MiRNA-574-5p modified mesenchymal stem cell outer vesicle and application thereof

Abstract

The invention discloses a mesenchymal stem cell outer vesicle modified by miRNA-574-5p and application of the mesenchymal stem cell outer vesicle, and relates to the technical field of biological medicine, and the vesicle is an extracellular vesicle secreted after the miRNA-574-5p is overexpressed by a mesenchymal stem cell. The invention also discloses a preparation method of the vesicle and application of the vesicle in preparation of a medicine for preventing or treating infectious lung inflammation injury. The miRNA-574-5p is overexpressed, so that the prevention and treatment effects of the mesenchymal stem cell outer vesicles on infectious lung inflammation injury are remarkably improved, and the miRNA-574-5p has an important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a miRNA-574-5p modified mesenchymal stem cell extravesicle and its application. Background Technology

[0002] Acute lung injury (ALI) induced by Staphylococcus aureus infection is a severe clinical condition, usually triggered by critical illnesses such as sepsis, aspiration of acidic substances, and trauma. Its main characteristic is a severe acute inflammatory response, manifested as massive neutrophil infiltration, alveolar edema, increased secretion of inflammatory mediators, and accumulation of inflammatory cells. Early ALI can progress to severe pulmonary dysfunction, namely acute respiratory distress syndrome (ARDS). ARDS and ALI are key pathological processes associated with high morbidity and mortality in critically ill patients. Therefore, exploring new clinical management strategies for ALI is urgently needed.

[0003] Mesenchymal stem cells (MSCs) are a type of stromal cell with multi-lineage differentiation potential, capable of differentiating into various lineages such as chondrocytes, osteoblasts, and adipocytes after specific induction. Due to their low immunogenicity and strong paracrine activity, MSCs, with their immunosuppressive efficacy and anti-inflammatory properties, are increasingly becoming one of the most ideal seed cells for the clinical treatment of inflammatory diseases. Notably, numerous animal model studies have confirmed that MSCs can secrete key paracrine factors (such as growth factors, anti-inflammatory cytokines, and antimicrobial peptides), effectively preventing ALI caused by pneumonia or sepsis. These findings highlight the broad application prospects of MSCs in the clinical treatment of acute pulmonary inflammation. Recent studies indicate that the regulatory role of MSCs in inflammation may mainly depend on the extracellular vesicles (EVs) they secrete. These membranous vesicles, ranging from 30 nm to 2000 nm in diameter, contain complex components such as proteins, non-coding RNA, and lipids, and can be transferred to effector cells to participate in intercellular functional regulation. Notably, a series of studies have confirmed the significant therapeutic effects of EVs (especially MSC-derived EVs) in the clinical treatment of various inflammatory diseases. Therefore, promoting the clinical application of MSC-EVs may become an important strategy for treating acute lung inflammation. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a miRNA-574-5p-modified mesenchymal stem cell extravesicle and its application. By overexpressing miRNA-574-5p, the present invention significantly enhances the preventive and therapeutic effects of mesenchymal stem cell extravesicles on infectious lung inflammation, and has important clinical application value.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a miRNA-574-5p modified mesenchymal stem cell extracellular vesicle is provided, which is an extracellular vesicle secreted by mesenchymal stem cells after overexpression of miRNA-574-5p.

[0006] Furthermore, the nucleotide sequence of miRNA-574-5p is shown in SEQ ID NO.1.

[0007] Furthermore, mesenchymal stem cells are derived from women's menstrual blood.

[0008] This invention also provides a method for preparing extracellular vesicles of mesenchymal stem cells modified with the above-mentioned miRNA-574-5p, comprising the following steps:

[0009] (1) Based on the nucleotide sequence of miRNA-574-5p, a double-stranded DNA was synthesized and ligated into a lentiviral vector to obtain a lentiviral expression vector.

[0010] (2) Transform the lentiviral expression vector obtained in step (1) into competent Escherichia coli cells, screen positive clones, culture and extract plasmids to obtain overexpression plasmids;

[0011] (3) The overexpression plasmid obtained in step (2) was transfected into HEK293T cells for packaging, and the viral supernatant was collected;

[0012] (4) Culture mesenchymal stem cells, then add the viral supernatant obtained in step (3) for culture, collect the cell supernatant, centrifuge, and obtain mesenchymal stem cell vesicles modified with miRNA-574-5p.

[0013] Furthermore, in step (1), the nucleotide sequence of the DNA double strand is shown in SEQ ID NO.2.

[0014] Furthermore, in step (1), the lentiviral vector is pHBLV-U6.

[0015] Furthermore, the nucleotide sequence of pHBLV-U6 is shown in SEQ ID NO.3.

[0016] The present invention also provides the application of the above-mentioned miRNA-574-5p modified mesenchymal stem cell extravesicles or preparation method in the preparation of drugs for the prevention or treatment of infectious pulmonary inflammatory damage.

[0017] Furthermore, in the above applications, the infectious pulmonary inflammatory damage is caused by Staphylococcus aureus infection.

