Application of denucleated mesenchymal stem cells in the preparation of drugs for the prevention or treatment of calcified aortic valve disease

Abstract

This invention discloses the application of denucleated mesenchymal stem cells in the preparation of drugs for the prevention or treatment of calcified aortic valve disease (CAVD). Addressing the potential drawbacks of mesenchymal stem cell therapy for CAVD, such as increased osteogenic differentiation leading to calcification, pulmonary retention, and poor targeting, this invention reveals that denucleated mesenchymal stem cells, lacking a nucleus, lose their osteogenic differentiation capacity while retaining functional organelles, metabolic activity, targeted migration ability, protein translation, and paracrine function. They can exert anti-CAVD effects by inhibiting local valve inflammation, blocking osteogenic differentiation of valve interstitial cells, and reducing calcium salt deposition. This invention also provides a method for preparing denucleated mesenchymal stem cells and corresponding pharmaceutical compositions. After intravenous infusion, these denucleated mesenchymal stem cells can target and migrate to calcified aortic valve lesions without the risk of osteogenic differentiation, exhibiting high safety and providing a novel non-surgical treatment for CAVD with excellent clinical translational prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of denucleated mesenchymal stem cells in the preparation of drugs for the prevention or treatment of calcified aortic valve disease. Background Technology

[0002] Calcific aortic valve disease (CAVD) is a valvular disease characterized by abnormal collagen and calcium salt deposition in the aortic valve, leading to thickening, hardening, and narrowing of the aortic valve. This results in outflow obstruction and regurgitation, ultimately causing adverse outcomes such as heart failure and death. With an aging population, the prevalence of CAVD is rising rapidly, becoming the third leading cardiovascular disease after coronary heart disease and hypertension. Currently, there are no effective drug interventions for CAVD; therefore, finding early drug interventions for CAVD is of significant clinical importance.

[0003] The development of CAVD is a complex, multifactorial, and multi-step pathological process involving mechanical stress injury, lipid deposition, endothelial dysfunction, and chronic inflammation activation. Its core pathological mechanism involves osteoblast-like differentiation of aortic valvular interstitial cells (VICs) under pathological stimulation, leading to high expression of osteogenic markers such as Runx2 and ALP, and consequently, abnormal calcium salt deposition in the valve tissue. Currently, there are no effective early drug interventions for CAVD. Statins and angiotensin-converting enzyme inhibitors were once considered promising, but numerous clinical trials have demonstrated their inability to effectively slow disease progression. In advanced stages, treatment is limited to surgical aortic valve replacement or transcatheter aortic valve replacement. However, surgical treatment has many drawbacks, including significant trauma, high risk, numerous postoperative complications, the need for secondary surgery for bioprosthetic valves, and lifelong anticoagulation for mechanical valves, resulting in a significant decline in patients' postoperative quality of life. Therefore, finding safe and effective early intervention drugs for CAVD is a major clinical problem urgently needing to be solved in this field.

[0004] Existing technologies also disclose the application of small molecule compounds such as BRD4 inhibitors, ginsenoside F4, and HIPK2 inhibitors in the treatment of CAVD, but they all suffer from problems such as single target, short in vivo half-life, and unknown side effects of long-term administration. Mesenchymal stem cells (MSCs), on the other hand, are adult stem cells with multi-lineage differentiation potential. Due to their low immunogenicity, inflammatory chemotaxis, strong tissue repair capacity, and immunomodulatory activity, they have shown great potential in the treatment of various diseases such as myocardial infarction, arthritis, and spinal cord injury, and multiple clinical studies have confirmed their safety and efficacy. However, MSCs still face significant technical bottlenecks in the treatment of CAVD. On the one hand, osteoblastic differentiation is the core pathogenesis of CAVD. When surviving MSCs are infused into the body, they are highly susceptible to osteoblastic differentiation under the stimulation of the local pathological microenvironment of the valve, which can actually worsen valve calcification and completely deviate from the therapeutic goal. On the other hand, surviving MSCs are relatively large, and more than 90% of them remain in the pulmonary microcirculation after intravenous infusion, making it difficult for them to target and migrate to the aortic valve lesion to exert a therapeutic effect. Furthermore, surviving MSCs also suffer from tumorigenicity, senescence and inactivation, and stringent storage and transportation requirements, severely limiting their application in CAVD treatment. In other words, there is currently no effective solution to address the core shortcomings of MSCs in CAVD, namely, their osteoblastic differentiation exacerbating calcification and poor targeting. Summary of the Invention

