Stem-cell-derived apoptotic extracellular vesicles having iron homeostasis regulatory effect and use thereof in preparation of drug for treating iron deficiency anemia

Abstract

The present invention belongs to the technical field of biomedicine. Disclosed are stem-cell-derived apoptotic extracellular vesicles (ApoEVs) having an iron homeostasis regulatory effect and the use thereof in the preparation of a drug for treating iron deficiency anemia. ApoEVs are extracellular vesicles isolated from apoptotic cells derived from human periodontal ligament stem cells, and can effectively restore low intracellular iron levels induced by deferoxamine mesylate (DFOM), upregulate the expression of iron storage proteins and regulate the levels of iron export proteins, thereby improving cellular iron homeostasis. Moreover, the ApoEVs have good biocompatibility and safety, and provide a new strategy for the treatment of iron deficiency anemia.

Description

Apoptotic extracellular vesicles derived from stem cells with iron homeostasis regulation and application thereof in preparation of a drug for treating iron deficiency anemia

[0001] This application claims priority to the Chinese patent application No. 202411355959.7, filed on September 27, 2024, and entitled "Apoptotic extracellular vesicles derived from stem cells with iron homeostasis regulation and application thereof in preparation of a drug for treating iron deficiency anemia", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and specifically relates to an apoptotic extracellular vesicle derived from stem cells with iron homeostasis regulation and application thereof in preparation of a drug for treating iron deficiency anemia. BACKGROUND

[0003] Iron metabolism abnormalities include two major categories: iron excess and iron deficiency. Among them, diseases caused by iron deficiency include subclinical iron deficiency (up to 50% of women of childbearing age) and iron deficiency anemia. At present, the treatment of iron deficiency anemia mainly relies on iron supplement, but this method has many limitations, such as low absorption efficiency, large side effects, poor patient compliance, and long-term iron therapy may lead to iron overload and other complications.

[0004] Apoptosis is a key physiological process for maintaining physiological homeostasis of the body, and plays an important role in development, immune regulation and disease prevention. Apoptotic extracellular vesicles (ApoEVs) as bioactive vesicles released during apoptosis carry a variety of signaling molecules and may play a role in cell-to-cell communication and material exchange. Although research on apoptosis and ApoEVs in multiple fields has made progress, their role in iron deficiency anemia has not been fully explored.

[0005] SUMMARY

[0006] Therefore, the purpose of the present application is to provide an apoptotic extracellular vesicle derived from stem cells, which can improve the absorption and utilization of iron by regulating iron homeostasis in cells, thereby achieving the purpose of treating iron deficiency anemia.

[0007] The present application provides an apoptotic extracellular vesicle derived from stem cells, which is an extracellular vesicle isolated from apoptotic cells derived from stem cells.

[0008] Preferably, the stem cells include human periodontal ligament stem cells;

[0009] The extracellular vesicle comprises at least one of the following apoptosis-related proteins: Fas, Caspase 3 and cleaved-caspase 3.

[0010] Preferably, the diameter of the outer vesicle is 300-1500nm.

[0011] The application provides a preparation method of the stem cell-derived apoptotic cell outer vesicle, comprising the following steps:

[0012] Collecting the apoptotic cells after the stem cells are subjected to apoptosis induction;

[0013] Lysing the apoptotic cells, removing cell debris, and collecting the supernatant;

[0014] Separating the outer vesicle from the supernatant to obtain the stem cell-derived apoptotic cell outer vesicle.

[0015] Preferably, the method for inducing apoptosis is to inoculate human periodontal ligament stem cells into a cell culture medium containing an apoptosis inducer for apoptosis culture.

[0016] The apoptosis inducer comprises staurosporine; and the working concentration of the staurosporine is 250nM.

[0017] The apoptosis culture time is 10-14h.

[0018] Preferably, the method for separating the outer vesicle comprises gradient centrifugation.

[0019] The procedure of the gradient centrifugation comprises 800RCF centrifugation for 10min, and 4℃, 16000RCF centrifugation of the collected centrifugal supernatant for 30min.

[0020] The application provides an application of the stem cell-derived apoptotic cell outer vesicle or the stem cell-derived apoptotic cell outer vesicle prepared by the preparation method in preparing a drug for treating iron deficiency anemia.

