Application of endometrial mesenchymal stem cell exosome in atherosclerosis

Abstract

The invention provides application of endometrial mesenchymal stem cell exosomes in atherosclerosis, and belongs to the technical field of biological medicine. The invention provides a bioactive preparation which is derived from specific tissues, has good safety and definite treatment directivity, can effectively intervene in the progress of atherosclerotic plaques and relieve pathological changes of blood vessels, and the treatment convenience and patient tolerance are remarkably improved through a non-invasive administration mode of the bioactive preparation. A brand new solution with excellent application potential is provided for clinical management of atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of endometrial mesenchymal stem cell exosomes in atherosclerosis. Background Technology

[0002] Atherosclerosis is a chronic cardiovascular disease that seriously endangers human health. Its pathological process involves multiple stages, including lipid metabolism disorders, vascular endothelial damage, chronic inflammatory responses, and fibrosis, ultimately leading to thickening, hardening, and narrowing of the arterial wall, causing fatal events such as myocardial infarction and stroke. Currently, first-line clinical treatment mainly relies on statins for intensive lipid-lowering, antiplatelet drugs to prevent thrombosis, and endovascular interventions or surgical bypass surgery for end-stage lesions. Although these methods can control the disease or restore blood flow to some extent, they cannot reverse existing plaques, and long-term medication is accompanied by side effects, while surgical treatment carries the risks of trauma and recurrence.

[0003] With the deepening of regenerative medicine, stem cell-based biotherapy has brought hope for repairing damaged blood vessels and regulating the local microenvironment. Mesenchymal stem cells (MSCs), as pluripotent stem cells, have been extensively studied due to their ease of acquisition, immunomodulatory properties, and multi-lineage differentiation potential. The traditional approach involves directly transplanting stem cells into the lesion area, hoping they will exert therapeutic effects through differentiation into vascular cells or secretion of active factors. However, direct cell transplantation faces numerous challenges, including cell survival rate in vivo, homing efficiency, uncontrollable directed differentiation, potential tumorigenic risks, and ethical concerns. Furthermore, the production, quality control, storage, and transportation of live cell preparations are costly and technically complex, significantly limiting their clinical translation and large-scale application. Therefore, exploring novel therapeutic strategies that can preserve the therapeutic effects of stem cells while avoiding the limitations of live cell applications has become a pressing area for breakthroughs in this field. Summary of the Invention

[0004] The purpose of this invention is to provide the application of endometrial mesenchymal stem cell exosomes in atherosclerosis, which solves the technical problems of complex preparation of biological agents, poor targeting, easy immune rejection and limited efficacy in existing atherosclerosis treatment methods.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a specification describing the application of endometrial mesenchymal stem cell exosomes. This invention provides the application of endometrial mesenchymal stem cell exosomes in the preparation of drugs for the treatment of atherosclerosis.

[0006] The present invention also provides the use of endometrial mesenchymal stem cell exosomes in any of the following: in the preparation of a medicament for inhibiting the development of atherosclerotic plaques; in the preparation of a medicament for reducing lipid deposition in atherosclerotic plaques; in the preparation of a medicament for inhibiting macrophage aggregation in atherosclerotic plaques.

[0007] Preferably, in the above applications, the endometrial mesenchymal stem cell exosomes are rich in let-7 family microRNAs; Alternatively, the diameter of the endometrial mesenchymal stem cell exosomes is 30~200 nm.

[0008] Preferably, in the above applications, the drug is administered via nebulized inhalation.

[0009] Preferably, in the above application, the endometrial mesenchymal stem cell exosomes are obtained by a preparation method comprising the following steps: obtaining an endometrial tissue sample and digesting it to isolate endometrial mesenchymal stem cells; culturing and expanding the endometrial mesenchymal stem cells; collecting the cell culture supernatant; and subjecting the supernatant to differential centrifugation and ultrafiltration to obtain the endometrial mesenchymal stem cell exosomes.

[0010] Preferably, in the above applications, the digestion process uses a complex enzyme comprising collagenase III and trypsin; the complex enzyme also comprises DNase; the concentration of collagenase III in the complex enzyme is 5-10 mg / ml, the concentration of trypsin is 0.01%-0.1%; the concentration of DNase in the complex enzyme is 15%-25%; the digestion process is carried out at 35-39°C, and the total digestion time is 50-130 minutes; the digestion process includes two digestion steps, after which the digestive fluid is collected and mixed with an equal volume of cell culture medium to terminate the enzyme reaction; after obtaining the endometrial tissue sample, the tissue is cut into pieces of 0.5-2 mm. 3 Small pieces.

