Stem cell-derived apoptotic extracellular vesicle with iron homeostasis regulation effect and application of stem cell-derived apoptotic extracellular vesicle in preparation of medicine for treating iron-deficiency anemia

Abstract

The invention provides stem cell-derived apoptotic extracellular vesicles (ApoEVs) with an iron homeostasis regulation effect and application of the stem cell-derived apoptotic extracellular vesicles in preparation of a medicine for treating iron-deficiency anemia, and belongs to the technical field of biological medicines. The ApoEVs is an outer vesicle separated from apoptotic cells derived from human periodontal ligament stem cells, and can effectively recover the iron level in low-iron cells induced by DFOM, up-regulate the expression of iron storage protein and regulate the level of iron export protein, thereby improving the iron homeostasis of the cells. Meanwhile, the ApoEVs have good biocompatibility and safety, and a new strategy is provided for treatment of iron-deficiency anemia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a stem cell-derived apoptotic extracellular vesicle with an iron homeostasis regulating effect and an application thereof in preparing a drug for treating iron deficiency anemia. Background Art

[0002] Abnormal iron metabolism includes two major categories: iron excess and iron deficiency. Diseases caused by iron deficiency include subclinical iron deficiency (up to 50% in women of childbearing age) and iron deficiency anemia. Currently, the treatment of iron deficiency anemia mainly relies on iron supplementation, but this method has many limitations, such as low absorption efficiency, large side effects, poor patient compliance, and long-term iron treatment may lead to iron overload and other complications.

[0003] Apoptosis is a key physiological process to maintain the physiological homeostasis of the body, and it plays an important role in development, immune regulation and disease defense. Apoptotic extracellular vesicles (ApoEVs), as biologically active vesicles released during apoptosis, carry a variety of signaling molecules and may play a role in intercellular communication and material exchange. Although research on apoptosis and ApoEVs has made progress in many fields, their role in iron deficiency anemia has not been fully explored. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a stem cell-derived apoptotic extracellular vesicle, which improves the absorption and utilization of iron by regulating iron homeostasis in cells, thereby achieving the purpose of treating iron deficiency anemia.

[0005] The present invention provides a stem cell-derived apoptotic extracellular vesicle, which is an extracellular vesicle separated from apoptotic cells derived from stem cells.

[0006] Preferably, the stem cells include human periodontal ligament stem cells;

[0007] The extracellular vesicles contain at least one of the following apoptosis-related proteins: Fas, Caspase 3 and cleaved-caspase3.

[0008] Preferably, the diameter of the exosomes is 300 to 1500 nm.

[0009] The present invention provides a method for preparing the stem cell-derived apoptotic extracellular vesicles, comprising the following steps:

[0010] After inducing apoptosis of stem cells, apoptotic cells were collected;

[0011] Lysing the apoptotic cells, removing cell debris, and collecting the supernatant;

[0012] The extracellular vesicles are separated from the supernatant to obtain stem cell-derived apoptotic extracellular vesicles.

[0013] Preferably, the method of inducing apoptosis is to inoculate human periodontal ligament stem cells into a cell culture medium containing an apoptosis inducer for apoptosis culture;

[0014] The apoptosis inducing agent includes staurosporine; the working concentration of staurosporine is 250 nM

[0015] The apoptosis culture time is 10 to 14 hours.

[0016] Preferably, the method for isolating extracellular vesicles comprises gradient centrifugation;

[0017] The gradient centrifugation procedure includes centrifugation at 800 RCF for 10 min, and centrifuging the collected supernatant at 4° C. and 16,000 RCF for 30 min.

[0018] The present invention provides the use of the stem cell-derived apoptotic extracellular vesicles or the stem cell-derived apoptotic extracellular vesicles prepared by the preparation method in preparing a drug for treating iron deficiency anemia.

[0019] The present invention provides the use of the stem cell-derived apoptotic extracellular vesicles or the stem cell-derived apoptotic extracellular vesicles prepared by the preparation method in improving cellular iron homeostasis in vitro.

