Preparation BMSCs-sec for repairing scar uterus and preparation method thereof

Abstract

The invention discloses a preparation BMSCs-sec for repairing scar uterus and a preparation method thereof.The preparation method comprises the steps that primary BMSCs growing well are subcultured to the third generation, and BMSC cells are collected; the method comprises the following steps: inoculating BMSCs into a culture bottle, culturing in a complete culture medium, when the cell adhesion fusion rate reaches 80%, discarding the supernatant, cleaning cells with PBS, adding DMEM / F12, culturing, collecting the cell supernatant, centrifuging, removing cell debris and dead cells, filtering with a filter, and collecting the filtrate; and transferring the filtrate into an ultrafiltration tube, and centrifuging to obtain a concentrated cell supernatant, thereby obtaining the preparation BMSCs-sec. The prepared preparation BMSCs-sec is used for repairing the scar uterus, and a new thought is provided for treating the uterus scar injury by using cell secretions.

Description

Technical Field

[0001] This invention belongs to the field of biological technology, specifically relating to a preparation BMSCs-sec for repairing scarred uterus and its preparation method. Background Technology

[0002] A scarred uterus refers to a uterus that has been surgically removed after a cesarean section or myomectomy. It is mainly caused by defects in the healing of the uterine myometrium after surgery, which leads to the formation of uterine scars and related complications, seriously affecting the patient's quality of life and fertility.

[0003] Currently, treatments for uterine scars mainly include medication, such as taking anti-progestin drugs like progesterone capsules to promote scar healing; and surgical treatment, such as hysteroscopic surgery, to remove the scar tissue and restore normal uterine function. However, these traditional treatments have limited effectiveness, with high recurrence rates, especially for moderate to severe cases.

[0004] Therefore, new and effective alternative methods are urgently needed to repair and treat uterine scars, and stem cell therapy is one of the main research directions. However, most stem cells currently have weak proliferation and directed migration to the endometrium, making them unable to effectively exert a repair effect. Currently, there are no reports of using BMSCs-derived secretome for the repair and treatment of uterine scars. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing BMSCs-sec, a preparation for repairing scarred uterus.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing a preparation of BMSCs-sec for repairing scarred uterus, comprising, Well-grown primary BMSCs were passaged to the third generation, and BMSCs were collected. BMSCs were seeded in culture flasks and cultured in complete culture medium. When the cell adhesion and confluence rate reached 80%, the supernatant was discarded, the cells were washed with PBS, DMEM / F12 was added for culture, the cell supernatant was collected, centrifuged, cell debris and dead cells were removed, filtered, and the filtrate was collected. The filtrate was transferred to an ultrafiltration tube, centrifuged, and concentrated cell supernatant was obtained to produce the formulation BMSCs-sec.

[0009] In a preferred embodiment of the preparation method described in this invention, the primary BMSCs are derived from rat bone marrow.

[0010] In a preferred embodiment of the preparation method described in this invention, the incubation time with DMEM / F12 is 24-48 hours.

[0011] In a preferred embodiment of the preparation method described in this invention, the centrifugation and removal of cell debris and dead cells are performed at a speed of 1000×g for a time of 15~20min.

[0012] In a preferred embodiment of the preparation method described in this invention, the filter is used for filtration, wherein the filter particle size is 0.22µm.

[0013] In a preferred embodiment of the preparation method described in this invention, the ultrafiltration tube has a specification of 3K.

[0014] As a preferred embodiment of the preparation method described in this invention, the centrifugation to obtain concentrated cell supernatant is carried out at a centrifugation temperature of 4°C and a centrifugation speed of 5000×g.

[0015] Another object of the present invention is to overcome the shortcomings of the prior art and provide a formulation BMSCs-sec.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of BMSCs-sec in the preparation of a medicament for repairing scarred uterus.

