Exosome rich in miRNA-21-5p and preparation method and application thereof

Abstract

The application discloses a kind of miRNA-21-5p-rich exosomes and its preparation method and application.The preparation method includes the following steps: culturing mesenchymal stem cells in culture medium containing promoting agent, collecting supernatant, and obtaining miRNA-21-5p-rich exosomes by separation;The promoting agent includes at least one of vascular cell adhesion molecule-1, tumor necrosis factor alpha and interleukin-6.The method can obtain miRNA-21-5p-rich exosomes;The exosome has a therapeutic effect on diabetic foot ulcers, and the therapeutic effect is significantly better than that of exosomes prepared by traditional methods.

Description

Technical Field

[0001] This invention relates to the field of ulcer treatment, and in particular to an exosome rich in miRNA-21-5p, its preparation method, and its application. Background Technology

[0002] Diabetic foot ulcer (DFU) is a serious complication of DM. The risk of DFU in DM patients is 19% to 34%, and the recurrence rate within 1 year is as high as 40%[1]. However, traditional treatments, advanced surgical procedures and even percutaneous transluminal angioplasty are not very effective for DFU patients. The amputation rate of DFU patients is very high, which causes a significant decline in the quality of life of patients and also places a great burden on patients' families and society[2].

[0003] At present, stem cell transplantation technology has been widely used in the clinical treatment of DFU. It mainly utilizes the differentiation potential of stem cells to induce the generation of angiogenic factors and local microvessels to promote the restoration of blood circulation in necrotic wounds. Studies have shown that stem cell transplantation technology is even better than general surgical treatment in promoting the healing of refractory skin wounds and reducing complications[3]. However, there are also reports suggesting that stem cells have certain carcinogenic risks when used for clinical disease treatment. According to a report on the website of the British magazine New Scientist, a new study conducted by scientists at the Hebrew University of Jerusalem found that more than 1 / 5 of the experimental stem cells cultured in the regenerative medicine research laboratory had carcinogenic mutations[4].

[0004] Mesenchymal stem cell (MSC)-derived exosomes (Exo) function similarly to stem cells. They can also promote angiogenesis, antioxidation, and inhibit cell apoptosis by regulating the inflammatory microenvironment of the wound, thereby promoting DFU wound healing [5] and reducing scar tissue formation [6]. Compared with cell-based therapies, Exo has the advantage of fewer side effects and a wide range of applications [7]. It can overcome adverse reactions associated with cell transplantation, such as immune rejection [6], and can therefore be used as an effective treatment for DFU.

[0005] However, our investigation revealed that current research on Exo and Exo-miR for treating DFU is relatively limited and still in its early stages, not involving the development of Exo products suitable for DFU treatment. While there is research on modifying Exo, it remains at the basic research stage. If it were considered a product for treating DFU, it would have disadvantages such as imperfect manufacturing processes, complex operation, poor stability, and excessively high costs.

[0006] References: [1] Reardon R, Simring D, Kim B, et al. Thediabetic foot ulcer. Aust JGen Pract, 2020, 49: 250⁃255. [2] Muhs BE, Gagne P, Sheehan P. Peripheral arterial disease: clinical assessment and indications for revascularization in the patient with diabetes. Curr Diab Rep. 2005 Feb;5(1):24-9. [3] Vu NB, Nguyen HT, Palumbo R, Pellicano R, Fagoonee S, Pham PV. Stemcell-derived exosomes for wound healing: current status and promising directions. Minerva Med. 2021 Jun;112(3):384-400. [4] Liu Xia. Research found that more than 1 / 5 of experimental stem cells have oncogenic mutations. Science and Technology Daily, February 21, 2024, page 004. [5] DING J, WANG X, CHEN B, et al. Exosomes derived from human bonemarrowmesenchymal stem cells stimulated by deferoxamine accelerate cutaneouswound healing by promoting angiogenesis. Biomed Res Int, 2019, 2019: 9742765. [6] RAGHAV A, TRIPATHI P, MISHRA BK, et al. Mesenchymal stromalcell-derived tailored exosomes treat bacteria-associated diabetes foot ulcers: Acustomized approach from bench to bed. Front Microbiol, 2021, 12: 712588. [7] Lotfy A, AboQuella NM, Wang H. Mesenchymal stromal / stem cell(MSC)-derived exosomes in clinical trials. Stem Cell Res Ther. 2023 Apr 7;14(1):66. Summary of the Invention

[0007] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing exosomes rich in miRNA-21-5p.

[0008] A second objective of the present invention is to provide exosomes rich in miRNA-21-5p prepared by the above method.

