contains a linker binding site in the C-terminal extension, which is beneficial for linker connection.

[0031] 4. The amino acid sequence of the active GLP1 fragment is H. G EGTF P SDVSSYLEGQAAKEFIAWL

[0032] Compared with the natural GLP1 (7-37) (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR) sequence, the mutations at positions 2 (A→G) and 7 (T→P) of VKGR significantly enhance its resistance to DPP-4 enzymatic degradation, extending its half-life from 2 minutes to 45 minutes and prolonging its duration of action in vivo.

[0033] The above components are sequentially linked by a linker to form a GLP1 fusion protein that is pancreatic islet-targeting and has a long-lasting effect, the amino acid sequence of which is shown in SEQ ID NO.2.

[0034] Example 2: Preparation and testing of exosomes modified with GLP1 fusion protein targeting pancreatic islet

[0035] I. Construction and Validation of Lentiviral Vectors

[0036] 1. Experimental Materials and Reagents

[0037] (1) Fusion protein coding sequence: Based on human cell codon preference optimization, the full-length coding sequence of GLP1 fusion protein with optimized codons was artificially synthesized (SEQ ID NO.3).

[0038] (2) Vectors and tool enzymes: pLenti-CMV-Lamp2b-Puro vector (containing CMV promoter and puromycin resistance gene); EcoRI and XhoI restriction endonucleases; Phanta Max DNA polymerase.

[0039] (3) Primers, synthesized by bioengineering, as follows:

[0040] Upstream primer: 5'-CGGAATTCATGTGTCCAAAGACCCGTCG-3';

[0041] Downstream primer: 5'-CCGCTCGAGTCGACCCTTGACCAGCCAGG-3'.

[0042] 2. Experimental Procedure

[0043] Target gene synthesis and vector ligation: GenScript was commissioned to synthesize the full-length coding sequence, and EcoRI (5') and XhoI (3') restriction sites were introduced at both ends, respectively; the digested target gene was ligated with the linearized pLenti vector at 16°C overnight using T4 DNA ligase (insertion fragment: vector molar ratio = 3:1).

[0044] (2) PCR verification: The reaction system is the same as before, and the amplification conditions are strictly controlled: 98℃ pre-denaturation for 30s; 30 cycles (98℃ denaturation for 10s, 62℃ annealing for 15s, 72℃ extension for 73s); 72℃ final extension for 5min.

[0045] (3) Enzyme digestion verification: Take 5 μg of recombinant plasmid and digest it with EcoRI (10 U) and XhoI (10 U) in a water bath at 37℃ for 2 h. The digestion system contains 1×CutSmart Buffer and the total reaction volume is 50 μL.

[0046] (4) Electrophoresis detection: 1% agarose gel (containing 0.5 μg / mL EB), 1×TAE buffer, 120V constant voltage electrophoresis for 20 min, observed by gel imaging system, the target fragment size is about 885 bp (consistent with the theoretical value).

[0047] (5) Cloning screening and sequencing: DH5α competent cells were transformed, plated on LB plates containing ampicillin (100 μg / mL), and cultured at 37℃ for 16 h; 6 single clones were picked, and plasmids were extracted using Tianprep Mini Kit after shaking; the clones were sent to Qingke Biotechnology for Sanger sequencing to confirm that the sequence matched the design sequence 100%.

[0048] II. Lentiviral Packaging and hUC-MSCs Transduction

[0049] 1. Experimental Materials

[0050] (1) Cells: HEK293T cells, human umbilical cord mesenchymal stem cells (hUC-MSCs, isolated from umbilical cord tissue or purchased commercially, and identified in accordance with the standards of the International Cell Therapy Association).

[0051] (2) Reagents: DMEM high glucose medium (Gibco), exosome-free fetal bovine serum (Gibco), PEI (Polysciences, 1 mg / mL stock solution, pH adjusted to 7.0 with 1 M HCl), Polybrene (Sigma)

[0052] 2. Experimental Procedure

[0053] (1) HEK293T cell preparation: 24 hours before transfection, HEK293T cells were seeded in 10cm culture dishes, cultured in DMEM containing 10% FBS, and cultured at 37℃ in a 5% CO2 incubator until confluence reached 70%.

