A preparation method and application of apoptotic bodies of deciduous dental pulp stem cells regulating macrophage polarization

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a preparation method and application of apoptotic bodies of deciduous dental pulp stem cells that regulate macrophage polarization. The present invention provides a method for preparing apoptotic bodies of deciduous dental pulp stem cells: step one, separation, culture and amplification of deciduous dental pulp stem cells; step two, extraction and identification of apoptotic bodies of deciduous dental pulp stem cells; step three, evaluation of the therapeutic effect of apoptotic bodies of deciduous dental pulp stem cells on spinal cord injury in rats. Deciduous dental pulp stem cells are induced by staurosporine, and more apoptotic bodies are obtained by differential centrifugation. The induced extraction scheme is simpler, easier, more time-saving, and more efficient, and does not require high instrument accuracy. The apoptotic bodies of deciduous dental pulp stem cells obtained by this method can regulate macrophage polarization, and are used to prepare drugs for treating spinal cord injury diseases, providing a new idea for the treatment of spinal cord injury diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of apoptotic bodies of deciduous dental pulp stem cells that regulate macrophage polarization. Background Art

[0002] Mesenchymal stem cells (MSCs) transplantation has been shown to be effective in treating spinal cord injury (SCI), but the risks of cell dedifferentiation, immune rejection, and tumor formation after direct MSC transplantation limit its clinical application. Recent studies have shown that paracrine mechanisms may underlie the beneficial effects of MSCs in tissue regeneration, and the release of extracellular vesicles is considered to be the most important mediator of the therapeutic effects of MSCs. More importantly, MSCs-derived extracellular vesicles can be reproducibly manufactured, are more convenient to use, and exhibit therapeutic effects that have been largely confirmed in relevant in vitro and in vivo disease models.

[0003] Extracellular vesicles are involved in the transport of biomolecules, including miRNA, lncRNA, mRNA, and proteins, while protecting them from degradation. Extracellular vesicle transplantation can achieve similar therapeutic effects and functional characteristics as direct stem cell transplantation while avoiding harmful effects. Recent studies have shown that mesenchymal stem cell-derived extracellular vesicles have therapeutic potential in tissue regeneration and the treatment of myocardial infarction and stroke. Studies have also shown that these nano-sized mesenchymal stem cell-derived extracellular vesicles can successfully cross the blood-brain barrier and promote functional recovery after spinal cord injury. Among them, apoptotic bodies are the most numerous type of extracellular vesicles, which are a collection of concentrated substances such as various proteins after cell apoptosis, which can greatly improve the convenience of treatment.

[0004] Dental pulp stem cells (SHEDs) are mesenchymal stem cells in the early stages of development and can be obtained from exfoliated deciduous teeth. From the perspective of regenerative medicine, SHEDs are closest to the characteristics of embryonic cells and have stronger stemness. They are a type of stem cell that is easily accessible and has high proliferation and multipotential differentiation capabilities. The effective contents of extracellular vesicles such as apoptotic bodies (ABs) derived from SHEDs and their modified applications have not been reported, and there are few existing apoptotic body induction extraction protocols, most of which are tumor cells. The induction process is not easy to operate, and the extraction process is time-consuming and has higher requirements. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing apoptotic bodies of deciduous dental pulp stem cells that regulate macrophage polarization and their application in treating spinal cord injury diseases.

[0006] The technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a method for preparing apoptotic bodies of deciduous dental pulp stem cells comprises the following steps:

[0007] S1. Isolation, culture and expansion of dental pulp stem cells of deciduous teeth: obtaining dental pulp tissue of exfoliated deciduous teeth, isolating and culturing dental pulp stem cells of deciduous teeth, and culturing and expanding the dental pulp stem cells of deciduous teeth with culture medium;

[0008] S2. Extraction and identification of apoptotic bodies of deciduous dental pulp stem cells: Prepare staurosporine (STS) stock solution, dilute the stock solution, induce cell apoptosis, collect the cell culture supernatant, perform differential centrifugation, collect the centrifugal precipitate, obtain apoptotic bodies of deciduous dental pulp stem cells, use transmission electron microscopy and dynamic light scattering to identify apoptotic bodies of deciduous dental pulp stem cells, and use immunofluorescence (IF) staining, protein immunoblotting (WB) and flow cytometry analysis to verify apoptotic bodies of deciduous dental pulp stem cells;

[0009] S3. Evaluation of the therapeutic effect of apoptotic bodies of deciduous tooth pulp stem cells on spinal cord injury in rats: The apoptotic body proteins of deciduous tooth pulp stem cells were lysed and their protein concentration was determined. A spinal cord injury model at the level of the tenth thoracic vertebra of the rat spine was established using a spinal cord impactor. Quantitative administration was performed by tail vein injection to evaluate the therapeutic effect of apoptotic bodies of deciduous tooth pulp stem cells on spinal cord injury in rats.