[0017] Beneficial effects of this invention: This invention provides a preparation BMSCs-sec for repairing scarred uterus. BMSCs-sec promotes the proliferation, migration, and chemotaxis of USMCs, reduces apoptosis and necrosis of USMCs after injury, regulates macrophage state, promotes macrophage M2 polarization, promotes angiogenesis, and secreted proteins are the main bioactive components of BMSCs-sec in regulating USMCs. It can activate the PI3K / AKT pathway in USMCs and has a good effect on repairing scarred uterus. The formulation BMSCs-sec prepared in this invention is used to repair scarred uterus, providing a new approach for treating uterine scar damage using cell secretions. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 This is an overview diagram of the experimental flowchart in an embodiment of the present invention.

[0019] Figure 2 This is a graph showing the proliferation of USMCs after BMSCs-sec treatment at different concentrations, as detected by CCK-8 in an embodiment of the present invention.

[0020] Figure 3 The image shown is a fluorescence image of USMCs proliferation detected by EDU cell proliferation assay in an embodiment of the present invention. Red fluorescence represents the nuclei of EDU-positive proliferating cells, and blue fluorescence represents the nuclei of Hoechst33342-positive cells. Scale bar = 400µm.

[0021] Figure 4 This is a percentage chart of USMCs with positive EDU in this embodiment of the invention, where n=3, one-way ANOVA, the ratio of USMC+BMSC-sec 10× group to 1× group*p<0.05, the ratio of USMC+BMSC-sec 10× group to USMC group**p<0.01.

[0022] Figure 5 For the transwell experiment used in this embodiment of the invention to detect the chemotactic effect of BMSCs-sec on USMCs, the scale bar is 400µm.

[0023] Figure 6 The figure shows the statistical analysis of chemotaxis of USMCs in this embodiment of the invention, where n=3, one-way ANOVA, the ratio of USMC+BMSC-sec 10× group to × group *p<0.05, the ratio of USMC+BMSC-sec 10× group to 1× group and USMC group **p<0.01.

[0024] Figure 7 The image shown in this embodiment of the invention displays the migration of USMCs at 2 hours, 24 hours, 48 ​​hours, and 72 hours. The red dashed line represents the outline of the USMC migration, and the scale bar is 1000µm.

[0025] Figure 8 For the statistics of the remaining area after the migration of USMCs in this embodiment of the invention, n=3, two-way ANOVA, *p<0.05, **p<0.01, ***p<0.001.

[0026] Figure 9 This is a full-field scan of the tube forming experiment in an embodiment of the present invention.

[0027] Figure 10 This is a quantitative analysis diagram of the pipe formation experiment in the embodiment of the present invention. In this diagram, the ratio of the total network coverage area USMC+BMSC-sec10× group to the 1× group and the USMC group is **p<0.01, and the ratio of the total pipe length USMC+BMSC-sec10× group to the 1× group and the USMC group is **p<0.01, ***p<0.001.

[0028] Figure 11 This figure shows the expression of IL-1α (A), TNF-α (B) and Bax (C) genes in USMCs after LPS treatment, detected by PCR in an embodiment of the present invention. In the figure, n=3, t test, LPS treatment group vs. control group, *p<0.05, **p<0.01, ***p<0.001.

[0029] Figure 12 The images shown are fluorescence images of USMCs necrosis and apoptosis detected by Hoechst33342 / PI double staining experiment in this embodiment of the invention. Strong red fluorescence represents the nuclei of necrotic cells, and strong blue fluorescence represents the nuclei of apoptotic cells. The scale bar is 200µm.

[0030] Figure 13 The percentage of necrotic USMCs in this embodiment of the invention, n=3, wherein, in one-way ANOVA, the comparison between the LPS+BMSC-sec 10× group and the LPS group showed ***p<0.001, and the comparison between the LPS+BMSC-sec 5× group and the LPS group showed **p<0.01; the percentage of apoptotic USMCs, n=3, in one-way ANOVA, the comparison between the LPS+BMSC-sec 10× group and the LPS group showed ***p<0.001, and the comparison between the LPS+BMSC-sec 5× group and the LPS group showed **p<0.01.