[0054] (2) Lentiviral packaging: Prepare plasmids (10 μg pLenti recombinant plasmid, 7.5 μg pPAX2, 2.5 μg pMD2.G) in a 4:3:1 ratio, dissolve them in 250 μL Opti-MEM (Gibco), and mix gently; take 60 μL PEI stock solution, dissolve it in 250 μL Opti-MEM, and let it stand at room temperature for 5 min; combine the plasmid mixture with the PEI solution, incubate at room temperature for 15 min, slowly add it to HEK293T cell culture medium, and shake gently; replace the culture medium with fresh medium 6 h after transfection, and collect the viral supernatant 48 h later.

[0055] (3) Virus purification and titer determination: The viral supernatant was filtered through a 0.45 μm PES filter membrane, and the titer was determined using a p24 ELISA kit (Cell Biolabs). The result was (1.2 ± 0.1) × 10⁻⁶. 8 TU / mL.

[0056] (4) hUC-MSCs transduction: log-growing hUC-MSCs were selected and transduced at a rate of 1×10⁻⁶. 6 Cells were seeded in 10cm culture dishes and cultured for 24h; virus solution (MOI=50) and Polybrene (final concentration 8μg / mL) were added and incubated at 37℃ for 24h; the medium was replaced with DMEM medium (10% v / v) containing exosome-free FBS and cultured for another 72h; after transduction, hUC-MSCs were screened for 72h with puromycin (2μg / mL, the optimal screening concentration was determined by preliminary experiments) to obtain stable hUC-MSCs expressing the fusion protein.

[0057] III. Exosome Extraction and Identification

[0058] 1. Experimental Materials and Instruments

[0059] (1) Instruments: Eppendorf 5810R centrifuge, Beckman Optima XPN-100 ultracentrifuge, Amicon Ultra-15 ultrafiltration tube (Millipor).

[0060] (2) Reagents: PBS, 4% paraformaldehyde, 2% phosphotungstic acid (pH 7.4).

[0061] 2. Exosome extraction steps

[0062] (1) Collect 40 mL of culture supernatant of stably transduced hUC-MSCs and perform gradient centrifugation in sequence: 300×g, 10 min, 4℃ (to remove live cells); 2000×g, 20 min, 4℃ (to remove cell debris); 10000×g, 30 min, 4℃ (to remove apoptotic bodies and large vesicles).

[0063] (2) Transfer the supernatant to an Amicon Ultra-15 ultrafiltration tube, centrifuge at 3000×g for 15 min at 4℃, repeat 3 times, and concentrate to 1 mL.

[0064] (3) Transfer the concentrate to an ultracentrifuge tube, 100,000×g, 2h, 4℃.

[0065] (4) Discard the supernatant, resuspend the precipitate in 200 μL of pre-cooled PBS, aliquot and store at -80℃.

[0066] 3. Exosome identification

[0067] (1) Transmission electron microscopy (TEM) observation: 10 μL of exocrine body suspension was added to a copper grid and allowed to stand at room temperature for 5 min; excess liquid was removed, and 2% phosphotungstic acid (pH 7.4) was added for negative staining for 5 min, and then allowed to air dry at room temperature; TEM observation was performed with an accelerating voltage of 80 kV and a magnification of 100,000×. The results showed that ( Figure 2 The typical cup-shaped vesicle structure is visible, with a diameter of 80-150 nm and a clear membrane structure.

[0068] (2) Nanoparticle tracking analysis (NTA): Exosome samples were diluted 100-fold with PBS and injected into the instrument; the detection parameters were: temperature 25℃, detection time 60s / time, repeated 3 times; the results showed ( Figure 3 The average particle size was 113.2 ± 5.7 nm, and the particle concentration was (1.04 ± 0.08) × 10⁻⁶. 11 The particle size distribution was concentrated (PDI = 0.18 ± 0.03).