[0011] Preferably, in the above application, the step of isolating endometrial mesenchymal stem cells includes filtering and centrifuging the digested cell suspension sequentially to obtain a cell pellet; the step of isolating endometrial mesenchymal stem cells further includes treating the cell pellet with erythrocyte lysis buffer and centrifuging again to obtain the endometrial mesenchymal stem cells; the filtration is performed using a cell sieve with a pore size of 3-10 μm; the step of treating the cell pellet with erythrocyte lysis buffer includes incubating the cell pellet and diluted erythrocyte lysis buffer at 2-8°C for 3-10 minutes, then adding phosphate buffer for dilution and centrifuging.

[0012] Preferably, in the above applications, when culturing and expanding endometrial mesenchymal stem cells, DMEM medium containing fetal bovine serum and antibiotics is used; when culturing and expanding endometrial mesenchymal stem cells, the cell passages used are from passage 1 to passage 5; during the culture and expansion process, the medium is changed every 2 to 5 days; the step of collecting the cell culture supernatant is as follows: when the cell confluence reaches 60% to 85%, the medium is replaced with serum-free medium and cultured for another 40 to 56 hours before collecting the supernatant.

[0013] Preferably, in the above application, the differential centrifugation and ultrafiltration treatment includes the following steps performed sequentially: performing a first centrifugation on the supernatant to remove cell debris; performing a second centrifugation on the supernatant after the first centrifugation to remove large particulate impurities; filtering the supernatant after the second centrifugation; and ultrafiltration concentration on the filtered liquid; wherein the centrifugal force of the first centrifugation is 200~400 ×g and the centrifugation time is 5~15 minutes; the centrifugal force of the second centrifugation is 1500~2500 ×g and the centrifugation time is 15~25 minutes; the filtration is performed using a filter membrane with a pore size of 0.20~0.25 μm; and the ultrafiltration concentration is performed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 80~120 kDa, centrifuged at a centrifugal force of 4000~6000 ×g for 20~40 minutes.

[0014] The present invention also provides the use of a pharmaceutical composition in the preparation of a medicament for treating atherosclerosis, said pharmaceutical composition comprising endometrial mesenchymal stem cell exosomes, a pharmaceutically acceptable carrier, and a device for nebulized inhalation administration.

[0015] The beneficial effects of this invention are: This invention provides a uniquely derived, precisely prepared, and non-invasively administered endometrial mesenchymal stem cell exosome formulation. This formulation exhibits significant comprehensive therapeutic advantages in an atherosclerosis model: firstly, it effectively inhibits the overall development of atherosclerotic plaques and significantly reduces abnormal lipid deposition within blood vessels, demonstrating superior efficacy compared to similar formulations from other sources; secondly, this formulation improves plaque stability, specifically by reducing the necrotic core within the plaque, helping to maintain the structure of the fibrous cap, and effectively inhibiting the abnormal aggregation of pro-inflammatory macrophages within the plaque, thereby "inhibiting progression." This invention plays an interventional role in two key aspects: "enhancing stability." Furthermore, the nebulized inhalation delivery method employed in this invention provides a convenient, safe, and highly patient-compliant systemic delivery route for this biological agent, overcoming the challenges of poor targeting and significant side effects associated with traditional formulations. Simultaneously, the complete preparation method provided by this invention, from tissue processing and cell culture to exosome extraction, has clear steps and well-defined parameters, exhibiting excellent reproducibility and standardization potential. This lays a solid technical foundation for the stable and large-scale production of this therapeutic product, ultimately providing a novel solution for the clinical treatment of atherosclerosis. Attached Figure Description

