Enhanced stem cells overexpressing IL-10 and / or CCR2, their construction method and applications

Abstract

This invention belongs to the field of biomedical technology, specifically relating to an enhanced mesenchymal stem cell overexpressing IL-10 and / or CCR2, its construction method, and the application of this enhanced stem cell in antiviral and anti-inflammatory treatments. Addressing the lack of reports in the prior art regarding enhanced mesenchymal stem cells overexpressing IL-10 and / or CCR2, their construction methods, and their effects, this invention provides an enhanced mesenchymal stem cell overexpressing IL-10 and / or CCR2, wherein the enhanced mesenchymal stem cell stably overexpresses IL-10, or stably overexpresses CCR2, or simultaneously stably overexpresses both IL-10 and CCR2. This invention also provides a method for constructing the aforementioned enhanced mesenchymal stem cell, and exhibits better antiviral and anti-inflammatory effects, providing a theoretical basis for the subsequent development of stem cell drugs for treating viral infections, acute lung injury, intestinal inflammation, and other diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an enhanced mesenchymal stem cell overexpressing IL-10 and / or CCR2, a method for constructing the same, and the application of the enhanced stem cell in antiviral and anti-inflammatory treatments. Background Technology

[0002] Mesenchymal stem cells (MSCs) possess multi-lineage differentiation potential, immune regulation, tissue repair, and anti-inflammatory and antiviral functions, showing great promise for applications in infectious diseases, inflammatory diseases, and organ damage repair. However, MSCs generally suffer from low survival rates, insufficient homing ability, and weak immune regulation and antiviral functions when used in vivo, limiting their clinical efficacy.

[0003] Currently, functionally enhanced MSCs constructed through gene modification or chemical induction have shown promising results. Upregulation of PD-L1 and CTLA4 has enhanced the immunomodulatory function of MSCs. Combined treatment of MSCs with IFN-γ and kynurenic acid significantly enhances IDO-1 expression and secretion, resulting in stronger immunomodulatory and anti-fibrotic capabilities.

[0004] However, research on functionally enhanced MSCs in the fields of antiviral and anti-inflammatory therapy still needs to be improved. In particular, there is a lack of relevant studies that systematically evaluate their antiviral and anti-inflammatory effects in complex organoid models that simulate the in vivo microenvironment. Therefore, it is particularly important to develop a stable engineered MSC with strong homing ability, significant immune regulation, and synergistic enhancement of antiviral and anti-inflammatory functions. Summary of the Invention

[0005] The technical problem to be solved by this invention is that there are no reports in the prior art of enhanced mesenchymal stem cells expressing IL-10 and / or CCR2 and their construction methods, and the application prospects of enhanced mesenchymal stem cells expressing IL-10 and / or CCR2 are not clear, which is an urgent problem to be developed.

[0006] The technical method of the present invention to solve the above-mentioned technical problems is as follows: an enhanced mesenchymal stem cell overexpressing IL-10 and / or CCR2 is provided, wherein the enhanced mesenchymal stem cell stably overexpresses IL-10, or stably overexpresses CCR2, or stably overexpresses both IL-10 and CCR2 simultaneously.

[0007] Furthermore, the enhanced mesenchymal stem cells overexpressing IL-10 and / or CCR2 mentioned above are obtained by introducing a recombinant vector containing the coding sequences of IL-10 and / or CCR2 into mesenchymal stem cells.

[0008] Furthermore, the mesenchymal stem cells are derived from sources including umbilical cord, umbilical cord blood, bone marrow, fat, dental pulp, amnion, and stem cells artificially induced from adult cells.

[0009] The enhanced mesenchymal stem cells described herein possess trilineage differentiation capabilities, including adipogenesis, osteoogenesis, and chondrogenesis.

[0010] Among them, the enhanced mesenchymal stem cells showed significantly higher mRNA and protein levels of IL-10 and CCR2 compared to unmodified MSCs.