[0005] This invention addresses the problems existing in the application of MSCs in the treatment of CAVD. Research has found that denucleated mesenchymal stem cells can lose their proliferative and differentiation capabilities, but can retain their metabolic and protein synthesis functions for a certain period of time to continue producing therapeutic substances. Therefore, denucleated mesenchymal stem cells have the advantage of both retaining the immunomodulatory function of MSCs to inhibit calcification and not causing the aggravation of CAVD calcification due to osteogenic differentiation of MSCs. Based on this, the application of denucleated mesenchymal stem cells in the preparation of drugs for the prevention or treatment of calcific aortic valve disease is proposed.

[0006] To achieve the above objectives, this technical solution provides the application of denucleated mesenchymal stem cells in the preparation of drugs for the prevention or treatment of calcified aortic valve disease.

[0007] Preferably, the denucleated mesenchymal stem cells are prepared from human mesenchymal stem cells through denucleation treatment.

[0008] Furthermore, the human mesenchymal stem cells are selected from any one of human bone marrow mesenchymal stem cells, human umbilical cord mesenchymal stem cells, and human adipose mesenchymal stem cells.

[0009] Preferably, human mesenchymal stem cells are human bone marrow mesenchymal stem cells.

[0010] It should be noted that the denucleated mesenchymal stem cells obtained after denucleation treatment lose their nucleus, proliferative capacity, and osteogenic differentiation capacity, but retain complete cell membrane structure, cytoplasm, and functional organelles, and possess metabolic activity, protein synthesis capacity, directional migration capacity, paracrine function, and immune regulatory activity.

[0011] Preferably, denucleated mesenchymal stem cells are prepared by combining cytochalasin B with Ficoll density gradient ultracentrifugation.

[0012] More preferably, the final concentration of cytochalasin B is 10 μg / ml, that is, cytochalasin B is added to each layer of Ficoll solution, and the actual concentration of cytochalasin B in the entire gradient solution used for denucleation is stable at 10 μg / mL.

[0013] More preferably, the centrifugation parameters for ultracentrifugation are 26000g, 35℃ for 1h.

[0014] More preferably, the Ficoll density gradient from bottom to top is 25%, 17%, 16%, 15%, and 12.5%, where the Ficoll density gradient refers to the mass percentage concentration (w / w, mass / mass) of the Ficoll PM400 solution, that is, it means that every 100 grams of Ficoll solution contains 25 g / 17 g / 16 g / 15 g / 12.5 g of Ficoll PM400 dry powder, with the remainder being solvent (PBS or serum-free culture medium).

[0015] Preferably, the drug is used to achieve at least one of the following functions: inhibiting osteoblast-like differentiation of aortic valve interstitial cells, reducing aortic valve calcium salt deposition, alleviating aortic valve leaflet thickening, reducing peak transvalvular flow velocity of aortic valve, and reducing mean transvalvular pressure gradient of aortic valve.

[0016] Preferably, denucleated mesenchymal stem cells exert their pharmacological effects by inhibiting local inflammatory responses in the aortic valve, promoting the repair of aortic valve endothelial cells, and regulating osteogenic differentiation of aortic valve interstitial cells, thereby achieving the effect of preventing or treating calcified aortic valve disease.

[0017] Preferably, the effective dose of denucleated mesenchymal stem cells in the drug is 2.0 × 10⁻⁶. 6 Cells per kilogram of body weight, administered once every 2 weeks. That is, 2 million denucleated mesenchymal stem cells are used per kilogram of body weight, based on the patient's weight.

[0018] Preferably, the drug further comprises pharmaceutically acceptable excipients selected from one or more of diluents, buffers, suspensions, isotonic adjusters, cryoprotectants, and pH adjusters.

[0019] Preferably, the dosage form of the drug is an injection.

[0020] More preferably, the drug is in the form of an intravenous injection. Accordingly, this protocol provides a solution for delaying the development and progression of calcific aortic valve disease by injecting denucleated mesenchymal stem cells.

[0021] Preferably, the calcific aortic valve disease is selected from at least one of aortic valve calcification, calcific aortic stenosis, aortic valve calcification induced by a high-fat / high-cholesterol diet, and rheumatic valvular disease.

[0022] In addition, this protocol also provides a drug for the prevention or treatment of calcific aortic valve disease, including denucleated mesenchymal stem cells and pharmaceutically acceptable excipients.

