Preparation method and application of mesenchymal stem cell polymer for activating HIF-1alpha pathway

Abstract

The invention relates to a preparation method and application of a mesenchymal stem cell polymer for activating an HIF-1alpha pathway, and the method comprises the following steps: culturing mesenchymal stem cells until the confluence degree is 90%, adding a polymer induction culture medium for induction culture, and adding a drug deferoxamine mesylate for treatment to obtain the mesenchymal stem cell polymer for activating the HIF-1alpha pathway; the polymer induction culture medium is prepared according to the following steps: adding fetal calf serum, dexamethasone, double antibodies and vitamin C into an alpha-MEM culture medium. The mesenchymal stem cell polymer is constructed, the novel polymer is obtained through drug induced culture, the cell function can be obviously improved, and the mesenchymal stem cell polymer has a better clinical application prospect. The mesenchymal stem cell polymer-derived extracellular vesicles extracted by the invention have a good blood vessel promoting function, and the specific effect of the mesenchymal stem cell polymer-derived extracellular vesicles is to regulate endothelial cells to form H-type blood vessels, so that bone tissue regeneration is promoted, and a feasible strategy is provided for treatment of bone tissue injury.

Description

Technical Field

[0001] The present invention relates to the technical field of regenerative medicine, and specifically to a preparation method and application of a mesenchymal stem cell aggregate for activating the HIF-1α pathway. Background Art

[0002] Bone defect is a relatively common skeletal disease in current clinical work. Bone defects are usually caused by factors such as trauma, infection, tumors, or bone diseases, resulting in bone tissue damage that affects the normal physiological activities of the body and increases the risk of fractures. According to data, with the aggravation of population aging, the incidence of bone defects has been increasing year by year. In particular, osteoporosis patients in the elderly population often suffer from bone defects. Currently, bone defects have become a major challenge to global public health, and the research on the treatment of bone defects has gradually developed from traditional surgical repair methods to emerging directions such as molecular biology and cell therapy. Currently, bone regeneration, stem cell therapy, and growth factors are used to promote the repair and regeneration of bone defects. However, these treatment methods still face problems such as improving treatment effects, reducing complications, and reducing costs, as well as challenges in clinical applications.

[0003] Mesenchymal stem cells (MSCs) have the potential for self-renewal and differentiation into various cell types such as osteoblasts, chondrocytes, and adipocytes, showing broad application prospects in bone defect repair. Research has found that MSCs can not only directly promote bone tissue repair by differentiating into osteoblasts, but also indirectly promote the bone repair process by secreting growth factors, cytokines, and exosomes to regulate the local microenvironment. Therefore, MSCs have been widely used in clinical research on bone defect repair, showing good effects. In recent years, MSC aggregates, as a new form of cell therapy, have demonstrated stronger biological functions than single MSCs. MSC aggregates are three-dimensional structures formed by MSCs through specific means, which can simulate the natural microenvironment of tissues, provide more space for cell growth and differentiation, enhance the interaction between cells, and have great development prospects in clinical treatment.

[0004] The HIF-1α pathway is a key signaling pathway for cells to respond to the hypoxic environment. As a transcription factor, HIF-1α regulates physiological processes such as cell metabolism, proliferation, and angiogenesis by activating multiple downstream genes under hypoxic conditions, helping tissues adapt to the hypoxic environment. During bone repair, the hypoxic microenvironment is one of the common physiological characteristics, especially in the initial stage of fracture healing or bone defect areas. The activation of HIF-1α can regulate the proliferation, differentiation, migration, and osteogenic ability of bone marrow mesenchymal stem cells, thereby promoting the repair of bone defects. Existing studies have shown that the activation of HIF-1α can promote bone repair through multiple mechanisms such as increasing angiogenesis, regulating osteoblast differentiation, and inhibiting bone resorption. Specifically, HIF-1α can promote angiogenesis in the bone repair area, providing sufficient oxygen and nutrients for the bone defect area; at the same time, the activation of HIF-1α can enhance the differentiation ability of osteoblasts and improve the synthesis and mineralization levels of the bone matrix. In addition, HIF-1α can further promote the bone repair process by upregulating some key factors such as VEGF, IGF, and BMP.

