Mesenchymal stem cell and exosome, preparation method and application

Abstract

The present application relates to the technical field of biotechnology, in particular to a mesenchymal stem cell and an exosome, a preparation method and an application. Nrf2 The present application provides a mesenchymal stem cell (Nrf2-hUMSC) overexpressing a gene, and the survival rate and function stability of the mesenchymal stem cell in an oxidative stress environment are significantly enhanced; the exosome (Nrf2-Exos) secreted by the Nrf2-hUMSC cell inherits the enhanced characteristics of the parent cell, and through verification of a diabetic chronic wound animal model, application of the Nrf2-hUMSC or the Nrf2-Exos can effectively activate the Nrf2 antioxidant defense pathway of a wound site, synergistically promote tissue repair, and significantly accelerate wound healing, and the curative effect is better than that of unmodified mesenchymal stem cells. The Nrf2-hUMSC or the Nrf2-Exos of the present application provides a complete innovative treatment scheme from a genetically engineered cell to a cell-free exosome for the treatment of chronic refractory skin wounds.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a mesenchymal stem cell and exosome, its preparation method, and its application. Background Technology

[0002] As the body's primary physiological barrier, the integrity of the skin's structure and the soundness of its function play a crucial role in maintaining homeostasis. Long-term exposure to the external environment makes the skin susceptible to physical trauma, thermal damage, infection, and systemic diseases such as diabetes, leading to acute and chronic wounds. While current routine clinical treatments such as local dressing changes, debridement and suturing, and flap transplantation can repair wounds to some extent, they generally suffer from long healing periods, susceptibility to secondary infections, and scar hyperplasia, severely impacting patients' quality of life and functional recovery.

[0003] Stem cell-based regenerative medicine offers new avenues for wound repair. Mesenchymal stem cells, such as umbilical cord mesenchymal stem cells (hUMSCs), are considered ideal "seed cells" for repair due to their ease of acquisition, strong proliferative capacity, low ethical controversy, and immunomodulatory functions. However, studies have shown that unmodified hUMSCs, after transplantation, exhibit significantly limited survival and paracrine activity in the face of high oxidative stress and inflammatory microenvironment at the wound site, hindering the full realization of their repair potential.

[0004] In recent years, exosomes, as an important medium for intercellular communication, have become a research hotspot in cell-free therapy. Mesenchymal stem cell-derived exosomes (MSCs-Exos) can deliver functional nucleic acids, proteins, and other bioactive molecules, regulate the proliferation, migration, differentiation, and immune phenotype of recipient cells, and play an important role in tissue repair. In skin injury repair, MSCs-Exos can promote the proliferation and migration of epithelial cells and fibroblasts, regulate macrophage polarization to reduce inflammation, induce angiogenesis, and promote the orderly remodeling of the extracellular matrix, thereby accelerating wound healing, improving repair quality, and avoiding the potential risks of live cell transplantation (see: Zhang, B. et al. Hype or Hope: The role of exosomes inskin wound healing and regeneration. Theranostics 2018, 8(1): 169-184). However, how to enhance the function of the blast cells and their secreted exosomes in advance through engineering means, so that they can actively resist and improve the local pathological microenvironment of the wound, remains a technical bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0005] Therefore, the first technical problem to be solved by this invention is to overcome the defect that the survival rate and repair function of unmodified human umbilical cord mesenchymal stem cells (hUMSCs) are significantly limited after transplantation into the wound microenvironment of high oxidative stress and inflammation, and to provide a functionally enhanced, overexpressing Nrf2 Gene-derived mesenchymal stem cells and their preparation methods.

[0006] The second technical problem to be solved by this invention is to provide an overexpression of a substance secreted by the aforementioned functionally enhanced stem cells, which has superior repair efficacy. Nrf2 Gene exosomes.

[0007] The third technical problem to be solved by the present invention is to provide the above-mentioned overexpression Nrf2 The application of gene-derived mesenchymal stem cells and their exosomes in the treatment of skin lesions (especially chronic, refractory wounds), as well as corresponding drug compositions and treatment methods.