[0023] Compared with existing technologies, this technical solution has the following characteristics and beneficial effects: This study is the first to discover that denucleated mesenchymal stem cells (MSCs) can be effectively used for the prevention and treatment of chronic aortic valve disease (CAVD). Research has shown that when preventing or treating CAVD, denucleated MSCs not only retain functional organelles and metabolic and protein synthesis capabilities, but also exert multiple pharmacological effects through paracrine mechanisms, including immunomodulation, endothelial repair, anti-inflammatory, and anti-apoptotic effects. Furthermore, because denucleated MSCs completely lose their nuclei, they lose their proliferative capacity and osteogenic differentiation potential, thus completely avoiding the risk of osteogenic differentiation and exacerbation of calcification at the valve site after infusion of surviving MSCs. Moreover, denucleated MSCs retain their complete cell membrane structure and chemotactic migration ability, effectively avoiding pulmonary retention after intravenous infusion and targeting and accumulating at the calcified aortic valve lesion site, demonstrating excellent clinical translational prospects. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of denucleated mesenchymal stem cells obtained by centrifugation in Example 1, where the white precipitate in the middle is the purified denucleated mesenchymal stem cells.

[0025] Figure 2 This is a fluorescence microscope image showing the observation results of denucleated mesenchymal stem cells, in which... Figure 2 In this context, Cytoplast represents denucleated mesenchymal stem cells, MSCs represent mesenchymal stem cells, Calcein AM represents calcein AM, Phalloidin represents phalloidin, Mitochondria represents mitochondria, Lysosome represents lysosome, and Hoechst represents Hearst dye.

[0026] Figure 3This is a diagram showing the results of the Transwell experiment characterizing migration ability, where Cytoplast represents denucleated mesenchymal stem cells and MSCs represent mesenchymal stem cells.

[0027] Figure 4 The figure shows the results of endothelial cell adhesion experiments under static and dynamic adhesion. Control represents the control group, Cytoplast represents the denucleated mesenchymal stem cell group, MSCs represents the mesenchymal stem cell group, and TNF-α represents the inflammatory factor TNF-α stimulation group.

[0028] Figure 5 In the diagram, A represents the endothelial cell migration results, and B represents the results of the experiment inhibiting the expression of macrophage inflammatory factors (IL6, IL-1β, TNF-α). Control represents the control group, Cytoplast represents denucleated mesenchymal stem cells, MSC represents mesenchymal stem cells, LPS represents lipopolysaccharide stimulation, LPS+MSC represents the addition of mesenchymal stem cells to LPS stimulation, and LPS+Cytoplast represents the addition of denucleated mesenchymal stem cells to LPS stimulation.

[0029] Figure 6 This is a diagram showing the results of denucleated mesenchymal stem cells inhibiting osteogenic differentiation of valve mesenchymal cells. The upper left image is a schematic diagram of cell culture, the upper right image is a diagram of Alizarin Red staining results, the lower left image is a diagram of Western blot results of cell proteins, and the lower right image is a statistical diagram of Western blot results of cell proteins. Control is the control group, OM indicates only osteogenic medium stimulation, OM+MSC indicates the addition of mesenchymal stem cells to osteogenic medium stimulation, and OM+Cytoplast indicates the addition of denucleated mesenchymal stem cells to osteogenic medium stimulation.

[0030] Figure 7 This is a diagram showing the results of an in vivo experiment demonstrating the active migration of denucleated mesenchymal stem cells to calcified valves in mice. Figure 7 The A markers are located in the lungs, heart, and aorta of mice. Figure 7 In the figure, B represents the labeling in the aortic intima and aortic valve of mice. ND represents the normal diet group; HFD represents the high-fat diet-induced valve calcification model group; ND+Cytoplast represents the normal diet group with added denucleated mesenchymal stem cells; and HFD+Cytoplast represents the high-fat diet group with added denucleated mesenchymal stem cells.

[0031] Figure 8 This is a diagram showing the results of an in vivo therapeutic experiment using denucleated mesenchymal stem cells. Figure 8Figure A represents an image of aortic valve HE staining (for observing valve thickness) and Von Kossa staining (for observing calcium salt deposition). Figure 8 In the diagram, B represents the peak transvalvular flow velocity map of the aortic valve detected by ultrasound in small animals. ND represents the normal diet group; HFD represents the high-fat diet model group; and HFD+Cytoplast represents the high-fat diet model group with added denucleated mesenchymal stem cell intervention. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, those skilled in the art can understand the advantages and effects of the solution from the content disclosed in this specification. Technical details that are modified or changed without departing from the concept of the solution are also within the protection scope of the solution.