[0021] The application provides an application of the stem cell-derived apoptotic cell outer vesicle or the stem cell-derived apoptotic cell outer vesicle prepared by the preparation method in improving cell iron homeostasis in vitro.

[0022] The application provides a stem cell-derived apoptotic cell outer vesicle, which is an outer vesicle separated from a culture solution of stem cell-derived cells subjected to apoptosis treatment. Compared with traditional oral iron agents, intravenous iron agents and blood transfusion treatment, the stem cell-derived apoptotic cell outer vesicle provided by the application has the following advantages:

[0023] ①Reducing iron overload: traditional iron supplement agents can cause non-specific accumulation of iron in the body, leading to iron overload and further causing organ damage, especially to the liver and heart. Due to its biological targeting property, the stem cell-derived apoptotic cell outer vesicle can more accurately deliver iron to the cells and tissues in need, reducing non-specific distribution and accumulation of iron.

[0024] ②Reducing oxidative stress: Excess free iron can catalyze the generation of reactive oxygen species, increasing oxidative stress, while stem cell-derived apoptotic extracellular vesicles may help regulate the oxidative stress response and protect cells from oxidative damage through their bioactive components;

[0025] ③Avoiding transfusion-related risks: Blood transfusion can pose risks of infectious diseases and immune response issues. Stem cell-derived apoptotic extracellular vesicles, as a cell-free therapy, do not pose the risk of infectious diseases and have very low immunogenicity due to their origin from the patient's own cells;

[0026] ④Improving patient compliance: Long-term oral iron agents may reduce patient compliance due to gastrointestinal side effects; stem cell-derived apoptotic extracellular vesicles can be administered through intravenous injection, avoiding gastrointestinal discomfort and improving patient compliance;

[0027] ⑤Reducing allergic reactions: Intravenous iron supplements can cause allergic reactions. Stem cell-derived apoptotic extracellular vesicles, due to their biocompatibility, reduce the risk of allergic reactions, especially for patients who cannot tolerate traditional iron supplements;

[0028] ⑥Regulating iron metabolism: Stem cell-derived apoptotic extracellular vesicles may carry signaling molecules that regulate iron metabolism, helping to regulate iron balance at the molecular level and optimize iron utilization efficiency;

[0029] ⑦Improving iron absorption and utilization: Compared to oral iron agents, stem cell-derived apoptotic extracellular vesicles can more effectively cross biological barriers, improve iron absorption rates, and improve iron utilization by directly acting on iron metabolism pathways;

[0030] ⑧Potential for personalized treatment: Due to the biological activity and controllability of stem cell-derived apoptotic extracellular vesicles, they can be individually adjusted according to the specific circumstances of patients, providing more precise treatment;

[0031] ⑨Reducing long-term complications: Long-term iron supplementation therapy can lead to chronic iron overload and other related complications. Stem cell-derived apoptotic extracellular vesicles, through their precise targeting and biological regulation, can reduce the risk of complications associated with long-term treatment.

[0032] In addition, stem cell-derived apoptotic extracellular vesicles, derived from human periodontal ligament stem cells, have inherent biocompatibility, reducing the immune response that may be caused by heterologous substances, and have good biocompatibility and safety. In addition, stem cell-derived apoptotic extracellular vesicles carry specific surface molecules that help bind to specific cells or receptors in the body, improving tissue and organ bioavailability, and have higher targeting.

[0033] The results of this application demonstrate that, at the in vitro cellular level, stem cell-derived apoptotic extracellular vesicles can effectively restore DFOM-induced low intracellular iron levels, upregulate the expression of iron storage proteins, and regulate the level of iron export proteins, thereby improving cellular iron homeostasis. Simultaneously, at the animal level, administration of stem cell-derived apoptotic extracellular vesicles to mice resulted in the repair of tissue structural damage and improved iron levels, particularly in key iron-regulating organs such as the liver and small intestine, indicating that stem cell-derived apoptotic extracellular vesicles provide a novel strategy for the treatment of iron deficiency anemia. Attached Figure Description

[0034] Figure 1 shows the morphology of apoptotic cells derived from human periodontal ligament stem cells (PDLSCs);

[0035] Figure 2 is a schematic diagram of the process for extracting external vesicles from PDLSCs;

[0036] Figure 3 shows the characterization results of stem cell-derived apoptotic extracellular vesicles extracted in this application, where the left figure shows the size of the extracellular vesicle particles and the right figure shows the Zeta value of the extracellular vesicles.