[0010] Furthermore, in S1, the isolation and culture of the deciduous tooth pulp stem cells are carried out by enzyme digestion method combined with tissue block method.

[0011] Furthermore, in S2, the staurosporine stock solution is prepared by dissolving in dimethyl sulfoxide (DMSO).

[0012] Furthermore, in S2, the differential centrifugation includes the following steps: centrifuging the supernatant at a low speed to remove cell debris and then centrifuging at a high speed to collect the precipitate, washing the precipitate at least once with sterile phosphate buffered saline (PBS), resuspending the precipitate with PBS, and centrifuging the resuspended solution at a high speed to remove foreign proteins to obtain apoptotic bodies as precipitates, and the differential centrifugation is performed under low temperature conditions.

[0013] Further, in S3, the preparation method of the spinal cord injury model at the level of the tenth thoracic vertebra of the rat spine is as follows: take the prepared 10% sodium pentobarbital solution, inject it intraperitoneally at a dose of 0.3 mL / 100 g to anesthetize the experimental rat, and at the same time give 2 mL / 0.9% sodium chloride solution for fluid replacement through the same route to maintain the normal metabolism of the experimental rat. After completion, the experimental rat is tied to the operating board in a prone position, and the skin is routinely disinfected and prepared. The operation range is from the level of the ninth thoracic vertebra of the experimental rat spine to the level of the eleventh thoracic vertebra of the experimental rat spine. The tenth thoracic vertebra of the experimental rat spine is the center, and a longitudinal incision of about 3 cm is made. After separating the fascia and muscles of the experimental rat layer by layer, the spinous forceps are used to cut off the spinous processes and lamina of the tenth and eleventh thoracic vertebrae of the experimental rat spine to expose the spinal cord of the experimental rat, and then the spinal cord beater is adjusted, and the 40 g striking rod of the spinal cord beater is raised to a height of 6.0 cm. After the head and tail sides of the experimental rat are clamped and stabilized, the spinal cord at the level of the tenth thoracic vertebra of the experimental rat spine is struck to complete the preparation of the spinal cord injury model.

[0014] The second aspect of the present invention is the use of apoptotic bodies of deciduous dental pulp stem cells as active substances in inducing macrophage polarization.

[0015] Specifically, the apoptotic bodies are 500-1000 nm in size and spherical in shape.

[0016] Specifically, the apoptotic bodies induce macrophage polarization toward the M2 type.

[0017] Specifically, the apoptotic bodies affect the biological processes of endothelial cells (HUVECs) to promote angiogenesis.

[0018] The apoptotic body of deciduous tooth pulp stem cells prepared by the above method can be used in the preparation of drugs for treating spinal cord injury diseases.

[0019] A complex inflammatory response will occur after spinal cord injury. The inflammatory response is an important cause of secondary spinal cord injury, which further destroys neuronal function and aggravates nerve cell damage. The central nervous system has limited ability to repair damaged axons and neurons. If it is not treated in time to restore its function, it will lead to the loss of movement, sensation and autonomic nerve function in patients, causing paralysis. The inflammatory response is aggravated by the release of immune cells and inflammatory cytokines. In the treatment of spinal cord injury, controlling the inflammatory response can reduce secondary damage and help nerve cells recover.

[0020] The apoptotic bodies of deciduous dental pulp stem cells prepared by the method of the present invention are used as active ingredients to downregulate inflammatory factors such as IL-1β and iNOS, and increase the levels of anti-inflammatory cytokines such as CD206 and ARG1, so as to reduce local inflammatory reactions, accelerate cell recovery, and thus promote spinal cord injury. ABs are mixed with other excipients to prepare a drug (preparation) for treating spinal cord injury diseases.

