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CN-121991900-A - Adiponectin gene modified mesenchymal stem cell, and preparation method, medicine and application thereof

CN121991900ACN 121991900 ACN121991900 ACN 121991900ACN-121991900-A

Abstract

The invention relates to the field of stem cell culture, in particular to an adiponectin gene modified mesenchymal stem cell, a preparation method, a medicine and application thereof. The mesenchymal stem cell modified by the adiponectin gene can overexpress the adiponectin to stimulate islet cells, regulate and control sugar stimulation reaction and improve insulin secretion. The preparation method of the adiponectin gene modified mesenchymal stem cells provided by the invention is simple, and the prepared adiponectin gene modified mesenchymal stem cells have higher insulin secretion and longer acting time in an INS-1 cell sugar stimulation experiment.

Inventors

  • ZHAO JIANZHI
  • TAO YULIN
  • YAN XIAOHUA
  • NI YA

Assignees

  • 浙江赛源细胞生物工程有限公司

Dates

Publication Date
20260508
Application Date
20260409

Claims (10)

  1. 1. A mesenchymal stem cell, characterized in that the mesenchymal stem cell has only a genetic modification over-expressing adiponectin and glucagon-like peptide-1.
  2. 2. The mesenchymal stem cell of claim 1, wherein the nucleotide sequence of the adiponectin gene is shown in SEQ ID No. 12 and the amino acid sequence of adiponectin is shown in SEQ ID No. 11.
  3. 3. The mesenchymal stem cell of claim 1, wherein the glucagon-like peptide-1 has a nucleotide sequence as shown in SEQ ID No. 4 and the glucagon-like peptide-1 has an amino acid sequence as shown in SEQ ID No. 3.
  4. 4. The mesenchymal stem cell of claim 1, wherein the type of mesenchymal stem cell comprises at least one of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, and bone marrow mesenchymal stem cells.
  5. 5. The mesenchymal stem cell of claim 1, wherein the adiponectin gene-modified mesenchymal stem cell further comprises an expression vector, wherein the expression vector further comprises a functional element and a connecting peptide, wherein the functional element comprises at least one of IgG4-Fc, T2A and SP, and wherein the connecting peptide comprises a linker; The adiponectin and the IgG4-Fc are connected through a connecting peptide sequence, the IgG4-Fc fusion protein consists of a signal peptide, an adiponectin protein, a connecting peptide and an IgG4-Fc structural domain from the N end to the C end, and the N end of the adiponectin protein is connected with the signal peptide.
  6. 6. The method for preparing mesenchymal stem cells according to any one of claims 1 to 5, comprising the steps of: 1) Cloning the adiponectin gene to a lentivirus transfer plasmid to obtain a recombinant expression plasmid; 2) Transfecting the assembled product of the recombinant expression plasmid, the lentivirus packaging auxiliary plasmid and the lentivirus packaging envelope plasmid into cells to obtain recombinant lentivirus; 3) And infecting the mesenchymal stem cells by the recombinant lentivirus to obtain the genetically modified mesenchymal stem cells stably expressing adiponectin.
  7. 7. The method of claim 6, wherein the lentiviral transfer plasmid of step 1) comprises pCDH; The lentiviral packaging helper plasmid in the step 2) comprises pSPAX < 2 >, and the lentiviral packaging envelope plasmid comprises pMD2.G; And before the recombinant lentivirus in the step 3) infects the mesenchymal stem cells, transfecting 293T cells for amplification, wherein the complex infection range of the recombinant lentivirus in the step 3) is 10-40 pfu/cell.
  8. 8. Use of the mesenchymal stem cell of any one of claims 1-5, the mesenchymal stem cell obtained by the preparation method of claim 6 or 7, in at least one of the following; 1) Preparing a medicament for improving the glucose stimulation response capability of islet cells; 2) Preparing a medicament for improving insulin secretion of islet cells; 3) Improving the insulin secretion capacity of islet cells or improving the insulin stimulation response capacity of islet cells.
  9. 9. A medicament for improving the ability of islet cell sugar to stimulate a reaction and/or for improving the secretion of insulin from islet cells, comprising the mesenchymal stem cells of any one of claims 1 to 5 or the mesenchymal stem cells obtained by the preparation method of claim 6 or 7 and a pharmaceutically acceptable adjuvant.
  10. 10. The medicament according to claim 9, wherein the dosage form of the medicament comprises one or more than two of injection, oral administration, slow release agent and targeting agent; The auxiliary materials comprise any one or a combination of at least two of carriers, diluents, excipients, fillers, adhesives, wetting agents, disintegrants, emulsifying agents, cosolvent, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, bacteriostats or buffers.