[0008] Therefore, the present invention provides the following technical solution: This invention provides an overexpression Nrf2 Mesenchymal stem cells of the gene, the overexpression Nrf2 Mesenchymal stem cells contain exogenous nucleotide sequences encoding the human Nrf2 protein. This invention has found that after umbilical cord mesenchymal stem cells are transplanted into wounds, their survival rate and repair function decrease due to the high oxidative stress and inflammatory wound microenvironment.

[0009] To solve the above problems, the present invention will... Nrf2 Genes are introduced into umbilical cord mesenchymal stem cells. This invention has found that: On the one hand, one of the core mechanisms by which cells respond to oxidative stress is to maintain redox balance by activating endogenous antioxidant pathways. These endogenous antioxidant pathways involve many regulatory genes, such as common NAD(P)H-related genes, enzymes that scavenge reactive oxygen species (ROS), and genes related to glutathione synthesis or metabolism. Based on previous experiments, this invention uses recombinant lentiviruses to deliver human... NFE2L2 (Nuclear factor erythroid 2-related factor 2, Nrf2 Genes are introduced into umbilical cord mesenchymal stem cells to achieve... Nrf2The gene is stably and efficiently expressed in umbilical cord mesenchymal stem cells. Genetically modified umbilical cord mesenchymal stem cells exhibit significantly enhanced antioxidant and anti-inflammatory functions, while also improving the cells' tolerance to oxidative stress, thus significantly improving cell survival and functional durability after transplantation to wound sites. Among these, Nrf2 is a key regulator of the endogenous antioxidant pathway, inactive under homeostasis by binding to the Keap1 protein. Upon exposure to oxidative stress or electrophilic substances, Nrf2 dissociates from Keap1 and enters the nucleus, binding to antioxidant response elements (AREs) and initiating a series of downstream protective genes (such as...). HO-1 , NQO1 , GCLC The expression of (etc.) can systematically enhance the antioxidant, anti-inflammatory and detoxification capabilities of cells (see: Baird L, Yamamoto M. The Molecular Mechanisms Regulating the KEAP1-NRF2 Pathway. Mol Cell Biol. 2020;40(13):e00099-20).

[0010] On the other hand, in chronic injury models such as diabetic wounds, impaired or insufficiently activated Nrf2 signaling pathway function is considered a significant factor hindering repair. More importantly, the innovative findings of this invention will... Nrf2 When genes are organically combined with umbilical cord mesenchymal stem cells, they exhibit a significant synergistic effect, enabling the construction of a synergistic treatment system that can both resist the pathological microenvironment and actively promote repair. Under this synergistic therapeutic effect, epidermal cell proliferation and migration can be systematically promoted, capillary angiogenesis can be stimulated, and fibroblasts can be guided to synthesize and orderly arrange the extracellular matrix (collagen, etc.), thereby achieving accelerated wound closure and high-quality functional repair.

[0011] Finally, it was also discovered that... Nrf2 Gene delivery into umbilical cord mesenchymal stem cells can remodel the function of these stem cells and overexpress their gene expression. Nrf2 The exosomes secreted by umbilical cord mesenchymal stem cells undergo specific changes in their contents and biological activity, becoming rich in more antioxidant and anti-inflammatory signaling molecules. This "mother cell-exosome" functional transmission mechanism enables a cascade amplification of therapeutic effects.

[0012] Preferably, the nucleotide sequence encoding the human Nrf2 protein is shown in SEQ ID NO:1, and the nucleotide sequence of SEQ ID NO:1 is as follows:

[0013] This invention provides a method for preparing mesenchymal stem cells overexpressing the Nrf2 gene, comprising: infecting mesenchymal stem cells with a recombinant lentivirus containing the nucleotide sequence encoding the human Nrf2 protein, and screening to obtain stably overexpressing Nrf2 gene-encoding mesenchymal stem cells. Nrf2 Gene-derived mesenchymal stem cells.