[0021] These pharmaceutical excipients can be those conventionally used in various preparations, such as but not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, adhesives, disintegrants and lubricants, etc.; they can also be selected for use in order to adapt to the substance, such as emulsifiers, solubilizers, antibacterial agents, analgesics and antioxidants, etc. Such excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, thereby improving the metabolism of various compounds in the body, and then enhancing the administration effect of the composition. In aqueous solution injections, excipients generally include isotonic agents and buffers, as well as necessary emulsifiers, solubilizers and antibacterial agents, etc. In addition, other pharmaceutically acceptable pharmaceutical excipients are also included, such as antioxidants, pH regulators and analgesics, etc. Excipients used to prepare liquid preparations generally include solvents, water, oil, emulsifiers, and necessary preservatives, etc. Various excipients and the vesicles of the present invention are made into dosage forms that are conducive to drug administration, such as but not limited to aqueous solution injections, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and oral preparations. In addition, excipients such as but not limited to gelatin, albumin, chitosan, polyether and polyester polymer materials, such as but not limited to polyethylene glycol, polyurethane, polycarbonate and its copolymers, etc., can be used to achieve specific drug administration purposes or methods, such as sustained-release drug administration, controlled-release drug administration and pulse drug administration. The main manifestations of the so-called "conducive to drug administration" include but not limited to improving therapeutic effects, improving bioavailability, reducing toxic side effects and improving patient compliance.

[0022] The addition of ABs can upregulate the levels of anti-inflammatory cytokines such as CD206 and ARG1, induce the polarization of M1 phenotype to M2 phenotype, and polarized macrophages can reduce inflammation and promote angiogenesis by affecting the biological processes of endothelial cells.

[0023] The beneficial effects of the present invention are as follows: the present invention provides a method for preparing apoptotic bodies of deciduous dental pulp stem cells, inducing deciduous dental pulp stem cells with staurosporine, and obtaining more apoptotic bodies by differential centrifugation, and the induction extraction scheme is simpler, easier, more time-saving, and more efficient, and does not require high instrument accuracy. The apoptotic bodies of deciduous dental pulp stem cells obtained by this method can regulate macrophage polarization and are used to treat spinal cord injury diseases, providing a new idea for the treatment of spinal cord injury diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0025] Figure 1 Schematic diagram of the comparison of deciduous dental pulp stem cells before and after STS induction in the present invention ((a) is before induction, (b) is after induction);

[0026] Figure 2 This is a schematic diagram of the extraction process of apoptotic bodies of deciduous dental pulp stem cells in the present invention;

[0027] Figure 3 Schematic diagram of the WB detection results of apoptotic bodies of deciduous dental pulp stem cells and deciduous dental pulp stem cells in the present invention ((a) is Western blot, and (b) is protein content);

[0028] Figure 4 Schematic diagram of immunofluorescence (IF) detection results in the present invention ((a) is Annexin V, (b) is C1q);

[0029] Figure 5 It is a schematic diagram of the flow cytometry results in the present invention;

[0030] Figure 6 Schematic diagram of transmission electron microscopy and dynamic light scattering results in the present invention ((a) is transmission electron microscopy, (b) is dynamic light scattering);

[0031] Figure 7 This is a schematic diagram of the changing trend of motor function scores of experimental rats at different time periods after treatment in the present invention;

[0032] Figure 8 This is a schematic diagram of the footsteps of the experimental rats in each group after 21 days of treatment in the present invention;

[0033] Fig. 9 This is a schematic diagram of the results of the phagocytosis of ABs by macrophages in the present invention;

[0034] Fig.10 This is a schematic diagram of the DAPI fluorescence staining results of each group of M1 macrophages in the present invention;

[0035] Fig.11 This is a schematic diagram of the DAPI fluorescence staining results of each group of M2 macrophages in the present invention. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0037] Example 1

[0038] This embodiment provides a method for preparing apoptotic bodies of deciduous tooth dental pulp stem cells, comprising the following steps:

[0039] S1. Isolation, culture and expansion of dental pulp stem cells from deciduous teeth:

[0040] 1) Obtaining pulp tissue of exfoliated deciduous teeth: The ex vivo deciduous teeth of clinical children were stored in PBS containing 300 U / mL penicillin and 300 U / mL streptomycin (3% double antibiotics). The ex vivo teeth were rinsed with PBS containing 3% double antibiotics, the surface soft tissue was scraped off, and the ex vivo teeth were split with a hammer under sterile conditions; the pulp was removed using micro forceps or pulp extraction needles in the clean bench, and the apical 1 / 3 of the pulp and the congested pulp were cut off.

