Adiponectin gene-modified mesenchymal stem cells, their preparation method, drugs, and applications

Abstract

This invention relates to the field of stem cell culture, specifically to an adiponectin gene-modified mesenchymal stem cell, its preparation method, drug, and applications. The adiponectin gene-modified mesenchymal stem cell provided by this invention can overexpress adiponectin to stimulate pancreatic islet cells, regulate glucose stimulation response, and increase insulin secretion. The method for preparing the adiponectin gene-modified mesenchymal stem cell provided by this invention is simple, and the prepared adiponectin gene-modified mesenchymal stem cell exhibits higher insulin secretion and a longer duration of action in INS-1 cell glucose stimulation experiments.

Description

Technical Field

[0001] This invention relates to the field of stem cell culture, specifically to an adiponectin gene-modified mesenchymal stem cell, its preparation method, drug, and application. Background Technology

[0002] A common method used in cellular therapy for diabetes is to differentiate pluripotent stem cells into pancreatic islets. However, due to the potential for tumorigenicity of pluripotent stem cells, the instability caused by their heterogeneity, and the cumbersome culture process, the industrial production of pluripotent stem cells is limited and it is not easy to expand production on a large scale.

[0003] Umbilical cord-derived mesenchymal stem cells (MSCs) have emerged as promising candidates for diabetes treatment due to their low immunogenicity and high safety profile. They not only possess the capacity for self-renewal and multi-lineage differentiation but also exhibit significant immunomodulatory effects. Stable overexpression of specific functional proteins in these cells could further enhance their potential for treating diabetes.

[0004] GLP-1 and FGF21 are common hormone-like proteins used to treat diabetes. Related literature and patents have reported that their co-expression can effectively regulate glucose-stimulated responses; however, they are easily degraded both in vivo and in vitro. Therefore, there is an urgent need to construct mesenchymal stem cells with a long-lasting effect and the ability to effectively regulate glucose-stimulated responses. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the easy degradation defect of GLP-1 and FGF21 in regulating glucose stimulation response in the prior art, thereby providing a mesenchymal stem cell modified with adiponectin gene that can effectively regulate glucose stimulation response and the adiponectin in the mesenchymal stem cell has a long duration of action.

[0006] Another technical problem that this invention aims to solve is to overcome the defects of GLP-1 and FGF21 in regulating glucose-stimulated responses in the prior art, which are easily degraded and have short-lasting effects, thereby providing a simple method for preparing adiponectin gene-modified mesenchymal stem cells.

[0007] Therefore, the present invention provides the following technical solution: This invention provides a mesenchymal stem cell modified with an adiponectin gene, wherein the mesenchymal stem cell overexpresses adiponectin.

[0008] Preferably, the nucleotide sequence of the adiponectin gene is shown in SEQ ID NO:12, and the amino acid sequence of the adiponectin is shown in SEQ ID NO:11. The adiponectin can regulate glucose-stimulated responses, resulting in higher insulin secretion.

[0009] Preferably, the type of mesenchymal stem cells includes at least one of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, and bone marrow mesenchymal stem cells.

[0010] Preferably, the adiponectin gene-modified mesenchymal stem cells further include an expression vector; the expression vector further includes a functional element and a linker peptide; the functional element includes at least one of IgG4-Fc, T2A, and SP, and the linker peptide includes a linker; The adiponectin and IgG4-Fc are linked by a linker peptide sequence. The IgG4-Fc fusion protein consists of a signal peptide, adiponectin protein, a linker peptide, and an IgG4-Fc domain from the N-terminus to the C-terminus. The N-terminus of the adiponectin protein is linked to a signal peptide.

[0011] Preferably, the adiponectin gene-modified mesenchymal stem cells also overexpress glucagon-like peptide-1 (GLP-1), the nucleotide sequence of which is shown in SEQ ID NO:4, and the amino acid sequence of which is shown in SEQ ID NO:3.

[0012] In a specific embodiment of the present invention, recombinant plasmid YL011 co-expresses GLP-1 and adiponectin: the recombinant plasmid YL011 is composed of SP-IgG4-Fc-linker-GLP-1-T2A-adiponectin; recombinant plasmid YL011 is formed by linking IgG4-Fc-GLP-1 fusion protein and adiponectin via the T2A peptide; wherein, the IgG4-Fc-GLP-1 fusion protein is composed of signal peptide SP, IgG4-Fc, (GGGGS)3linker, and GLP-1 sequentially from the N-terminus to the C-terminus. Co-expression of GLP-1 and adiponectin enhances the glucose stimulation effect on pancreatic islet cells.

[0013] In a specific embodiment of the present invention, the recombinant plasmid YL006 expresses only adiponectin, thereby increasing the expression level of adiponectin.

[0014] Preferably, the adiponectin gene-modified mesenchymal stem cells further include an expression vector; the expression vector further includes a functional element and a linker peptide; the functional element includes at least one of IgG4-Fc, T2A, and SP, and the linker peptide includes a linker; The adiponectin and IgG4-Fc are linked by a linker peptide sequence. The IgG4-Fc fusion protein consists of a signal peptide, adiponectin protein, a linker peptide, and an IgG4-Fc domain from the N-terminus to the C-terminus. The N-terminus of the adiponectin protein is linked to a signal peptide.