[0037] Figure 4 shows the detection results of apoptotic proteins in stem cell-derived apoptotic extracellular vesicles extracted in this application;

[0038] Figure 5 shows the results of optimizing the safe concentration of stem cell-derived apoptotic extracellular vesicles;

[0039] Figure 6 shows the results of qPCR detection of the effect of stem cell-derived apoptotic extracellular vesicles on the expression levels of iron metabolism-related genes in cells, where A. transferrin gene (TF); B. transferrin receptor gene (TFR); C. heavy iron chain protein gene (FTH); D. light iron chain protein gene (FTL); E. iron export protein gene (FPN).

[0040] Figure 7 shows the effect of stem cell-derived apoptotic extracellular vesicle treatment on the expression levels of iron metabolism-related proteins in cells, including A. transferrin (TF); B. transferrin receptor (TFR); C. heavy iron chain protein (FTH); D. light iron chain protein (FTL); and E. iron export protein (FPN).

[0041] Figure 8 shows the effect of treatment with stem cell-derived apoptotic extracellular vesicles on the expression and distribution of iron metabolism-related proteins in cells, as detected by fluorescence immunoassay.

[0042] Figure 9 shows the effect of RerroOrange on Fe in cells after treatment with extracellular vesicles for stem cell-derived apoptosis. 2+ The effect of content is shown in the figure, where A represents the RerroOrange detection results, and B represents the Fe content in different treatment groups. 2+ Content statistical results;

[0043] Figure 10 is a graph showing the effect of injection of stem cell-derived apoptotic extracellular vesicles on the body weight of mice;

[0044] Figure 11 is a graph showing the effect of injection of stem cell-derived apoptotic extracellular vesicles on blood routine indexes, wherein A. red blood cell count (RBC); B. hemoglobin (Hb); C. hematocrit (HCT); D. mean corpuscular volume (HCV); E. mean corpuscular hemoglobin content (HCH); F. mean corpuscular hemoglobin concentration (HCHC); G. red blood cell variation coefficient (RDW);

[0045] Figure 12 is a graph showing the effect of injection of stem cell-derived apoptotic extracellular vesicles on serum iron levels, wherein A. serum iron (SI); B. total iron binding capacity (TIBC); C. transferrin saturation (TSAT); D. serum ferritin (SF);

[0046] Figure 13 is a graph showing the H&E staining results of mouse tissues after injection of stem cell-derived apoptotic extracellular vesicles;

[0047] Figure 14 is a graph showing the Prussian blue staining results of mouse tissues after injection of stem cell-derived apoptotic extracellular vesicles. DETAILED DESCRIPTION

[0048] The present application provides a stem cell-derived apoptotic extracellular vesicle, which is an extracellular vesicle isolated from apoptotic cells derived from stem cells.

[0049] In the present application, the stem cells preferably include human periodontal ligament stem cells. The extracellular vesicle preferably contains at least one of the following apoptosis-related proteins: Fas, Caspase 3 and cleaved-caspase 3. The diameter of the extracellular vesicle is preferably 300-1500 nm, and more preferably 600-1200 nm, as determined by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA).

[0050] In the present application, the cell culture method of the human periodontal ligament stem cells preferably includes culturing the human periodontal ligament stem cells in a humidified environment at 37°C and 5% carbon dioxide, and replacing the culture medium containing 1% double antibodies every 48h. The source of the human periodontal ligament stem cells preferably includes isolation from the periodontal ligament of a healthy person or purchase from a commercial source.

[0051] The present application provides a preparation method of the stem cell-derived apoptotic extracellular vesicle, which comprises the following steps:

[0052] Collecting apoptotic cells after inducing apoptosis in stem cells;

[0053] Lysing the apoptotic cells, removing cell debris, and collecting the supernatant;

[0054] isolating exosomes from the supernatant to obtain stem cell-derived apoptotic cell exosomes.

[0055] The present application collects apoptotic cells after inducing apoptosis of stem cells.