[0005] Therefore, studying a mesenchymal stem cell aggregate that activates the HIF-1α pathway can better promote bone injury repair and bone regeneration, which has important significance for the regenerative treatment of bone tissue injuries. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for preparing and applying a mesenchymal stem cell aggregate that activates the HIF-1α pathway to solve the technical problems mentioned in the background art.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] A method for preparing a mesenchymal stem cell aggregate that activates the HIF-1α pathway, the method comprising the following steps:

[0009] When the mesenchymal stem cells are cultured to 90% confluence, add aggregate induction medium for induction culture, and add the drug deferoxamine mesylate for treatment to obtain a mesenchymal stem cell aggregate that activates the HIF-1α pathway;

[0010] The aggregate induction medium is prepared according to the following steps: Add fetal bovine serum, dexamethasone, double antibody, and vitamin C to α-MEM medium; and based on 250 mL of the aggregate induction medium, the concentration of dexamethasone is 10 μM, the concentration of penicillin is 100 U / mL, the concentration of streptomycin is 100 g / mL, the concentration of vitamin C is 0-100 μg / mL, and the volume concentration of fetal bovine serum is 5-10%, and make up with α-MEM medium.

[0011] As a further technical solution of the present invention, the step of culturing mesenchymal stem cells to 90% confluence, adding a polymer induction medium for induction culture, and adding the drug deferoxamine mesylate for treatment to obtain a mesenchymal stem cell polymer activating the HIF-1α pathway includes:

[0012] When the mesenchymal stem cells are cultured to 90% confluence, discard the culture medium, wash twice with PBS, add the polymer induction medium, and place it in an incubator at 37°C and 5% CO 2的 2. Culture in an incubator, change the induction medium every 2 - 3 days, and after continuous induction for 14 - 21 days, the morphology of the polymer can be observed with the naked eye and under a microscope. The polymer formed after continuous culture is spherical;

[0013] When the cells are cultured for 7 days, add 10 μmol of deferoxamine mesylate to the induction medium to activate the HIF-1α pathway in the cells. For the constructed cell polymers, detect the expression levels of osteogenic markers and angiogenic markers by WB.

[0014] As a further technical solution of the present invention, it also includes the cell isolation and culture of mesenchymal stem cells;

[0015] Among them, the cell isolation of the mesenchymal stem cells includes MSC isolation culture and MSC subculture. The MSC isolation culture includes the following steps:

[0016] a. Take out the tissue in a laminar flow hood and place it in a culture dish. After repeatedly rinsing it clean with 1x PBS, transfer it to another sterile cell culture dish;

[0017] b. Repeatedly rinse the tissue with PBS until there is no obvious blood on the surface, then place it in a culture dish, and use ophthalmic scissors to cut it into tissue fragments of 5 mm × 5 mm;

[0018] c. Digestion: Add the culture medium to the minced tissue and further mince it;

[0019] d. Resuspend with a double volume of ordinary α-MEM medium containing 20% fetal bovine serum and culture in a 37°C constant temperature incubator with a CO2 saturation of 5% to allow the tissue blocks to adhere.

[0020] As a further technical solution of the present invention, the MSC subculture includes:

[0021] a. Place the primary cells in an incubator for culture, change the culture medium every three days, add 5 ml of culture medium for full culture after one week, and passage when the cell confluence reaches 80 - 90%;

[0022] b. Remove the original culture solution, add PBS and wash 2 - 3 times;

[0023] c. After removing PBS, add 1.5 mL of 2.5% trypsin and digest at 37°C in an incubator for 1 - 2 min;

[0024] d. After observing the digested cells under the microscope, add 1.5 mL of medium to stop digestion, and pipette to obtain a single-cell suspension;

[0025] e. Collect the single-cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 r / min for 5 min;

[0026] f. Discard the supernatant, add fresh medium, and pipette repeatedly to resuspend the cells;

[0027] g. Perform cell counting and inoculate for passage at a density of not less than 6×10 5 / cm 2 and change the medium regularly.

[0028] As a further technical solution of the present invention, it further includes cell function identification, and the cell function identification includes flow cytometry identification of the surface markers of SHED;

[0029] Take the MSC P2-generation cells and use flow cytometry to detect the surface markers of MSC cells. The antibodies for the detected indicators are: positive indicators CD73, CD90, CD105, and negative indicators CD14, CD19, CD45;

[0030] a. Culture MSC cells of the corresponding passage number, observe whether the cell state is good, digest with trypsin for 1 min, add ordinary α-MEM medium containing 20% fetal bovine serum to terminate, collect the cell suspension, centrifuge, add PBS to resuspend and then perform cell counting. The cell density is 1×10 6 cell / mL;

[0031] b. Take 100 μL of the cell suspension into each 1.5 mL tube, and add the target antibodies (CD73, CD90, CD105, CD14, CD19, CD45) to each EP tube at the recommended concentration of 0.5 μg / test, and incubate at 4°C in the dark for 30 min;

[0032] c. After incubation, add PBS containing 1% fetal bovine serum to stop, centrifuge at 1000 rpm for 5 min, wash 3 times with PBS, and resuspend with 200 μL of PBS;

[0033] d. Perform detection on the machine.