[0015] This invention provides a method for preparing mesenchymal stem cells as described in the above technical solution, comprising the following steps: 1) The adiponectin gene was cloned into a lentiviral transfer plasmid to obtain a recombinant expression plasmid; 2) The recombinant expression plasmid, lentiviral packaging helper plasmid, and lentiviral packaging envelope plasmid were assembled into cells and transfected to obtain recombinant lentivirus; 3) The recombinant lentivirus was used to infect mesenchymal stem cells to obtain gene-modified mesenchymal stem cells that stably express adiponectin.

[0016] Preferably, the lentiviral transfer plasmid in step 1) includes pCDH.

[0017] Preferably, the lentiviral packaging helper plasmid in step 2) includes pSPAX2; the lentiviral packaging envelope plasmid includes pMD2.G; Before infecting mesenchymal stem cells with the recombinant lentivirus described in step 3), 293T cells were transfected for expansion; Preferably, in step 3), the infection multiplicity of recombinant lentivirus and mesenchymal stem cells is in the range of 10~40 pfu / cell.

[0018] The present invention provides the application of adiponectin gene-modified mesenchymal stem cells as described in the above technical solution, and adiponectin gene-modified mesenchymal stem cells obtained by the preparation method described in the above technical solution in at least one of the following: 1) To prepare drugs that enhance the glucose-stimulated response of pancreatic islet cells; 2) To prepare drugs that increase insulin secretion from pancreatic islet cells; 3) Enhance the ability of pancreatic islet cells to respond to glucose stimulation or increase the amount of insulin secreted by pancreatic islet cells.

[0019] This invention provides a drug that enhances the ability of pancreatic islet cells to respond to glucose stimulation and / or increases the amount of insulin secreted by pancreatic islet cells, comprising adiponectin gene-modified mesenchymal stem cells as described in the above technical solution, adiponectin gene-modified mesenchymal stem cells obtained by the preparation method described in the above technical solution, and pharmaceutically acceptable excipients.

[0020] Preferably, the dosage form of the drug includes one or more of the following: injection, oral, sustained-release, and targeted formulation; The excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0021] The technical solution of this invention has the following advantages: 1. The present invention provides a mesenchymal stem cell modified with adiponectin gene, which can overexpress adiponectin to stimulate pancreatic islet cells, regulate glucose stimulation response, increase insulin secretion, and maintain a longer duration of action.

[0022] 2. The method for preparing adiponectin gene-modified mesenchymal stem cells provided by this invention is simple. In the INS-1 cell glucose stimulation experiment, insulin secretion is high and the effect is long-lasting. The lentivirus prepared by the recombinant plasmid of this invention does not destroy the core stemness characteristics of mesenchymal stem cells during transfection, and the transfection system is safe and applicable.

[0023] 3. The drug provided by this invention for improving the ability of pancreatic islet cells to respond to glucose stimulation and / or increasing the amount of insulin secreted by pancreatic islet cells has an active ingredient that is adiponectin gene-modified mesenchymal stem cells. Adiponectin gene-modified mesenchymal stem cells can continuously stimulate cells to secrete insulin and have a long duration of action. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of the components of the YL001 recombinant plasmid vector; Figure 2 This is a fluorescence microscope image of HEK293T transfected with YL001 72 hours later. Figure 3 A fluorescence image taken 72 hours after YL001 lentivirus was packaged and used to infect UC-MSCs. Figure 4 The image shows a flow cytometry analysis of UC-MSCs after YL001 lentivirus packaging and infection, with the horizontal axis representing the MOI value of lentivirus-infected cells and the vertical axis representing EGFP% (EGFP positivity rate). Figure 5 Schematic diagram of the recombinant plasmid vectors pCDH-YL004, pCDH-YL006, pCDH-YL009, pCDH-YL010, and pCDH-YL011; Figure 6 The expression diagram of adiponectin protein, FGF21 protein and IgG4-Fc fusion protein in the supernatant of MSC-YL004, MSC-YL006, MSC-YL009, MSC-YL010, MSC-YL011 and MSC-NC cell lines; Figures 7-10Flow cytometry analysis of cell surface markers for MSC-YL006 and MSC-YL011 cells; Figure 11 Images of MSC-YL006 and MSC-YL011 stained with lipogenic oil red O and osteogenic alizarin red; Figure 12 The graph shows the human adiponectin ELISA analysis of MSC-NCMSC-YL004, MSC-YL006, MSC-YL009, MSC-YL010 and MSC-YL011 cell lines, with the vertical axis representing adiponectin content. Figure 13 The graph shows the amount of insulin secreted by INS-1 cells at different culture times after stimulation of INS-1 cells with the supernatant of MSC-YL004, MSC-YL006, MSC-YL009, MSC-YL010 and MSC-YL011 cells. Figure 5 , Figure 6 and Figure 12 In this context, Adiponectin represents adiponectin. Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0028] In the following examples, the YL004 plasmid has been reported in the prior art and is used as a positive control in this invention. See Example 1 of Chinese Patent CN111518770A, "A Stem Cell Expressing GLP1 and FGF21 and Its Use Thereof," for the prepared pCDH-FG plasmid, as shown in the schematic diagram below. Figure 2 As shown.

[0029] The recombinant plasmids used in the following examples were synthesized, constructed, and sequenced by Beijing Qingke Biotechnology Co., Ltd.