[0011] The present invention also provides a method for constructing enhanced mesenchymal stem cells overexpressing IL-10 and / or CCR2, comprising the following steps:

[0012] a. Construct and package lentiviral overexpression vectors: Construct IL-10 overexpression lentiviral vector, CCR2 overexpression lentiviral vector, and IL-10 and CCR2 bicistronic overexpression lentiviral vector, respectively;

[0013] b. Lentiviral packaging and concentration: The lentiviral vector from step a and the packaging plasmid were co-transfected into packaging cells, the supernatant was collected and concentrated to obtain high-titer lentivirus;

[0014] c. Viral infection of MSCs: Lentiviral cells are infected with MSCs, and stable strains are selected by adding screening antibiotics to obtain stable enhanced mesenchymal stem cells that overexpress IL-10 and / or CCR2.

[0015] The present invention also provides the use of the above-mentioned enhanced mesenchymal stem cells overexpressing IL-10 and / or CCR2 in the preparation of antiviral and anti-inflammatory drugs.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention, for the first time, constructs functionally enhanced mesenchymal stem cells overexpressing IL-10, CCR2, and simultaneously overexpressing both IL-10 and CCR2. These stem cells retain the trilineal differentiation potential of stem cells and possess trilineal differentiation capabilities (adipogenic, osteogenic, or chondrogenic), exhibiting higher safety and functional stability. Furthermore, functional validation was performed using lung and intestinal organoids, demonstrating that these functionally enhanced mesenchymal stem cells overexpressing IL-10, CCR2, and simultaneously overexpressing both IL-10 and CCR2 exhibit better antiviral and anti-inflammatory effects, and can be used to develop stem cell drugs for treating viral infections, acute lung injury, and intestinal inflammation. Attached Figure Description

[0018] Figure 1The results show the identification of functionally enhanced stem cells; where A represents the expression of IL-10 in MSC-NC, MSC-IL-10, and MSC-IL-10-CCR2 cells detected by quantitative PCR; B represents the expression of IL-10 in the culture supernatant of MSC-NC, MSC-IL-10, and MSC-IL-10-CCR2 cells detected by ELISA; C represents the expression of CCR2 in MSC-NC, MSC-CCR2, and MSC-IL-10-CCR2 cells detected by quantitative PCR; D represents the expression of CCR2 in MSC-NC, MSC-CCR2, and MSC-IL-10-CCR2 cells detected by Western blotting, with GAPDH as an internal control; EG represents the collection of MSC-NC, MSC-IL-10, MSC-CCR2, and MSC-IL-10-CCR2 cells for trilineage differentiation experiments, and then the adipogenic, osteogenic, and chondrogenic differentiation abilities of functionally enhanced MSCs were detected by Oil Red O staining, Alizarin Red staining, and Alcian Blue staining, respectively. Scale bar = 200 μm.

[0019] Figure 2 The results show the evaluation of the antiviral function of functionally enhanced stem cells based on organoids; where A represents the white light image after organoid treatment, scale bar = 200 μm; B and C represent the expression of hemagglutinin (HA) and neuraminidase (NA) in the "vascular-T cell-lung" organoids of each treatment group detected by quantitative PCR, n=3, **, p<0.01. Figure 2 (Only AC in the middle)

[0020] Figure 3 The results show the anti-inflammatory function of functionally enhanced stem cells based on organoids; where A represents the white light image after organoid treatment, scale bar = 200 μm; B and C represent the expression of IL-6 and TNF-α in "vascular-T cell-lung" organoids of each treatment group detected by quantitative PCR, n=3, *, p<0.05; **, p<0.01.

[0021] Figure 4 The results show the anti-inflammatory function of organoid-based functional enhancement stem cells. A represents the white light image after organoid treatment, scale bar = 200 μm; B and C represent the expression of IL-6 and TNF-α in organoids of each treatment group detected by quantitative PCR, n=3, **, p<0.01. Detailed Implementation

[0022] This invention provides enhanced mesenchymal stem cells that overexpress IL-10 and / or CCR2. The IL-10 and / or CCR2 genes are transduced into the genome of the mesenchymal stem cells via a lentiviral vector to obtain functionally enhanced mesenchymal stem cells.