[0034] As a further technical solution of the present invention, the cell function identification further includes detection of the proliferation and cloning ability and differentiation ability of MSC and HE staining analysis:

[0035] a. Proliferation detection:

[0036] Seed 1×10 5 cells of MSC in a culture dish, and evenly distribute the cells. Replace the culture medium every 2 days. After culturing for 14 days, discard the culture medium, wash 2 - 3 times with PBS, add 3 mL of 4% paraformaldehyde by mass concentration to fix for 30 min, wash 2 times with PBS, add 10 mL of crystal violet staining solution, discard after incubating at room temperature for 20 min, wash 2 times with PBS, and observe the total amount of colony formation and the morphology of single colonies under a microscope;

[0037] b. Detection of MSC differentiation ability;

[0038] Seed 1×10 5 cells of MSC in a 6 - well plate, add 2 mL of culture medium to each well for culture; when the confluence of MSC cells reaches 80%, replace the normal culture medium with osteogenic / adipogenic induction differentiation medium, replace it every 2 days, and perform staining after continuous culture for 21 days. Remove the culture medium, wash 2 times with PBS, add 3 mL of 4% paraformaldehyde by mass concentration to fix for 30 min, wash 2 times with PBS, add 2 mL of alizarin red / Oil Red O to stain at room temperature for 30 min, remove the staining solution, wash thoroughly with PBS until the staining solution is completely washed away, and observe under a microscope.

[0039] c. HE staining of the polymer;

[0040] After constructing the polymer treated with the drug, remove the culture medium, wash 2 times with PBS, fix the polymer with 4% paraformaldehyde for 30 min, wash 2 times with PBS, perform dehydration treatment and then paraffin embedding, and perform HE staining after sectioning. Remove the staining solution, wash thoroughly with PBS until the staining solution is completely washed away, and observe under a microscope.

[0041] Application of a mesenchymal stem cell polymer prepared by the method for preparing a mesenchymal stem cell polymer activating the HIF - 1α pathway as described above in the treatment of bone defects, the application comprising the following steps:

[0042] Prepare a mouse bone defect model, prepare a defect site with a diameter of 1 mm * 1 mm at the metaphyseal end of the mouse femoral shaft, and place the constructed polymer at the defect site for treatment.

[0043] Compared with the prior art, the beneficial effects of the present invention are:

[0044] 1. By constructing a mesenchymal stem cell polymer, the novel polymer obtained by drug - induced culture can significantly improve cell function and has better clinical application prospects.

[0045] 2. The extracellular vesicles derived from the mesenchymal stem cell aggregates obtained by the present invention have good blood vessel promoting functions. Their specific role is to regulate endothelial cells to form H-type blood vessels, thereby promoting bone tissue regeneration, providing a feasible strategy for the treatment of bone tissue injuries.

[0046] 3. The present invention constructs drug-treated mesenchymal stem cell aggregates, with simple and clear steps, convenient operation, low equipment usage cost, short preparation cycle, more convenient for actual operation and preparation, and more suitable for industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 are the MSC positive markers CD73, CD90 and CD105 (A); the MSC negative markers CD19, CD14 and CD45 (B);

[0048] Figure 2 are the general views of MSC and functional detection experiments;

[0049] Figure 3 are the general view of MSC before aggregation, the HE staining map of MSC-CA and the general view of MSC-CA;

[0050] Figure 4 is the detection of the levels of osteogenic and angiogenic markers of CA in different groups by Western blotting;

[0051] Figure 5 is the detection of the levels of osteogenic and angiogenic genes of CA in different groups by RT-PCR;

[0052] Figure 6 is the detection of the extracellular matrix and cell adhesion levels of the ECM of aggregates in different groups by RT-PCR;

[0053] Figure 7 is the immunofluorescence staining map of endothelial cell lumen formation treated with the supernatant of CA in different groups;

[0054] Figure 8 is the immunofluorescence staining map of different groups of CA treating femoral defects for 1 day;

[0055] Figure 9 is the immunofluorescence staining map of different groups of CA treating femoral defects for 3 days;

[0056] Figure 10 is the immunofluorescence staining map of different groups of CA treating femoral defects for 7 days;

[0057] Figure 11 is the immunofluorescence staining map of different groups of CA treating femoral defects for 14 days. DETAILED DESCRIPTION OF THE INVENTION

[0058] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clear and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0059] The present invention provides a method for preparing a mesenchymal stem cell aggregate that activates the HIF-1α pathway, and the method includes the following steps:

[0060] (1) Cell separation and culture;

[0061] 1) The MSC separation and culture includes the following steps:

[0062] After being approved by the Medical Ethics Committee of the Stomatological Hospital of the Fourth Military Medical University and with the informed consent of the patient and his / her guardian, the umbilical cord was collected and transferred to the Tissue Engineering Center of the Stomatological Hospital of the Fourth Military Medical University with a culture medium. The mesenchymal stem cells of the present invention are extracted from the umbilical cord.