[0030] Example 1: Determining the Multiple of Infection 1. Plasmid pCDH-YL001 is a tool plasmid used to determine the correctness of the viral infection assay system. It was extracted using the OMEGA endotoxin-free plasmid mini-extraction kit. YL001 is: pCDH-EF1α-GFP-T2A-Puro (Addgene#72263).

[0031] 2. Packaging and Concentration of YL001 Lentiviral Virus The mass ratio of the three plasmids (pCDH-YL001 transfer plasmid, pSPAX2, and pMD2.G packaging helper plasmids) in each 100mm diameter culture dish was 4:3:1. Lipofectamine 3000 was used as the transfection reagent, and the specific preparation method was as follows: 1.5mL of Opti-MEM I serum-depleted medium and 27μL of Lipofectamine 3000 were added to Tube A; 1.5mL of Opti-MEM I serum-depleted medium, 27μL of P3000 Enhancer reagent, and 7.875 μg of pSPAX2, 2.625 μg of pMD2.G, and 10.5μg of pCDH-YL001 lentiviral transfer plasmid were added to Tube B. Tube A and Tube B were mixed thoroughly and incubated at room temperature for 20min to form a lipid-DNA complex. Then, the mixture was added to HEK293T cell culture plates and cultured at 37℃ and 5% CO2 for 8h. After transfection, the medium was replaced with fresh DMEM complete medium, and the virus supernatant was collected after 48h and 72h of culture, and temporarily stored at 4℃. After collection, the supernatant was filtered through a 0.45μm filter to remove cell debris, yielding the viral solution. The viral solution was then mixed with lentiviral concentrate (Sangon Biotech (Shanghai) Co., Ltd.) at a volume ratio of 4:1, gently inverted to mix, and incubated at 4℃ for 45min. The mixture was then centrifuged at 4℃, 7000×g for 45min. The supernatant was discarded, and the white precipitate was retained. The precipitate was resuspended in 1×PBS at 1 / 100 to 1 / 10 the volume of the original viral solution to obtain the high-concentration lentiviral solution pCDH-YL001. The viral titer of the high-concentration lentiviral solution pCDH-YL001 was determined using a p24 rapid ELISA kit (Spectrum Biotech). Finally, the solution was aliquoted according to the needs of a single experiment and stored at –80℃ to avoid repeated freeze-thaw cycles affecting viral activity.

[0032] 3. Lentiviral YL001 infection of UC-MSC cells 1) Cell resuscitation: After removing UC-MSC cells from liquid nitrogen, they were immediately thawed rapidly in a 37°C water bath. The cell suspension was then transferred to a centrifuge tube containing 10 mL of preheated serum-free MSC complete medium (purchased from Shenzhen Dakewei Biotechnology Co., Ltd.), and centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in fresh serum-free MSC complete medium and seeded into T75 culture dishes. The cells were then cultured in a 37°C, 5% CO2 incubator. When the UC-MSC cell confluence reached 80%, plating preparation was performed: the old medium was discarded, and the cells were washed with PBS and then digested with 2 mL of TrypLE digestion solution for 2-5 min. After observing complete cell detachment under a microscope, the digestion was immediately terminated with serum-free MSC medium. The cell suspension was collected and centrifuged again. Finally, the cells were resuspended in serum-free MSC complete medium and counted, at a ratio of 1×10⁶ cells / mL. 6 Cells were seeded at a density of 100 cells / dish in 100 mm culture dishes, and 15 mL of serum-free MSC medium was added for further culture for 16 h in preparation for viral infection.

[0033] 2) Viral infection: Preparation of infection working solution YL001: An infection working solution containing 40 MOI lentivirus pCDH-YL001 and 8 μg / mL polybrene was prepared using α-MEM basal medium (Pronosai) in a volume of 5 mL.

[0034] Viral infection was performed on the day after UC-MSC cells were plated. Cells were washed with PBS before infection. Then, infection working solution YL001 was added to the 100 mm culture dish obtained in step (1). The multiplicity of infection of the recombinant lentivirus was 40 pfu / cell. The cells were infected for 8 h at 37°C in a 5% CO2 incubator.

[0035] After infection, the viral fluid was discarded, and the cells were thoroughly washed with PBS and then replaced with serum-free MSC complete medium for further culture. When the cells re-aggregated to 80%, they were digested with TrypLE, centrifuged, resuspended, and counted, and then photographed using a fluorescence microscope.

[0036] Figure 1 A schematic diagram of the vector elements of the pCDH-YL001 recombinant plasmid is shown. After transfection into HEK293T cells for 72 hours, the driving expression capacity of the pCDH-EF1α promoter was detected by fluorescence microscopy. The results are as follows: Figure 2 As shown in the figure. The results indicate that the EF1α promoter exhibits high transcriptional activity. Therefore, YL001 can be selected as the control vector for subsequent infection experiments, and the pCDH-EF1α backbone is suitable for the construction of subsequent lentiviral packaging systems.