[0069] (3) Western Blot validation: Exosomes were lysed on ice for 30 min using RIPA lysis buffer (containing protease inhibitor), and protein concentration was quantified by BCA method; 20 μg of protein was loaded onto a 12% SDS-PAGE gel (80V stacking gel for 30 min, 120V separating gel for 90 min); wet transfer was performed to a PVDF membrane (300mA, 1.5h); blocking was performed with 5% skim milk powder TBST solution for 1h; primary antibody was incubated overnight at 4℃ (CD63: Abcam, 1:1000; CD9: Cell Signaling, 1:1000; Lamp2b: Abcam, 1:500; GLP1: self-made rabbit polyclonal antibody, 1:800); HRP-labeled secondary antibody (1:5000) was incubated at room temperature for 1h; ECL chemiluminescence imaging was performed. The results showed that ( Figure 4 CD63 and CD9 were positively expressed; Lamp2b and GLP1 were strongly positively expressed, confirming that the fusion protein was integrated into the exosome membrane.

[0070] IV. Verification of pancreatic islet cell targeting and function

[0071] 1. Targeted validation

[0072] (1) Fluorescent labeling of exosomes: Take 100 μg of exosomes, add PKH26 dye, and incubate in PBS at 37℃ for 5 min at a ratio of 1:10 (exosomes: dye, v / v); add an equal amount of 1% BSA to terminate the reaction; centrifuge at 100,000×g for 30 min (4℃) and wash twice to remove free dye.

[0073] (2) Cell co-incubation: MIN6 cells (mouse pancreatic β cells) and HepG2 cells (human liver cancer cells) were incubated with 1×10⁻⁶ cells per cell line. 5 Cells / wells were seeded in confocal culture dishes and allowed to adhere for 24 hours;

[0074] (3) Add PKH26-labeled fusion exosomes (final concentration 50 μg / mL) and co-incubate in DMEM containing 1% FBS at 37°C for 4 h;

[0075] (4) Fix with 4% paraformaldehyde for 15 min, stain the nucleus with DAPI for 5 min, and wash with PBS 3 times.

[0076] (5) Imaging and quantification: observation by laser confocal microscopy; ImageJ software was used to randomly select 5 fields of view, and the average fluorescence intensity (integrated density) of 100 cells in each field of view was analyzed.

[0077] (6) Results: The average fluorescence intensity of MIN6 cells was (2856±312) and that of HepG2 cells was (842±95), the former being 3.4 times that of the latter (P<0.01), confirming the targeting of pancreatic β cells mediated by TP peptide.

[0078] 2. GLP-1 Functional Testing

[0079] (1) Cell treatment: MIN6 cells were treated with 1×10 5 Cells / wells were seeded in 24-well plates and cultured to 80% confluence; then the plates were starved with sugar-free DMEM for 2 hours, with 3 replicates per group, and incubated at 37°C for 24 hours. The groups are as follows:

[0080] Control group: PBS;

[0081] Negative control group: Unmodified exosomes (50 μg / mL);

[0082] Positive control group: Natural GLP1 (100 nM);

[0083] Experimental group: Fusion exosomes (containing 100 nM GLP1 equivalent).

[0084] (2) Insulin secretion detection: The supernatant was collected and detected using an insulin ELISA kit. The procedure was strictly performed according to the instructions. The results are shown in Table 1:

[0085] Table 1 Insulin secretion test results

[0086] Grouping Insulin release (μIU / mL, mean ± SD) Compared with the PBS group (P value) PBS 4.2±0.5 — Unmodified exosomes 4.5±0.6 ns Natural GLP1 8.8±1.2 <0.01 Fusion exosomes 14.7±1.4 <0.001

[0087] (3) Duration of function test: Insulin levels were detected by collecting supernatant at 0, 2, 4, 6, 8 and 12 h according to the above grouping; the results showed that the fusion exosome group still maintained 75% of the maximum activity at 8 h, while the activity of the natural GLP1 group dropped to below 30% after 2 h (P<0.01).

[0088] V. Experimental Analysis and Summary

[0089] 1. Fusion protein design: TP peptide (β-turn structure) mediates pancreatic islet targeting, Lamp2b ensures exosome membrane anchoring, and GLP1 mutant (A2G / T7P) significantly resists DPP-4 digestion (half-life extended by 22.5 times).

[0090] 2. Preparation process: Lentiviral-mediated stable expression of hUC-MSCs, combined with gradient centrifugation and ultrafiltration for efficient extraction of modified exosomes, yielding (2.6±0.3)×10⁻⁶ cells / mL. 10 particles / 10 6 cell;

[0091] 3. Functional advantages: In vitro experiments have confirmed its targeting of pancreatic β cells (3.4 times specific uptake) and its potent and long-lasting insulin secretion-promoting effect (activity lasts for more than 8 hours).