[0063] a. Take out the tissue in a laminar flow hood and place it in a culture dish. After rinsing it repeatedly with 1X PBS until clean, transfer it to another sterile cell culture dish;

[0064] b. Rinse the tissue repeatedly with PBS until there is no obvious blood on the surface, then place it in a culture dish and cut it into tissue fragments of 5 mm × 5 mm with ophthalmic scissors;

[0065] c. Digestion: Add a culture medium to the cut tissue and further cut it;

[0066] d. Add a normal α-MEM culture medium containing 20% fetal bovine serum and culture it in a 37°C incubator with a CO2 saturation of 5% to make the tissue block adhere to the wall.

[0067] 2) The MSC subculture includes:

[0068] a. The primary cells are placed in an incubator for culture, and the culture medium is changed every three days. After one week, 5 mL of culture medium is added for sufficient culture. When the cell confluence reaches 80-90%, subculture is carried out;

[0069] b. Remove the original culture medium and add PBS to rinse 3 times;

[0070] c. After removing the PBS, add 1.5 mL of 2.5% trypsin and digest it in a 37°C incubator for 2 min;

[0071] d. After observing the cell digestion under a microscope, add 1.5 mL of culture medium to stop the digestion, and pipette to obtain a single cell suspension;

[0072] e. Collect the single-cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 r / min for 5 min;

[0073] f. Discard the supernatant, add fresh medium, and pipette repeatedly to resuspend the cells;

[0074] g. Perform cell counting and passage the cells at a density of not less than 6×10 5 / cm 2 , and change the medium regularly.

[0075] (2) Cell function identification;

[0076] 1) Flow cytometry identification of surface markers of SHED;

[0077] Take the MSC P2-generation cells and detect the surface markers of MSC cells by flow cytometry. The antibodies for the detected indicators are as follows: positive indicators CD73, CD90, CD105; negative indicators CD14, CD19, CD45;

[0078] a. Culture MSC cells at the corresponding passage number, observe whether the cell state is good, digest with trypsin for 1 min, add medium containing 20% fetal bovine serum to terminate, collect the cell suspension, centrifuge, resuspend with PBS, and perform cell counting. The cell density is 1×10 6 cell / mL;

[0079] b. Take 100 μL of the cell suspension into 1.5 mL tubes respectively. Add the target antibodies (CD73, CD90, CD105, CD14, CD19, CD45) to each EP tube at the recommended concentration of 0.5 μg / test, and incubate at 4°C in the dark for 30 min;

[0080] c. After incubation, add 2 mL of PBS containing 1% fetal bovine serum to terminate, centrifuge at 1000 rpm for 5 min, wash 3 times with PBS, and resuspend with 200 μL of PBS;

[0081] d. Perform detection on the machine.

[0082] The results are as Figure 1 shown. The flow cytometry results show that MSC cells show positive expression of CD73, CD90, and CD105, and the positive rates reach 99.03%, 99.39%, and 98.12% respectively. At the same time, they show negative expression of CD14, CD19, and CD45.

[0083] 2) Detection of MSC proliferation and cloning ability and differentiation ability:

[0084] a. Proliferation detection:

[0085] Seed the MSC at a density of 1×10 5Cells were seeded in a culture dish to ensure uniform distribution. The culture medium was changed every 2 days. After 14 days of culture, the culture medium was discarded, and the cells were washed 3 times with PBS. Then, 3 mL of 4% paraformaldehyde was added to fix the cells for 30 min. After that, the cells were washed 2 times with PBS, 10 mL of crystal violet staining solution was added, and the cells were incubated at room temperature for 20 min and then discarded. The cells were washed 2 times with PBS, and the total number of colony-forming colonies and the morphology of individual colonies were observed under a microscope.

[0086] b. Detection of MSC differentiation ability;

[0087] 1×10 5 MSC cells were seeded in a 6-well plate, and 2 mL of culture medium was added to each well for culture. When the confluence of MSC cells reached 80%, the normal culture medium was replaced with osteogenic / adipogenic induction differentiation medium, which was changed every 2 days. After continuous culture for 21 days, staining was performed. The culture medium was removed, and the cells were washed 2 times with PBS. Then, 3 mL of 4% paraformaldehyde was added to fix the cells for 30 min. After that, the cells were washed 2 times with PBS, 2 mL of alizarin red / oil red O was added for room temperature staining for 30 min. After removing the staining solution, the cells were washed thoroughly with PBS until the staining solution was completely washed away, and the cells were observed under a microscope. The osteogenic / adipogenic induction differentiation medium included osteogenic induction solution and adipogenic induction solution respectively.