[0020] The present invention provides a stem cell-derived apoptotic extracellular vesicle, which is an extracellular vesicle separated from the culture medium of stem cell-derived cells after apoptosis treatment. The stem cell-derived apoptotic extracellular vesicle provided by the present invention has the following advantages over traditional oral iron preparations, intravenous iron supplements and blood transfusion therapy:

[0021] ① Reduce iron overload: Traditional iron supplements may lead to non-specific accumulation of iron in the body, causing iron overload, which in turn leads to organ damage, especially the liver and heart. Due to their biological targeting, stem cell-derived apoptotic extracellular vesicles can more accurately deliver iron to the cells and tissues in need, reducing the non-specific distribution and accumulation of iron;

[0022] ② Reduce oxidative stress: Excess free iron can catalyze the generation of reactive oxygen species and increase oxidative stress, while stem cell-derived apoptotic extracellular vesicles may help regulate oxidative stress responses and protect cells from oxidative damage through their bioactive components;

[0023] ③Avoid transfusion-related risks: blood transfusion may bring the risk of infectious diseases and immune response problems. As a cell-free treatment method, stem cell-derived apoptotic extracellular vesicles do not have the risk of infectious diseases, and because they are derived from the patient's own cells, they have extremely low immunogenicity;

[0024] ④ Improve patient compliance: Long-term oral iron supplements may reduce patient compliance due to gastrointestinal side effects; stem cell-derived apoptotic extracellular vesicles may be administered by intravenous injection, avoiding gastrointestinal discomfort and improving patient compliance;

[0025] ⑤ Reduce allergic reactions: Intravenous iron supplements may cause allergic reactions. Stem cell-derived apoptotic extracellular vesicles reduce the risk of allergic reactions due to their biocompatibility, especially for patients who cannot tolerate traditional iron supplements;

[0026] ⑥Regulate iron metabolism: Stem cell-derived apoptotic extracellular vesicles may carry signaling molecules that regulate iron metabolism, which helps regulate iron balance at the molecular level and optimize iron utilization efficiency;

[0027] ⑦ Improve iron absorption and utilization: Compared with oral iron preparations, stem cell-derived apoptotic extracellular vesicles may more effectively cross biological barriers, increase iron absorption, and improve iron utilization by directly acting on iron metabolic pathways;

[0028] ⑧ Personalized treatment potential: Due to the biological activity and controllability of stem cell-derived apoptotic extracellular vesicles, they can be personalized according to the specific conditions of the patient to provide more precise treatment;

[0029] ⑨ Reduce long-term complications: Long-term iron supplementation may lead to chronic iron overload and other related complications. Stem cell-derived apoptotic extracellular vesicles may reduce the risk of complications of long-term treatment through their precise targeting and biological regulation.

[0030] In addition, since stem cell-derived apoptotic extracellular vesicles are derived from human periodontal ligament stem cells, they have inherent biocompatibility, reduce the immune response that may be caused by xenologous substances, and have good biocompatibility and safety; in addition, stem cell-derived apoptotic extracellular vesicles carry specific surface molecules, which help to bind to specific cells or receptors in the body, improve the bioavailability of tissues and organs, and have higher targeting.

[0031] The results of the examples of the present invention show that, at the in vitro cellular level, stem cell-derived apoptotic extracellular vesicles can effectively restore the intracellular iron level of DFOM-induced low iron cells, upregulate the expression of iron storage proteins, and regulate the level of iron export proteins, thereby improving cellular iron homeostasis. At the same time, at the animal level, after administration of stem cell-derived apoptotic extracellular vesicles, the tissue structure damage of mice was repaired, and the iron level was improved, especially in the liver and small intestine, which are key iron-regulated organs. The repair is good, indicating that stem cell-derived apoptotic extracellular vesicles provide a new strategy for the treatment of iron deficiency anemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1This is a morphological diagram of apoptotic cells derived from human periodontal ligament stem cells (PDLSCs);

[0033] Figure 2 Schematic diagram of the process for extracting extracellular vesicles from PDLSCs;

[0034] Figure 3 The results of characterization of stem cell-derived apoptotic extracellular vesicles extracted by the present invention, wherein the left figure shows the particle size of the extracellular vesicles, and the right figure shows the Zeta value of the extracellular vesicles;

[0035] Figure 4 This is a graph showing the detection results of apoptotic proteins in stem cell-derived apoptotic extracellular vesicles extracted by the present invention;

[0036] Figure 5 Optimizing results for safe concentrations of stem cell-derived apoptotic extracellular vesicles;

[0037] Figure 6 The results of qPCR detection of the effect of stem cell-derived apoptotic extracellular vesicles on the expression of genes related to iron metabolism in cells, including A. transferrin gene (TF); B. transferrin receptor gene (TFR); C. iron heavy chain protein gene (FTH); D. iron light chain protein gene (FTL); E. iron export protein gene (FPN);