[0018] The present invention also provides a drug for the prevention or treatment of infectious pulmonary inflammatory damage, characterized in that it comprises mesenchymal stem cell extravesicles modified with miRNA-574-5p.

[0019] The present invention has the following beneficial effects:

[0020] This invention involves constructing an overexpression vector for miRNA-574-5p in vitro, then overexpressing miRNA-574-5p in mesenchymal stem cells (MSCs), and finally extracting extracellular vesicles (EVs) secreted by these cells using ultracentrifugation. Intervention in MRSA-induced lung inflammation in mice revealed that EVs secreted by miRNA-574-5p-modified MSCs more effectively inhibited Staphylococcus aureus-induced lung inflammation. Attached Figure Description

[0021] Figure 1 A comparison of miR-574-5p expression levels in mesenchymal stem cells;

[0022] Figure 2 Image showing H&E staining of mouse lung tissue;

[0023] Figure 3 A graph showing the statistical results of lung tissue damage scores in mice;

[0024] Figure 4 This serves as a comparative study of changes in the expression of inflammatory factors in mouse lung tissue.

[0025] Figure 5 A comparison chart of total protein content in bronchoalveolar lavage fluid;

[0026] Figure 6 This is a comparison chart of inflammatory factor levels in bronchoalveolar lavage fluid. Detailed Implementation

[0027] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0028] Example 1

[0029] A method for preparing a miRNA-574-5p-modified mesenchymal stem cell extravesicle includes the following steps:

[0030] (1) Based on the RNA sequence of miR-574-5p (i.e. miRNA-574-5p, as shown in SEQ ID NO.1), the DNA double strand of miR-574-5p was synthesized using a DNAchem192 synthesizer according to the solid-phase ammonium phosphite triester method. The synthesized product was then purified by passing it through a C18 simple reverse-phase column. The synthesized product was given two restriction enzyme sites, BamHI and EcoRI, at its 5' and 3' ends, respectively (the synthesized DNA sequence is shown in SEQ ID NO.2).

[0031] The miR-574-5p double-stranded DNA product synthesized by the company was ligated into the pHBLV-U6 lentiviral empty vector by double digestion at two sites, BamHI and EcoRI (the digestion reaction system is shown in Table 1). The lentiviral vector is shown as SEQ ID NO.3, and the T4 DNA ligase reaction system is shown in Table 2.

[0032] Table 1 Enzyme digestion reaction system

[0033]

[0034] Table 2 Ligase Reaction System

[0035]

[0036] (2) Take 5 μL of the ligation reaction system and add it to 50 μL of Escherichia coli DH5α competent cells for transformation (place on ice for 5 minutes, heat shock at 42°C for 90 seconds, place on ice for 10 minutes); spread the heat-shocked transformant on an ampicillin-resistant LB plate using a spreader, place it at 37°C overnight, pick single colonies, place them in ampicillin-resistant LB medium, and incubate them on a shaker at 37°C and 200 rpm overnight; extract plasmid DNA from Escherichia coli and screen for positive clone plasmids;

[0037] (3) Seed HEK293T cells into 6-well plates. When the cell density reaches 70%, transfect commercial lentiviral packaging plasmids pSPAX2 and pMD2G, along with overexpression miR-574-5p plasmid or empty vector control, into HEK293T cells using Lipofectamine 3000 (plasmid ratio: pSPAX2 0.5 μg, pMD2G 0.5 μg, overexpression plasmid or empty vector 1 μg). After 48 hours of transfection, collect the viral supernatant, which can be used directly to infect cells or frozen in a -80°C ultra-low temperature freezer for later use.

[0038] (4) Mesenchymal stem cells (MenSCs) isolated from menstrual blood samples of healthy young women were seeded into six-well plates and cultured. When the cell density reached 50%, the original culture medium was discarded. 1 mL of the supernatant of the empty vector and the overexpression virus of miR-574-5p were added to the MenSCs, along with 1 mL of fresh culture medium. Finally, 2 μL of Polybrene at a concentration of 4 μg / μL was added. After 8 hours, the culture medium was replaced with fresh medium. After 48 hours of cell infection, a portion of the cells were taken to extract total RNA and the expression efficiency of overexpression of miR-574-5p was detected by real-time PCR. The cell supernatant of the remaining cells was collected and centrifuged at 100,000 g for 2 hours to obtain unmodified extracellular vesicles (MenSC-EVs) and miR-574-5p-modified mesenchymal stem cell extracellular vesicles (miR-574-5p-MenSC-EVs).

[0039] The expression of miR-574-5p was detected by quantitative real-time PCR as follows: Figure 1 As shown in the figure. The results showed that after infection of cells with the supernatant of miR-574-5p overexpression virus, the expression level of miR-574-5p in MenSCs increased significantly (*** represents significance P value <0.001), proving the successful preparation of miR-574-5p modified mesenchymal stem cell vesicles.

[0040] Example 2

[0041] 1×10 6 MenSC-EVs and miR-574-5p-MenSC-EVs prepared in Example 1 were dissolved in 50 μL of PBS and injected intratracheally into the lung tissue of C57 / BL6 mice. Four hours later, methicillin-resistant Staphylococcus aureus (MRSA) strains (3 × 10⁻⁶ per mouse) were introduced. 8 CFU was also used to infect mouse lung tissue via tracheal instillation to examine the effects of different treatment groups of EVs on lung inflammation and damage in mice.