[0088] Osteogenic induction solution: In 125 mL of α-MEM medium, add the following filtered solutions: 0.765 g of sodium β-glycerophosphate, 0.2085 mL of VC with a concentration of 30 mg / mL, and 25 μL of dexamethasone with a concentration of 0.02 mg / mL, 5% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, and store at 4°C in the dark.

[0089] Adipogenic induction solution: In 125 mL of α-MEM medium, add the following filtered solutions: 5% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Dissolve 4.4715 mg of indomethacin with 125 μL of methanol, and dissolve three drugs including 13.89 mg of IBMX (3-isobutyl-1-methylxanthine), 1.25 mL of insulin (1 mg / mL), and 125 μL of dexamethasone (0.391 mg / mL) with 250 μL of dimethyl sulfoxide, and then add them to the medium, and store at 4°C in the dark.

[0090] The results were as Figure 2 shown, Figure 2In this, A is the normal culture state of MSC; B is the macroscopic photograph of monoclonal crystal violet staining of MSC, scale bar = 50 μm; C is the alizarin red staining of MSC; D is the oil red O staining of MSC, scale bar = 50 μm; The MSC has good cell proliferation ability and the colonies are evenly distributed. After osteogenic differentiation induction of MSC, dark red mineralized nodules appear, indicating good osteogenic ability; after adipogenic differentiation induction, orange-red lipid droplets appear, indicating its adipogenic ability.

[0091] (3) Polymer induction and drug treatment: When the mesenchymal stem cells are cultured to 90% confluence, add the polymer induction medium for induction culture, and add the drug deferoxamine mesylate for treatment to obtain the mesenchymal stem cell polymer that activates the HIF-1α pathway;

[0092] The polymer induction medium is prepared according to the following steps: Add fetal bovine serum, dexamethasone, double antibody, and vitamin C to the α-MEM medium; and taking 250 mL of the polymer induction medium, the concentration of dexamethasone is 10 μM, the concentration of penicillin is 100 U / mL, the concentration of streptomycin is 100 g / mL, the concentration of vitamin C is 100 μg / mL, and the volume concentration of fetal bovine serum is 10%, and make up with α-MEM medium.

[0093] The steps of culturing the mesenchymal stem cells to 90% confluence, adding the polymer induction medium for induction culture, and adding the drug deferoxamine mesylate for treatment to obtain the mesenchymal stem cell polymer that activates the HIF-1α pathway include:

[0094] When the mesenchymal stem cells are cultured to 90% confluence, discard the medium, wash twice with PBS, add the polymer induction medium, and place it in an incubator at 37°C and 5% CO 2的 culture box for culture, change the induction medium every 2 days, and after continuous induction for 21 days, the morphology of the polymer can be observed with the naked eye and under the microscope. The polymer formed after continuous culture is spherical;

[0095] At 7 days of cell culture, add 10 μMol of deferoxamine mesylate to the induction medium to activate the HIF-1α pathway in the cells. For the constructed cell polymer,

[0096] The results are as Figure 3 shown, Figure 3 In this, A is the normal culture state of MSC; B is the HE staining photograph of the polymer, scale bar = 50 μm; C is the macroscopic photograph of the polymer; D is the flow chart of MSC induction polymerization and drug treatment.

[0097] (4) Protein extraction and identification of MSC polymer

[0098] a. Protein extraction of MSC polymer

[0099] After adding the MSC aggregates constructed by induced polymerization and subjecting them to drug treatment, they were collected into a centrifuge tube and centrifuged at 400 g for 5 min. After centrifugation, the supernatant was discarded, 100 μL of lysis buffer was added to resuspend, and it was placed on ice for 5 min for lysis. After lysis, it was centrifuged at 12,000 g at 4 °C for 10 min, and the supernatant was aspirated and transferred to a 1.5 mL EP tube.

[0100] b. Detection of MSC aggregate protein concentration

[0101] Prepare a 1 mg / mL BSA standard solution. Add different volumes of standard samples to a 96-well plate, and make up the total volume to 20 μL with normal saline. Add 2 μL of the sample and 18 μL of normal saline. Both the standard and the sample are set with three replicates. Subsequently, mix solution A and solution B in the BCA kit at a ratio of 50:1, and add 200 μL to each well. After incubating the plate in an incubator at 37 °C for 30 min, measure the absorbance at 560 nm, and calculate the sample concentration through the formula.

[0102] c. Loading analysis of MSC aggregate protein concentration

[0103] According to the calculated sample concentration, add the corresponding volume of loading buffer and normal saline to dilute the sample protein concentration to 1 μg / μL. After boiling for 10 min, it can be used for western blot loading analysis.