[0016] Figure 1 The images show the results of the extraction and identification of uterine mesenchymal stem cells, where: A is a cell morphology image of P2 generation; B is a flow cytometry image of cell surface markers. Figure 2 The images show the results of the identification of the microstructure and size of exosomes from uterine mesenchymal stem cells, where: A is a transmission electron microscope image; B is a nanoparticle size tracking analysis image. Figure 3 The following figures illustrate the overall effect of uterine mesenchymal stem cell exosomes on atherosclerosis: Figure A shows macroscopic images of the aorta stained with Oil Red O in different treatment groups, and Figure B shows the percentage of plaque area in different treatment groups. Figure 4 The image shows the pathological evaluation results of uterine mesenchymal stem cell exosome therapy for atherosclerosis. A shows a pathological section of the aortic root stained with HE, Oil Red O, and Masson staining; B shows a statistical chart of lipid deposition stained with Oil Red O; and C shows a statistical chart of collagen area stained with Masson staining. Figure 5 The image shows the results of the assessment of plaque stability by uterine mesenchymal stem cell exosomes, where: A is the immunofluorescence staining image of the plaque site; B is the statistical graph of the CD68 positive area within the plaque; and C is the statistical graph of the α-SMA positive area within the plaque. Figure 6Figure 1 shows the evaluation results of the effective components and application mechanism of uterine mesenchymal stem cell exosomes. Figure 2 shows the comparison of LYN gene expression in normal tissue and plaque. Figure 3 shows the enrichment analysis of LYN expression in cells within the plaque. Figure 4 shows the abundance of miRNA expression in exosomes. Figure 5 shows the regulatory network of miRNAs and target genes. Figure 6 shows the co-localization of LYN and macrophages with immunofluorescence in different treatment groups. Figure 7 shows the statistical graph of LYN expression in macrophages. Detailed Implementation

[0017] This invention provides the application of endometrial mesenchymal stem cell exosomes in the preparation of drugs for treating atherosclerosis. In this invention, the term "exosome" refers to a bilayered membrane vesicle, approximately 30-200 nanometers in diameter, actively secreted by cells, carrying various bioactive molecules such as proteins, lipids, and RNA (e.g., microRNA), and serving as a key medium for intercellular communication. The term "endometrial mesenchymal stem cells" refers to adult stem cells with self-renewal and multi-lineage differentiation potential found in endometrial tissue, characterized by relatively readily available sources, strong proliferative capacity, and low immunogenicity. This invention discovers that exosomes derived from mesenchymal stem cells of this specific tissue exhibit unique and superior therapeutic activity in regulating the pathological process of atherosclerosis.

[0018] This invention also provides the application of endometrial mesenchymal stem cell exosomes in any of the following: application in the preparation of drugs for inhibiting the development of atherosclerotic plaques; application in the preparation of drugs for reducing lipid deposition within atherosclerotic plaques; and application in the preparation of drugs for inhibiting macrophage aggregation within atherosclerotic plaques. Wherein, "atherosclerotic plaque development" includes the process of initial plaque formation, increase in size, decrease in stability, and even rupture; "lipid deposition" mainly refers to the abnormal accumulation of cholesterol and its esters under the arterial intima; and "macrophage aggregation" is a core link in the inflammatory response, where aggregated macrophages phagocytose lipids to form foam cells, which are one of the main constituent cells of plaques. Through the above applications, atherosclerosis can be intervened at multiple pathological stages.

[0019] Preferably, in the above applications, the endometrial mesenchymal stem cell exosomes are rich in let-7 family microRNAs. This invention has found that the exosomes are rich in let-7 family members (e.g., hsa-let-7a-5p), which may be one of the key molecular mechanisms by which they exert their therapeutic effect. Further, the total relative expression level of let-7 family miRNAs in the exosomes is preferably among the top 10% of all detected miRNAs, more preferably the top 5%.

[0020] Preferably, in the above applications, the diameter of the endometrial mesenchymal stem cell exosomes is 30-200 nm. More preferably, the diameter ranges from 50 to 150 nm; even more preferably, it ranges from 80 to 120 nm; and most preferably, the average diameter is about 100 nm. This size range is characteristic of typical exosomes and facilitates their transport, delivery, and uptake by target cells in the body.

[0021] Preferably, in the above applications, the drug is administered via nebulized inhalation. Nebulized inhalation is a non-invasive drug delivery method that converts the drug into aerosol particles, which act directly on the lungs through respiration and are rapidly absorbed into the bloodstream through the rich capillary network of the alveoli, thereby delivering the drug systemically. This method has high patient compliance and is suitable for the management of chronic diseases requiring long-term medication. The device used for nebulized inhalation can be any type conventional in the art, including but not limited to jet nebulizers, ultrasonic nebulizers, and vibrating sieve nebulizers.