[0023] IL-10 has significant anti-inflammatory properties. This invention aims to enhance the anti-inflammatory and antiviral effects of MSCs by overexpressing IL-10, and experiments have confirmed the significant effect. Simultaneously, this invention discovered high expression of CCL2 at the site of pathogen-mediated lung injury; therefore, overexpression of CCR2 was chosen to enhance the chemotaxis of MSCs towards target organs.

[0024] Furthermore, this invention also employs complex organoids to validate the function of MSCs overexpressing IL-10 and / or CCR2. Compared to other models, organoids have higher physiological relevance and higher time efficiency.

[0025] The functionally enhanced mesenchymal stem cells constructed by this invention, which overexpress IL-10, overexpress CCR2, and simultaneously overexpress both IL-10 and CCR2, retain the trilineal differentiation potential of stem cells and possess trilineal differentiation capabilities of adipogenesis, osteoogenesis, or chondrogenesis. They exhibit better antiviral and anti-inflammatory effects and can be used to develop stem cell drugs for the treatment of viral infections, acute lung injury, intestinal inflammation, and other diseases.

[0026] The specific implementation of the present invention will be further explained and described below through examples, but this does not mean that the scope of protection of the present invention is limited to the scope described in the examples.

[0027] Example 1: Construction and Identification of Functionally Enhanced Stem Cells

[0028] To construct functionally enhanced stem cells stably overexpressing IL-10 and CCR2, we purchased a lentivirus-mediated IL-10 and / or CCR2 overexpression system constructed by Shanghai Gemma Biopharmaceutical Co., Ltd. This overexpression system uses IL-10 and / or CCR2 as the target genes, LV6 (EF-1a / Puro) as the cloning vector, and NotI / NsiI as the cloning site. Lentiviral viruses were added at a multiplicity of infection of 20:1. The medium was changed 24 hours after lentivirus addition, and 48 hours after lentivirus addition, puromycin (2 μ / ml) and hygromycin (2 μ / ml) were added for resistance selection. After two passages (with puromycin and hygromycin added for resistance selection), MSCs stably overexpressing IL-10 and / or CCR2 were obtained, named MSC-NC, MSC-IL-10, MSC-CCR2, and MSC-IL10-CCR2, respectively.

[0029] To clarify the expression of IL-10 in the above cell types, we collected MSC-NC, MSC-IL-10, and MSC-IL10-CCR2 cells, extracted total mRNA using the Trizol method, and then detected IL-10 expression in these cells using reverse transcription-quantitative PCR. The results showed that compared to the MSC-NC group, the expression levels of IL-10 mRNA in MSC-IL-10 and MSC-IL-10-CCR2 cells were significantly increased. Figure 1 A). Based on this, we evenly seeded the same number of MSC-NC, MSC-IL-10, and MSC-IL10-CCR2 cells in 6-well plates, added 2 ml of cell culture medium to each, and cultured the cells for 48 hours. Then, we collected the cell culture supernatant and used an ELISA kit to detect the expression of IL-10 protein in the cell culture supernatant. The results showed that the expression level of IL-10 protein in the culture supernatant of MSC-IL-10 and MSC-IL-10-CCR2 cells was significantly increased (…). Figure 1 B).