[0104] The results are as Figure 4 shown, Figure 4 It is the detection of the levels of osteogenic and angiogenic markers in different groups by immunoblotting. Among them, CON is normal MSC-CA, DFM10 μM 2d is MSC-CA treated with 10 μM DFM for 2 d, DFM10 μM 14d is MSC-CA treated with 10 μM DFM for 14 d, DFM1 μM 14d is MSC-CA treated with 1 μM DFM for 14 d. After treating the MSC aggregates with 10 μMol DFM, the HIF-1α pathway is upregulated, the osteogenic marker RUNX2 is upregulated, and the angiogenic marker ANG is upregulated, indicating that this treatment group can significantly enhance the osteogenic and angiogenic abilities of the aggregates.

[0105] (5)RNA extraction and identification of MSC aggregates

[0106] After adding the MSC aggregates constructed by induced aggregation to the drug treatment, 1 mL of Trizol reagent was added to each sample, collected in a RNase-free centrifuge tube, and centrifuged at 400 g for 5 min. Chloroform was used for phase separation to extract RNA, the supernatant was collected, and RNA was precipitated with isopropanol. The RNA precipitate was washed and dissolved in RNase-free water. The concentration and purity of RNA were measured. Using a reverse transcription kit, the extracted RNA was reverse transcribed into cDNA. Target genes were selected and primers were designed. Using real-time quantitative PCR (qRT-PCR) technology and SYBR Green dye for quantitative analysis, the amplification curve, standard curve, and the intensity of fluorescence signals were analyzed to evaluate the expression level of the target genes.

[0107] The results are as Figure 5 shown Figure 5 in the RT-PCR detection of the osteogenic and angiogenic gene levels of aggregates in different groups, where CON is normal MSC-CA, DFM 10 μM 14d is MSC-CA treated with 10 μM DFM for 14 days, and DFM 1 μM 14d is MSC-CA treated with 1 μM DFM for 14 days.

[0108] The results showed that the angiogenic gene VEGF and the osteogenic gene RUNX2 increased significantly in the DFM 10 μM group, further indicating that treating MSC-CA with 10 μM DFM for 14 days can significantly enhance the angiogenic and osteogenic functions of the aggregates.

[0109] The results are as Figure 6 shown Figure 6 in the RT-PCR detection of the extracellular matrix (ECM) and cell adhesion levels of aggregates in different groups, where CON is normal MSC-CA, DFM 10 μM 14d is MSC-CA treated with 10 μM DFM for 14 days, and DFM 10 μM 2d is MSC-CA treated with 10 μM DFM for 2 days.

[0110] The results showed that the ECM genes TGB1, COL1A, LAM, and the cell adhesion genes CDH1, FN1 increased significantly in the DFM 10 μM 14d group, further indicating that treating MSC-CA with 10 μM DFM for 14 days can significantly increase the ECM and cell adhesion functions of the aggregates.

[0111] (6) Tube formation experiment

[0112] a. Place the Matrigel in the 4 °C refrigerator overnight for thawing and prepare for use 24 h in advance. Place the angiogenesis slides and tips in the -20 °C refrigerator for pre-cooling 1 h before the experiment, and prepare an ice box. Add 10 μL of Matrigel to the bottom of each well of the angiogenesis slides and place them in the incubator for 300 min. Digest the endothelial cells and perform cell counting. Seed 1×104 Cells were used, and endothelial cells were treated with supernatants from different groups of cells and cultured in an incubator for 4 h, followed by photographing and observation.

[0113] The results are as Figure 7 shown. Figure 7 Figure shows the immunofluorescence staining images of endothelial cell lumen formation treated with supernatants from different groups of CA. CON is normal MSC-CA, DFM10μM 14d is MSC-CA treated with 10 μM DFM for 14 d, DFM1μM 14d is MSC-CA treated with 1 μM DFM for 14 d, and the supernatant of MSC aggregates was used to treat EC. The results show that the addition of 10 μMol DFM can significantly enhance the lumen formation ability of EC, that is, the treatment of aggregates with 10 μMol DFM can significantly promote angiogenesis.

[0114] (7) Treat bone defects after the construction of aggregates;

[0115] A mouse bone defect model was prepared, and a defect with a diameter of 1 mm * 1 mm was prepared at the metaphyseal end of the femoral shaft of the mouse. The constructed aggregates were placed at the defect site for treatment. The femurs of mice treated for 1 d, 3 d, 7 d, and 14 d were harvested. After fixation for 4 h, they were washed 3 times with PBS and then decalcified. After decalcification, they were dehydrated in a 30% sucrose solution, and then embedded in OCT and frozen sections were prepared for immunofluorescence staining to label H-type blood vessels and RUNX2-positive cells in the bone defect area for analysis.