[0014] The overexpression Nrf2 Molecular and functional verification of the gene-modified umbilical cord mesenchymal stem cells (Nrf2-hUMSC) showed that the expression levels of Nrf2 protein and its downstream key antioxidant proteins (such as heme oxygenase-1, HO-1) in the cells were significantly higher than those in the unmodified control cells; and the cell survival rate was significantly improved under in vitro oxidative stress conditions.

[0015] This invention provides an overexpression Nrf2 Gene exosomes, resulting from overexpression Nrf2 The gene is secreted by mesenchymal stem cells.

[0016] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic mesenchymal stem cells, or dental pulp mesenchymal stem cells.

[0017] This invention provides overexpression Nrf2 Methods for preparing gene exosomes include: culturing overexpressing genes. Nrf2 Mesenchymal stem cells containing the gene were collected in conditioned medium and then isolated and purified to obtain the exosomes.

[0018] This invention provides overexpression Nrf2 The application of gene-derived mesenchymal stem cells or their secreted exosomes in the preparation of drugs for promoting tissue damage repair.

[0019] Preferably, the tissue damage repair includes skin damage repair. The skin damage includes chronic, non-healing wounds accompanied by abnormal oxidative stress. More preferably, the chronic, non-healing wounds include diabetic chronic wounds, venous ulcers, or pressure injuries.

[0020] This invention provides a pharmaceutical composition for skin damage repair, comprising a therapeutically effective amount of overexpressed... Nrf2 The gene-derived mesenchymal stem cells and a pharmaceutically acceptable carrier. Preferably, the dosage form of the pharmaceutical composition is an injection.

[0021] Preferably, the injectable includes a cell suspension for local wound injection.

[0022] This invention provides a pharmaceutical composition for skin damage repair, comprising a therapeutically effective amount of overexpressed... Nrf2 Gene exosomes and pharmaceutically acceptable vectors or delivery systems.

[0023] Preferably, the dosage form of the pharmaceutical composition is a gel, a spray, or a medicated dressing patch. Preferably, the gel comprises a hydrogel, and the hydrogel is a thermosensitive hydrogel.

[0024] The beneficial effects and innovativeness of this invention: 1. An overexpression method provided by the present invention Nrf2 Mesenchymal stem cells containing an exogenous nucleotide sequence encoding the human Nrf2 protein. Overexpression Nrf2 Gene-enhanced mesenchymal stem cells as stem cell products: overexpression Nrf2 Mesenchymal stem cells (MSCs) can activate the Nrf2 pathway, exhibiting sustained endogenous antioxidant capacity and improving cell survival. The contents of their secreted exosomes (Nrf2-Exos) are correspondingly altered, becoming richer in antioxidant and anti-inflammatory signals, thus producing therapeutic exosomes with active regulatory capabilities.

[0025] Furthermore, this invention provides a novel treatment approach: by combining enhanced cellular antioxidant capacity with the repair function of stem cells / exosomes, this invention offers a feasible strategy for improving the local microenvironment for chronic wound repair.

[0026] Furthermore, suitable local application formulations have been developed: In view of the characteristics of chronic skin wounds, this invention provides application methods such as locally injected cell suspensions and exosome preparations loaded in thermosensitive hydrogels.

[0027] Furthermore, a standardized preparation process has been established: This invention forms a complete process from gene-modified cell construction to exosome preparation, and this process is reproducible.

[0028] In summary, this invention addresses the insufficient efficacy of conventional stem cell therapy in treating chronic oxidative stress wounds by providing a method that... Nrf2 An optimized scheme for enhancing repair function through genetic engineering. The core of this invention lies in... Nrf2 The antioxidant pathway was engineered and integrated with the repair function of mesenchymal stem cells / exosomes. The resulting... Nrf2 Genetically modified stem cells (Nrf2-hUMSC) and their exosomes (Nrf2-Exos) not only have enhanced antioxidant capacity, but can also synergistically regulate the wound microenvironment and activate host repair cells through direct action and secretion signals, thereby promoting the effective healing of chronic wounds at multiple levels. Attached Figure Description

[0029] 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.