[0041] 2) Isolation and culture of dental pulp stem cells from deciduous teeth: After rinsing the dental pulp tissue with PBS containing 3% double antibody for 5 times, transfer it into an EP tube containing collagenase (collagenase I and collagenase II mixed at a ratio of 1:1, 1 mg / mL) and chop it with sterile scissors. After mixing, digest it in a 37°C constant temperature incubator for about 10 minutes, and then terminate the digestion with an equal volume of α-MEM culture medium containing 10% fetal bovine serum (FBS). Set the centrifuge program to 1500 rpm and centrifuge for 5 minutes. After centrifugation, discard the supernatant, resuspend the precipitate with a small amount of α-MEM culture medium containing 20% ​​FBS, transfer it to a culture dish, and after the tissue block adheres to the wall, add an appropriate amount of α-MEM culture medium containing 20% ​​FBS, and place it in a 5% CO2, 37°C, saturated humidity incubator for culture. After 3-7 days, cells will crawl out. When the cell density reached 90%, discard the old culture medium, rinse the cells twice with PBS, add an appropriate amount of 0.25% trypsin (containing EDTA) to digest for about 2 min, observe the cells shrink and round under a microscope, add 2 times the volume of trypsin containing 10% FBS culture medium to terminate digestion, gently blow the adherent cells that have not yet fallen off to make them fall off, transfer the cell suspension to a centrifuge tube, set the centrifuge program to 1000 rpm, centrifuge for 3 min, discard the supernatant, retain the cell pellet, add α-MEM culture medium containing 10% FBS, 100 U / mL penicillin and 100 U / mL streptomycin to resuspend the cell pellet and blow and mix, and aspirate the cell suspension into a culture dish and pass it at a ratio of 1:3. Select 3-5 generations of SHEDs cells with good growth status for the experiment.

[0042] The pulp can also be removed under sterile conditions, and about 2 mm of the pulp tissue at the apex of the tooth is removed and cut into pieces. Type I collagenase (3 mg / ml) is used to digest the pulp at 37°C for 10 min, and complete culture medium is added to terminate the digestion. The suspension is centrifuged and the supernatant is discarded. A small amount of complete culture medium is added to prepare a suspension and inoculated into a 6-well plate. A cover glass is pressed on the pulp tissue, and an appropriate amount of culture medium is added. The suspension is placed in a constant temperature incubator at 37°C and 5% CO2 for culture. The medium is changed once after 24 hours, and then every 3 days until the cells in the culture flask are fused into a monolayer (70%~80% confluence rate), and then 0.25% trypsin is added to digest at room temperature for 3~5 min, which is the primary culture of SHEDs. Add α-MEM culture medium containing 10% FBS or 20% FBS high-glucose DMEM cell culture medium to completely eliminate the effect of 0.25% trypsin, centrifuge, and discard the supernatant; continue to add α-MEM culture medium containing 10% FBS or 20% FBS high-glucose DMEM cell culture medium to the precipitated cells at a ratio of 1:2 or 1:3 to perform subculture of SHEDs, and the 3rd to 5th generation cells can be used for subsequent experiments.

[0043] S2. Extraction and identification of apoptotic bodies of deciduous dental pulp stem cells:

[0044] 1) Prepare staurosporine stock solution: Dissolve staurosporine (STS) in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. Ultrasonicate for 10 min to assist dissolution (the temperature should not exceed 37°C). Aliquot and store at -80°C for long term.

[0045] 2) Dilute the stock solution to induce cell apoptosis: Dilute the stock solution to 0.5 uM with α-MEM medium at a ratio of 1:2000. Induce SHEDs cells with this medium for 24 h. Cells can be seen to transform into round, shiny droplets, which are apoptotic bodies (e.g. Figure 1 If the dosage is low, the 1 mM stock solution can be diluted 10 times with PBS and stored at -20℃ for a short period of time. When used, it can be diluted with culture medium to a final concentration of 0.5 uM.