[0033] Unless otherwise specified, the raw materials used in this invention are all commercially available products, and the equipment and testing methods used are all conventional equipment and methods in the field.

[0034] Example 1: Preparation and Characterization of Denucleated Mesenchymal Stem Cells 1.1 The main reagents used are as follows: commercially available human bone marrow mesenchymal stem cells, PBS buffer, Serum-free medium (i.e., high-glucose DMEM medium), fetal bovine serum, penicillin-streptomycin solution, 0.25% trypsin-EDTA solution, Ficoll PM 400, cytochalasin B (10 μg / ml).

[0035] It should be noted that the normal culture medium mentioned below is equivalent to the serum-containing culture medium, which is a culture medium in which 10% fetal bovine serum is added to high glucose DMEM medium.

[0036] 1.2 Preparation of denucleated mesenchymal stem cells: ① Gradient Ficoll solution preparation steps: Ficoll PM400 powder was dissolved in PBS buffer to prepare a 50% w / w Ficoll stock solution. The Ficoll stock solution was magnetically stirred and dissolved overnight. The Ficoll stock solution was diluted with serum-free cell culture medium to prepare gradient working solutions with mass fractions of 25% (w / w), 17% (w / w), 16% (w / w), 15% (w / w), and 12.5% ​​(w / w). Cytochalasin B was added to each layer of gradient working solution to a final concentration of 10 μg / mL. The gradient working solutions were added sequentially from bottom to top along the wall of the ultracentrifuge tube (volumes of 2 mL, 2 mL, 0.5 mL, 0.5 mL, and 2 mL, respectively). The tube was sealed and incubated overnight to form a stable and continuous density gradient.

[0037] ② Cell enucleation steps: Discard the cell culture supernatant, wash the cells 2-3 times with PBS buffer, add an appropriate amount of trypsin, observe under a microscope until the cells shrink and become round and the cytoplasm is clear, immediately add serum-containing culture medium to stop digestion, and centrifuge at 1000 rpm for 5 min, discard the supernatant and collect the cell pellet.

[0038] The cell pellet was resuspended in 3 ml of 12.5% ​​(w / w) Ficoll solution (containing 10 μg / mL cytochalasin B). The suspension was slowly added to the top of an ultracentrifuge tube, and 1 ml of serum-free culture medium was added on top to seal the tube. The tube was then transferred to an incubator and allowed to stand for 1 h. After standing, the tube was ultracentrifuged at 26000×g and 35℃ for 1 h, and the middle white precipitate was collected (this white precipitate is the nucleated mesenchymal stem cells). The middle white precipitate was resuspended in PBS and centrifuged at 300×g for 10 min. The supernatant was discarded, and the washing was repeated twice to obtain purified nucleated mesenchymal stem cells.

[0039] like Figure 1 As shown, Figure 1 The cell stratification after Ficoll density gradient ultracentrifugation is shown. A clear density gradient layer can be seen after ultracentrifugation, and the white precipitate layer in the middle is purified nucleated mesenchymal stem cells, indicating that this method can stably isolate nucleated mesenchymal stem cells.

[0040] 1.3 Characterization of denucleated mesenchymal stem cells: 1.3.1 Activity characterization: Cell activity was verified by calcein staining. Cytoplast group design: Purified nucleated mesenchymal stem cells were seeded into sterile well plates, added to serum-containing medium and cultured normally for 24 h. On the second day, the medium was discarded and 1 μM calcein staining working solution was added. After incubation at 37°C in the dark for 30 min, the staining solution was discarded. After washing 3 times with PBS, the cells were returned to serum-containing medium and cultured for another 30 min. Nucleated mesenchymal stem cells were observed using a fluorescence microscope. Design of MSCs group: Commercial human bone marrow mesenchymal stem cells were seeded into sterile well plates, added with serum-containing culture medium and cultured normally for 24 h. On the second day, the culture medium was aspirated and 1 μM calcein staining working solution was added. After incubation at 37°C in the dark for 30 min, the staining solution was discarded. After washing 3 times with PBS, the cells were replaced with serum-containing cell culture medium and cultured for another 30 min. Mesenchymal stem cells were observed using a fluorescence microscope.

[0041] The fluorescence results of the MCs group and the Cytoplast group are shown in the figure below. Figure 2 As shown, denucleated mesenchymal stem cells retain normal activity.