[0056] In the present application, the stem cells preferably include human periodontal ligament stem cells. The human periodontal ligament stem cells are preferably washed twice with PBS solution before inoculation to remove the residues of the culture medium. The method of inducing apoptosis is to inoculate the human periodontal ligament stem cells into a cell culture medium containing an apoptosis inducer for apoptosis culture. The apoptosis inducer includes staurosporine; the working concentration of the staurosporine is preferably 240-260 nM, and more preferably 250 nM. The cell culture medium is preferably serum-free medium (α-MEM). The time of apoptosis culture is preferably 10-14 h, and more preferably 12 h. The method of collecting apoptotic cells is preferably low-temperature differential centrifugation; the specific centrifugation program is preferably 800 RCF, 4°C, 10 min; 16000 RCF, 4°C, 30 min. The present application preferably uses TUNEL detection and flow cytometry analysis to confirm cell apoptosis.

[0057] After obtaining the apoptotic cells, the present application lysates the apoptotic cells, removes cell debris, and collects the supernatant.

[0058] In the present application, the method of lysing is to resuspend the apoptotic cells in a PBS solution. The method of removing cell debris is preferably centrifugation. The speed of centrifugation is preferably 130000-150000 rpm; and more preferably 140000 rpm. The time of centrifugation is preferably 18-22 min, and more preferably 20 min. The temperature of centrifugation is preferably 4°C.

[0059] After obtaining the supernatant, the present application isolates exosomes from the supernatant to obtain stem cell-derived apoptotic cell exosomes.

[0060] In the present application, the method of isolating exosomes preferably includes gradient centrifugation. The program of gradient centrifugation preferably includes 800 RCF centrifugation for 10 min, and centrifugation of the collected centrifugation supernatant at 4°C and 16000 RCF for 30 min.

[0061] In the present application, the evaluation of the effect of the stem cell-derived apoptotic extracellular vesicles prepared by the above preparation method on the low-iron cell under the action of iron chelator (Desferrioxamine, DFOM) in vitro is carried out, specifically, the influence of the stem cell-derived apoptotic extracellular vesicles on the expression of iron metabolism related genes in low-iron cells after treatment is detected by qPCR technology, at the same time, the expression level of iron metabolism related proteins is detected by Western blot experiment, in addition, the expression and distribution of iron metabolism related proteins are detected by immunofluorescence technology, and the results show that the treatment of stem cell-derived apoptotic extracellular vesicles can effectively up-regulate the expression of intracellular ferritin (FTH & FTL) genes and proteins, improve the expression of iron export protein (FPN), and thus restore the intracellular iron level. The FerroOrange detection results show that the stem cell-derived apoptotic extracellular vesicle treatment can significantly increase the intracellular Fe 2+ level.

[0062] The present application provides the application of the stem cell-derived apoptotic extracellular vesicles or the stem cell-derived apoptotic extracellular vesicles prepared by the preparation method in the preparation of a drug for treating iron deficiency anemia.

[0063] In the examples of the present application, the influence of stem cell-derived apoptotic extracellular vesicle administration on blood routine and biochemical indicators is carried out by taking the animal models of iron deficiency anemia caused by acute gastrointestinal hemorrhage and the animal models of iron deficiency anemia induced by low-iron feed as objects, and the results show that there is a significant difference in the blood routine indicators of the treatment group mice compared with the control group, and the specific numerical change reflects the improvement effect of ApoEVs on anemia symptoms. The influence of stem cell-derived apoptotic extracellular vesicle administration on the iron level in serum is also detected, and the serum iron level detection results show that the stem cell-derived apoptotic extracellular vesicles have a positive regulation effect on iron metabolism. In addition, the influence of stem cell-derived apoptotic extracellular vesicle administration on the changes of tissue structure and iron deposition is also detected, and the histopathological evaluation results show that the tissue structure damage of the treatment group mice is repaired, and the iron deposition is improved, especially in the key organs of iron regulation such as liver and small intestine. It can be seen that the stem cell-derived apoptotic extracellular vesicles can significantly improve the anemia symptoms of mice, reshape the body iron homeostasis, restore the tissue and circulating iron level, and have an improvement effect on the tissue structure and function of iron regulation organs. These results support the potential application value of stem cell-derived apoptotic extracellular vesicles in the treatment of iron deficiency anemia.

[0064] In view of the effect of the stem cell-derived apoptotic extracellular vesicles in effectively restoring the intracellular iron level of DFOM-induced low-iron cells, up-regulating the expression of iron storage proteins, and regulating the level of iron export proteins, the present application provides the application of the stem cell-derived apoptotic extracellular vesicles or the stem cell-derived apoptotic extracellular vesicles prepared by the preparation method in improving the cell iron homeostasis in vitro.