[0022] Preferably, in the above application, the endometrial mesenchymal stem cell exosomes are obtained by a preparation method comprising the following steps: obtaining an endometrial tissue sample and digesting it to isolate endometrial mesenchymal stem cells; culturing and expanding the endometrial mesenchymal stem cells; collecting the cell culture supernatant; and subjecting the supernatant to differential centrifugation and ultrafiltration to obtain the endometrial mesenchymal stem cell exosomes.

[0023] Preferably, in the above applications, the digestion process uses a complex enzyme comprising collagenase III and trypsin. Collagenase III specifically degrades type III collagen, effectively breaking down the extracellular matrix in endometrial tissue; trypsin hydrolyzes peptide chains, dispersing cells. More preferably, the complex enzyme also contains DNase at a concentration of 15%–25%, for example, about 20%, to degrade DNA released during digestion and reduce cell clumping. The concentration of collagenase III in the complex enzyme is 5–10 mg / ml, for example, about 8 mg / ml; the concentration of trypsin is 0.01%–0.1%, for example, about 0.05%. The digestion process is carried out at 35–39°C (preferably 37°C) for a total digestion time of 50–130 minutes (preferably 120 minutes). Further, the digestion process includes two digestion steps, after which the digestate is collected and mixed with an equal volume of cell culture medium (such as serum-containing DMEM complete medium) to terminate the enzymatic reaction, which helps to improve cell yield and maintain cell viability. After obtaining endometrial tissue samples, routine cleaning and trimming of the tissue into 0.5–2 mm pieces are required. 3 Small pieces, preferably about 1 mm 3 This increases the contact area and improves digestion efficiency.

[0024] Preferably, in the above application, the step of isolating endometrial mesenchymal stem cells includes sequentially filtering and centrifuging the digested cell suspension to obtain a cell pellet. The filtration is performed using a cell sieve with a pore size of 3-10 μm (preferably 5 μm) to remove undigested tissue fragments and cell clusters. The centrifugation force can be 800-1200 rpm (approximately 100-200 × g) for 5-10 minutes. The step of isolating endometrial mesenchymal stem cells further includes treating the cell pellet with red blood cell lysis buffer and centrifuging again to obtain relatively pure endometrial mesenchymal stem cells. The steps for treating the cell pellet with erythrocyte lysis buffer include incubating the cell pellet with diluted erythrocyte lysis buffer (such as commercial lysis buffer diluted with deionized water or buffer at a ratio of 1:9 to 1:11) at 2 to 8°C (preferably 4°C) for 3 to 10 minutes (preferably 5 minutes), then adding a large amount (such as more than 10 times the volume) of phosphate-buffered saline (1×PBS) to terminate the lysis, and centrifuging (such as at 1200 rpm for 5 minutes) to collect the cell pellet.

[0025] Preferably, in the above applications, when culturing and expanding endometrial mesenchymal stem cells, DMEM medium containing fetal bovine serum and antibiotics is used. DMEM (Dulbecco's Modified Eagle Medium) is one of the most commonly used basal media in the art; the amount of fetal bovine serum (FBS) added is typically 5%-15%, preferably 10%, to provide the nutrients and growth factors required for cell growth; the "antibiotics" typically refer to a mixture of penicillin and streptomycin, used to prevent bacterial contamination, and the commonly used concentration is 1%. When culturing and expanding endometrial mesenchymal stem cells, to ensure the stability of cell biological characteristics and the uniformity of exosome quality, the cell passages used are preferably from generation 1 to 5, more preferably from generation 2 to 4. During the culture and expansion process, the medium is replaced with fresh medium every 2-5 days (preferably every 3-4 days) according to the cell growth rate. The procedure for collecting cell culture supernatant is as follows: when cell confluence reaches 60%–85% (preferably 70%–80%), discard the original culture medium, gently wash the cells with sterile PBS, replace with serum-free culture medium (such as DMEM without exosome serum), and continue culturing for 40–56 hours (preferably 48 hours) before collecting the supernatant. This step aims to induce cells to secrete exosomes while avoiding interference from exogenous exosomes in the serum.