[0038] Figure 7 The results show the effect of stem cell-derived apoptotic extracellular vesicles on the expression of iron metabolism-related proteins in cells, including A. transferrin (TF); B. transferrin receptor (TFR); C. iron heavy chain protein (FTH); D. iron light chain protein (FTL); E. iron export protein (FPN);

[0039] Figure 8 The results of the fluorescent immunoblotting technique to detect the effect of stem cell-derived apoptotic extracellular vesicles on the expression and distribution of proteins related to iron metabolism in cells;

[0040] Fig. 9 RerroOrange is used to detect the effect of stem cell-derived apoptotic extracellular vesicles on the Fe 2+ The results of the influence of Fe content in different treatment groups are shown in Figure 1, where A is the RerroOrange test result and B is the Fe content in different treatment groups. 2+ Content statistics results;

[0041] Fig.10 This is a graph showing the effect of stem cell-derived apoptotic extracellular vesicle injection on mouse body weight;

[0042] Fig.11The results of the effect of stem cell-derived apoptotic extracellular vesicle injection on routine blood indexes include 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 coefficient of variation (RDW);

[0043] Fig.12 The results of the effect of stem cell-derived apoptotic extracellular vesicle injection on serum iron levels are shown in Figure 1, including A. serum iron (SI); B. total iron binding capacity (TIBC); C. transferrin saturation (TSAT); D. serum ferritin (SF);

[0044] Fig.13 This is the result of H&E staining of mouse tissue after injection of stem cell-derived apoptotic extracellular vesicles;

[0045] Fig.14 This is the result of Prussian blue staining of mouse tissue after injection of stem cell-derived apoptotic extracellular vesicles. DETAILED DESCRIPTION

[0046] The present invention provides a stem cell-derived apoptotic extracellular vesicle, which is an extracellular vesicle separated from apoptotic cells derived from stem cells.

[0047] In the present invention, the stem cells preferably include human periodontal ligament stem cells. The exosomes preferably contain at least one of the following apoptosis-related proteins: Fas, Caspase 3 and cleaved-caspase 3. The present invention is measured by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), and the diameter of the exosomes is preferably 300 to 1500 nm, more preferably 600 to 1200 nm.

[0048] In the present invention, the cell culture method of the human periodontal ligament stem cells preferably includes culturing the human periodontal ligament stem cells in a humidified environment of 37°C and 5% carbon dioxide, and replacing the culture medium containing 1% double antibody every 48 hours. The source of the human periodontal ligament stem cells preferably includes isolation from healthy human periodontal ligament, or commercial purchase.

[0049] The present invention provides a method for preparing the stem cell-derived apoptotic extracellular vesicles, comprising the following steps:

[0050] After inducing apoptosis of stem cells, apoptotic cells were collected;

[0051] Lysing the apoptotic cells, removing cell debris, and collecting the supernatant;

[0052] The extracellular vesicles are separated from the supernatant to obtain stem cell-derived apoptotic extracellular vesicles.

[0053] The present invention collects apoptotic cells after inducing apoptosis of stem cells.

[0054] In the present invention, the stem cells preferably include human periodontal ligament stem cells. Before inoculation of the human periodontal ligament stem cells, the cells are preferably washed twice with PBS solution to remove the residues of the culture medium. The method of inducing apoptosis is to inoculate 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 staurosporine is preferably 240 to 260 nM, more preferably 250 nM. The cell culture medium is preferably a serum-free culture medium (α-MEM). The time of apoptosis culture is preferably 10 to 14 h, more preferably 12 h. The method for collecting apoptotic cells is preferably low-temperature differential centrifugation; the specific centrifugation procedure is preferably 800 RCF, 4 ° C, 10 min; 16000 RCF, 4 ° C, 30 min. The present invention preferably uses TUNEL detection and flow cytometry analysis to confirm cell apoptosis.

[0055] After obtaining the apoptotic cells, the present invention lyses the apoptotic cells, removes cell fragments, and collects the supernatant.

[0056] In the present invention, the lysis method is to resuspend the apoptotic cells in a PBS solution. The method for removing cell debris is preferably centrifugation. The speed of the centrifugation is preferably 130,000 to 150,000 rpm; more preferably 140,000 rpm. The time of the centrifugation is preferably 18 to 22 minutes, more preferably 20 minutes. The temperature of the centrifugation is preferably 4°C.