[0065] The application provides a stem cell-derived apoptotic extracellular vesicle with iron homeostasis regulation and application thereof in preparation of a drug for treating iron deficiency anemia. The application is described in detail below in combination with examples, but they should not be understood as limiting the scope of the application.

[0066] Example 1

[0067] Preparation and identification method of human periodontal ligament stem cell (PDLSCs)-derived apoptotic extracellular vesicles (ApoEVs)

[0068] 1. Human stem cells (PDLSCs) were cultured at 37°C in a humidified atmosphere of 5% carbon dioxide, and the medium containing 1% double antibody was replaced every 48 h, and the cells were passaged in time to maintain the activity and proliferation ability of the stem cells.

[0069] 2. Cell culture and apoptosis induction: the stem cells were cultured to about 80% confluence, the old culture medium was aspirated, and PBS was used to wash twice to remove the residues. Serum-free medium (a-MEM) containing apoptosis inducer Staurosporine (STS) was added, the concentration of the STS stock solution was 1 mM, and it was diluted to 5 mL / dish according to the volume ratio of 1000:1, and the cells were induced to undergo apoptosis for 12 h. The results are shown in FIG. 1. The apoptotic cells were generally atrophic, the volume was reduced, the cell membrane showed local bubbling phenomenon, lost the connection with other cells, showed the separation between cells, and would be further fragmented to form smaller apoptotic vesicles.

[0070] 3. Extraction method: after the apoptotic cells were collected, PBS was used to rinse and collect into a centrifuge tube, and the cell fragments were removed by centrifugation, and the supernatant was collected.

[0071] 4. ApoEVs separation: ApoEVs were separated by using an optimized gradient centrifugation protocol, including low-speed centrifugation at 800 RCF, 4°C for 10 minutes to remove cell fragments, followed by high-speed centrifugation at 16,000 RCF, 4°C for 30 minutes to collect ApoEVs (see FIG. 2 for the process).

[0072] 5. ApoEVs resuspension and concentration determination: ApoEVs were resuspended with PBS, and the protein concentration was determined by using a BCA kit.

[0073] 6. Identification method: the size distribution of ApoEVs was determined by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), which provided basic data for subsequent biological applications.

[0074] The results are shown in Figure 3. The ApoEVs prepared in the present application mainly have a particle size of 300-1600 nm, with the highest distribution at 600 nm. Nanoparticle tracking analysis showed that the Zeta potential value of the ApoEVs was -33.67 mV.

[0075] The expression of apoptosis-related proteins such as Fas, Caspase 3, cleaved-caspase 3 was analyzed by Western blot to evaluate the characterization of ApoEVs. The results are shown in Figure 4. The apoptosis-related protein Caspase 3 was expressed in PDLSCs and apoptotic PDLSCs, but not in ApoEVs. Cleaved-caspase 3 was expressed in apoptotic PDLSCs and ApoEVs. Fas was expressed in ApoEVs.

[0076] Example 2

[0077] Evaluation of the iron homeostasis effect of stem cell-derived apoptotic extracellular vesicles (ApoEVs) on iron-chelator Desferrioxamine (DFOM)-induced low-iron cells

[0078] 1. Determination of the dose of DFOM for inducing low-iron cells: CCK-8 cell proliferation experiment was used to test the cell activity of different concentrations of DFOM, and the control group was only solvent-treated cells.

[0079] The results are shown in Figure 5. It was determined that 150 μM DFOM was a suitable low-iron inducing dose, which could effectively reduce the intracellular iron level without significantly affecting the cell activity.

[0080] 2. Treatment of ApoEVs on low-iron cells: ApoEVs were added to the cell culture medium treated with DFOM (150 μM) for a treatment period of 48 h, which was recorded as the ApoEVs group. Control group, DFOM group were also set up, respectively. The control group was only treated with cell culture medium, and the DFOM group was treated with cell culture medium containing 150 μM DFOM.