[0030] Figure 1 This is a map of the pLVX-EF1α-Nrf2 recombinant expression plasmid from Example 1; Figure 2 This is a graph validating the overexpression efficiency of Nrf2-hUMSC cells in Example 1, where a represents the result of qPCR detection. Nrf2 mRNA level diagram; b is a Western blotting diagram showing the expression of Nrf2 and its downstream gene HO-1 protein. Figure 3 This is a comparison of cell survival rates between Nrf2-hUMSC and NC-hUMSC cells under oxidative stress conditions in Example 1. Figure 4 The following are the characterization results of the Nrf2-Exos and NC-Exos exosomes from Example 2; where a is the result of transmission electron microscopy observation, scale bar is 100 nm; b is the result of NTA detection particle size; c is the result of Western blotting. Figure 5 The images show the wound healing status of the four groups of mice in Example 3, where a is a photograph of the wound at different time points; and b is a curve showing the change in the wound closure rate. Figure 6 This is an image showing the HE staining results of full-thickness wound tissue from four groups of mice on day 6 of modeling in Example 3; Figure 7 This is a graph showing the hydrogen peroxide (H2O2) level in the wound margin tissue of the four groups of mice on day 6 of modeling in Example 3; Figure 8 This is a graph showing the expression of genes related to oxidative stress pathways in the wound tissue of the four groups of mice on day 6 of modeling in Example 3. In the above attached figures, p <0.05; p <0.01; p <0.001. Detailed Implementation

[0031] 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.

[0032] 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.

[0033] Example 1 Nrf2 Construction and identification of genetically modified human umbilical cord mesenchymal stem cells (Nrf2-hUMSC) 1.1 Cell source and culture Commercially available human umbilical cord mesenchymal stem cells (hUMSCs) conforming to ethical standards, catalog number PCS-500-010 (ATCC), were used in the experiments. Cell culture was performed using a dedicated mesenchymal stem cell culture medium, catalog number PT-3001 (Lonza), at 37°C and 5% CO2 for resuscitation and expansion. All subsequent experiments used cells in good growth condition, at passage P3.

[0034] 1.2 Preparation of Nrf2 overexpression lentivirus (1) Construction of pLVX-EF1α-Nrf2 recombinant expression plasmid: human NFE2L2 ( Nrf2 The complete coding sequence of the gene (its nucleotide sequence is shown in SEQ ID NO: 1, which is based on the NCBI reference sequence NM_006164.5 and encodes a 605-amino acid Nrf2 protein) was inserted into the lentiviral expression vector pLVX-EF1α-IRES-Puro containing the EF-1α promoter and a puromycin resistance selection marker to construct the recombinant plasmid pLVX-EF1α- Nrf2 ( Figure 1 This vector utilizes the strong EF-1α promoter to drive efficient expression of the target gene and includes a puromycin resistance gene for subsequent screening.

[0035] (2) Lentiviral packaging and production: The recombinant plasmid pLVX-EF1α- Nrf2Virus packaging and production were performed using a mature third-generation lentiviral packaging system. The specific procedure was as follows: recombinant plasmids, packaging plasmid psPAX2 (Addgene plasmid #12260), and envelope plasmid pMD2.G (Addgene plasmid #12259) were co-transfected into HEK293T cells. After culturing, supernatant collection, concentration, purification, and titer determination, high-titer Nrf2 protein overexpressing lentiviral particles were finally obtained, with a titer not less than 1 × 10⁻⁶. 8 TU / mL, stored at -80℃ for later use. Simultaneously, pLVX-EF1α-IRES-Puro containing only the empty vector was prepared. pLVX-EF1α-IRES-Puro has the exact same backbone as the recombinant expression vector, except that it lacks the target gene insert at the multiple cloning site. This control is used to exclude non-specific effects of the vector backbone, selection markers, and viral transduction process on experimental results, and is a standard negative control virus (NC-lentivirus) for gene function studies.