[0031] Figure 14 Here are the following examples from the present invention: (A) Flowchart of primary rat BMDMs isolation and culture; (B) Representative optical micrograph of BMDMs 7 days after induced differentiation, scale bar = 50µm; (C) Identification diagram of BMDMs surface marker (CD68).

[0032] Figure 15 This is a graph showing the expression of CD86 and CD163 in BMDMs under different treatment conditions as detected by flow cytometry in an embodiment of the present invention.

[0033] Figure 16For the quantitative analysis and intergroup comparisons by flow cytometry in this embodiment of the invention, n=3, one-way ANOVA, CD86: LPS group vs. GS group, p<0.0001, BMSCs-sec group vs. LPS group, p<0.01; CD163: BMSCs-sec group vs. LPS group, p<0.0001.

[0034] Figure 17 These are representative images of CD86 and CD163 expression in the uterine wound of rats in the BMSCs-sec group and NR group 2 weeks post-surgery, as described in this embodiment of the invention. Brown indicates immunohistochemically positive areas, and the scale bar is 200 µm.

[0035] Figure 18 For the quantitative analysis of CD86 and CD163 positive areas of uterine wounds in the BMSCs-sec group and NR group in the embodiments of the present invention, n=5, t test, CD86: BMSCs-sec group vs. NR group **p<0.01, CD163: BMSCs-sec group vs. NR group **p<0.01.

[0036] Figure 19 In this embodiment of the invention, a transwell experiment was used to detect the chemotactic effect of P0 BMSCs-sec and P3 BMSCs-sec on USMCs. The scale bar is 400µm.

[0037] Figure 20 For the statistical analysis of USMC chemotaxis in this embodiment of the invention, n=3, one-way ANOVA, the ratio of 24-hour P3 BMSC-sec 5x group to P0 BMSC-sec 5x group *p<0.05, the ratio of 48-hour P3 BMSC-sec 5x group to P0 BMSC-sec 5x group **p<0.01.

[0038] Figure 21 These are images showing the state of uterine scarring observed when the BMSCs-sec / RGD-hydrogel system was transplanted into a rat uterine injury model in this invention. A shows the constructed full-thickness rat uterine injury model, B shows the natural healing process after uterine injury, and C shows the gross morphology of the uterus in the BMSCs-sec / RGD-hydrogel group. Detailed Implementation

[0039] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0040] The primary cells used in this embodiment of the invention are derived from rats, and the preparation method of BMSCs (bone marrow mesenchymal stem cells) is as follows: After euthanasia of rats, bilateral femurs and tibias were removed under aseptic conditions, surrounding muscles and tissues were removed, and the cells were soaked in PBS. The bone marrow cavity was flushed with DMEM / F12 medium using a No. 4 needle. The flushing fluid was collected, gently mixed by pipetting, and a single-cell suspension was prepared. The suspension was centrifuged at 300×g for 5 min, and the supernatant was discarded. DMEM / F12 complete medium containing 10% fetal bovine serum (FBS, v / v) and 1% (v / v) penicillin / streptomycin was added, and the cells were seeded into sterile T25cm culture flasks and incubated in a 37℃, 5% CO2 incubator. The medium was changed every 24 hours for the first 3 days after seeding, and then every 48-72 hours thereafter. The growth status of the cells was observed and recorded. When the cells reached 80%-90% confluence, they were passaged, and the well-grown third-generation cells were selected for experiments.