[0081] 3. Evaluation of the recovery of intracellular iron level:

[0082] 3.1 qPCR experiment: Total RNA was extracted from cells and cDNA was synthesized using reverse transcription reagents. Specific primers were designed for ferritin (FTH & FTL). Total RNA was extracted from each group of cell samples, reverse transcribed using a reverse transcription kit to obtain cDNA, and qPCR detection was performed using a fluorescent quantitative PCR kit. The detection primers for TF, TFR, FTH, TFL, and FPN are shown in Table 1. Two-step PCR amplification: The reaction system was set up as 10 μL: SYBR Premix Ex Taq II (5 μL), PCR reverse primer (0.4 μL), PCR forward primer (0.4 μL), cDNA solution (1 μL), DEPC water (3.2 μL), and three duplicate wells were set up. After the reaction, the amplification and melting curve of qRT-PCR were determined, and a standard curve was prepared during PCR quantification. qPCR analysis was performed to evaluate the effect of ApoEVs on the expression of iron metabolism-related genes.

[0083] Table 1 Detection primer sequences

[0084] 3.2 Western blot experiment: Cell proteins were extracted using protein extraction reagents. The proteins were separated using SDS-PAGE and transferred to PVDF membranes. Immunodetection was performed using specific antibodies for FTH, FTL, and iron export protein (FPN) (see Table 2) to evaluate changes in protein expression levels.

[0085] Table 2 Antibody source explanation for Western blot experiment

[0086] 3.3 Immunofluorescence experiment: Cells were fixed and permeabilized, and fluorescently labeled antibodies against FTH, FTL, and FPN were used for staining. Fluorescence microscopy was used to observe and record the expression and distribution of proteins within the cells.

[0087] 3.4 FerroOrange detection: FerroOrange fluorescent probe was used according to the manufacturer's instructions to detect changes in intracellular Fe 2+ levels.

[0088] 4. Results

[0089] qPCR (Figure 6), Western blot (Figure 7) and Immunofluorescence (Figure 8) experimental results showed that ApoEV treatment can up-regulate the expression of intracellular ferritin (FTH & FTL) genes and proteins, improve the expression of iron export protein (FPN), and restore intracellular iron levels. FerroOrange detection results showed that ApoEV treatment can significantly increase intracellular Fe 2+ levels (Figure 9).

[0090] Conclusion: The results of this example show that ApoEVs can effectively restore the low intracellular iron level induced by DFOM, up-regulate the expression of iron storage proteins, and regulate the level of iron export proteins, thereby improving cellular iron homeostasis. These findings provide new strategies for ApoEVs in the treatment of diseases related to iron metabolism disorders.

[0091] Example 3

[0092] Evaluation of the iron supplementing effect of stem cell-derived apoptotic extracellular vesicles (ApoEVs) on iron deficiency anemia mice

[0093] ① Evaluation of the therapeutic effect of stem cell-derived apoptotic extracellular vesicles (ApoEVs) on low-iron diet-induced iron deficiency anemia

[0094] Low-iron diet feeding: Mice were fed for 4 weeks in a controlled environment, during which only low-iron diet of a specific formula was provided to ensure that the iron intake was controlled at a low level. ApoEVs tail vein injection: From the 5th week, the treatment group mice were also injected with ApoEVs resuspension through the tail vein under sterile conditions, with a dose of 200 μg / ml, injected every 3 days for a total of 10 times, recorded as IDA-ApoEVs. At the same time, IDA-PBS and control groups were set up. The IDA-PBS group was injected with PBS through the tail vein under sterile conditions, with the same dose as the IDA-ApoEVs group. The control group was not treated.

[0095] ② Sample collection and processing

[0096] Blood collection: At the end of the experiment, the mice were lightly anesthetized, and the orbital blood collection method was used to collect venous blood, followed by cervical dislocation.

[0097] Organ collection: After the mice were sacrificed, the abdominal hair and skin were disinfected, and the skin and muscle layers were cut vertically using sterile surgical scissors to open the chest and abdominal cavities and expose the required organs.

[0098] Organ fixation: The heart, liver, spleen, lung, kidney, small intestine, and lumbar spine were isolated, rinsed with physiological saline, and fixed with paraformaldehyde solution.

[0099] ③ Blood routine and biochemical index detection

[0100] Routine blood test: The collected blood samples were subjected to routine blood tests to determine RBC, Hb, HCT, and other indicators. Automated blood analyzers were used for the operation.

[0101] ④ Serum separation and iron level detection: The blood samples were centrifuged to separate the serum, and specific kits were used to perform quantitative detection of serum iron (SI), serum ferritin (SF), total iron binding capacity (TIBC), and transferrin saturation (TS) according to the manufacturer's guidelines.