Description

Adiponectin gene modified mesenchymal stem cell, and preparation method, medicine and application thereof Technical Field The invention relates to the field of stem cell culture, in particular to an adiponectin gene modified mesenchymal stem cell, a preparation method, a medicine and application thereof. Background The common method for treating diabetes by using a cell method is that the pluripotent stem cells are differentiated into islet pellets, but the pluripotent stem cells are limited in industrial production due to the fact that the pluripotent stem cells are possibly tumorigenic and differentiation instability caused by heterogeneity of the pluripotent stem cells is remained, and the culturing operation of the pluripotent stem cells is complicated, so that the pluripotent stem cells are not easy to expand in large scale. Umbilical cord-derived mesenchymal stem cells are powerful candidates for diabetes treatment due to their low immunogenicity and high safety. They have not only self-renewing and multi-directional differentiation capabilities but also exhibit a marked immunomodulatory effect. Stable overexpression of specific functional proteins in these cells can further enhance their potential for the treatment of diabetes. GLP-1 and FGF21 are common hormone-like proteins for the treatment of diabetes, and their combined overexpression has been reported in the literature and patents to be effective in regulating the glucose stimulation response, but are readily degraded both in vivo and in vitro. Therefore, there is an urgent need to construct a mesenchymal stem cell that has a long-lasting action time and can effectively regulate the sugar stimulation response. Disclosure of Invention Therefore, the technical problem to be solved by the invention is to overcome the defect of easy degradation of GLP-1 and FGF21 in the prior art for regulating and controlling sugar stimulation reaction, so as to provide an adiponectin gene modified mesenchymal stem cell which can effectively regulate and control sugar stimulation reaction and has lasting action time of adiponectin in the mesenchymal stem cell. The invention aims to solve the other technical problem of overcoming the defects of easy degradation and non-lasting effect of GLP-1 and FGF21 in the prior art for regulating and controlling sugar stimulation reaction, thereby providing a preparation method of adiponectin gene modified mesenchymal stem cells, and the preparation method is simple. For this purpose, the invention provides the following technical scheme: the invention provides a mesenchymal stem cell modified by an adiponectin gene, which overexpresses the adiponectin. Preferably, the nucleotide sequence of the adiponectin gene is shown as SEQ ID NO. 12, and the amino acid sequence of the adiponectin is shown as SEQ ID NO. 11. The adiponectin can regulate and control sugar stimulation reaction, and insulin secretion amount is higher. Preferably, the type of the mesenchymal stem cells includes at least one of umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and bone marrow mesenchymal stem cells. Preferably, the adiponectin gene modified mesenchymal stem cell further comprises an expression vector, wherein the expression vector further comprises a functional element and a connecting peptide, the functional element comprises at least one of IgG4-Fc and T2A, SP, and the connecting peptide comprises a linker; The adiponectin and the IgG4-Fc are connected through a connecting peptide sequence, the IgG4-Fc fusion protein consists of a signal peptide, an adiponectin protein, a connecting peptide and an IgG4-Fc structural domain from the N end to the C end, and the N end of the adiponectin protein is connected with the signal peptide. Preferably, the adiponectin gene modified mesenchymal stem cells also overexpress glucagon-like peptide-1 (GLP-1), the nucleotide sequence of glucagon-like peptide-1 is shown as SEQ ID NO. 4, and the amino acid sequence of glucagon-like peptide-1 is shown as SEQ ID NO. 3. In a specific embodiment of the invention, recombinant plasmid YL011 simultaneously co-expresses GLP-1 and adiponectin, wherein the recombinant plasmid YL011 comprises SP-IgG 4-Fc-linker-GLP-1-T2A-adiponectin, the recombinant plasmid YL011 is formed by connecting an IgG4-Fc-GLP-1 fusion protein and adiponectin through T2A peptide, and the IgG4-Fc-GLP-1 fusion protein sequentially comprises signal peptides SP, igG4-Fc, (GGGGS) 3 linker and GLP-1 from N end to C end. Co-expression of GLP-1 and adiponectin enhances sugar stimulation of islet cells. In a specific embodiment of the invention, recombinant plasmid YL006 only expresses adiponectin, and the expression level of adiponectin is improved. Preferably, the adiponectin gene modified mesenchymal stem cell further comprises an expression vector, wherein the expression vector further comprises a functional element and a connecting peptide, the functional element comprises at least one