[0042] (1) The effect of H&E histological staining on lung inflammation damage in mice was observed, and the results are as follows: Figure 2As shown in the figure. The results showed that, compared with the PBS group without EVs (MRSA), the EVs group (MRSA+MenSC-EVs) significantly inhibited MRSA infection-induced lung tissue swelling, alveolar wall thickening, and neutrophil infiltration, indicating that MenSC-EVs significantly inhibited MRSA-induced lung inflammatory damage. More importantly, the EVs group overexpressing miR-574-5p (MRSA-miR-574-5p-MenSC-EVs) further significantly inhibited lung inflammatory damage in mice compared with the EVs group (MRSA+EVs).

[0043] The lung injury scores of mice were statistically analyzed as follows: Figure 3 As shown in the figure. The results showed that the lung tissue damage in mice treated with EVs overexpressing miR-574-5p was significantly reduced (* indicates significance P value <0.05).

[0044] (2) The expression changes of inflammatory factors in mouse lung tissue were detected by qPCR, and the results are as follows: Figure 4 As shown.

[0045] The results showed that, compared with the PBS group without EVs (MRSA), the EVs group (MRSA+MenSC-EVs) could significantly inhibit the secretion of inflammatory factors in lung tissue induced by MRSA infection, and the EVs group overexpressing miR-574-5p (MRSA-miR-574-5p-MenSC-EVs) further significantly inhibited the secretion of inflammatory factors in lung tissue compared with the EVs group (MRSA+EVs) (* indicates significance P value <0.05).

[0046] (3) Using bronchoalveolar lavage, the changes in total protein content and inflammatory factor levels in the bronchoalveolar lavage fluid were observed, and the results are as follows: Figure 5 and Figure 6 As shown.

[0047] The results showed that, compared with the PBS group without EVs (MRSA), the EVs group (MRSA+MenSC-EVs) significantly inhibited the infiltration of proteins and the secretion of inflammatory factors in bronchoalveolar lavage fluid induced by MRSA infection. More importantly, the EVs group overexpressing miR-574-5p (MRSA-miR-574-5p-MenSC-EVs) further significantly inhibited the infiltration of proteins and the secretion of inflammatory factors in bronchoalveolar lavage fluid compared with the EVs group (MRSA+MenSC-EVs) (* indicates a significant P value <0.05, ** indicates a significant P value <0.01).

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A miRNA-574-5p-modified mesenchymal stem cell extravesicle, characterized in that, These are extracellular vesicles secreted by mesenchymal stem cells after overexpression of miRNA-574-5p.

2. The miRNA-574-5p-modified mesenchymal stem cell extravesicles as described in claim 1, characterized in that, The nucleotide sequence of the miRNA-574-5p is shown in SEQ ID NO.

1.

3. The mesenchymal stem cell extravesicles modified with miRNA-574-5p as described in claim 1, characterized in that, The mesenchymal stem cells mentioned are derived from women's menstrual blood.

4. The method for preparing miRNA-574-5p-modified mesenchymal stem cell extravesicles according to any one of claims 1-3, characterized in that, Includes the following steps: (1) A double-stranded DNA was synthesized according to the nucleotide sequence of miRNA-574-5p, and the double-stranded DNA was ligated to a lentiviral vector to obtain a lentiviral expression vector. (2) Transform the lentiviral expression vector obtained in step (1) into competent Escherichia coli cells, screen positive clones, culture and extract plasmids to obtain overexpression plasmids; (3) The overexpression plasmid obtained in step (2) was transfected into HEK293T cells for packaging, and the viral supernatant was collected; (4) Culture mesenchymal stem cells, then add the viral supernatant obtained in step (3) for culture, collect the cell supernatant, centrifuge, and obtain mesenchymal stem cell vesicles modified with miRNA-574-5p.

5. The method for preparing miRNA-574-5p-modified mesenchymal stem cell extravesicles as described in claim 4, characterized in that, In step (1), the nucleotide sequence of the DNA double strand is shown in SEQ ID NO.

2.

6. The method for preparing miRNA-574-5p-modified mesenchymal stem cell extravesicles as described in claim 4, characterized in that, In step (1), the lentiviral vector is pHBLV-U6.

7. The method for preparing miRNA-574-5p-modified mesenchymal stem cell extravesicles as described in claim 6, characterized in that, The nucleotide sequence of pHBLV-U6 is shown in SEQ ID NO.

3.

8. The use of the miRNA-574-5p modified mesenchymal stem cell extravesicles according to any one of claims 1-3 or the preparation method according to any one of claims 4-7 in the preparation of a drug for the prevention or treatment of infectious pulmonary inflammatory injury.

9. A drug for the prevention or treatment of infectious pulmonary inflammatory damage, characterized in that, Mesenchymal stem cell extravesicles modified with miRNA-574-5p as described in any one of claims 1-3.

Images