[0036] 1.3 Viral infection and stable strain screening of hUMSC Experimental group: hUMSC cells in logarithmic growth phase were added to an Nrf2-overexpressing lentivirus suspension at an optimized multiplicity of infection (MOI=15). To improve infection efficiency, polybrene was added to the infection system at a final concentration of 6 μg / mL. After 8 hours of infection, the medium was replaced with fresh complete medium. After 72 hours of infection, cells were seeded at an appropriate density in fresh complete medium and then continuously screened using complete medium containing 1.5 μg / mL puromycin (Thermo Fisher Scientific, A1113803) for 12 days, with regular medium changes, until stable Nrf2-hUMSC resistant cell clones were formed in the experimental group.

[0037] Control group: Same as experimental group, except that NC-lentivirus suspension was added. The screening period was 12 days, during which the medium was changed regularly until the negative control cells that were not infected with the virus died completely, forming stable NC-hUMSC resistant cell clones.

[0038] Surviving resistant cells from both the experimental and control groups were screened and cultured on a large scale. After multiple passages to ensure genetic stability, two stable cell lines were obtained: the experimental group was the Nrf2-hUMSC cell line, and the control group was the NC-hUMSC cell line transfected with an empty viral vector. The resulting cell lines were cryopreserved in liquid nitrogen for long-term storage.

[0039] 1.4 Molecular biological identification of gene-modified cells qRT-PCR detection Nrf2mRNA expression: Total RNA was extracted from the Nrf2-hUMSC and NC-hUMSC cell lines obtained in step 1.3 using the TRIzol method and reverse transcribed into cDNA. Using a human-specific... Nrf2 Gene-specific primers were used for quantitative PCR analysis: forward primer: 5'-GAGACGGCCATGACTGATTT-3' (SEQ ID NO: 2) and reverse primer: 5'-CTGCGACAAAAGCTGCATTT-3' (SEQ ID NO: 3). β-actin was used as an internal reference gene for quantitative PCR analysis, and the β-actin primers were: forward primer 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID NO: 4) and reverse primer: 5'-CTCCTTAATGTCACGCACGAT-3' (SEQ ID NO: 5).

[0040] The results showed that, compared with NC-hUMSC, Nrf2-hUMSC... Nrf2 mRNA expression levels were upregulated approximately 8-fold. p <0.001), see Figure 2 a.

[0041] Western Blot Analysis: Total protein was extracted from Nrf2-hUMSC and NC-hUMSC cells and subjected to SDS-PAGE electrophoresis. Immunoblot analysis was performed using anti-Nrf2 antibody, anti-heme oxygenase-1 (HO-1) antibody, and anti-β-actin antibody. Anti-Nrf2 antibody and HO-1 serve as markers of Nrf2 pathway activation. Results confirmed that the expression levels of Nrf2 protein and its downstream effector protein HO-1 in Nrf2-hUMSC were significantly higher than those in the NC-hUMSC control group, demonstrating successful activation of the Nrf2 pathway. (See [link to relevant documentation]). Figure 2 b. qRT-PCR and Western Blot analysis confirmed that the expression levels of Nrf2 and HO-1 proteins in the cells were significantly higher than those in the unmodified control cells (NC-hUMSC).

[0042] 1.5 Validation of in vitro antioxidant stress function To evaluate the effect of Nrf2 overexpression on enhancing cellular resistance to damage, an oxidative stress experiment was conducted. Two groups of Nrf2-hUMSC and NC-hUMSC cells were cultured at 1×10⁻⁶ cells / year. 4Cells were seeded at a density of 100 μL / well in 96-well plates containing complete culture medium. After adhesion, the medium was replaced with 100 μL of complete culture medium containing 300 μM H2O2 per well, and the treatment was carried out for 12 h. Cell viability was detected using a CCK-8 assay kit. The results showed that under H2O2 stimulation, the cell viability of the Nrf2-hUMSC group was significantly higher than that of the NC-hUMSC group. p <0.001); however, under normal conditions, there was no statistically significant difference in cell viability between the two groups. See Figure 3 The result indicates that Nrf2 Overexpression of the protein significantly enhanced the survival ability of hUMSC in oxidative stress microenvironments.