[0116] The results are as Figures 8 - 11 shown. Figure 8 Figure shows the immunofluorescence staining images of different groups of CA in treating femoral defects for 1 d; Figure 9 Figure shows the immunofluorescence staining images of different groups of CA in treating femoral defects for 3 d; Figure 10 Figure shows the immunofluorescence staining images of different groups of CA in treating femoral defects for 7 d; where CON is normal MSC-CA, DFM10μM 2d is MSC-CA treated with 10 μM DFM for 2 d, DFM10μM 14d is MSC-CA treated with 10 μM DFM for 14 d, scale bar = 100 μm; in the first 7 d of aggregate treatment of bone defects, the activation of cell aggregate function by treating aggregates with 10 μMol deferoxamine mesylate for 14 d is significantly better than that of treating aggregates with 10 μMol deferoxamine mesylate for 2 d and untreated aggregates. H-type blood vessels are formed faster in the bone defect area, and the expression level of osteogenesis-related RUNX2 increases significantly, promoting bone regeneration in the defect area.

[0117] As Figure 11 shown. Figure 11It is an immunofluorescence staining image of different groups of CA for treating femoral defects for 14 days. Scale bar = 100 μm. Among them, con is normal MSC-CA, DFM10 μM 2d means MSC-CA is treated with 10 μM DFM drug for 2 days, and DFM10 μM 14d means MSC-CA is treated with 10 μM DFM drug for 14 days. In the 14 days of polymer treating bone defects, the blood vessels in the group of polymer treated with 10 μMol deferoxamine mesylate for 14 days have significantly recovered, and the extremely high expression of RUNX2 promotes bone formation, which is significantly better than other groups. The above results indicate that treatment with 10 μMol deferoxamine mesylate for 14 days can better promote the mesenchymal stem cell differentiation of cell aggregates and regulate the related functions of angiogenesis and bone regeneration, can effectively treat bone injuries and promote bone tissue regeneration, successfully prepare a stem cell drug for treating bone defects, and is expected to play an important role in clinical treatment.

[0118] It should be noted that in this article, the term "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising a..." does not exclude the existence of additional identical elements in the process, method, article or device comprising such element.

[0119] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

Claims

1. A method for preparing a mesenchymal stem cell aggregate that activates the HIF-1α pathway, characterized in that, The method includes the following steps: When the mesenchymal stem cells are cultured to 90% confluence, a polymer induction medium is added for induction culture, and deferoxamine mesylate is added for treatment to obtain a mesenchymal stem cell polymer that activates the HIF-1α pathway. The polymer induction medium is prepared according to the following steps: fetal bovine serum, dexamethasone, double antibiotics, and vitamin C are added to the α-MEM medium; and taking 250 mL of the polymer induction medium, the concentration of dexamethasone is 10 μM, the concentration of penicillin is 100 U / mL, the concentration of streptomycin is 100 μg / mL, the concentration of vitamin C is 0 - 100 μg / mL, and the volume concentration of fetal bovine serum is 5 - 10%, and it is made up with the α-MEM medium.

2. The method for preparing a mesenchymal stem cell aggregate that activates the HIF-1α pathway according to claim 1, wherein The step of culturing the mesenchymal stem cells to 90% confluence, adding the polymer induction medium for induction culture, and adding deferoxamine mesylate for treatment to obtain a mesenchymal stem cell polymer that activates the HIF-1α pathway includes: When the mesenchymal stem cells are cultured to 90% confluence, discard the culture medium, wash twice with PBS, add the polymer induction medium, and place it in an incubator at 37°C and 5% CO 2的 2 incubator for culture. Replace the induction medium every 2 - 3 days. After continuous induction for 14 - 21 days, the morphology of the polymer can be observed macroscopically and microscopically. The polymer formed after continuous culture is spherical; When the cells are cultured for 7 days, 10 μMol of deferoxamine mesylate is added to the induction medium to activate the HIF-1α pathway in the cells.

3. The method for preparing a mesenchymal stem cell aggregate for activating the HIF-1α pathway according to claim 1, wherein It also includes the cell separation and culture of mesenchymal stem cells; Among them, the cell separation of the mesenchymal stem cells includes MSC isolation culture and MSC subculture. The MSC isolation culture includes the following steps: a. Place the tissue in a sterile culture dish in a laminar flow hood, repeatedly rinse it with 1x PBS until clean, and then transfer it to another sterile cell culture dish. b. Repeatedly rinse the tissue with PBS until there is no obvious blood on the surface, then place it in the culture dish, and use ophthalmic scissors to cut it into tissue fragments of 5 mm × 5 mm. c. Digestion: Add the medium to the minced tissue and further mince it. d. Add an equal volume of ordinary α-MEM medium containing 20% fetal bovine serum for resuspension, and place it in a 37°C incubator with a CO2 saturation of 5% for culture to allow the tissue blocks to adhere to the wall.