[0042] 1.3.2 Integrity Characterization: Cell integrity was verified by staining the cellular skeleton with phalloidin. Cytoplast group design: Purified nucleated mesenchymal stem cells were seeded into sterile well plates, cultured in serum-containing medium for 24 hours. On the second day, the medium was aspirated, and the cells were washed once with PBS buffer. Then, 4% paraformaldehyde was added for fixation for 15 minutes. After discarding the fixative, the cells were washed three times with PBS buffer. Then, phalloidin staining working solution was added and incubated in the dark for 15 minutes. After discarding the staining solution, the cells were washed three times with PBS buffer. Nucleated mesenchymal stem cells were observed using a fluorescence microscope.

[0043] Design of MSCs group: Commercial human bone marrow mesenchymal stem cells were seeded into sterile well plates, added with serum-containing culture medium and cultured normally for 24 h. On the second day, after aspirating the culture medium, the cells were washed once with PBS buffer, fixed with 4% paraformaldehyde for 15 min, discarded the fixative, and washed three times with PBS buffer. Then, phalloidin staining working solution was added and incubated in the dark for 15 min. After discarding the staining solution, the cells were washed three times with PBS buffer and observed using a fluorescence microscope.

[0044] The fluorescence results of the MCs group and the Cytoplast group are shown in the figure below. Figure 2 As shown, denucleated mesenchymal stem cells retain a complete cytoskeleton.

[0045] 1.3.3 Organelle Characterization: The presence of normal organelles in denuclearized mesenchymal stem cells was verified by fluorescent dyes in mitochondria and lysosomes. Cytoplast group design: Purified nucleated mesenchymal stem cells were seeded into sterile well plates, cultured in serum-containing medium for 24 hours. On the second day, the medium was aspirated and live cell fluorescent dyes for mitochondria and lysosomes were added. The cells were incubated at 37°C in the dark for 30 minutes. After the staining solution was discarded, the cells were washed three times with PBS buffer and observed under a fluorescence microscope.

[0046] Design of MSCs group: Commercial human bone marrow mesenchymal stem cells were seeded into sterile well plates, added with serum-containing culture medium and cultured normally for 24 h. On the second day, the culture medium was aspirated and live cell fluorescent dyes for mitochondria and lysosomes were added. The cells were incubated at 37°C in the dark for 30 min. After the staining solution was discarded, the cells were washed three times with PBS buffer and observed using a fluorescence microscope.

[0047] The fluorescence results of the MCs group and the Cytoplast group are shown in the figure below. Figure 2 As shown, denucleated mesenchymal stem cells retain mitochondria and lysosomal organelles.

[0048] In summary, as Figure 2 As shown, bright nuclear fluorescence was observed in the MSCs group, while no nuclear fluorescence was observed in the Cytoplast group, confirming that the enucleated mesenchymal stem cells prepared by this protocol had been successfully enucleated. The Cytoplast group showed positive Calcein AM fluorescence, confirming that the enucleated mesenchymal stem cells prepared by this protocol retained viable cell activity after enucleation. The Cytoplast group showed uniform Phalloidin fluorescence, confirming that the cytoskeleton structure of the enucleated mesenchymal stem cells prepared by this protocol was intact. Clear organelle fluorescence was observed in the Mitochondria / Lysosome group of the Cytoplast group, confirming that the enucleated mesenchymal stem cells prepared by this protocol retained functional organelles.

[0049] 1.3.4 Characterization of Transwell experimental transferability: Commercially available human bone marrow mesenchymal stem cells (MSCs) and purified nucleated mesenchymal stem cells (NMSCs) were resuspended in serum-free medium and seeded into upper transwell chambers. The chambers containing commercially available MSCs were designated as the MSCs group, and the chambers containing NMSCs were designated as the Cytoplast group. 10% FBS medium was added to the lower well plate, and the cells were cultured for 24 h. The upper transwell chambers were then removed, washed once with PBS buffer, fixed with 4% paraformaldehyde for 15 min, and the fixative was discarded. The cells were washed three times with PBS buffer, stained with crystal violet working solution for 10 min, washed three times with PBS buffer, and the cells in the chambers were gently wiped away with cotton swabs. After appropriate air drying, the cells were observed under a microscope.

[0050] The results are as follows Figure 3 As shown, the number of cells that migrated in vitro from denucleated mesenchymal stem cells was comparable to that of intact mesenchymal stem cells, with no significant difference. This demonstrates that denucleation treatment does not impair the chemotactic migration ability of cells, and that denucleated mesenchymal stem cells retain the potential to migrate to the lesion site.