[0046] 3) Collect the cell culture supernatant, perform differential centrifugation, collect the centrifugal precipitate, and obtain the apoptotic bodies of deciduous dental pulp stem cells: Use a Pasteur pipette to blow the bottom of the culture bottle to make the apoptotic bodies fall off completely. Collect all the culture medium and use differential centrifugation to obtain SHED-ABs. Collect the supernatant and centrifuge it at 300 G for 10 min at 4°C to remove cells and cell debris; collect the supernatant and centrifuge it at 3000 G for 50 min at 4°C. The resulting precipitate is the extracellular vesicles of ABs size. After gently washing with PBS, repeat the above centrifugation steps to remove impurities and obtain the precipitate. Resuspend the precipitate in 100 µL PBS and store it in a -80°C refrigerator for subsequent experiments (such as Figure 2 as shown).

[0047] 4) Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to observe the size distribution of apoptotic bodies of deciduous dental pulp stem cells. Immunofluorescence (IF) staining, Western blotting (WB) and flow cytometry analysis were used to verify the apoptotic bodies of deciduous dental pulp stem cells: DLS results showed that the diameter of the apoptotic bodies ranged from 500 to 1000 nm, and TEM showed that the apoptotic bodies of deciduous dental pulp stem cells were spherical in shape and about 700 nm in size. Annexin V and C1q were detected by IF and were positively expressed in the apoptotic bodies. Cleaved Caspase-3, an apoptotic-specific indicator, was detected by WB and was expressed in large quantities in the apoptotic bodies, while SHEDs were expressed in small quantities or not at all. Flow cytometry also detected a large number of apoptotic indicators (such as Figures 3 to 6 as shown).

[0048] S3. Evaluation of the therapeutic effect of apoptotic bodies of deciduous dental pulp stem cells on spinal cord injury in rats:

[0049] 1) Experimental groups: 90 rats were randomly divided into the following three groups, 30 rats in each group: sham operation group, spinal cord injury model group, and deciduous dental pulp stem cell apoptotic body treatment group. The sham operation group did not receive any special treatment after the lamina was removed, while the other two groups used the striking method to prepare the spinal cord injury model after the lamina was removed and the spinal cord was exposed.

[0050] 2) Preparation of rat spinal cord injury model: Take the prepared 10% sodium pentobarbital solution and inject it intraperitoneally at a dose of 0.3 mL / 100g to anesthetize the experimental rats. At the same time, give 2 mL / 0.9% sodium chloride solution to maintain normal metabolism. After completion, tie them to the operating board in a prone position, disinfect and prepare the skin routinely, and make a longitudinal incision of about 3 cm from thoracic 9 to thoracic 11 with thoracic 10 as the center. After separating the fascia and muscles layer by layer, the bone rongeurs cut off the spinous processes and lamina of the thoracic 10 and thoracic 11 segments to expose the spinal cord. Then adjust the spinal cord striker, raise its 40 g striking rod to a height of 6.0 cm, clamp the spine to stabilize the head and tail sides, and strike the spinal cord at the thoracic 10 position to complete the preparation of the spinal cord injury model.

[0051] 3) Treatment of spinal cord injury in rats: Before use, the apoptotic bodies of deciduous dental pulp stem cells should be washed with sterile PBS at least once, centrifuged at 16000 G, 4°C for 50 min, and then lysed with an appropriate amount of protein lysis buffer. The protein concentration was determined by the BCA quantitative method for quantitative administration. It was suspended in sterile PBS to a 1 mg / mL solution for standby use and can be stored at 4°C for 1-3 days. After the modeling was completed, except for the sham operation group, the apoptotic bodies of deciduous dental pulp stem cells group was given 200 μL of apoptotic bodies of deciduous dental pulp stem cells solution (1 mg / mL), and the spinal cord injury model group was given 200 μL of 0.9% sodium chloride solution. Routine postoperative care was performed, and the bladder was pressed 3 times a day to promote urination until the normal urination reflex was restored.