[0037] MOI (Multiplicity of Infection) represents the number of viral particles (usually referring to infectious viral particles, i.e., TU, Transducing Unit) that infect each host cell. For example, MOI=10 means that on average, each cell will be exposed to 10 infectious viral particles. After 72 hours of infection with YL001 lentivirus, the titer measured was 1.11E+10 PP / mL ~ 1.11E+08 TU / mL. At this time, UC-MSC-P1 cells showed clearly visible fluorescence, as shown in the results. Figure 3 As shown, the corresponding flow cytometry quantitative analysis data are as follows: Figure 4 As shown. Conclusion: Under the conditions of MOI=40, Polybrene 8μg / mL, and α-MEM medium, the EGFP positivity rate was >90%.

[0038] Example 2 Plasmid Construction 1. Constructing plasmids Using pCDH as the base vector and EF1α as the promoter, plasmids pCDH-YL004, pCDH-YL006, pCDH-YL009, pCDH-YL010, and pCDH-YL011 were constructed. YL004 was synthesized based on sequence information provided by Beijing Jiyuan. The remaining plasmids were designed by the inventors and their synthesis, construction, and sequencing were outsourced to Beijing Qingke Biotechnology. The specific construction strategy is as follows: 1) The YL004 plasmid encodes a co-expressed protein, which is formed by linking FGF21V1 and GLP-1-Fc fusion protein via a T2A peptide; wherein the GLP-1-Fc fusion protein consists of a signal peptide SP, GLP-1, (GGGGS)3 linker, and IgG4-Fc sequentially from the N-terminus to the C-terminus. YL-004: FGF21V1-T2A-SP-GLP-1-linker-IgG4-Fc.

[0039] 2) The YL006 plasmid encodes an adiponectin-Fc fusion protein, whose structure consists of adiponectin, (GGGGS)3 linker, and IgG4-Fc from the N-terminus to the C-terminus. YL-006: Adiponectin-linker-IgG4-Fc.

[0040] 3) The YL009 plasmid encodes a co-expressed protein, which is formed by linking FGF21V2 and adiponectin-Fc fusion protein via a T2A peptide; wherein the adiponectin-Fc fusion protein is composed of adiponectin fused with IgG4-Fc via a (GGGGS)3 linker. YL-009: FGF21V2-T2A-adiponectin-linker-IgG4-Fc.

[0041] 4) The YL010 plasmid encodes a trifunctional single fusion protein whose structure, from the N-terminus to the C-terminus, consists of adiponectin, a first (GGGGS)3 linker, FGF21V2, a second (GGGGS)3 linker, and IgG4-Fc linked together. YL-010: Adiponectin-linker-FGF21V2-linker-IgG4-Fc.

[0042] 5) YL011: SP-IgG4-FC-linker-GLP-1-T2A-adiponectin. The YL011 plasmid encodes a co-expressed protein, which is composed of an IgG4-Fc-GLP-1 fusion protein and adiponectin linked by a T2A peptide; wherein the IgG4-Fc-GLP-1 fusion protein is composed of a signal peptide SP, IgG4-Fc, (GGGGS)3linker and GLP-1 from the N-terminus to the C-terminus.

[0043] Schematic diagrams of the recombinant plasmid vectors pCDH-YL004, pCDH-YL006, pCDH-YL009, pCDH-YL010, and pCDH-YL011 are shown below. Figure 5 Compared to the recombinant plasmid vector pCDH-YL004, the only difference between pCDH-YL006, pCDH-YL009, pCDH-YL010, and pCDH-YL011 is that, according to... Figure 5 Replace the corresponding component in the middle.

[0044] The amino acid sequence of T2A is shown in SEQ ID NO:1: EGRGSLLTCGDVEENPGP; The nucleotide sequence of T2A is shown in SEQ ID NO:2: gagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggacct.

[0045] The amino acid sequence of GLP-1 is shown in SEQ ID NO:3: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG.

[0046] The nucleotide sequence of GLP-1 is shown in SEQ ID NO:4: catggcgaagggacctttaccagtgatgtaagttcttatttggaagagcaagctgccaaggaattcattgcttggctggtgaaaggcggcgga.

[0047] The amino acid sequence of linker (GGGGS)3 is shown in SEQ ID NO:5: GGGGSGGGGSGGGGS.

[0048] The nucleotide sequence of linker (GGGGS)3 is shown in SEQ ID NO:6: ggcggaggcggaagcggaggcggaggaagcggcggtggcggcagc.

[0049] The amino acid sequence of IgG4 Fc is as SEQ ID NO:7 shows: ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG*.

[0050] The nucleotide sequence of IgG4 Fc is shown in SEQ ID NO:8: gagtccaaatatggtcccccatgcccaccctgcccagcacctgaggccgccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggaaagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggttga。

[0051] The amino acid sequence of FGF21V1 is shown in SEQ ID NO:9: MDSDETGFEHSGLWVSVLAGLLLGACQADSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS*。

[0052] The nucleotide sequence of FGF21V1 is shown in SEQ ID NO:15: atggactcggacgagaccgggttcgagcactcaggactgtgggtttctgtgctggctggtcttctgctgggagcctgccaggcagactccagtcctctcctgcaattcgggggccaagtccggcagcggtacctctacacagatgatgcccagcagacagaagcccacctggagatcagggaggatgggacggtggggggcgctgctgaccagagccccgaaagtctcctgcagctgaaagccttgaagccgggagttattcaaatcttgggagtcaagacatccaggttcctgtgccagcggccagatggggccctgtatggatcgctccactttgaccctgaggcctgcagcttccgggagctgcttcttgaggacggatacaatgtttaccagtccgaagcccacggcctcccgctgcactgcccagggaacaagtccccacaccgggaccctgcaccccgaggaccatgccgcttcctgccactaccaggcctgccccccgcactcccggagccacccggaatcctggccccccagccccccgatgtgggctcctcggaccctctggccatggtgggaccttcccagggccgaagccccagctacgcttcc。

