CN-122012402-A - Mesenchymal stem cells and extracellular vesicles over-expressing Midkine and application thereof

Abstract

The invention provides mesenchymal stem cells and extracellular vesicles over-expressing Midkine and application thereof. The mesenchymal stem cells integrate gene elements for over-expressing Midkine, realize local and continuous over-expression of Midkine recombinant protein by utilizing the long-term survival and tissue chemotactic capability of the mesenchymal stem cells, and the engineered mesenchymal stem cells integrate the advantages of the Midkine recombinant protein and the mesenchymal stem cells, play the synergistic effect of the two, have great potential in promoting tissue regeneration and repair, especially cartilage tissue regeneration and skin wound repair, and in addition, the extracellular vesicles secreted by the mesenchymal stem cells over-expressing Midkine contain Midkine recombinant protein or mRNA and are also used for promoting cartilage injury repair.

Inventors

• WANG YAN
• WANG HONGLI
• DONG LIU
• WEI CHEN

Assignees

• 复旦大学附属华山医院

Dates

Publication Date

20260512

Application Date

20260214

Claims (7)

1. 1. The mesenchymal stem cell over-expressing Midkine is characterized by integrating gene elements over-expressing Midkine, and realizing local sustained high-concentration release of Midkine by utilizing the long-term survival and tissue chemotaxis of the mesenchymal stem cell and playing the synergistic effect of the mesenchymal stem cell and the Midkine.
2. 2. A method for preparing a mesenchymal stem cell over-expressing Midkine according to claim 1, comprising the steps of: Step 1), inserting a coding sequence of Midkine shown as SEQ ID NO.1 and a coding sequence of T2A polypeptide shown as SEQ ID NO.2 in front of EGFP sequence of pRRLSIN plasmid to obtain an over-expression Midkine lentiviral vector pRRLSIN-MK shown as SEQ ID NO. 3; Step 2), co-transfecting the lentiviral expression vector pRRLSIN-MK over-expressing Midkine with packaging plasmids pMD2.G and psPAX to 293T cells to prepare lentiviral particles over-expressing Midkine; Step 3) transfecting the slow virus which overexpresses Midkine into the mesenchymal stem cells.
3. 3. The method for preparing mesenchymal stem cells overexpressing Midkine according to claim 2, wherein in the step 3), the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.
4. 4. Use of a Midkine-overexpressing mesenchymal stem cell according to claim 1, for promoting tissue regeneration repair.
5. 5. The use of a Midkine-overexpressing mesenchymal stem cell of claim 4, wherein the tissue regeneration repair is cartilage regeneration and skin wound repair.
6. 6. An extracellular vesicle, characterized in that it is secreted by the Midkine-overexpressing mesenchymal stem cell of claim 1.
7. 7. Use of an extracellular vesicle according to claim 6 for promoting cartilage damage repair.

Description

Mesenchymal stem cells and extracellular vesicles over-expressing Midkine and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to mesenchymal stem cells and extracellular vesicles over-expressing Midkine and application thereof. Background Mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCS) are widely considered to have powerful immunoregulatory activity and stimulate repair and regeneration of diseased or damaged tissues, and the main mechanisms include secretion of various nutritional factors (such as VEGF, PDGF, FGF and the like) to promote angiogenesis and cell proliferation, secretion of various immunoregulatory factors (such as PGE2, IDO and TGF-beta) to inhibit excessive inflammation, regulate immunity and generate anti-apoptotic factors to protect damaged cells, secretion of chemokines to recruit endogenous stem cells to participate in repair. Therefore, MSCs have potential in wound healing (diabetic foot ulcers, pressure sores, large area burns), promotion of bone and cartilage repair, cardiovascular repair and angiogenesis, neural tissue repair, and the like. However, since MSCs are affected by sources (bone marrow, fat or umbilical cord), age of donor, culture method, etc., and their functions are affected by damaged microenvironment after transplantation into the body, current research is being conducted to overcome these shortages by strategies such as genetic engineering (e.g., overexpression of functional factors), pretreatment (hypoxia or drug pretreatment), or combination with novel biomaterials, etc., and to enhance their therapeutic effects. Mesenchymal stem cells can exert their immunomodulatory and tissue injury repair effects through Extracellular Vesicles (EVs) secreted by the mesenchymal stem cells. EVs are a group of structures produced by cells, consisting of lipid bilayer membranes, containing biologically active proteins, lipids, RNA, etc. inside, and carrying important information transfer between cells. According to their biological differences, EVs are generally divided into subgroups, such as exosomes (40-150 nm in diameter), microcapsules (150-1000 nm in diameter) and apoptotic bodies (50-2000 nm in diameter), and recent studies have shown that EVs can perform a series of biological functions instead of MSCs and become substitutes for cell therapy in certain application scenarios. Midkine (MK) is a heparin-binding growth factor that has multiple functions of promoting cell proliferation, migration, survival and tissue repair. Midkine plays an important role in embryonic development, tissue repair and tumorigenesis, and particularly exhibits a prominent aspect of cartilage regeneration. Midkine can stimulate the proliferation of chondrocyte and the synthesis of matrix, promote the repair of cartilage defect, so has wide prospect in the treatment of osteoarthritis, cartilage injury and other diseases. In addition, midkine can accelerate the wound healing process by promoting proliferation and migration of key repair cells, stimulating neovascularization, regulating inflammation, preventing cell death and other multiple mechanisms, and has great application potential in the treatment of chronic wounds which are difficult to heal. However, midkine has a short half-life in vivo, and local administration is difficult to maintain effective concentration, limiting its clinical application. Therefore, the development of a sustained release Midkine delivery system is of great clinical importance. Disclosure of Invention In order to solve the technical problems, the invention provides mesenchymal stem cells and extracellular vesicles which overexpress Midkine and application thereof. The invention provides mesenchymal stem cells and extracellular vesicles over-expressing Midkine and application thereof, which adopts the following technical scheme: In a first aspect of the present invention, there is provided a mesenchymal stem cell overexpressing Midkine, wherein the mesenchymal stem cell integrates a gene element overexpressing Midkine, and utilizes the long-term survival and tissue chemotactic ability of the mesenchymal stem cell to realize local sustained high-concentration release of Midkine and exert the synergistic effect of both the mesenchymal stem cell and the Midkine. In a second aspect of the present invention, there is provided a method for preparing the above mesenchymal stem cells overexpressing Midkine, comprising the steps of: Step 1), inserting a coding sequence of Midkine shown as SEQ ID NO.1 and a coding sequence of T2A polypeptide shown as SEQ ID NO.2 in front of EGFP sequence of pRRLSIN plasmid to obtain an over-expression Midkine lentiviral vector pRRLSIN-MK shown as SEQ ID NO. 3; Step 2), co-transfecting the lentiviral expression vector pRRLSIN-MK over-expressing Midkine with packaging plasmids pMD2.G and psPAX to 293T cells to prepare lentiviral particles over-expressing Midkine; Step 3) tran