[0026] Preferably, in the above application, the differential centrifugation and ultrafiltration process includes the following steps performed sequentially: a first centrifugation of the supernatant to remove cell debris; a second centrifugation of the supernatant after the first centrifugation to remove large particulate impurities; filtration of the supernatant after the second centrifugation; and ultrafiltration concentration of the filtered liquid. The first centrifugation uses a centrifugal force of 200-400 ×g (preferably 300 ×g) and a centrifugation time of 5-15 minutes (preferably 10 minutes), performed at 4°C. The second centrifugation uses a centrifugal force of 1500-2500 ×g (preferably 2000 ×g) and a centrifugation time of 15-25 minutes (preferably 20 minutes), performed at 4°C. The filtration is performed using a filter membrane (such as PVDF or PES material) with a pore size of 0.20-0.25 μm (preferably 0.22 μm) to remove any remaining small particles and some bacteria. The ultrafiltration concentration is carried out using an ultrafiltration centrifuge tube with a molecular weight cutoff of 80-120 kDa (preferably 100 kDa), centrifuged at a centrifugal force of 4000-6000 ×g (preferably 5000 ×g) for 20-40 minutes (preferably 30 minutes) at 4°C, thereby concentrating the exosomes and retaining them above the filter membrane.

[0027] This invention also provides the use of a pharmaceutical composition in the preparation of a medicament for treating atherosclerosis, the pharmaceutical composition comprising endometrial mesenchymal stem cell exosomes, a pharmaceutically acceptable carrier, and a device for nebulized inhalation administration. The "pharmaceutically acceptable carrier" refers to an excipient that is non-toxic to the body and does not interact harmfully with the active ingredient, used to help in the formulation, stabilization, delivery, or improvement of compliance of the active ingredient. For nebulized inhalation formulations, the carrier may include, but is not limited to: isotonic agents (such as sodium chloride, at a concentration of about 0.9%), buffers (such as phosphate buffer, used to maintain pH at 6.0-7.5, preferably 6.5-7.0), stabilizers (such as human serum albumin, trehalose), etc. This pharmaceutical composition can be prepared as a ready-to-use nebulized inhalation solution or a lyophilized powder that needs to be reconstituted with a specific solvent before use.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example This embodiment provides endometrial mesenchymal stem cell exosomes rich in let7 family miRNAs (such as hsa-let7a-5p); the exosome diameter is 50-150 nm.

[0030] Preparation method: 1) Obtain clinical samples; 2) Prepare a compound enzyme: 5 ml each of 20% DNase, 8 mg / ml collageIII enzyme, and 0.05% trypsin; 3) Cut the specimen into 1×1×1m pieces. 3 Size, wash twice with PBS, add compound enzyme and incubate in a water bath at 37°C for 60 min twice. After each digestion, add an equal volume of DMEM complete medium to inactivate the enzyme. 4) Collect the digestion fluid, filter it through a 5µm cell sieve, and centrifuge it at 1000rpm for 5min to obtain approximately 500µl of cell suspension; 5) Add 5 ml of 10-fold diluted red blood cell lysis buffer to the suspension, gently invert and mix, and let stand at 4°C for 5 min; after standing, add 45 ml of 1×PBS to dilute, and centrifuge at 1200 rpm for 5 min; obtain the cell pellet, culture, and change the medium every 3-5 days. 6) Endometrial mesenchymal stem cells were passaged using DMEM + 10% FBS + 1% P / S, using only cells before passage P5; 7) When the cells reach 70%-80% confluence, replace the medium with serum-free medium and culture for 48 hours, then collect the supernatant. 8) Dispense the supernatant into 50ml centrifuge tubes and place them in a pre-cooled centrifuge at 4℃. After balancing, centrifuge at 300×g for 10min. 9) Transfer the supernatant from centrifugation into a new 50ml centrifuge tube and place it in the centrifuge. Balance the mixture and centrifuge at 2000×g for 20min. 10) Collect the supernatant after centrifugation and filter it using a 0.22 mm filter membrane; 11) Place the filtered supernatant into the Amicon@ Ultra 100K device filter and centrifuge at 5000×g for 30min; 12) Collect the upper layer of liquid from the filter, which is the endometrial mesenchymal stem cell exosome. Collect the lower layer of liquid and repeat step 11) to obtain more output. The obtained endometrial mesenchymal stem cell exosomes are used as a biological agent.

[0031] The biological agent is administered via inhalation through the mouth and nose using a jet nebulizer, with a dosage of 1×10⁻⁶. 11 The dosage was 100 particles / kg, and the administration period was 7 days.

[0032] Results of uterine mesenchymal stem cell extraction and identification: Figure 1 As shown.