[0053] The amino acid sequence of FGF21V2 is shown in SEQ ID NO:16: MDSDETGFEHSGLWVSVLAGLLLGACQADSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGNSTGRSPSYAS*。

[0054] The nucleotide sequence of FGF21V2 is shown in SEQ ID NO:10: atggactcggacgagaccgggttcgagcactcaggactgtgggtttctgtgctggctggtcttctgctgggagcctgccaggcagactccagtcctctcctgcaattcgggggccaagtccggcagcggtacctctacacagatgatgcccagcagacagaagcccacctggagatcagggaggatgggacggtggggggcgctgctgaccagagccccgaaagtctcctgcagctgaaagccttgaagccgggagttattcaaatcttgggagtcaagacatccaggttcctgtgccagcggccagatggggccctgtatggatcgctccactttgaccctgaggcctgcagcttccgggagctgcttcttgaggacggatacaatgtttaccagtccgaagcccacggcctcccgctgcactgcccagggaacaagtccccacaccgggaccctgcaccccgaggaccatgccgcttcctgccactaccaggcctgccccccgcactcccggagccacccggaatcctggccccccagccccccgatgtgggctcctcggaccctctggcaatggtgggaaactccacgggccgaagccccagctacgcttcctga。

[0055] The amino acid sequence of adiponectin is shown in SEQ ID NO:11: MLLLGAVLLLLALPGHDQETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDGTPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN*。

[0056] The nucleotide sequence of adiponectin is shown in SEQ ID NO:12: atgctgttgctgggagctgttctactgctattagctctgcccggtcatgaccaggaaaccacgactcaagggcccggagtcctgcttcccctgcccaagggggcctgcacaggttggatggcgggcatcccagggcatccgggccataatggggccccaggccgtgatggcagagatggcacccctggtgagaagggtgagaaaggagatccaggtcttattggtcctaagggagacatcggtgaaaccggagtacccggggctgaaggtccccgaggctttccgggaatccaaggcaggaaaggagaacctggagaaggtgcctatgtataccgctcagcattcagtgtgggattggagacttacgttactatccccaacatgcccattcgctttaccaagatcttctacaatcagcaaaaccactatgatggctccactggtaaattccactgcaacattcctgggctgtactactttgcctaccacatcacagtctatatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataatgtggaccaggcctccggctctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgctgataatgacaatgactccaccttcacaggctttcttctctaccatgacaccaactga。

[0057] The amino acid sequence of the signal peptide SP is shown in SEQ ID NO:13: MRALLARLLLCVLVVSDSKG。

[0058] The nucleotide sequence of the signal peptide SP is shown in SEQ ID NO:14: atgagagccctgctggcgcgcctgcttctctgcgtcctggtcgtgagcgactccaaaggc。

[0059] 2. Plasmid transformation: Remove *E. coli* DH5α competent cells (Sangon Biotech (Shanghai) Co., Ltd.) from a -80°C freezer and quickly thaw them in an ice box for 8 minutes. In centrifuge tubes, add 1 μL each of pCDH-YL004, pCDH-YL006, pCDH-YL009, pCDH-YL010, and pCDH-YL011 plasmids (100 ng / μL concentration) to 100 μL of the thawed DH5α cells, mix gently, and incubate on ice for 30 minutes. Then, quickly transfer the centrifuge tubes to a 42°C water bath for 45 seconds and immediately return them to ice for approximately 2 minutes. Add 10 μL of the transformed DH5α cells to 10 mL of TS B ampicillin medium and incubate at 37°C with shaking for 16 hours to obtain the revived bacterial culture.

[0060] 3. Plasmid extraction: The revived bacterial culture was plated on TSB ampicillin medium and cultured to obtain positive colonies. The plasmids of the positive colonies were extracted using the OMEGA endotoxin-free plasmid mini-extraction kit to obtain recombinant plasmids pCDH-YL004, pCDH-YL006, pCDH-YL009, pCDH-YL010, and pCDH-YL011.

[0061] Example 3 Lentiviral Packaging and Infection HEK293T cells were derived from the stem cell bank of the Chinese Academy of Sciences Cell Bank.

[0062] HEK 293T complete medium is: DMEM basal medium supplemented with 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin-amphoteric acid B solution.

[0063] 1. Cell Resuscitation and Plating: Remove frozen HEK293T cells from liquid nitrogen and thaw rapidly in a 37°C water bath until the ice completely disappears. Transfer the cell suspension to a 15 mL centrifuge tube containing 10 mL of pre-warmed HEK293T complete medium and centrifuge at 1000 rpm for 3 min. Discard the supernatant, resuspend the cells in HEK293T complete medium, and seed them into T75 cell culture flasks. Incubate at 37°C, 5% CO2, and saturated humidity. When cell confluence reaches 80% or higher, discard the old medium, wash the cells once with 10 mL of sterile PBS and discard the PBS. Add 2 mL of TrypLE digestion solution and digest for 2-5 min until the cells are completely detached. Add HEK293T complete medium to stop digestion, collect the cell suspension, and centrifuge at 1000 rpm for 3 min. Discard the supernatant after centrifugation, resuspend the cells in HEK293T complete medium, and plate at 7 × 10⁶ cells per 100 mm culture dish. 6The cells were plated at a density of 10 mL each, and 10 mL of HEK293T complete medium was added to each plate. The plates were then incubated at 37°C and 5% CO2 for 16 h to prepare for virus packaging experiments the following day.