[0041] USMCs (Uterine Smooth Muscle Cells): After euthanasia of rats, the uterus was removed; the serosa and endometrium were peeled off under a dissecting microscope; the tissue was immersed in PBS buffer containing penicillin and streptomycin for 5 min, and then transferred to 1.5 mL centrifuge tubes (EP tubes); the uterine muscle strips were cut into small pieces (less than 1 mm in volume) using ophthalmic scissors. 3 Centrifuge at 1500 rpm for 5 min, then transfer the tissue to a small conical flask; Next, add 2 mL of 0.2% type II collagenase solution and digest for 45-60 minutes, blowing the solution evenly every 15 minutes during this period; Subsequently, 2 mL of HG-DMEM containing 10% fetal bovine serum was added, and the mixture was filtered through a 200-mesh cell sieve. The filtrate was collected and transferred to a centrifuge tube. After centrifugation, the cells were seeded in T25 culture flasks and cultured in an incubator containing 5% CO2 at 37°C until the cells reached confluence.

[0042] The specific methods for isolating and culturing bone marrow-derived macrophages (BMDMs) are as follows: Adult male SD rats, weighing 220-250g, were euthanized and then immersed in 70% ethanol for 10 minutes to remove... After removing the skin and exposing the lower limbs, the muscles and fascia around the hip joint were removed, exposing the hip joint. The muscles covering the femur and tibia were removed, and the lower femur and tibia were cut off. The epiphyses of the femur and tibia were removed, and the bone marrow cavity was entered from the excised epiphyseal ends using a 5 mL syringe. Each bone was slowly rinsed 3 times with 2-3 mL PBS each time. The bone marrow rinsing fluid was collected in a 50 mL test tube (placed on ice). The above operation was repeated for all the obtained long bones. The bone marrow fluid was repeatedly pipetted with a 1 mL pipette and filtered through a 200-mesh cell sieve. The collected bone marrow fluid was centrifuged at 1000×g at 4℃ for 5 min. The collected cell pellet was resuspended in a medium containing recombinant rat macrophage colony-stimulating factor (30 ng / mL, M-CSF) and seeded in 6-well plates. The cells were cultured at 37℃ in a 5% CO2 incubator. Half of the medium was replaced every 72 h, and cell growth was observed under a microscope every 24-48 h. BMDMs do not proliferate in vitro. The initial cell density should be adjusted until the cells reach approximately 75% confluence before proceeding to the next stage of the experiment.

[0043] Human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC). Unless otherwise specified, all cells were cultured in complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO2.

[0044] Cell proliferation detection method in this embodiment of the invention: (a) CCK8 experiment: USMCs cells were injected at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of 100 µL of culture medium in 96-well plates and cultured for 24 h. The supernatant was discarded, and the cells were treated with 10×, 5×, and 1× BMSCs-sec solutions, respectively. The plates were incubated for 24 h. Using a repeat pipette, 10 µL of CCK-8 solution was added to each well of the plate, and the plates were incubated for 4 h. The cells were then gently mixed for 1 min using a shaker. The mixture was then analyzed using Multiskan Go. The ELISA reader measures at a wavelength of 450 nm, and the absorbance reflects cell proliferation.

[0045] (II) EdU cell proliferation assay: USMCs in logarithmic growth phase were used, with 1×10⁶ cells per well. 5 After inoculating in 96-well plates and culturing for 24 hours, USMCs were treated with different concentrations of BMSCs-sec according to the above method, and then EdU experiments were performed.

[0046] (III) Transwell Experiment: Transwell experiments were conducted using 8µm pore size chambers to evaluate the chemotactic effect of BMSCs-sec on USMCs.

[0047] (1) Seeding: Select the Transwell chamber corresponding to the 24-well plate, take well-grown USMCs, resuspend them in serum-free DMEM-HG medium and count them, then add 200µL of cell suspension (containing 1×10⁻⁶ cells / well). 5 USMCs Seeds were placed in the upper chamber of a Transwell plate, and the chamber was gently shaken to ensure even distribution of cells. In the lower chamber of a 24-well plate... Add 500µL of serum-free DMEM-HG medium, and divide the mixture into groups according to the groups already containing DMEM-HG medium. Add 100µL of BMSCs-sec at different concentrations to the lower chamber, and then incubate the prepared 24-well plate at 37°C and 5% CO2 for 24 hours.