[0043] Validated using an in vitro oxidative stress model, Nrf2-hUMSC showed significantly enhanced cell viability and survival rate compared to NC-hUMSC, confirming its enhanced antioxidant capacity.

[0044] Example 2: Preparation and characterization of Nrf2-hUMSC-derived exosomes (Nrf2-Exos) 2.1 Collect cell culture medium Cells were cultured in a standard cell culture incubator at 37°C, 5% CO2, and saturated humidity. When Nrf2-hUMSC and NC-hUMSC cells reached 80% confluence, they were gently washed three times with PBS and then cultured for another 48 hours under the same conditions using exosome-free medium. The exosome-free medium was primarily used for exosome collection: exosome-free fetal bovine serum (FBS) EXO-FBS-50A-1 (System Biosciences) was mixed with DMEM / F12 basal medium at a final concentration of 10% (v / v). The mixture was then filtered through a 0.22 μm filter for sterilization before use.

[0045] 2.2 Exosome Isolation and Purification After culture, the conditioned medium was collected and centrifuged sequentially at low speed (400×g, 10 min; 2000×g, 20 min) to remove cells and debris, followed by centrifugation at high speed (10000×g, 30 min) to remove large vesicles. The supernatant obtained after centrifugation was filtered through a 0.22 μm filter membrane to obtain a clear conditioned medium supernatant. Subsequently, in accordance with the guidelines of the International Society for Extracellular Vesicles, the filtered supernatant was purified by size exclusion chromatography for exosome separation (see: Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014guidelines. Journal of Extracellular Vesicles. 2018;7(1):1535750).

[0046] The specific procedures for exosome separation and purification using size exclusion chromatography were as follows: A HiPrep S-500 HR column (Cytiva, catalog number 28935606) was used with pre-cooled 1×PBS (pH 7.4) as the mobile phase. Size exclusion chromatography (Cytiva, ÄKTA pure 25) was performed on the clarified conditioned medium supernatant. The exosome fraction corresponding to the first major UV absorption peak was collected. This fraction was transferred to a 100 kDa ultrafiltration centrifuge tube and concentrated by centrifugation at 4℃ and 4000 g. After further concentration with fresh 1×PBS, exosome suspensions were obtained and named Nrf2-Exos and NC-Exos, respectively.

[0047] 2.3 Systematic characterization of exosomes The obtained exosomes Nrf2-Exos and NC-Exos were characterized in multiple dimensions: 1) The structure and diameter distribution of exosomes were observed using transmission electron microscopy, see... Figure 4 As shown in a, both Nrf2-Exos and NC-Exos vesicles exhibit a typical bilayered cup structure.

[0048] 2) NTA nanoparticle tracking analysis was performed to determine its main particle size distribution peaks and particle concentration. The results are shown in [Figure number missing]. Figure 4 From b, we can see that the particle size is mainly distributed in the range of 120-150 nm.

[0049] 3) Western Blot Detection: Western blotting was used to identify biomarkers in exosomes. Results are as follows: Figure 4 As shown in Figure c, both Nrf2-Exos and NC-Exos showed high expression of the characteristic positive markers of exosomes, CD63 and TSG101, while the endoplasmic reticulum marker Calnexin was not detected. This result is consistent with the exosome characterization criteria recommended by the International Society for Extracellular Vesicles (ISEV) (see: Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): aposition statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles.2018;7(1):1535750), indicating that the obtained samples were rich in exosomes, and the main vesicle components originated from the cell membrane / endosome system, with minimal contamination from endoplasmic reticulum debris.

[0050] Example 3: Treatment evaluation in an animal model of diabetic chronic wounds 3.1 Establishing an animal model Eight-week-old male db / db spontaneous type 2 diabetic mice (SPF grade, Jackson Lab, strain number 000642) were selected. After anesthetizing with intraperitoneal injection of 1% sodium pentobarbital solution, the skin on the back was prepared and disinfected on a sterile operating table. Two circular full-thickness skin defects with a diameter of 8 mm were created symmetrically on both sides of the spine using a sterile biopsy piercing instrument.