[0064] 2. Virus Packaging and Concentration Lentiviral transfer plasmids pCDH-YL004, pCDH-YL006, pCDH-YL009, pCDH-YL010 and pCDH-YL011, constructed based on the pCDH vector, were prepared.

[0065] Process 1: Same as step 2 of Example 1, lentivirus packaging and concentration, to obtain high-concentration lentivirus solution pCDH-YL004, the difference being that the multiplicity of infection is now determined.

[0066] Treatment 2: Same as Treatment 1, except that the pCDH-YL004 lentivirus transfer plasmid is replaced with the pCDH-YL006 lentivirus transfer plasmid to obtain a high-concentration lentivirus solution pCDH-YL006.

[0067] Treatment 3: Same as Treatment 1, except that the pCDH-YL004 lentivirus transfer plasmid is replaced with the pCDH-YL009 lentivirus transfer plasmid to obtain a high-concentration lentivirus solution pCDH-YL009.

[0068] Treatment 4: Same as Treatment 1, except that the pCDH-YL004 lentivirus transfer plasmid is replaced with the pCDH-YL010 lentivirus transfer plasmid to obtain a high-concentration lentivirus solution pCDH-YL010.

[0069] Treatment 5: Same as Treatment 1, except that the pCDH-YL004 lentivirus transfer plasmid is replaced with the pCDH-YL011 lentivirus transfer plasmid to obtain a high-concentration lentivirus solution pCDH-YL011.

[0070] 3. Lentiviral infection of umbilical cord mesenchymal stem cells (UC-MSCs) Human umbilical cord mesenchymal stem cells (UC-MSCs) were purchased from Shangen Biotechnology, catalog number SNP-H198.

[0071] Treatment group: UC-MSC cells were infected in the same way as in step 3 of Example 1, except that pCDH-YL001 was replaced with pCDH-YL004, pCDH-YL006, pCDH-YL009, pCDH-YL010, and pCDH-YL011, respectively.

[0072] After infection with YL004, YL006, YL009, YL010, and YL011, the viral fluid was discarded, and the cells were thoroughly washed with PBS and then cultured in serum-free MSC complete medium. When the cells re-aggregated to 80%, they were digested with TrypLE, centrifuged, resuspended, and counted. Then, they were cultured at a concentration of 1×10⁻⁶ cells / mL. 6 The cells were passaged and expanded at a density of 100 cells / T175 culture flask, and the infected cells were named MSC-YL004, MSC-YL006, MSC-YL009, MSC-YL010, and MSC-YL011, respectively, to obtain the P3 generation MSC-YL004, MSC-YL006, MSC-YL009, MSC-YL010, and MSC-YL011 cell lines. Serum-free MSC complete culture medium was purchased from Shenzhen Dakewei Biotechnology Co., Ltd.

[0073] 1) The number of lentivirus particles (TU / mL) after packaging YL004, YL006, YL009, YL010 and YL011 were 2.93E+08, 2.47E+08, 2.61E+08, 5.28E+08 and 4.29E+08, respectively.

[0074] 2) Analysis of the secretion levels of various proteins after infection MSC supernatant preparation: Lentivirally infected P6 generation MSCs-YL004, MSC-YL006, MSC-YL009, MSC-YL010, MSC-YL011, and untreated MSC-NC cells were seeded in 100 mm culture dishes and cultured. When the cell confluence reached 80%, the original serum-free medium was aspirated and replaced with 10 mL of fresh α-MEM basal medium. The cells were then cultured for another 72 h at 37°C, 5% CO2, and saturated humidity. After culture, the culture supernatant from each group was collected and concentrated using an ultrafiltration tube (such as the Amicon® Ultra series) to approximately 1 / 10 of the original volume, achieving a 10-fold concentration. The concentrated supernatants MSC-YL004, MSC-YL006, MSC-YL009, MSC-YL010, MSC-YL011 and MSC-NC obtained can be stored at 4℃ for a short period of time; if long-term storage is required, they should be aliquoted and stored in a -80℃ freezer to avoid repeated freeze-thaw cycles.

[0075] Expression analysis of YL004, YL006, YL009, YL010, and YL011 proteins in concentrated supernatants: Cell culture supernatants from P6 generation MSCs (YL004, YL006, YL009, YL010, YL011, and MSC-NC) concentrated 10-fold were subjected to 12% SDS-PAGE gel electrophoresis and wet-transferred to PVDF membranes. The membranes were blocked with 5× protein-free rapid blocking buffer (Shanghai Yamei Biomedical Technology Co., Ltd.), washed three times with TBST, and incubated overnight at 4°C with FGF21 antibody (1:2000, Abcam) and adiponectin antibody (1:2000, Abcam). Mouse anti-human IgG4 Fc(HRP) (1:20000, Abcam) was then added, and the membranes were incubated for 1 hour. The membranes were washed three times with TBST. After three TBST washes, the membranes were developed using a chemiluminescence assay kit and exposed using an Imaje Lab computer image analysis system.