[0102] ⑤ Histopathological evaluation

[0103] H&E staining: The fixed tissue samples were subjected to paraffin embedding, sectioning, and H&E staining. The changes in tissue structure were observed and recorded.

[0104] Prussian blue staining: The tissue sections were subjected to Prussian blue staining to evaluate the iron level in the tissue. Optical microscopy was used for observation and recording.

[0105] ⑥ Detailed description of experimental results

[0106] Body weight results showed that the treatment group mice improved in body weight (see Figure 10).

[0107] Blood test results showed that the blood test indicators of the treatment group mice were significantly different from the control group, and the specific numerical changes reflected the improvement effect of ApoEVs on anemia (see Figure 11).

[0108] Serum iron level detection results: The serum iron indicators of the treatment group mice showed a trend of returning to near normal levels, and the specific numerical changes confirmed the positive regulatory effect of ApoEVs on iron metabolism (see Figure 12).

[0109] Histopathological evaluation results: The results of H&E staining (see Figure 13) and Prussian blue staining (see Figure 14) showed that the tissue structure damage of the treatment group mice was repaired, and the iron level was improved, especially in the liver and small intestine, which are key organs for iron regulation.

[0110] The above is only the preferred embodiment of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. A stem cell-derived apoptotic extracellular vesicle, characterized in that, An exovesicle isolated from apoptotic cells derived from stem cells.

2. The stem cell-derived apoptotic extracellular vesicle of claim 1, wherein, The stem cells comprise human periodontal ligament stem cells; The exovesicle comprises at least one of the following apoptosis-related proteins: Fas, Caspase 3 and cleaved-caspase 3.

3. The stem cell-derived apoptotic extracellular vesicle of claim 1, wherein, The exovesicle has a diameter of 300-1500 nm.

4. The method of producing stem cell-derived apoptotic extracellular vesicles according to claims 1 to 3, characterized in that, The method comprises the following steps: Collecting apoptotic cells after inducing apoptosis of stem cells; Lysing the apoptotic cells, removing cell debris and collecting supernatant; Isolating exovesicles from the supernatant to obtain stem cell-derived apoptotic cell exovesicles.

5. The preparation method according to claim 4, characterized in that, The method for inducing apoptosis comprises culturing human periodontal ligament stem cells in a cell culture medium containing an apoptosis inducer.

6. The preparation method according to claim 5, characterized in that, The apoptosis inducer comprises staurosporine; and the working concentration of the staurosporine is 250 nM.

7. The preparation method according to claim 5, characterized in that, The apoptosis culture time is 10-14 h.

8. The preparation method according to claim 4, characterized in that, The method for isolating exovesicles comprises gradient centrifugation. The gradient centrifugation program comprises 800 RCF centrifugation for 10 min, and centrifugation of the collected centrifugal supernatant at 4℃ and 16000 RCF for 30 min.

9. Use of the stem cell-derived apoptotic cell exovesicle of any one of claims 1-3 or the stem cell-derived apoptotic cell exovesicle prepared by the method of any one of claims 4-8 in the preparation of a medicament for treating iron deficiency anemia.

10. Use of the stem cell-derived apoptotic cell exovesicle of any one of claims 1-3 or the stem cell-derived apoptotic cell exovesicle prepared by the method of any one of claims 4-6 in improving cellular iron homeostasis in vitro.

11. Use of the stem cell-derived apoptotic cell exovesicle of any one of claims 1-3 or the stem cell-derived apoptotic cell exovesicle prepared by the method of any one of claims 4-8 in the treatment of iron deficiency anemia.

12. Use according to claim 11, characterized in that, The stem cell-derived apoptotic cell exovesicle is administered by intravenous injection.

13. The use according to claim 11, characterized in that, The treatment of iron deficiency anemia comprises at least one of the following: improving the tissue structure and function of iron regulatory organs, remodeling the body's iron homeostasis, and restoring tissue and circulating iron levels.

14. Use according to claim 13, characterized in that, The iron regulatory organs comprise the liver and / or the small intestine.

15. A method of modulating cellular iron homeostasis, comprising contacting a cell with a compound of any one of claims 1-14. 5 The stem cell-derived apoptotic cell exovesicle of any one of claims 1-3 or the stem cell-derived apoptotic cell exovesicle prepared by the method of any one of claims 4-8 is used to treat cells with reduced iron levels.

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