[0033] Uterine mesenchymal stem cells were cultured at passages P2-P5. During passage, the cells exhibited normal morphology and moderate density, making them ideal cells for exosome extraction. Figure 1A) Identification of CD90 in uterine mesenchymal stem cells by flow cytometry. + CD105 + CD44 + CD73 + The positive rate was above 95% ( Figure 1 B), and differentiated from hematopoietic stem cell marker Negative (CD45) + CD34 + The positive rate of the cells was identified, and the results showed that the positive rate was 0.13%. Overall, the cells met the requirements for mesenchymal stem cells.

[0034] The results of the identification of the microstructure and size of uterine mesenchymal stem cell exosomes are as follows: Figure 2 As shown.

[0035] The extracted exosomes were imaged using transmission electron microscopy (TEM), revealing their intact morphology. Figure 2 A) These are normal vesicles, without any breakage or incompleteness. Nanoparticle size tracking (NAT) analysis shows that the exosome diameter is mainly 99 nm, with an average diameter of 133.4 + / - 1.8 nm. Figure 2 B) meets the requirement of exosome diameter of 30-150nm.

[0036] Evaluation results of the overall effect of uterine mesenchymal stem cell exosomes on atherosclerosis: Figure 3 As shown.

[0037] APO-E mice were fed a high-fat diet for 12 weeks and then treated with nebulized inhalation of uterine mesenchymal stem cell exosomes and umbilical cord mesenchymal stem cell exosomes, respectively. 1X phosphate-buffered saline was used as a control group. Seven days after treatment, the mice were dissected, and the exosomes were removed from the aortic root to the bifurcation of the abdominal aorta. Figure 3 A). The artery was stained with Oil Red O and longitudinally dissected from the midpoint, and its gross appearance was analyzed.

[0038] The results showed that the inhalation treatment groups of uterine-derived and umbilical cord-derived exosomes had significantly fewer plaque formations in the arteries than the control group, and the treatment effect of uterine-derived exosomes was superior to that of umbilical cord-derived exosomes, indicating that uterine-derived exosomes are more effective in alleviating arterial lipid deposition. Figure 3 B).

[0039] Pathological evaluation results of uterine mesenchymal stem cell exosome therapy for atherosclerosis: Figure 4 As shown.

[0040] Pathological staining results of the aortic root section of the heart ( Figure 4A) HE staining showed that exosome treatment reduced the formation of necrotic cores; after Oil Red O staining, the results of the two exosome treatments differed from the gross effects on the aorta. Although uterine-derived exosome treatment was more statistically significant than umbilical cord-derived exosome treatment, there was no statistically significant difference between the two groups. Figure 4 B); Masson staining results showed that both exosome treatments maintained the stability of the plaque fibrous cap structure, and the statistical results were consistent with the trend of Oil Red O statistical results. Figure 4 C).

[0041] The results of the assessment of plaque stability by uterine mesenchymal stem cell exosomes are as follows: Figure 5 As shown.

[0042] Atherosclerosis is mainly caused by macrophages engulfing lipids and forming foam cells, which in turn lead to plaque formation. CD68 and α-SMA, as typical markers of macrophages and smooth muscle cells, respectively, can reflect the degree of progression of atherosclerosis.

[0043] Immunofluorescence ( Figure 5 A) shows that, compared to umbilical cord-derived mesenchymal stem cell exosomes, uterine-derived exosome therapy can achieve significantly better inhibition of macrophage aggregation at atherosclerotic plaque sites. Figure 5 B).

[0044] Evaluation results of effective components and application mechanisms of uterine mesenchymal stem cell exosomes are as follows: Figure 6 As shown.

[0045] Transcriptomic data from normal tissues and atherosclerotic plaques showed elevated expression of the key marker LYN in the plaques. Figure 6 A), its expression location is mainly enriched in macrophages ( Figure 6 B).

[0046] Exosomal transcriptome data showed that the expression abundance of miRNAs was among the highest for the let7 family. Figure 6 C), indicating that it plays a major role in atherosclerosis. The let7 family and LYN have a significant regulatory relationship (C). Figure 6 D); Exosomes can inhibit LYN expression ( Figure 6 E and 6F effectively inhibit macrophage recruitment in plaques and suppress plaque formation.

[0047] As can be seen from the above embodiments, the present invention provides a novel application of a bioactive preparation derived from endometrial mesenchymal stem cells in the treatment of atherosclerosis. Experiments show that this preparation can effectively intervene in key pathological processes in disease models, exhibiting excellent overall therapeutic activity and safety characteristics, thus verifying its feasibility as a novel therapeutic tool for atherosclerosis.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of endometrial mesenchymal stem cell exosomes in the preparation of drugs for the treatment of atherosclerosis.