[0076] Adiponectin protein, FGF21 protein, and IgG4-Fc fusion protein were detected in the concentrated supernatant. Results are shown below. Figure 6 It can be seen that adiponectin protein is secreted normally in pCDH-YL006, pCDH-YL009, pCDH-YL010, and pCDH-YL011, and FGF21 protein is secreted normally in pCDH-YL004 and pCDH-YL009. In pCDH-YL004 and pCDH-YL011, IgG4-Fc is fused with GLP-1 and expressed together. The expression of IgG4-Fc protein also represents the expression of GLP-1 protein. Therefore, GLP-1 protein is secreted normally in pCDH-YL004 and pCDH-YL011. In parallel experiments, we found that GLP-1 was occasionally undetectable, which is related to its easily degraded characteristics; sometimes it cannot be captured because it has already degraded.

[0077] 3) Validation of the biological function of UC-MSCs after lentiviral infection (1) Cell phenotype identification: The P3 generation MSC-YL006 and MSC-YL011 cell lines obtained in Example 3 were passaged and expanded stepwise to obtain P7 generation cells. The above P7 generation cell lines were selected, digested with 2 mL of TrypLE, washed twice with PBS, and then labeled with mouse anti-human CD19-PE, CD34-PE, CD45-PE, CD73-PerCp-Cy5.5, CD90-FITC, and CD105-APC (Becton, Dickinson and Company) antibodies. Approximately 1 × 10⁻⁶ cells were used for each sample. 6Cells were incubated at room temperature in the dark for 30 minutes, washed twice with PBS, and then analyzed by flow cytometry. The results showed that both MSC-YL006 and MSC-YL011 met the phenotypic identification requirements for mesenchymal stem cells, stably and highly expressing mesenchymal stem cell markers such as CD73, CD90, and CD105, while typical negative markers such as CD19, D34, and CD45 were all below the detection limit. This indicates that lentiviral transfection did not destroy the core stem cell characteristics, and the transfection system is safe and suitable. Figures 7-10 The antibody staining results for MSC-YL006 and MSC-YL011 are shown.

[0078] (2) Identification of adipogenic and osteogenic capacity: P6 generation MSC-YL006 and MSC-YL011 cell lines were digested with 2 mL of 0.05% (w / v) TrypLE and then divided into 2 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. When cell confluence reached 100%, the adipogenic induction medium was replaced, and an alternating induction regimen of adipogenic solution A and solution B was used (induction with solution A for 3 days, maintenance with solution B for 1 day) for cyclic induction until sufficient lipid droplets of suitable morphology were formed within the cells. The adipogenic differentiation effect was then assessed using Oil Red O staining. Results are shown in [figure missing]. Figure 11 ,according to Figure 11 It was found that both MSC-YL006 and MSC-YL011 cells infected with lentiviruses possessed adipogenic differentiation capacity. P6 generation MSC-YL006 and MSC-YL011 cell lines were digested with 2 mL of 0.05% (w / v) TrypLE solution and then divided into two wells at a ratio of 2 × 10⁶ cells / well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates. When confluence reached 70%, the osteogenic induction medium was replaced. The induction medium was changed every 3 days during osteogenic induction, and the cells were cultured for 23 days before alizarin red staining for identification. Results are shown below. Figure 11 ,according to Figure 11 It was found that both MSC-YL006 and MSC-YL011 cells infected with lentivirus possessed osteogenic differentiation capacity. These two experiments together demonstrate that lentivirus transfection did not destroy the core stem cell characteristics, and that the transfection system is safe and suitable.

[0079] Example 4: Effect of concentrated supernatant on insulin secretion after glucose stimulation 1. MSC Supernatant Preparation: Lentivirally infected P6 generation MSCs (YL004, YL006, YL009, YL010, YL011) and untreated MSC-NC cells were seeded in 100 mm culture dishes and cultured. When the cell confluence reached 80%, the original serum-free medium was discarded and replaced with 10 mL of fresh α-MEM basal medium. The cells were then cultured for another 72 h at 37°C, 5% CO2, and saturated humidity. After culture, the culture supernatant from each group was collected and concentrated using an ultrafiltration tube (such as the Amicon® Ultra series) to approximately 1 / 10 of the original volume, achieving a 10-fold concentration. The concentrated supernatants MSC-YL004, MSC-YL006, MSC-YL009, MSC-YL010, MSC-YL011, and MSC-NC can be stored at 4°C for a short period of time. If long-term storage is required, they should be aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles.

[0080] The concentrated supernatant was analyzed using a human adiponectin (ADP / Acrp30) ELISA kit. Results are shown below. Figure 12 It can be seen that the expression level of human adiponectin is high in the supernatant of MSC-YL006, MSC-YL009, MSC-YL010 and MSC-YL011. MSC-NC and MSC-YL004 do not integrate exogenous adiponectin expression genes; therefore, adiponectin is not expressed in their concentrated supernatant.

[0081] 2. INS (Insulin-secreting Cells) Culture The composition of the INS-1 complete medium is as follows: 1640 medium with the addition of 10% FBS, 1mM sodium pyruvate, 2mM glutamine, and 50μM mercaptoethanol.