[0057] After obtaining the supernatant, the present invention separates extracellular vesicles from the supernatant to obtain stem cell-derived apoptotic extracellular vesicles.

[0058] In the present invention, the method for isolating extracellular vesicles preferably comprises gradient centrifugation. The procedure of the gradient centrifugation preferably comprises centrifugation at 800 RCF for 10 min, and centrifuging the collected supernatant at 4°C and 16000 RCF for 30 min.

[0059] In the present invention, the effect of stem cell-derived apoptotic extracellular vesicles prepared by the above preparation method on iron chelator (Desferrioxamine, DFOM)-induced low iron cells in vitro was evaluated. Specifically, qPCR technology was used to detect the effect of stem cell-derived apoptotic extracellular vesicle treatment on the expression of iron metabolism-related genes in low iron cells. At the same time, Western blot experiments were used to detect changes in the expression levels of iron metabolism-related proteins. In addition, immunofluorescence technology was used to detect the expression and distribution of iron metabolism-related proteins. The results showed that the treatment of stem cell-derived apoptotic extracellular vesicles can effectively upregulate the expression of intracellular ferritin (FTH&FTL) genes and proteins, improve the expression of iron export protein (FPN), and thus restore intracellular iron levels. FerroOrange test results show that stem cell-derived apoptotic extracellular vesicle treatment can significantly increase intracellular Fe 2+ level.

[0060] The present invention provides the use of the stem cell-derived apoptotic extracellular vesicles or the stem cell-derived apoptotic extracellular vesicles prepared by the preparation method in preparing a drug for treating iron deficiency anemia.

[0061] In an embodiment of the present invention, an animal model of iron deficiency anemia caused by acute gastrointestinal blood loss and an animal model of iron deficiency anemia induced by low iron feed were used as the subjects, and the effects of stem cell-derived apoptotic extracellular vesicle administration on blood routine and biochemical indicators were carried out. The results showed that the blood routine indicators of the mice in the treatment group were significantly different from those in the control group, and the specific numerical changes reflected the improvement effect of ApoEVs on anemia symptoms. The effect of stem cell-derived apoptotic extracellular vesicle administration on serum iron levels was also detected, and the results of serum iron level detection showed that stem cell-derived apoptotic extracellular vesicles had a positive regulatory effect on iron metabolism. In addition, the effects of stem cell-derived apoptotic extracellular vesicle administration on changes in tissue structure and iron deposition were also detected. The results of histopathological evaluation showed that the tissue structure damage of the mice in the treatment group was repaired, and the iron deposition was improved, especially in the liver and small intestine, which were not obvious in key iron-regulated organs. It can be seen that the stem cell-derived apoptotic extracellular vesicles can significantly improve the anemia symptoms of mice, reshape the body's iron homeostasis, restore tissue and circulating iron levels, and improve the tissue structure and function of iron-regulated organs. These results support the potential application value of stem cell-derived apoptotic extracellular vesicles in the treatment of iron deficiency anemia.

[0062] In view of the fact that the stem cell-derived apoptotic extracellular vesicles have the effects of effectively restoring DFOM-induced low iron intracellular iron levels, upregulating the expression of iron storage proteins, and regulating the levels of iron export proteins, the present invention provides the use of the stem cell-derived apoptotic extracellular vesicles or the stem cell-derived apoptotic extracellular vesicles prepared by the preparation method in improving cellular iron homeostasis in vitro.

[0063] The following is a detailed description of the stem cell-derived apoptotic extracellular vesicles with iron homeostasis regulation provided by the present invention and their use in the preparation of a drug for the treatment of iron deficiency anemia in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] Preparation and identification of apoptotic extracellular vesicles (ApoEVs) derived from human periodontal ligament stem cells (PDLSCs)

[0066] 1. Human stem cells (PDLSCs) were cultured in a humidified environment at 37°C and 5% carbon dioxide. The culture medium containing 1% double antibody was replaced every 48 hours, and cell passage was performed in a timely manner to maintain the activity and proliferation ability of stem cells.