[0092] This method provides a highly targeted GLP-1 delivery system with a long half-life for diabetes treatment, and has clinical translational potential.

[0093] Example 3: Experiment on the treatment of diabetic SD rats with fused exosomes (Lamp2b-GLP1-TP-Exos)

[0094] 1. Laboratory animals and grouping

[0095] (1) Animal selection: 40 SPF-grade male SD rats, weighing 220±20g, 6-8 weeks old; the rearing temperature was 23±2℃, the humidity was 50±5%, the light and dark cycle was 12h, and the rats had free access to food and water (standard rat food + sterile water).

[0096] (2) Adaptation feeding: Rats were adapted to feeding for 7 days. Their activity status, food and water intake and fecal characteristics were observed daily, and healthy individuals with abnormalities were excluded.

[0097] 2. Establishment of a diabetes model

[0098] (1) STZ preparation: Strawtozotocin (STZ, Sigma) is freshly prepared with 0.1M citrate buffer (pH 4.5) at a concentration of 11 mg / mL. It should be stored on ice in the dark and used within 30 min.

[0099] (2) Modeling method: Rats in the model group, unmodified exosomes and fused exosomes group were fasted for 12h (free access to water) and injected intraperitoneally with STZ (55mg / kg, injection volume calculated according to body weight, 1mL / kg); the normal control group was injected with an equal volume of 0.1M citrate buffer.

[0100] (3) Successful model establishment judgment: After model establishment, blood was collected from the tail tip daily (blood glucose meter) and monitored for 3 consecutive days; if the fasting blood glucose for 3 days was >16.7mmol / L, the diabetes model was determined to be successful; a total of 24 models were successfully established and randomly divided into 3 groups (8 models in each group, with 2 models reserved as backup to prevent death during the process).

[0101] 3. Dosing regimen and group adjustments: Four groups were ultimately determined, with six animals in each group (excluding individuals that failed to establish the model or exhibited abnormal conditions):

[0102] (1) Normal control group: No model was established, and PBS (0.2 mL / 100 g body weight) was injected into the tail vein.

[0103] (2) Model control group: Modeling was successful, and PBS (0.2 mL / 100 g body weight) was injected into the tail vein.

[0104] (3) Unfused exosome group: Modeling was successful. Unmodified hUC-Exos (200 μg / animal, diluted with PBS to 0.2 mL / 100 g body weight) was injected into the tail vein.

[0105] (4) Fusion exosome group: Modeling was successful. Lamp2b-GLP1-TP-Exos (200 μg / animal, diluted with PBS to 0.2 mL / 100 g body weight) was injected into the tail vein.

[0106] Dosage frequency: Administer once every 3 days for a total of 4 times (total dosing cycle of 12 days). Dosage time is 9:00-10:00 AM daily. Observe the rats for 15 minutes after tail vein injection and record whether allergic or stress reactions occur.

[0107] 4. Detection Indicators and Methods

[0108] (1) Blood glucose monitoring

[0109] Time points: Day 0 after successful model establishment (before drug administration), Day 3 after drug administration, Day 7, Day 10, and Day 14 (2 days after the last drug administration).

[0110] Procedure: Rats were fasted for 6 hours, and approximately 2 μL of blood was collected from the tail tip. Blood glucose was measured using a blood glucose meter. Each rat was tested twice, and the average value was taken.

[0111] (2) Serum insulin detection

[0112] After the last blood glucose test on Day 14, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL / kg), and 5 mL of blood was collected from the abdominal aorta. The serum was separated by centrifugation at 3000×g for 15 min (4℃) and stored at -80℃. The insulin concentration was calculated by using a rat insulin ELISA kit, strictly following the instructions.

[0113] 5. Experimental Results

[0114] (1) General condition observation: On the 3rd day after modeling, the rats in the model group showed obvious polydipsia, polyphagia, polyuria and weight loss (by Day 14, the weight decreased by 21.3±3.5% compared with before modeling); the above symptoms were slightly relieved in the non-fused exosome group (weight decreased by 14.5±2.8%); the symptoms of the fused exosome group were significantly improved (weight decreased by 8.7±1.9%), and the activity level was close to that of the normal group.