[0048] (2) Fixation and staining: Gently wash the chamber with PBS, and wipe away any unmigrated cells from the upper chamber surface with a cotton swab, taking care to avoid contact with the bottom membrane. Add 600µL of 4% paraformaldehyde for fixation for 15 min, and wash 3 times with PBS. After air drying, add 600µL of crystal violet to the lower chamber for staining for 20 min, and wash 3 times with PBS to remove unbound dye.

[0049] (3) Imaging and data analysis: Images were taken under a fluorescence microscope at 5 randomly selected fields of view. ImageJ was used to calculate the number of cells that crossed the membrane, count the number of migrating cells, and perform data analysis.

[0050] (iv) Scratch test Scratch assays were performed on USMCs to assess the effect of different concentrations of BMSCs-sec treatment on the cell migration ability of USMCs based on wound healing.

[0051] USMCs are set to 1×10 6 Cells were seeded per well in 6-well plates and cultured routinely until 80% confluence. Longitudinal scratches were made in the center of each well using a 200 μL pipette tip. After washing 1-2 times with PBS to remove cell debris, serum-free DMEM-HG medium containing different concentrations of BMSCs-sec was added to the scratched wells. Images of the USMCs were captured under a microscope at 2, 24, 48, and 72 hours. The remaining scratch area was measured using ImageJ software.

[0052] (v) Tube forming experiment The in vitro angiogenesis-promoting effect of BMSCs-sec was detected using an endothelial cell tube-forming assay, following the methods provided in previous studies. The day before the experiment, the matrix gel was transferred from a -20°C freezer to a 4°C environment and allowed to thaw overnight, with the pipette tip pre-cooled. The following day, 50 μL of matrix gel was added to each well of a 96-well plate, avoiding air bubbles. The plate was then incubated at 37°C for 30 min to allow the matrix gel to solidify. Logarithmic growth phase HUVECs were harvested, digested, and resuspended in serum-free DMEM-HG medium, with 3 × 10⁶ cells per well. 4 Cells were seeded in bulk onto matrix gel. Four groups were included in the experiment, with one group receiving BMSCs-sec at three different concentrations and the other receiving DMEM-HG medium alone (negative control). After cell treatment, the wells were incubated at 37°C and 5% CO2 for 24 hours. Images of the cells were acquired using an EVOS microscope with low magnification.

[0053] (vi) RT-qPCR To simulate an in vitro model of USMC damage under inflammatory conditions, USMCs were treated with LPS (100 ng / mL) for 24 hours, and the expression levels of IL-1α, TNF-α, and Bax genes were detected by RT-qPCR. Primer sequences are shown in Table 1.

[0054] Table 1 Summary of PCR Primer Sequences

[0055] (vii) Hoechst 33342 / propidium iodide (PI) double staining experiment Healthy USMCs were seeded in 96-well plates and stimulated with LPS as described above. After treatment with different concentrations of BMSCs-sec for 24 hours according to the grouping, Hoechst 33342 / propidium iodide (PI) double staining was performed using the same method as before. ImageJ software was used to calculate the number of PI-positive cells, Hoechst 33342 strongly positive cells, and the total number of cells. Cell necrosis and apoptosis rates after treatment with different concentrations of BMSCs-sec were also calculated.

[0056] (viii) Flow cytometry (1) BMDMs marker detection: Cells were washed with pre-cooled PBS. After scraping off the cells, the culture dish was washed with PBS, and as many scattered cells as possible were aspirated. The cell concentration was adjusted to 1×10⁻⁶. 7Cells / mL, 100µL of cell suspension was mixed with fluorescently labeled CD68 antibody, incubated at 4°C in the dark for 30 min, resuspended in PBS, centrifuged at 300×g for 5 min, resuspended in 100µL PBS, and the cell suspension was collected into flow cytometry tubes for detection on a CytoFLEX flow cytometer. The results were analyzed using FlowJo software.