[0009] A third objective of this invention is to provide the application of the above-mentioned exosomes rich in miRNA-21-5p.

[0010] The objective of this invention is achieved through the following technical solution: A method for preparing exosomes rich in miRNA-21-5p includes the following steps: culturing mesenchymal stem cells (MSCs) in a culture medium containing a promoter, collecting the supernatant, and separating exosomes rich in miRNA-21-5p; wherein the promoter includes at least one of vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6).

[0011] Furthermore, the promoters are vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6).

[0012] Furthermore, the concentration of vascular cell adhesion molecule-1 in the culture medium containing the promoter is 20–400 ng / mL; even more specifically, it is 200 ng / mL.

[0013] Furthermore, the concentration of tumor necrosis factor α in the culture medium containing the promoter is 5–20 ng / mL; even more specifically, it is 10 ng / mL.

[0014] Furthermore, the concentration of interleukin-6 in the culture medium containing the promoter is 10–700 ng / mL; even more specifically, it is 100 ng / mL.

[0015] Furthermore, the culture medium containing the promoter is a serum-free DMEM-F12 culture medium containing the promoter.

[0016] Furthermore, the culture medium containing the promoter also contains human umbilical cord blood plasma and glucose.

[0017] Furthermore, the volume percentage concentration of human umbilical cord plasma in the culture medium containing the promoter is 2-20%.

[0018] Furthermore, the concentration of glucose in the culture medium containing the promoter is 1–3 mg / mL; even further, it is 2 mg / mL.

[0019] Furthermore, the culture is carried out for 36 to 60 hours, and even further, for 48 hours.

[0020] Furthermore, the cultivation conditions are: a temperature of 37°C, a saturated humidity of 95%, and a gaseous environment of 5% CO2.

[0021] Furthermore, the mesenchymal stem cells mentioned are human umbilical cord mesenchymal stem cells (hUC-MSCs).

[0022] Furthermore, the ratio of the culture medium to mesenchymal stem cells is 1 × 10⁻⁶ cells per 1 mL of culture medium. 4 ~1×10 6 Each mesenchymal stem cell; furthermore, 1×102 cells are cultured per 1 mL of culture medium. 5 Mesenchymal stem cells.

[0023] Exosomes rich in miRNA-21-5p were prepared using the method described above.

[0024] The above-mentioned exosomes rich in miRNA-21-5p are used in the preparation of products for the treatment of diabetic foot ulcers.

[0025] The products mentioned include daily necessities or medicines.

[0026] The present invention has the following advantages and effects compared with the prior art: The technical solution of the present invention can obtain exosomes rich in miRNA-21-5p by using the promoters VCAM-1, TNF-α and IL-6; these exosomes have a therapeutic effect on diabetic foot ulcers, and the therapeutic effect is significantly better than that of exosomes prepared by traditional methods. Attached Figure Description

[0027] Figure 1 This is a heatmap of Exo-miRNA expression.

[0028] Figure 2 This is a graph showing the particle size of exosomes and the results of flow cytometry analysis.

[0029] Figure 3 This is a diagram showing the results of culturing hUC-MSCs in exosome medium rich in miRNA-21-5p to promote secretion. Figure 4 This is a graph showing the results of treating a rat DFU model with Exo. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0031] Example 1 1. Mesenchymal stem cell culture Human umbilical cord mesenchymal stem cells (hUC-MSCs) were provided by the Guangdong Umbilical Blood Hematopoietic Stem Cell Bank (Guangzhou, China). Flow cytometry was used to detect the phenotype of MSCs, and cell surface antigen markers included CD34, CD45, CD73, CD90, and CD105. MSCs were cultured in serum-free (DMEM) F12 medium (HyClone, Logan, UT, USA) with different components according to their groups. After 48 hours of incubation at 37°C, 95% saturated humidity, and 5% CO2 atmosphere, the supernatant was collected. 1 × 10⁶ cells were cultured per 1 mL of medium. 5 Personal umbilical cord mesenchymal stem cells. Experimental groups are as follows: Control group: Added glucose to a final concentration of 2 mg / mL; VCAM-1 group: glucose was added to a final concentration of 2 mg / mL and VCAM-1 (ADP5, R&D, USA) to a final concentration of 20 ng / mL; TNF-α group: glucose and TNF-α (T6674, Sigma, Germany) were added to a final concentration of 2 mg / mL; IL6 group: glucose was added to a final concentration of 2 mg / mL and IL-6 was added to a final concentration of 20 ng / mL (200–06-20, PeproTech, USA).