[0051] 3.2 Experimental grouping and treatment The model mice were randomly divided into 4 groups, with 6 mice in each group: 1) PBS control group: 100 μL of sterile PBS was injected into the wound and edges of mice at 5 points.

[0052] 2) NC-hUMSC treatment group: Mice were injected with 1×10⁻⁶ mol / L at 5 points on the wound and edges. 6 A cell suspension of NC-hUMSC (100 μL).

[0053] 3) Nrf2-hUMSC treatment group: Mice were injected with 0.5×10 at 5 points on the wound surface and edges. 6100 μL of Nrf2-hUMSC cell suspension.

[0054] 4) Nrf2-Exos treatment group: 100 μg (quantified by total exosome protein, if BCA method is used, the dosage is 100 μg) of Nrf2-Exos was thoroughly mixed with 100 μL of thermosensitive chitosan and β-glycerophosphate sodium hydrogel precursor solution, injected to cover the wound, and gelled and fixed within 30s at 37℃.

[0055] The thermosensitive chitosan / sodium β-glycerophosphate hydrogel precursor solution was prepared according to a classic method in the art (see: Chenite et al., Novel injectable neutral solutions of chitosan formbiodegradable gels in situ. Biomaterials, 2000, 21(21), 2155-2161). A brief description is as follows: Chitosan with a degree of deacetylation ≥95% was dissolved in 0.1 M acetic acid to prepare a 2.0% (w / v) solution; sodium β-glycerophosphate was dissolved in water to prepare a 56% (w / v) solution. Both solutions were sterile filtered and stored at 4°C. Before use, equal volumes of the two solutions were mixed in an ice bath to obtain precursor solutions with final concentrations of 1.0% chitosan and 28% (w / v) sodium β-glycerophosphate, respectively. This solution rapidly transforms into a hydrogel at 37°C.

[0056] All treatments were administered immediately after model establishment (day 0) and repeated on day 3 and day 6, for a total of 3 treatments.

[0057] 3.3 Observation and evaluation of therapeutic effects (1) Dynamic monitoring of wound healing: The wound was photographed at fixed points on days 0, 4, 8 and 12 after modeling. The wound area was analyzed by ImageJ software and the wound closure rate was calculated.

[0058] See results Figure 5 Compared with the PBS control group, the wound healing rate in the NC-hUMSC treatment group was significantly faster. p <0.01, the Nrf2-hUMSC group and the Nrf2-Exos group showed more significant healing effects; their healing rate was significantly higher than that of the PBS control group ( p <0.001, which was also significantly higher than that of the NC-hUMSC treatment group ( p<0.001). By day 12, statistical analysis showed that the wound healing rates in the NC-hUMSC group, Nrf2-hUMSC group, and Nrf2-Exos group were significantly faster than those in the PBS control group ( p <0.01). Among them, the healing rate of the Nrf2-hUMSC group and the Nrf2-Exos group was significantly faster than that of the NC-hUMSC group ( p <0.001).

[0059] (2) On the 6th day of treatment, full-thickness wound tissue was taken, paraffin-embedded and sectioned, and HE stained. Figure 6 It can be seen that the Nrf2 treatment groups (Nrf2-hUMSC group and Nrf2-Exos group) had thicker granulation tissue and less inflammatory infiltration.

[0060] (3) Oxidative stress and pathway activation detection: On day 6 of treatment, tissue samples were collected from the wound margins for the following tests: 1) Hydrogen peroxide (H2O2) level detection: such as Figure 7 As shown, compared with the PBS control group, the H2O2 level in the NC-hUMSC group was significantly lower ( p <0.05 indicates that conventional stem cell therapy has a certain antioxidant effect. Furthermore, the H2O2 levels in the Nrf2 treatment groups (Nrf2-hUMSC group and Nrf2-Exos group) were further reduced, both significantly lower than those in the NC-hUMSC group ( p <0.05) and PBS group ( p <0.01), indicating that the Nrf2 treatment group had a significantly lower value than the control group, see Figure 7 .