[0051] 1.3.5 Static and dynamic adhesion experiments: Design Control group: Endothelial cells cultured in normal culture medium; Design of the TNF-α group: Endothelial cells were cultured in normal culture medium and then stimulated with the inflammatory factor TNF-α; Design of the Cytoplast group under static adhesion: First, endothelial cells were spread in a sterile well plate. Then, purified Dil-labeled denucleated mesenchymal stem cell suspension was added to the sterile well plate. The plate was incubated in a 37°C incubator with gentle shaking at 200 rpm for 15 min. The culture medium was then discarded, and the cells were washed three times with PBS. The cells adhering to the endothelial cells were observed under a fluorescence microscope. Design of MSC group under static adhesion: First, endothelial cells were spread in sterile well plate, and then Dil-labeled commercial human bone marrow mesenchymal stem cell suspension was added to the sterile well plate. The plate was incubated in a 37°C constant temperature incubator with gentle shaking at 200 rpm for 15 min. Then the culture medium was discarded, and the cells were washed three times with PBS. The cells adhering to the endothelial cells were observed under a fluorescence microscope. Design of Cytoplast arrays under dynamic adhesion: Endothelial cells were spread onto a substrate such as... Figure 4 The bottom surface of the microfluidic mold is shown. Then, the purified Dil-labeled denucleated mesenchymal stem cell suspension is added to the syringe. Under the injection pump, the cells flow through the surface of the endothelial cells. The cells are incubated in a 37°C constant temperature incubator with gentle shaking at 200 rpm for 15 min. Then, the culture medium is discarded, and the cells are washed three times with PBS. The cells adhering to the endothelial cells are observed under a fluorescence microscope. Designing a dynamic adhesion-based MSC group: First, endothelial cells were spread onto a substrate such as... Figure 4 The bottom surface of the microfluidic mold is shown. Then, Dil-labeled commercial human bone marrow mesenchymal stem cell suspension is added to the syringe. Under the injection pump, the cells flow through the surface of the endothelial cells. The cells are incubated in a 37°C constant temperature incubator with gentle shaking at 200 rpm for 15 min. Then, the culture medium is discarded, and the cells are washed three times with PBS. The cells adhering to the endothelial cells are observed under a fluorescence microscope. The results of static adhesion and dynamic adhesion are as follows Figure 4 As shown, denucleated mesenchymal stem cells have a certain degree of adhesion to endothelial cells, whether in a static or flowing state.

[0052] 1.3.6 Co-culture experiment of endothelial cells and macrophages: Design of the Cytoplast group: First, endothelial cells were seeded in the lower sterile plate. After the cells were confluent, a straight line was drawn with a pipette tip. The initial situation was photographed under a fluorescence microscope. Then, the purified nucleated mesenchymal stem cells were seeded in the upper transwell chamber and co-cultured for 24 hours before observing the migration of endothelial cells.

[0053] Design Control group: First, endothelial cells were seeded in the lower layer of sterile plate. After the cells were confluent, a straight line was drawn with a pipette tip. The initial situation was photographed under a fluorescence microscope. After co-culturing for 24 hours, the migration of endothelial cells was observed.

[0054] Design of the MSC group: First, endothelial cells were seeded in the lower layer of sterile plate. After the cells were confluent, a straight line was drawn with a pipette tip. The initial situation was photographed under a fluorescence microscope. Then, mesenchymal stem cells were seeded in the upper layer of transwell chambers and co-cultured for 24 hours before observing the migration of endothelial cells.

[0055] The results are as follows Figure 5 As shown in A, denucleated mesenchymal stem cells have the function of promoting endothelial cell migration.

[0056] The Control group was designed as follows: macrophages were first seeded in a sterile plate and cultured for 24 hours. Then, the macrophages were collected and mRNA was extracted. The expression of inflammatory molecules (TNF-α, IL-1β, IL6) was detected by qPCR.

[0057] Design of the LPS group: Macrophages were first seeded in the lower layer of sterile plates and stimulated with LPS. After culturing for 24 hours, macrophages were collected and mRNA was extracted. The expression of inflammatory molecules (TNF-α, IL-1β, IL6) was detected by qPCR.