[0082] Take one vial of cryopreserved INS-1 cells (Shangen Biotechnology) from liquid nitrogen and quickly place it in a 37°C water bath until the ice melts. Add the cells dropwise to a 15 mL centrifuge tube containing 10 mL of preheated INS-1 complete medium. Centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells in INS-1 complete medium, and seed them into T25 culture flasks. Incubate at 37°C with 5% CO2. When cell confluence reaches 80%, discard the old medium, add 5 mL of sterile PBS solution, gently agitate, wash the cells, discard the PBS solution, add 1 mL of LTrypLE for digestion, and digest for 5 min until the cells are completely digested. Add INS-1 complete medium to stop digestion, centrifuge the cell suspension at 1000 rpm for 3 min, resuspend the resulting cells in INS-1 complete medium, and seed 1 × 10⁶ cells per well in a 6-well plate. 6Add 2 mL of INS-1 complete medium to each cell and incubate at 37°C with 5% CO2 saturated humidity. When the cell density reaches 80%, it can be used for functional verification.

[0083] 3. Verification of sugar-stimulated function Low-glucose KRBH buffer: KRBH buffer with 0.2% (w / v) BSA and 0.4 mg / mL glucose added. High-glucose KRBH buffer: KRBH buffer with 0.2% (w / v) BSA and 3 mg / mL glucose added.

[0084] For functional validation, the original INS-1 complete culture medium in the 6-well plate was aspirated, and INS-1 cells were starved for 2 h by adding 2 mL of pre-warmed low-glucose KRBH buffer. The low-glucose KRBH buffer was then aspirated, and 1.5 mL of pre-warmed high-glucose KRBH buffer was added. Then, 500 μl of the concentrated supernatant obtained in step 1 (MSC-YL004, MSC-YL006, MSC-YL009, MSC-YL010, MSC-YL011, and MSC-NC) were added respectively. The cells were incubated at 37℃ and 5% CO2 saturated humidity for 0 h, 2 h, 4 h, 6 h, and 8 h. After incubation, the supernatant was collected by centrifugation, and the insulin content in the supernatant was detected using a rat insulin (INS) ELISA kit. The detection results for 0 h, 2 h, and 6 h of incubation are shown in the figure. Figure 13 The percentage changes in insulin content at different culture times are shown in Table 1.

[0085] Table 1. Changes in insulin content in supernatant at different culture times

[0086] It was found that the culture supernatants of the gene-modified UC-MSC cell lines MSC-YL004 (YL-004), MSC-YL006 (YL-006), MSC-YL009 (YL-009), MSC-YL010 (YL-0010), and MSC-YL011 (YL-0011) all significantly promoted insulin secretion from INS-1 cells, with significantly better effects than unmodified UC-MSC cells. Notably, in the glucose stimulation experiment, the supernatant from single-gene-modified YL-006 cells showed a similarly high level of insulin secretion promotion as the multi-gene-modified group. Furthermore, under the influence of YL-006 adiponectin, islet cells exhibited a more sustained islet secretion state, indicating that adiponectin's stimulatory effect on islet cells is more stable and prolonged, resulting in a longer-lasting therapeutic effect. Co-expression of GLP-1 and adiponectin can enhance the stimulation of pancreatic islet cells and significantly increase insulin secretion.

[0087] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A type of mesenchymal stem cell, characterized in that, The mesenchymal stem cells described herein are modified only to overexpress adiponectin and glucagon-like peptide-1. The mesenchymal stem cells include a recombinant plasmid, which is based on pCDH and uses EF1α as a promoter. The recombinant plasmid co-expresses GLP-1 and adiponectin, and its component composition is SP-IgG4-FC-linker-GLP-1-T2A-adiponectin. It is formed by linking an IgG4-Fc-GLP-1 fusion protein and adiponectin via a T2A peptide. The IgG4-Fc-GLP-1 fusion protein is composed of a signal peptide SP, IgG4-Fc, (GGGGS)3linker, and GLP-1 sequentially from the N-terminus to the C-terminus. The nucleotide sequence of the adiponectin gene is shown in SEQ ID NO:12, and the amino acid sequence of the adiponectin gene is shown in SEQ ID NO:

11. The nucleotide sequence of glucagon-like peptide-1 is shown in SEQ ID NO:4, and the amino acid sequence of the glucagon-like peptide-1 gene is shown in SEQ ID NO:

11. The nucleotide sequence of IgG4-Fc is shown in SEQ ID NO:3; the amino acid sequence of IgG4-Fc is shown in SEQ ID NO:8; the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

2. The use of the mesenchymal stem cells of claim 1 in at least one of the following: 1) To prepare drugs that enhance the glucose-stimulated response of pancreatic islet cells; 2) Prepare drugs that increase the amount of insulin secreted by pancreatic islet cells.

3. A drug that enhances the ability of pancreatic islet cells to respond to glucose stimulation and / or increases the secretion of insulin by pancreatic islet cells, characterized in that, It includes the mesenchymal stem cells as described in claim 1 and pharmaceutically acceptable excipients.

4. The drug according to claim 3, characterized in that, The dosage form of the drug includes one or more of the following: injection, oral, sustained-release, and targeted formulation. The excipients include any one or a combination of at least two of the following: carrier, diluent, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

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