[0052] 4) BMS motor function scoring and footprint analysis: BMS motor function scoring refers to a scoring system that observes the range of motion, coordination, paw posture, trunk stability and tail posture of the mouse hind limbs by crawling experimental rats. It is currently a relatively complete objective quantitative value for observing changes in motor function in rats with spinal cord injury. The score is 0-9 points, and the lower the score, the more severe the motor dysfunction. The present invention scores the motor function of rats in each group at different times. The scoring is performed independently by 2 people who are familiar with the scoring criteria but not the research team. Each mouse is observed for 5 min, and the average of the two is taken as the final score of the tested mouse. Graphpad Prism 9.0 was used for statistical analysis, and the data were expressed as mean ± standard error (mean ± SEM). The comparison of the two groups of data was analyzed using the Student t test, and the comparison of multiple groups of data was analyzed using one-way ANOVA. The BMS score was analyzed using repeated measures analysis of variance. P<0.05 was considered to be statistically significant. The results showed that the motor function of rats in each group was normal before surgery, with a score of 9 points. The scores of the sham operation group did not fluctuate significantly at each time period after surgery, and remained basically stable. Compared with the sham operation group, one day after modeling, the rats in the spinal cord injury model group and the deciduous tooth pulp stem cell apoptotic body treatment group were all paralyzed, with a score of 0 points; 3-21 days after surgery, the scores of the spinal cord injury model group and the deciduous tooth pulp stem cell apoptotic body treatment group increased, but there was no statistically significant difference between the groups (P>0.05). Footprint analysis was performed 21 days after surgery, showing that the sham operation group had a normal gait, the spinal cord injury model group had obvious foot dragging, and the deciduous tooth pulp stem cell apoptotic body treatment group had a lighter foot dragging phenomenon than the spinal cord injury model group; 28 days after surgery, the hind limb motor function of rats in each group gradually improved, the overall change in the spinal cord injury model group was small, while the score of the deciduous tooth pulp stem cell apoptotic body treatment group increased significantly, and there was a significant statistical difference with the spinal cord injury model group (P<0.05); the above results confirm that the deciduous tooth pulp stem cell apoptotic body treatment can restore the hind limb motor function of rats with spinal cord injury (such as Figures 7 and 8 as shown).

[0053] Example 2

[0054] Detection of apoptotic bodies-induced macrophage immunoregulation

[0055] THP-1 cells were cultured in complete medium (RPMI 1640 + 10% FBS + 1% antibiotics). The THP-1 cell concentration was adjusted to 5×105 cells / mL and inoculated in a 6-well plate. 2 mL of the adjusted cell suspension was added to each well. Phorbol ester (PMA, also known as TPA) was added to each well to a final concentration of 100 nM. Other inducers such as vitamin D3 can also be used to a final concentration of 100 nM. The culture plate was placed in a 5% CO2 incubator at 37°C for 2 days. Cell morphology changes were observed regularly. Differentiated cells adhered to the wall and presented a flat, stretched morphology. Subsequently, on the third day, the THP-1 cells were treated with the above method into three groups, namely the control group, the LPS group, and the LPS+ABs group. THP-1 cell-derived macrophages, LPS-treated THP-1 cell-derived macrophages, and LPS- and ABs-treated THP-1 cell-derived macrophages were collected and gently washed 2-3 times with pre-warmed PBS to remove non-adherent cells. The differentiated cells were used for further experiments.

[0056] Characterization of the process of macrophage phagocytosis of ABs:

[0057] Inducing apoptosis in deciduous dental pulp stem cells leads to the production of ABs, which are rich in DNA, RNA, proteins and organelles. Since ABs contain a range of components, they are believed to contribute to the therapeutic effect after SHEDs transplantation. In addition, once apoptosis occurs, the apoptotic signal is rapidly transmitted to phagocytes, resulting in the immediate clearance of apoptotic cells and debris. Macrophages are specialized phagocytes responsible for the elimination of apoptotic debris. They are also important immune cells that are essential for inflammation and tissue repair. Therefore, the present invention assumes that macrophages are the main target cells of ABs. Macrophages are crucial in mediating cell-to-cell interactions after stem cell transplantation and are equally important in activating the inflammatory regulatory ability of SHEDs. After being engulfed by macrophages, this cell capsule causes anti-inflammatory polarization of macrophages and secretes a series of cytokines that promote healing. Fluorescence microscopy was used to analyze and explore the phagocytosis of ABs by macrophages at the cellular level. Over time, red fluorescence was found to be encapsulated in the cells, meaning that the macrophages had engulfed the ABs (such as Fig. 9 as shown).