[0030] To clarify the expression of CCR2 in MSC-NC, MSC-CCR2, and MSC-IL10-CCR2 cells, we collected these cells, extracted total mRNA using the Trizol method, and then detected CCR2 expression in these cells using reverse transcription-quantitative PCR. The results showed that compared to the MSC-NC group, the expression level of CCR2 mRNA in MSC-CCR2 and MSC-IL-10-CCR2 cells was significantly increased. Figure 1 C). Based on this, we collected MSC-NC, MSC-CCR2, and MSC-IL10-CCR2 cells, extracted total protein after lysis using RIPA, quantified the protein using the BCA method, and then performed Western blotting. The results showed that the expression of CCR2 protein was significantly increased in MSC-CCR2 and MSC-IL-10-CCR2 cells (C). Figure 1 D). Trilineage differentiation experiments revealed that MSC-NC, MSC-IL-10, MSC-CCR2, and MSC-IL10-CCR2 cells all possessed the ability to differentiate into adipocytes, osteoblasts, and chondrocytes, indicating that the obtained functionally enhanced stem cells possessed the characteristics of stem cells. Figure 1 (EG). This experiment demonstrates that we have successfully constructed functionally enhanced stem cells that stably overexpress IL-10 and / or CCR2, laying a solid foundation for future research.

[0031] Example 2: Validation of the antiviral function of the functionally enhanced stem cells constructed in this invention.

[0032] To investigate the antiviral function of functionally enhanced stem cells MSC-NC, MSC-IL-10, MSC-CCR2, and MSC-IL10-CCR2 at the organoid level, we constructed human adult tissue-derived lung organoids using a Stem Cell kit and co-cultured them with vascular organoids and T cells to form complex organoids. Based on this, an H1N1 influenza virus infection model was established. Twenty-four hours after H1N1 virus addition, we co-cultured the aforementioned functionally enhanced stem cells, and after 48 hours of culture, we collected lung organoids and used quantitative PCR to detect H1N1 virus infection. The experimental results showed that H1N1 virus infection and MSC treatment had no significant effect on the morphology of the complex lung organoids. Figure 2 A). Quantitative PCR results revealed that: 1) Hemagglutinin (HA) and neuraminidase (NA) expression was significantly increased after H1N1 infection. Figure 2 B and Figure 2 C); 2) MSC-NC can significantly inhibit the content of HA and NA in lung organoids ( Figure 2 B and Figure 2 C); 3) Compared with MSC-NC, MSC-IL-10, MSC-CCR2 and MSC-IL-10-CCR2 have a more significant inhibitory effect on the expression of HA and NA in H1N1 infection state ( Figure 2 B and Figure 2 C). This part of the study shows that functionally enhanced MSCs overexpressing IL-10 and / or CCR2 have better antiviral activity.

[0033] Example 3: Validation of the anti-inflammatory function of the functionally enhanced stem cells of the present invention in lung organoids.

[0034] To investigate the anti-inflammatory functions of functionally enhanced stem cells MSC-NC, MSC-IL-10, MSC-CCR2, and MSC-IL10-CCR2 at the organoid level, we constructed an inflammation model using LPS treatment on complex lung organoids. Two hours after LPS treatment, the aforementioned functionally enhanced stem cells were added, and after 24 hours, the organoids were collected for anti-inflammatory function evaluation. Results showed that LPS treatment and MSC co-culture had no significant effect on the morphology of the lung organoids. Figure 3 A). Organoids were collected and quantitative PCR was performed, revealing (…). Figure 3(B and 3C): 1) LPS significantly promoted the expression of pro-inflammatory factors IL-6 and TNF-α in organoids; 2) MSC-NC significantly inhibited the expression of IL-6 and TNF-α; 3) Compared with MSC-NC, MSC-IL-10, MSC-CCR2, and MSC-IL-10-CCR2 had more significant inhibitory effects on the expression of IL-6 and TNF-α. This part of the study shows that functionally enhanced MSCs overexpressing IL-10 and / or CCR2 have better anti-inflammatory effects.

[0035] Example 4: Validation of the anti-inflammatory function of the functionally enhanced stem cells of the present invention in intestinal organoids.