[0032] 2. Isolation and purification of mesenchymal stem cell exosomes According to the references, exosomes were isolated from the collected culture medium supernatant using ultracentrifugation (Tooi et al., 2016). The supernatant was centrifuged at 300g for 5 min, 2000g for 10 min, and 10000g for 20 min to remove cell debris and proteins. The supernatant was then filtered through a 0.22 μm filter (UFC810096-1, Merck Millipore, Germany) to remove particles larger than 0.22 μm. The filtrate was ultracentrifuged at 100000g, 4℃ for 120 min (Optima XE-90 ultracentrifuge with a swing rotor, Type 70Ti; Beckman Coulter, CA, USA). The concentrated MSCs-exo were resuspended in PBS for further analysis.

[0033] 3. Sequencing analysis of exosomal miRNAs To investigate the miRNA composition of exosomes, BGISEQ-500 technology was used to analyze MSCs-exo (8×10⁻⁶). 10 Functional heatmaps were used to visualize hierarchical clustering analysis of miRNA expression. Then, based on a database (see http: / / www.geneontology.org / ), significantly abundant GO entries were analyzed using hypergeometric tests. The heatmap results for miRNA expression in MSCs-exo are shown below. Figure 1 As shown, exosomal miRNA-21-5p was highly expressed in the VCAM-1 group, TNF-α group, and IL6 group.

[0034] Electron microscopy images (scale bar, 200 nm) of MSCs-exo obtained from the control group, VCAM-1 group, TNF-α group, and IL6 group are shown below. Figure 2 As shown in (A), the size distribution of MSCs-exo in the control group, VCAM-1 group, TNF-α group, and IL6 group measured by NTA is as follows. Figure 2 As shown in (B), the exosome size distribution (mean diameter 154.1 ± 42.2 nm) and exosome concentration range (number per milliliter) are displayed.

[0035] Example 2 hUC-MSCs were cultured in a medium rich in miRNA-21-5p exosomes (miRNA-21-5p-MSC-Exo) to promote secretion (1×10⁻⁶ mcg). 5 live cells / cm 2 The culture supernatant was collected after culturing in an incubator at 37℃, 95% saturated humidity, and 5% CO2 atmosphere for 48 hours. The exosome medium rich in miRNA-21-5p for promoting secretion was prepared by adding human umbilical cord plasma, glucose, VCAM-1 (ADP5, R&D, USA), TNF-α (T6674, Sigma, Germany), and IL-6 (200-06-20, PeproTech, USA) to serum-free DMEM-F12 medium (HyClone, Logan, UT, USA). The final concentrations of human umbilical cord plasma, glucose, VCAM-1, TNF-α, and IL-6 were 8% (v / v), 2 mg / mL, 200 ng / mL, 10 ng / mL, and 100 ng / mL, respectively. Each 1 mL of medium was used for 1×10⁻⁶ culture. 5 Personal umbilical cord mesenchymal stem cells.

[0036] Exosomes were isolated from the collected culture medium supernatant using ultracentrifugation (Tooi et al., 2016). In short, the supernatant was centrifuged at 300g for 5 min, 2000g for 10 min, and 10000g for 20 min to remove cell debris and proteins. The supernatant was then filtered through a 0.22 μm filter (UFC810096-1, Merck Millipore, Germany) to remove particles larger than 0.22 μm. The filtrate was ultracentrifuged at 100000g, 4°C for 120 min (Optima XE-90 ultracentrifuge with a swing rotor, Type 70Ti; Beckman Coulter, CA, USA). The concentrated MSCs-exo were resuspended in PBS for further analysis.

[0037] In addition, cultured hUC-MSCs were collected and their related cell surface markers were analyzed by flow cytometry. The results are as follows: Figure 3 As shown in (A), CD90 is 99.67%, CD105 is 98.36%, CD73 is 99.70%, CD34 is only 0.1%, and CD45 is only 0.08%.

[0038] The flow cytometry results for MSCs-exo are as follows: Figure 3As shown in (B), CD63 is 63.6% and CD81 is 62.5%.

[0039] Confocal imaging of the interaction between human umbilical vein endothelial cells and stem cell exosomes, as shown in... Figure 3 As shown in (C), red represents tubulin, blue represents the cell nucleus, and green represents exosomes.

[0040] Example 3 Exosomes promote ischemic repair and angiogenesis in a rat model of diabetic foot.