4. The method for preparing a mesenchymal stem cell aggregate for activating the HIF-1α pathway according to claim 3, wherein The MSC subculture includes: a. The primary cells are placed in an incubator for culture, and the medium is changed every three days. After one week, 5 mL of medium is added for sufficient culture, and when the cell confluence reaches 80 - 90%, subculture is carried out. b. Remove the original culture medium, and add PBS to rinse 2 - 3 times. c. After removing the PBS, add 1.5 mL of 2.5% trypsin, and digest it in a 37°C incubator for 1 - 2 minutes. d. After observing the cell digestion under the microscope, add 1.5 mL of medium to stop the digestion, and use a pipette to pipette to obtain a single-cell suspension. e. Collect the single-cell suspension into a 15 mL centrifuge tube and centrifuge at a speed of 1000 r / min for 5 minutes. f. Discard the supernatant, add new medium, and pipette repeatedly to resuspend the cells. g. Perform cell counting and inoculate for subculture at a density of not less than 6×10 5 / cm 2 , and replace the culture medium regularly.

5. The method for preparing a mesenchymal stem cell aggregate that activates the HIF-1α pathway according to claim 4, characterized in that, It also includes cell function identification. The cell function identification includes flow cytometry identification of the surface markers of SHED. Take the MSC P2 generation cells, and use flow cytometry to detect the surface markers of MSC cells. The antibodies for the detected indicators are: positive indicators CD73, CD90, CD105, and negative indicators CD14, CD19, CD45. a. Culture the corresponding passage of MSC cells, observe whether the cell state is good, digest with trypsin for 1 minute, add normal α-MEM medium containing 20% fetal bovine serum to terminate digestion, then collect the cell suspension, centrifuge, resuspend with PBS, and perform cell counting. The cell density is 1×10 6 cells / mL; b. Take 100 μL of the cell suspension into a 1.5 mL tube, and add the target antibody to each EP tube at the recommended concentration of 0.5 μg / test. Incubate at 4°C in the dark for 30 min; c. After incubation, add PBS containing 1% fetal bovine serum to stop the reaction, centrifuge at 1000 rpm for 5 min, wash 3 times with PBS, and resuspend with 200 μL of PBS; d. Detect by machine.

6. The method for preparing a mesenchymal stem cell aggregate that activates the HIF-1α pathway according to claim 5, wherein The cell function identification also includes the detection of MSC proliferation and cloning ability, differentiation ability, and HE staining of the aggregate: a. Proliferation detection: Seed 1×10 5 cells in a culture dish to evenly distribute the cells. Replace the culture medium every 2 days. After 14 days of culture, discard the culture medium, wash 2 - 3 times with PBS, add 3 mL of 4% paraformaldehyde by mass concentration to fix for 30 min, wash 2 times with PBS, add 10 mL of crystal violet staining solution, discard it after incubating at room temperature for 20 min, wash 2 times with PBS, and observe the total number of colony-forming colonies and the morphology of individual colonies under a microscope; b. Detection of MSC differentiation ability; Inoculate 1×10 5 MSC cells in a 6-well plate, and add 2 mL of culture medium to each well for culture; when the confluence of MSC cells reaches 80%, replace the normal culture medium with osteogenic / adipogenic induction differentiation medium, add 2-3 mL of culture medium to each well for culture, change the medium every 2 days, and after continuous culture for 21 days, perform staining. Remove the culture medium, wash twice with PBS, add 3 mL of 4% paraformaldehyde by mass concentration to fix for 30 min, wash twice with PBS, add 2 mL of alizarin red / oil red O to stain at room temperature for 30 min. After removing the staining solution, wash thoroughly with PBS until the staining solution is completely washed away, and observe under a microscope; c. HE staining of the aggregate; After constructing the drug-treated aggregate, remove the culture medium, wash 2 times with PBS, fix the aggregate with 4% paraformaldehyde for 30 min, wash 2 times with PBS, perform dehydration treatment, followed by paraffin embedding, sectioning, and then HE staining. After removing the staining solution, wash thoroughly with PBS until the staining solution is completely removed, and observe under a microscope.

7. Use of a mesenchymal stem cell aggregate prepared by the method for preparing a mesenchymal stem cell aggregate activating the HIF-1α pathway according to any one of claims 1-6 in the treatment of bone defects, characterized in that, The application includes the following steps: Prepare a mouse bone defect model, create a defect site with a diameter of 1 mm * 1 mm at the metaphyseal end of the mouse femur, and place the constructed aggregate at the defect site for treatment.

Images