2. Application of endometrial mesenchymal stem cell exosomes in any of the following: Application in the preparation of drugs for inhibiting the development of atherosclerotic plaques; Application in the preparation of drugs for reducing lipid deposition in atherosclerotic plaques; Application in the preparation of drugs for inhibiting the aggregation of macrophages in atherosclerotic plaques.

3. The application according to claim 1 or 2, characterized in that, The endometrial mesenchymal stem cell exosomes are rich in let-7 family microRNAs; Alternatively, the diameter of the endometrial mesenchymal stem cell exosomes is 30~200 nm.

4. The application according to claim 1 or 2, characterized in that, The drug is administered via nebulized inhalation.

5. The application according to claim 1 or 2, characterized in that, The endometrial mesenchymal stem cell exosomes were obtained by a preparation method comprising the following steps: Endometrial tissue samples were obtained and digested to isolate endometrial mesenchymal stem cells; The endometrial mesenchymal stem cells were cultured and expanded. Collect cell culture supernatant; The supernatant was subjected to differential centrifugation and ultrafiltration to obtain the endometrial mesenchymal stem cell exosomes.

6. The application according to claim 5, characterized in that, The digestion process uses a complex enzyme containing collagenase III and trypsin; The complex enzyme also includes a DNA enzyme; The concentration of collagenase III in the complex enzyme is 5-10 mg / ml, and the concentration of trypsin is 0.01%-0.1%. The concentration of DNase in the complex enzyme is 15%~25%; The digestion process was carried out at 35~39℃, and the total digestion time was 50~130 minutes. The digestion process includes two digestion steps, after which the digestate is collected and mixed with an equal volume of cell culture medium to terminate the enzyme reaction. After obtaining endometrial tissue samples, the tissue is cut into pieces of 0.5–2 mm. 3 Small pieces.

7. The application according to claim 5, characterized in that, The steps for isolating endometrial mesenchymal stem cells include filtering and centrifuging the digested cell suspension sequentially to obtain cell pellet. The step of isolating endometrial mesenchymal stem cells further includes treating the cell pellet with erythrocyte lysis buffer and centrifuging again to obtain the endometrial mesenchymal stem cells. The filtration is performed using a cell sieve with a pore size of 3~10 μm; The steps for treating the cell pellet with erythrocyte lysis buffer include incubating the cell pellet and diluted erythrocyte lysis buffer at 2-8°C for 3-10 minutes, followed by dilution with phosphate buffer and centrifugation.

8. The application according to claim 5, characterized in that, When culturing and expanding endometrial mesenchymal stem cells, DMEM medium containing fetal bovine serum and antibiotics was used; When culturing and expanding endometrial mesenchymal stem cells, the cell passages used are from the 1st to the 5th generation. During the culture and amplification process, the culture medium should be changed every 2 to 5 days; The steps for collecting cell culture supernatant are as follows: when the cell confluence reaches 60%~85%, replace with serum-free medium and continue culturing for 40~56 hours before collecting the supernatant.

9. The application according to claim 5, characterized in that, The differential centrifugation and ultrafiltration process includes the following steps performed sequentially: the supernatant is subjected to a first centrifugation to remove cell debris; The supernatant after the first centrifugation is subjected to a second centrifugation to remove large particulate impurities. Filter the supernatant after the second centrifugation; And the filtered liquid is concentrated by ultrafiltration; The centrifugal force of the first centrifuge is 200~400 ×g, and the centrifugation time is 5~15 minutes; The centrifugal force for the second centrifugation is 1500~2500 ×g, and the centrifugation time is 15~25 minutes; The filtration is performed using a filter membrane with a pore size of 0.20~0.25 μm; The ultrafiltration concentration is achieved by centrifuging with an ultrafiltration centrifuge tube having a molecular weight cutoff of 80-120 kDa for 20-40 minutes at a centrifugal force of 4000-6000 ×g.

10. The use of a pharmaceutical composition in the preparation of a medicament for treating atherosclerosis, characterized in that, The pharmaceutical composition comprises endometrial mesenchymal stem cell exosomes, a pharmaceutically acceptable carrier, and a device for nebulized inhalation administration.

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