[0067] 2. Cell culture and apoptosis induction: Culture stem cells to about 80% confluence, discard the old culture medium, and wash twice with PBS to remove the residue. Add serum-free culture medium (α-MEM) containing apoptosis inducer Staurosporine (STS), where the STS stock solution concentration is 1mM, dilute to 5mL / dish at a volume ratio of 1000:1, and induce cell apoptosis for 12h. Results are shown in Figure 1 Apoptotic cells shrink as a whole, their volume decreases, local blistering occurs on the cell membrane, and they lose connection with other cells, which manifests as separation between cells. They further fragment and form smaller apoptotic vesicles.

[0068] 3. Extraction method: After apoptotic cells are collected, they are rinsed with PBS and collected in centrifuge tubes. Cell debris are removed by centrifugation and the supernatant is collected.

[0069] 4. ApoEVs isolation: ApoEVs were isolated using an optimized gradient centrifugation protocol, including low-speed centrifugation at 800 RCF, 4°C, for 10 minutes to remove cell debris, followed by high-speed centrifugation at 16,000 RCF, 4°C, for 30 minutes to collect ApoEVs (see the procedure). Figure 2 ).

[0070] 5. ApoEVs resuspension and concentration determination: ApoEVs were resuspended in PBS and the protein concentration was determined using a BCA kit.

[0071] 6. Identification method: The size distribution of ApoEVs was determined by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) to provide basic data for subsequent biological applications.

[0072] Results Figure 3The particle size of ApoEVs prepared by the present invention is mainly distributed in the range of 300 to 1600 nm, with the highest distribution in 600 nm. The results of nanoparticle tracking analysis show that the Zeta potential value of ApoEVs is -33.67 mV.

[0073] The expression of apoptosis-related proteins such as Fas, Caspase 3, and cleaved-caspase 3 was analyzed by Western blot to evaluate the characterization of ApoEVs. Figure 4 .

[0074] Example 2

[0075] Evaluation of the effect of stem cell-derived apoptotic extracellular vesicles (ApoEVs) on iron homeostasis in iron-deficient cells induced by desferrioxamine (DFOM)

[0076] 1. Determine the dose of DFOM that induces low iron in cells: Use CCK-8 cell proliferation assay to test cell activity at different concentrations of DFOM.

[0077] Results Figure 5 150 μM DFOM was determined to be the appropriate low iron induction dose, which could effectively reduce the intracellular iron level without significantly affecting cell activity.

[0078] 2. ApoEVs treatment of low iron cells: ApoEVs were added to the cell culture medium treated with DFOM (150 μM) for 48 hours. The experimental groups included control group, DFOM group, and ApoEVs group.

[0079] 3. Assessment of the restoration of intracellular iron levels:

[0080] 3.1qPCR experiment: Total RNA of cells was extracted and cDNA was synthesized using reverse transcription reagent. Specific primers for ferritin (FTH & FTL) were designed, total RNA of each group of cell samples was extracted, reverse transcription was performed 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 to 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 3 secondary wells were set. After the reaction was completed, the amplification and dissolution curves 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 genes related to iron metabolism.

[0081] Table 1 Detection primer sequences

[0082]

[0083] 3.2 Western blot experiment: Cellular proteins were extracted using protein extraction reagents. Proteins were separated using SDS-PAGE and transferred to PVDF membranes. Immunodetection was performed using specific antibodies against FTH, FTL, and iron export protein (FPN) (see Table 2) to evaluate changes in protein expression levels;

[0084] Table 2 Western blot antibody sources

[0085] Antibody Name brand Part Number TransferrinRabbitpAb ABclonal A1448 CD71 / TransferrinReceptorRabbitpAb ABclonal A5865 FerritinHeavyChainRabbitmAb ABclonal A19544 Anti-FerritinLightChain Antibody [8E1E7] abcam ab201975 Ferroportin / SLC40A1Antibody(8G10NB) NOVUS NBP2-45357 β-ActinRabbitmAb(HighDilution) ABclonal AC026

[0086] 3.3 Immunofluorescence experiment: Fix and permeabilize cells, stain with fluorescently labeled antibodies against FTH, FTL and FPN, and observe and record the expression and distribution of intracellular proteins using a fluorescence microscope.

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

[0088] 4. Results

[0089] qPCR( Figure 6 )、Westernblot( Figure 7 ) and Immunofluorescence( Figure 8 ) The experimental results showed that ApoEV treatment can upregulate the expression of intracellular ferritin (FTH&FTL) genes and proteins, improve the expression of iron export protein (FPN), and thus restore intracellular iron levels. FerroOrange test results showed that ApoEV treatment can significantly increase intracellular Fe 2+ level( Fig. 9 ).