[0041] Thirty-five male Sprague-Dawley rats (300-350 g; License No.: 1100111911009085) were purchased from Charles River Laboratory Animal Technology Co., Ltd (Beijing; License No.: SCXK 2016-0011) to establish a rat model of diabetes. All animal experiments were conducted in accordance with the requirements of the Animal Care Institution and Animal Use Committee of Jinan University (License No.: SYXK 2017-0174). The animal experimental procedures were approved by the Experimental Animal Ethics Committee of Jinan University. Diabetes in rats was induced by intraperitoneal injection of streptozotocin 40 mg / kg (containing 0.01 M sodium citrate, pH 4.3). Blood glucose levels were controlled between 11.1 and 31.5 mmol / L. Four weeks later, the femoral artery and its branches in the streptozotocin-injected diabetic rats were ligated (Long et al., 2016). In addition, a 10 mm full-thickness skin injury was created on the dorsum of the hind paw of diabetic rats to simulate a diabetic injury model (Shi et al., 2016; Li et al., 2018). Rats were randomly divided into three groups: a control group (PBS group), an MSC-Exo group, and a miRNA-21-5p-MSC-Exo group. Rats in the PBS group received an intramuscular injection of 1 mL of PBS at the DFU site in their hind limbs, while rats in the MSC-Exo group received an intramuscular injection of a mixture of 5 × 10⁻⁶ PBS and 5 × 10⁻⁶ MSC-Exo. 5 1 mL of PBS containing MSC-Exo was administered intramuscularly to the DFU site in the hind limb of rats in the miRNA-21-5p-MSC-Exo group, and the mixture contained 5 × 10⁻⁶ mg / mL of PBS. 5 1 mL of PBS was used to extract miRNA-21-5p-MSC-Exo. The MSC-Exo was obtained by culturing hUC-MSCs in serum-free and glucose-free DMEM-F12 medium at 37°C, 95% humidity, and 5% CO2 for 48 h, followed by collection of the culture supernatant and separation. 1 × 10⁻⁶ cells were cultured per 1 mL of medium. 5 Personal umbilical cord mesenchymal stem cells.

[0042] Photos were taken on postoperative day 0 and day 14 to analyze the healing status of the hind limbs in each group of rats, specifically by measuring the size of the paw ulcers. The results are as follows: Figure 4 As shown, both conventional Exo and Exo rich in miRNA-21-5p are effective against DFU, but Exo rich in miRNA-21-5p is particularly effective.

[0043] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing exosomes rich in miRNA-21-5p, comprising the following steps: Mesenchymal stem cells were cultured in a culture medium containing a promoter, and the supernatant was collected to isolate exosomes rich in miRNA-21-5p; the promoter included at least one of vascular cell adhesion molecule-1, tumor necrosis factor α and interleukin-6.

2. The preparation method according to claim 1, characterized in that, The promoters mentioned are vascular cell adhesion molecule-1, tumor necrosis factor-α, and interleukin-6.

3. The preparation method according to claim 1, characterized in that, The concentration of vascular cell adhesion molecule-1 in the culture medium containing the promoter is 20–400 ng / mL.

4. The preparation method according to claim 1, characterized in that, The concentration of tumor necrosis factor α in the culture medium containing the promoter is 5–20 ng / mL.

5. The preparation method according to claim 1, characterized in that, The concentration of interleukin-6 in the culture medium containing the promoter is 10–700 ng / mL.

6. The preparation method according to claim 1, characterized in that, The culture medium containing the promoter is a serum-free DMEM-F12 culture medium containing the promoter; The mesenchymal stem cells mentioned are human umbilical cord mesenchymal stem cells; The culture medium containing the promoter also contains human umbilical cord blood plasma and glucose. The density of the mesenchymal stem cells in the culture medium is [value missing].

7. The preparation method according to claim 6, characterized in that, The volume percentage concentration of human umbilical cord blood plasma in the culture medium containing the promoter is 2-20%. The concentration of glucose in the culture medium containing the promoter is 1-3.

8. The preparation method according to claim 1, characterized in that, The cultivation conditions are: a temperature of 37°C, a saturated humidity of 95%, and a gaseous environment of 5% CO2. The ratio of culture medium to mesenchymal stem cells is 1 × 10⁻⁶ cells per 1 mL of culture medium. 4 ~1×10 6 Each mesenchymal stem cell; furthermore, 1×102 cells are cultured per 1 mL of culture medium. 5 Mesenchymal stem cells.

9. Exosomes rich in miRNA-21-5p, prepared by the preparation method described in claims 1 to 8.

10. The use of the exosomes rich in miRNA-21-5p as described in claim 9 in the preparation of a product for treating diabetic foot ulcers.

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