[0058] Design of LPS+MSC group: Macrophages were first seeded in the lower sterile plate and stimulated with LPS. Then, purified mesenchymal stem cells were seeded in the upper transwell chamber and co-cultured for 24 h. Macrophages were collected and mRNA was extracted. The expression of inflammatory molecules (TNF-α, IL-1β, IL6) was detected by qPCR.

[0059] Design of LPS+Cytoplast group: Macrophages were first seeded in the lower sterile well plate and stimulated with LPS. Then, purified denucleated mesenchymal stem cells were seeded in the upper transwell chamber and co-cultured for 24 h. Macrophages were collected and mRNA was extracted. The expression of inflammatory molecules (TNF-α, IL-1β, IL6) was detected by qPCR.

[0060] The results are as follows Figure 5 As shown in B, denucleated mesenchymal stem cells can inhibit the transformation of M1 type inflammatory macrophages. These results together demonstrate that denucleated mesenchymal stem cells retain certain paracrine functions.

[0061] Example 2: Denucleated mesenchymal stem cells inhibit osteogenic differentiation of valve mesenchymal cells: 2.1 The main reagents used are as follows: β-glycerophosphate (Sigma-Aldrich), dexamethasone (Sigma-Aldrich), ascorbic acid (Sigma-Aldrich), and Alizarin Red S staining solution (Cyagen Biosciences).

[0062] 2.2 Design Experiment: The Control group was designed to culture human valvular interstitial cells in normal culture medium. After 3 days, cell proteins were collected and Western blot experiments were performed to detect the expression levels of ALPL and RUNX2. After 21 days, Alizarin Red staining was performed to detect calcium salt deposition.

[0063] Design of the OM group: Human valvular interstitial cells were added to osteogenic induction medium (the formula of which is a complete medium supplemented with 10 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone and 50 μmol / L ascorbic acid) for stimulation. After 3 days, cell proteins were collected for Western blot experiments to detect the expression levels of ALPL and RUNX2. After 21 days, Alizarin Red staining was performed to detect calcium salt deposition.

[0064] Design of the OM+MSC group: Commercial human bone marrow mesenchymal stem cells were co-cultured with human valvular mesenchymal cells, and osteogenic induction medium (a complete medium supplemented with 10 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone, and 50 μmol / L ascorbic acid) was added for stimulation. After 3 days, cell proteins were collected for Western blot experiments to detect the expression levels of ALPL and RUNX2. After 21 days, alizarin red staining was performed to detect calcium salt deposition.

[0065] Design of the OM+Cytoplast group: Purified denucleated mesenchymal stem cells were co-cultured with human valvular mesenchymal cells, and osteogenic induction medium (a complete medium supplemented with 10 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone, and 50 μmol / L ascorbic acid) was added for stimulation. After 3 days, cell proteins were collected for Western blot experiments to detect the expression levels of ALPL and RUNX2. After 21 days, Alizarin Red staining was performed to detect calcium salt deposition.

[0066] The results are as follows Figure 6 As shown, Figure 6 The top left image shows a schematic diagram of cell culture, the top right image shows the results of Alizarin Red staining of cells, the bottom left image shows the results of Western blot experiments on cell proteins, and the bottom right image shows a statistical graph of Western blot experiments on cell proteins. It can be seen that under the induction of osteogenic culture medium, mesenchymal stem cells cannot inhibit osteogenic differentiation of valve mesenchymal cells, while denucleated mesenchymal stem cells can effectively inhibit osteogenic differentiation of valve mesenchymal cells.

[0067] Example 3: Intravenous injection of denucleated mesenchymal stem cells for targeted migration to calcified mouse aortic valves: 3.1 The main reagents used are as follows: high-fat diet (TD.88137), fluorescent dye DiD, and Von Kossa stain (Saiwell Biotech, G1043).

[0068] 3.2 Experimental Methods: 3.2.1 In vivo experiment on the active migration of denucleated mesenchymal stem cells to calcified valves in mice: Design ND group: Mice fed a normal diet; Design of the HFD group: Mice fed a high-fat diet; Design of the ND+cytoplasts group: Aortic valve calcification model was established by feeding Ldlr- / - mice with a normal diet for 6 months. After labeling denucleated mesenchymal stem cells with the fluorescent dye DiD, the DiD-labeled denucleated mesenchymal stem cells were injected into the mice via the tail vein. 24 hours later, the aortic tissue of the mice was removed and in vivo imaging was performed. Subsequently, the tissue was embedded with OCT and sectioned for immunofluorescence imaging.