[0090] Conclusion: The results of this example show that ApoEVs can effectively restore DFOM-induced hypoferrogenic intracellular iron levels, upregulate the expression of iron storage proteins, and regulate the levels of iron export proteins, thereby improving cellular iron homeostasis. These findings provide a new strategy for the treatment of diseases related to iron metabolism disorders with ApoEVs.

[0091] Example 3

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

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

[0094] Low-iron diet feeding: Mice were raised in a controlled environment for 4 weeks, during which only a specially formulated low-iron diet was provided to ensure that iron intake was controlled at a low level. ApoEVs tail vein injection: From the 5th week onwards, mice in the treatment group were also injected with ApoEVs resuspension through the tail vein under sterile conditions at a dose of 200 μg / ml, once every 3 days, for a total of 10 times.

[0095] ②Sample collection and processing

[0096] Blood collection: At the end of the experiment, mice were lightly anesthetized and venous blood was collected by retro-orbital bleeding, followed by sacrifice by cervical dislocation.

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

[0098] Organ fixation: The heart, liver, spleen, lungs, kidneys, small intestine and lumbar spine were separated, rinsed with saline, and fixed with paraformaldehyde solution.

[0099] ③ Blood routine and biochemical index testing

[0100] Routine blood test: The collected blood samples are subjected to routine blood test to measure indicators such as RBC, Hb, and HCT, using an automated blood analyzer.

[0101] ④Serum separation and iron level detection: Blood samples were centrifuged to separate serum, and serum iron (SI), serum ferritin (SF), total iron binding capacity (TIBC), and transferrin saturation (TS) were quantitatively detected using specific kits according to the manufacturer's guidelines.

[0102] ⑤ Histopathological evaluation

[0103] H&E staining: The fixed tissue samples were embedded in paraffin, sliced, and stained with H&E to observe and record the changes in tissue structure.

[0104] Prussian Iron Stain: Tissue sections are stained with Prussian blue to assess iron levels in tissues and observed and documented using a light microscope.

[0105] ⑥ Detailed description of experimental results

[0106] The results of body weight showed that the body weight of mice in the treatment group improved (see Fig.10 ).

[0107] The blood routine test results showed that the blood routine indicators of the treated group mice were significantly different from those of the control group. The specific numerical changes reflected the improvement effect of ApoEVs on anemia symptoms (see Fig.11 ).

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

[0109] Histopathological evaluation results: H&E staining (see Fig.13 ) and Prussian blue staining (see Fig.14 ) showed that tissue structural damage in the treated mice was repaired and iron levels were improved, especially in key iron-regulating organs such as the liver and small intestine.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A stem cell-derived apoptotic extracellular vesicle, characterized in that: are extracellular vesicles isolated from apoptotic cells derived from stem cells.

2. The stem cell-derived apoptotic extracellular vesicles according to claim 1, characterized in that The stem cells include human periodontal ligament stem cells; The extracellular vesicles contain at least one of the following apoptosis-related proteins: Fas, Caspase 3 and cleaved-caspase 3.

3. The stem cell-derived apoptotic extracellular vesicles according to claim 1, characterized in that: The diameter of the exosomes is 300 to 1500 nm.

4. The method for preparing stem cell-derived apoptotic extracellular vesicles according to claims 1 to 3, characterized in that: The following steps are involved: After inducing apoptosis of stem cells, apoptotic cells were collected; Lysing the apoptotic cells, removing cell debris, and collecting the supernatant; The extracellular vesicles are separated from the supernatant to obtain stem cell-derived apoptotic extracellular vesicles.

5. The preparation method according to claim 4, characterized in that: The apoptosis induction method is to inoculate human periodontal ligament stem cells into a cell culture medium containing an apoptosis inducer for apoptosis culture.

6. The preparation method according to claim 5, characterized in that: The apoptosis inducing agent includes staurosporine; the working concentration of staurosporine is 250 nM.

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

8. The preparation method according to claim 4, characterized in that: The method for isolating extracellular vesicles comprises gradient centrifugation; The gradient centrifugation procedure includes centrifugation at 800 RCF for 10 min, and centrifuging the collected supernatant at 4° C. and 16,000 RCF for 30 min.

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

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

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