[0058] Example 3

[0059] Apoptotic bodies regulate macrophage polarization toward M2

[0060] Studies have shown that after SCI, the polarization of macrophage M1 phenotype increases, the inflammatory response intensifies, and neuronal cells are damaged. This provides ideas for regulating macrophages to treat SCI. The inflammatory response can be regulated by inhibiting the M1 macrophage phenotype, inducing the polarization of the M1 phenotype to the M2 phenotype, and transplanting beneficial macrophages, thereby promoting cell recovery.

[0061] After ABs were added to inflammatory cells and co-cultured for 24 hours, the phenotypic distribution of macrophages was detected by DAPI fluorescence staining. The fluorescence results showed that compared with the control group, the number of M1 (CD68 IL-1β) macrophages in the LPS group increased, and the number of M2 (CD68 CD206) macrophages decreased; after the addition of ABs, the number of M1 (CD68 IL-1β) macrophages decreased, and the number of M2 (CD68 CD206) macrophages increased. The results showed that compared with the control group, the inflammatory cytokine (IL-1β) in the LPS group was upregulated, while the anti-inflammatory cytokine (CD206) was downregulated, while the LPS+ABs group downregulated the inflammatory cytokine (IL-1β) and upregulated the anti-inflammatory cytokine (CD206), indicating that apoptotic bodies have the function of regulating the polarization of macrophages to M2 (such as Figure 10 to Figure 11 as shown).

[0062] Example 4

[0063] Verification of apoptotic bodies on endothelial cell migration

[0064] Different from the secretions of macrophages stimulated by LPS, the secretions of macrophages stimulated by ABs showed a strong effect on the migration of HUVECs, and the results are shown in Table 1.

[0065] Table 1

[0066]

[0067] After 2 days of culture, the data showed that the relative cell migration area of ​​the LPS+ABs group was 1.79 times higher than that of the control group, and the migration area was 1.96 times higher than that of the LPS group. The difference in the migration area of ​​HUVECs in each group was more obvious on the 3rd day, indicating that the secretions of macrophages stimulated by ABs contain substances that promote the migration of HUVECs.

[0068] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for preparing apoptotic bodies of deciduous dental pulp stem cells, wherein the prepared apoptotic bodies of deciduous dental pulp stem cells are used as active substances in the preparation of drugs for treating spinal cord injury diseases, characterized in that: The method for preparing apoptotic bodies of deciduous tooth dental pulp stem cells comprises the following steps: S1. Isolation, culture and expansion of dental pulp stem cells of deciduous teeth: obtaining dental pulp tissue of exfoliated deciduous teeth, isolating and culturing dental pulp stem cells of deciduous teeth, and culturing and expanding the dental pulp stem cells of deciduous teeth with culture medium; S2. Extraction and identification of apoptotic bodies of deciduous dental pulp stem cells: prepare staurosporine stock solution, dilute the stock solution, induce cell apoptosis, collect the cell culture supernatant, perform differential centrifugation, collect the centrifugal precipitate, obtain apoptotic bodies of deciduous dental pulp stem cells, identify apoptotic bodies of deciduous dental pulp stem cells using transmission electron microscopy and dynamic light scattering, and verify apoptotic bodies of deciduous dental pulp stem cells using immunofluorescence staining, protein immunoblotting, and flow cytometry analysis; In S1, the isolation and culture of the deciduous tooth pulp stem cells are carried out by enzyme digestion method combined with tissue block method; In S2, the staurosporine stock solution is prepared by dissolving in dimethyl sulfoxide; In S2, the differential centrifugation includes the following steps: centrifuging the supernatant at a low speed to remove cell debris and then centrifuging at a high speed to collect the precipitate, washing the precipitate at least once with a sterile phosphate-buffered saline solution, resuspending the precipitate with a phosphate-buffered saline solution, and centrifuging the resuspended solution at a high speed to remove foreign proteins to obtain apoptotic bodies as a precipitate, and the differential centrifugation is performed under low temperature conditions.

2. The use according to claim 1, characterized in that: The apoptotic bodies are 500-1000 nm in size and spherical in shape.

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