[0069] The HFD+cytoplasts group was designed as follows: an aortic valve calcification model was established by feeding Ldlr- / - mice with a high-fat diet for 6 months. After labeling denucleated mesenchymal stem cells with the fluorescent dye DiD, the DiD-labeled denucleated mesenchymal stem cells were injected into the mice via the tail vein. 24 hours later, the aortic tissue of the mice was removed and in vivo imaging was performed. Subsequently, the tissue was embedded with OCT and sectioned for immunofluorescence imaging.

[0070] The result is shown in the figure below. Figure 7 As shown, Figure 7 The A markers are located in the lungs, heart, and aorta of mice. Figure 7 B in the figure represents the labeling of the aortic intima and aortic valve in mice. The results show that denucleated mesenchymal stem cells can migrate to the calcified aortic valve.

[0071] 3.2.2 Validation of the in vivo therapeutic effect of denucleated mesenchymal stem cells: The ND group consisted of mice fed a normal diet without any cell injection intervention.

[0072] The HFD group consisted of mice fed a high-fat diet. HFD+Cytoplast: An aortic valve calcification model was established by feeding Ldlr- / - mice with a high-fat diet. Two months after model establishment, denucleated mesenchymal stem cells were injected via the tail vein. 10 5 Each mouse was injected with one dose every two weeks. After 6 months, the transvalvular aortic valve velocity was measured by small animal ultrasound. The mice were then euthanized, and aortic root tissue was collected. The tissue was dehydrated in a 30% sucrose solution for 24 hours before OCT embedding and frozen sectioning. The tissue sections were stained with hematoxylin and eosin (HE) and von Kossa stain to observe valve morphology, thickness, and calcium salt deposition. The results are shown in the figure below. Figure 8 As shown.

[0073] like Figure 8 As shown in Figure A, HE staining revealed significant thickening of the aortic valve in the model group, while the thickness was significantly reduced in the denucleated mesenchymal stem cell intervention group. Vonkossa staining showed a large amount of calcium salt deposition in the aortic valve of the model group, while this was significantly reduced in the denucleated mesenchymal stem cell intervention group.

[0074] like Figure 8 As shown in Figure B, the peak transvalvular flow velocity of the aortic valve significantly increased after aortic valve calcification, while the flow velocity decreased after supplementation with denucleated mesenchymal stem cells. All of these results suggest that intravenous injection of denucleated mesenchymal stem cells can alleviate aortic valve calcification in mice.

[0075] Those skilled in the art should understand that the technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0076] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. The application of denucleated mesenchymal stem cells in the preparation of drugs for the prevention or treatment of calcified aortic valve disease, characterized in that, Denucleated mesenchymal stem cells are obtained by denucleating human mesenchymal stem cells using a combination of cytochalasin B and Ficoll density gradient ultracentrifugation. Human mesenchymal stem cells are human bone marrow mesenchymal stem cells.

2. The application of the denucleated mesenchymal stem cells according to claim 1 in the preparation of drugs for the prevention or treatment of calcified aortic valve disease, characterized in that, The drug is used to achieve at least one of the following functions: inhibiting osteoblast-like differentiation of aortic valve interstitial cells, reducing aortic valve calcium salt deposition, alleviating aortic valve leaflet thickening, reducing peak transvalvular flow velocity of aortic valve, and reducing mean transvalvular pressure gradient of aortic valve.

3. The application of the denucleated mesenchymal stem cells according to claim 1 in the preparation of drugs for the prevention or treatment of calcified aortic valve disease, characterized in that, Denucleated mesenchymal stem cells exert their pharmacological effects through at least one pathway: inhibiting local inflammatory response of the aortic valve, promoting repair of aortic valve endothelial cells, inhibiting aortic valve interstitial cell apoptosis, and regulating osteogenic signaling pathways of the aortic valve, in order to prevent or treat calcified aortic valve disease.

4. The application of the denucleated mesenchymal stem cells according to claim 1 in the preparation of drugs for the prevention or treatment of calcified aortic valve disease, characterized in that, The drug is in the form of an injection.

5. The application of the denucleated mesenchymal stem cells according to claim 1 in the preparation of drugs for the prevention or treatment of calcified aortic valve disease, characterized in that, Calcific aortic valve disease is calcific aortic stenosis.

6. The application of the denucleated mesenchymal stem cells according to claim 1 in the preparation of drugs for the prevention or treatment of calcified aortic valve disease, characterized in that, Calcific aortic valve disease is aortic valve calcification induced by a high-fat / high-cholesterol diet.

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