[0036] To further evaluate the anti-inflammatory effect of functionally enhanced MSCs on intestinal organoids, we constructed an inflammation model using LPS treatment of complex intestinal organoids. Two hours after LPS treatment, MSC-NC, MSC-IL-10, MSC-CCR2, and MSC-IL10-CCR2 cells were added. Organoids were collected 24 hours after treatment for anti-inflammatory function evaluation. Results showed that LPS treatment and MSC co-culture had no significant effect on the morphology of complex intestinal organoids. Figure 4 A). Collecting intestinal organoids and performing quantitative PCR revealed ( Figure 4 A and Figure 4 B): 1) LPS significantly promoted the expression of pro-inflammatory factors IL-6 and TNF-α in intestinal organoids; 2) MSC-NC significantly inhibited the expression of IL-6 and TNF-α; 3) Compared with MSC-NC, MSC-IL-10, MSC-CCR2, and MSC-IL-10-CCR2 had more significant inhibitory effects on the expression of IL-6 and TNF-α. This part of the study indicates that functionally enhanced MSCs overexpressing IL-10 and / or CCR2 have better anti-inflammatory effects.

[0037] The above embodiments collectively demonstrate that this invention successfully constructed, for the first time, functionally enhanced MSCs that stably overexpress IL-10, stably overexpress CCR2, and simultaneously stably overexpress both IL-10 and CCR2. The stem cell characteristics of the constructed functionally enhanced MSCs were then clarified through trilineage differentiation. Next, this invention utilized a complex organoid model to evaluate the antiviral and anti-inflammatory functions of the functionally enhanced MSCs in vitro. It was determined that functionally enhanced MSCs overexpressing IL-10 and / or CCR2 exhibited stronger antiviral and anti-inflammatory effects than control MSCs, providing a fundamental basis for the application of functionally enhanced MSCs overexpressing IL-10 and / or CCR2 in antiviral and anti-inflammatory drugs, and demonstrating excellent application prospects.

Claims

1. An enhanced mesenchymal stem cell overexpressing IL-10 and / or CCR2, characterized in that: The enhanced mesenchymal stem cells stably overexpress IL-10, or stably overexpress CCR2, or stably overexpress both IL-10 and CCR2 simultaneously.

2. The enhanced mesenchymal stem cells overexpressing IL-10 and / or CCR2 according to claim 1, characterized in that: The enhanced mesenchymal stem cells are obtained by introducing a recombinant vector containing the coding sequences of IL-10 and / or CCR2 into mesenchymal stem cells.

3. The enhanced mesenchymal stem cells overexpressing IL-10 and / or CCR2 according to claim 2, characterized in that: The mesenchymal stem cells mentioned above are derived from umbilical cord, umbilical cord blood, bone marrow, fat, dental pulp, amnion, and stem cells artificially induced from adult cells.

4. The enhanced mesenchymal stem cells overexpressing IL-10 and / or CCR2 according to claim 2, characterized in that: The enhanced mesenchymal stem cells have trilineage differentiation capabilities: adipogenic, osteogenic, or chondrogenic.

5. The method for constructing enhanced mesenchymal stem cells overexpressing IL-10 and / or CCR2 as described in any one of claims 1 to 4, characterized in that, Includes the following steps: a. Construct and package lentiviral overexpression vectors: Construct IL-10 overexpression lentiviral vector, CCR2 overexpression lentiviral vector, and IL-10 and CCR2 bicistronic overexpression lentiviral vector, respectively; b. Lentiviral packaging and concentration: The lentiviral vector from step a and the packaging plasmid were co-transfected into packaging cells, the supernatant was collected and concentrated to obtain high-titer lentivirus; c. Viral infection of MSCs: Lentiviral cells are infected with MSCs, and stable strains are selected by adding screening antibiotics to obtain stable enhanced mesenchymal stem cells that overexpress IL-10 and / or CCR2.

6. Use of the enhanced mesenchymal stem cells overexpressing IL-10 and / or CCR2 as described in any one of claims 1 to 4 in the preparation of antiviral and anti-inflammatory drugs.

7. The drug according to claim 6, characterized in that: The drug is used to prevent or treat viral infections, acute lung injury, and intestinal inflammation.

Images