[0061] 2) qRT-PCR analysis is shown in [link to qRT-PCR analysis]. Figure 8 Nrf2 downstream target genes in wound tissue of Nrf2 treatment groups (Nrf2-hUMSC group and Nrf2-Exos group) HO-1 and NQO1 The mRNA expression level was significantly upregulated ( p <0.05, p <0.01, p<0.001), confirming that the local Nrf2 antioxidant pathway was effectively activated.

[0062] HO-1 (Hmox1) primer: F: 5'-AAGCCGAGAATGCTGAGTTCA-3' (SEQ ID NO: 6); R: 5'-GCCGTGTAGATATGGTACAAGGA-3' (SEQ ID NO: 7).

[0063] NQO1 primer: F: 5'-AGGATGGGAGGTACTCGAATC-3' (SEQ ID NO: 8); R: 5'-AGGCGTCCTTCCTTATATGCTA-3' (SEQ ID NO: 9).

[0064] In summary, this invention uses commercially available hUMSCs as starting material and genetically engineered a functionally enhanced cell line (Nrf2-hUMSCs) stably overexpressing Nrf2 and its derived exosomes (Nrf2-Exos). In a db / db mouse model of diabetic chronic wounds, this treatment regimen demonstrated significant healing-promoting effects by effectively activating the local Nrf2 antioxidant defense network, improving the repair microenvironment, and promoting angiogenesis and re-epithelialization. Its efficacy was superior to unmodified stem cell therapy. This invention provides solid preclinical evidence for developing therapeutic strategies for refractory wounds based on genetically engineered stem cells / exosomes.

[0065] 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. An overexpression Nrf2 Mesenchymal stem cells with genes, characterized by, The overexpression Nrf2 Gene-derived mesenchymal stem cells contain exogenous nucleotide sequences encoding the human Nrf2 protein.

2. The overexpression according to claim 1 Nrf2 Mesenchymal stem cells with genes, characterized by, The nucleotide sequence encoding the human Nrf2 protein is shown in SEQ ID NO:

1.

3. The overexpression according to claim 1 or 2 Nrf2 Mesenchymal stem cells with genes, characterized by, The mesenchymal stem cells are umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, amniotic mesenchymal stem cells, or dental pulp mesenchymal stem cells.

4. The overexpression as described in any one of claims 1-3 Nrf2 A method for preparing gene-derived mesenchymal stem cells, characterized in that, include: Mesenchymal stem cells were infected with a recombinant lentivirus containing the nucleotide sequence encoding the human Nrf2 protein, and then screened to obtain stably overexpressing [the protein]. Nrf2 Gene-derived mesenchymal stem cells.

5. An overexpression Nrf2 Gene exosomes, characterized by, The overexpression as described in any one of claims 1-3 Nrf2 The gene is obtained from mesenchymal stem cells or from the overexpression method described in claim 4. Nrf2 The gene is secreted by mesenchymal stem cells.

6. The overexpression as described in any one of claims 1-3 Nrf2 Gene overexpression obtained from mesenchymal stem cells or the preparation method described in claim 4 Nrf2 Mesenchymal stem cells of the gene or overexpression as described in claim 5 Nrf2 The application of gene exosomes in the preparation of drugs to promote tissue damage repair.

7. The application according to claim 6, characterized in that, The tissue damage repair includes skin damage repair; the skin damage includes chronic, non-healing wounds accompanied by abnormal oxidative stress.

8. The application according to claim 7, characterized in that, The chronic, non-healing wounds include diabetic chronic wounds, venous ulcers, or pressure injury wounds.

9. A pharmaceutical composition for tissue damage repair, characterized in that, The overexpression comprising a therapeutically effective amount as described in any one of claims 1-3 Nrf2 The gene is obtained from mesenchymal stem cells or from the overexpression method described in claim 4. Nrf2 Gene-derived mesenchymal stem cells and pharmaceutically acceptable vectors.

10. A pharmaceutical composition for tissue damage repair, characterized in that, The overexpression of claim 5 includes a therapeutically effective amount. Nrf2 Gene exosomes and pharmaceutically acceptable vectors or delivery systems.

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