[0115] (2) Blood glucose changes: On Day 14, the blood glucose levels in the fused exosome group were significantly lower than those in the model group (P<0.001) and the non-fused exosome group (P<0.01), and were close to the levels in the normal group. See Table 2 for details.

[0116] Table 2. Blood glucose changes (unit: mmol / L, x±s)

[0117]

[0118] (3) Serum insulin concentration: The insulin level in the fused exosome group was significantly higher than that in the model group (P<0.001) and the non-fused exosome group (P<0.01), which was 2.24 times that of the model group, as shown in Table 3.

[0119] Table 3 Serum insulin concentration (unit: ng / mL, x±s)

[0120] Group result normal group 2.94±0.11 Model group 0.94±0.12 Unfused exosomes 1.43±0.09 Fusion exosomes 2.11±0.11

[0121] 6. Results Analysis

[0122] In vivo experiments confirmed that modified exosomes can deliver GLP-1 to pancreatic islet tissue via TP peptide targeting, significantly reducing blood glucose in diabetic rats (down to 9.8±1.0 mmol / L on Day 14), with better results than unmodified exosomes; the restoration of insulin levels indicates that modified exosomes can improve pancreatic β-cell function and promote pancreatic islet tissue repair.

[0123] The results in this section are consistent with in vitro experiments, confirming that the fusion protein-modified exosomes have good application prospects in the treatment of diabetes.

[0124] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A GLP1 fusion protein with pancreatic islet-targeting properties, characterized in that, The GLP1 fusion protein structure is TP-linker-Lamp2b-linker-GLP1, which is composed of pancreatic islet-targeting polypeptide TP, first linker, Lamp2b anchoring sequence, second linker and GLP1 mutant linked sequentially. The amino acid sequence of TP is CPKTRRVY; the amino acid sequences of the first and second linkers are both GSGSGSGSGS; the amino acid sequence of the Lamp2b anchoring sequence is shown in SEQ ID NO.1; the amino acid sequence of the GLP1 mutant is HGEGTFPSDVSSYLEGQAAKEFIAWLVKGR.

2. The GLP1 fusion protein according to claim 1, characterized in that, The amino acid sequence of the GLP1 fusion protein is shown in SEQ ID NO.

2.

3. A nucleic acid molecule encoding the GLP1 fusion protein of claim 2, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO.

3.

4. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule of claim 3.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector is the pLenti-CMV-Lamp2b-Puro vector, and nucleic acid molecules are inserted into the vector through EcoRI and XhoI restriction sites.

6. A host cell, characterized in that, The host cell comprises the recombinant vector of claim 5.

7. The host cell according to claim 6, characterized in that, The host cells are HEK293T cells or human umbilical cord mesenchymal stem cells.

8. An exosome modified with a fusion protein, characterized in that, The exosomes have the fusion protein of claim 1 or 2 bound to their membranes.

9. A method for preparing exosomes modified with the fusion protein according to claim 8, characterized in that, The method includes the following steps: (1) Construct a recombinant lentiviral vector comprising the nucleic acid molecule of claim 3; (2) The recombinant lentiviral vector and packaging plasmid were co-transfected into HEK293T cells to package the lentivirus and determine the titer; (3) Human umbilical cord mesenchymal stem cells were transduced with the lentivirus from step (2), and human umbilical cord mesenchymal stem cells that stably expressed the fusion protein were obtained by screening with puromycin. (4) Collect the culture supernatant of human umbilical cord mesenchymal stem cells in step (3), and extract exosomes by gradient centrifugation and ultrafiltration to obtain the fusion protein modified exosomes.

10. The preparation method according to claim 9, characterized in that, In step (2), the ratio of plasmids co-transfected is recombinant lentiviral vector: psPAX2: pMD2.G = 4:3:1; in step (3), the MOI of lentiviral transduction is 50, and the puromycin screening concentration is 2 μg / mL; in step (4), the gradient centrifugation includes 300×g for 10 min, 2000×g for 20 min, and 10000×g for 30 min, followed by ultrafiltration and ultracentrifugation at 100,000×g for 2 h.

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