[0057] (2) Effect of BMSCs-sec on macrophage polarization: In order to detect whether BMSCs-sec can affect the phenotype of macrophages, an in vitro cell experiment was first conducted: 10×BMSCs-sec and BMDMs were obtained, and the specific method was the same as before.

[0058] Preparation of induction medium: ① LPS group: LPS (100ng / mL) + RPMI 1640 medium + 10% fetal bovine serum + antibiotics + M-CSF (30ng / mL). ②BMSCs-sec group: LPS (100ng / mL) + RPMI 1640 medium + 10% fetal bovine serum + penicillin antibiotics + M-CSF (30ng / mL) + 10×BMSCs-sec; ③ Blank control. BMDMs were treated with the different culture media described above. After 24 hours, cells were scraped off, a certain amount of PBS was added, and the cells were repeatedly mixed by blowing to adjust the cell concentration to 1×10⁻⁶. 7 Cells / mL were collected, CD86 antibody and CD163 antibody were added, and the mixture was incubated at 4°C in the dark for 30 minutes. Cells were resuspended in PBS, centrifuged at 300×g for 5 minutes, and the cells were collected into flow cytometry tubes. The instrument parameters were adjusted and the cells were analyzed.

[0059] (ix) Immunohistochemical analysis VitroGel 3D-RGD and HBSS were diluted at a 1:2 ratio to prepare a working solution. The VitroGel 3D-RGD working solution was then combined with 10× BMSCs-sec to prepare a BMSCs-sec / Hydrogel soft gel. The soft gel was drawn into a syringe, and a rat uterine injury model was reconstructed. The BMSCs-sec / Hydrogel was injected into the uterine wound. Three days post-surgery, the rat uterus was harvested for CD86 and CD163 immunohistochemical detection using the same method as before. Positive areas at the injury site were quantitatively analyzed using ImageJ software.

[0060] Data statistical analysis in this embodiment of the invention: One-way ANOVA was used for univariate analysis among multiple groups, while two-way ANOVA was used for multivariate analysis. Tukey's post-hoc test was used for multiple comparisons, and t-tests were used for comparisons between two groups. All data are expressed as mean ± standard deviation. Statistical analysis was performed in GraphPad Prism, and a statistical significance level was defined as a p-value less than 0.05.

[0061] Example 1 This embodiment provides a method for preparing BMSCs-sec, a stem cell preparation for repairing scarred uterus. The main steps are as follows: (1) Passage well-grown primary BMSCs to the third generation and collect BMSC cells; (2) BMSCs were divided into groups of 2×10⁻⁶ cells. 6 Cells were seeded in T75 culture flasks and cultured in complete medium (complete medium: DMEM / F12 containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin). When the cell adhesion and confluence reached 80%, the supernatant was discarded, and the cells were washed with PBS. 15 mL of DMEM / F12 was added to each flask (at this point, the cell count was approximately 2 × 10⁶ cells / mL). 7 After culturing the cells for 48 hours, collect the cell supernatant (the cell supernatant contains only a few cell debris and dead cells, and no other cells are present, because live cells are adherent cells and will not float in the liquid), centrifuge at 1000×g for 15 min to remove cell debris and dead cells, filter through a 0.22µm filter, and collect the filtrate. (3) Transfer the filtrate into a 3K ultrafiltration tube, centrifuge at 4°C and 5000×g, and obtain cell supernatants with a concentration of 5 times (5×) and 10 times (10×) by adjusting the centrifugation time. After collection, store at -80°C to obtain the cell preparation BMSCs-sec.

[0062] (4) Detection of BMSCs-sec protein components: Label-free quantitative proteomics analysis was performed on BMSCs-sec, identifying a total of 470 proteins and 2089 peptides, including 195 secretory proteins. These secretory proteins were qualitatively and quantitatively analyzed, and sorted by mean. The top 30 proteins were selected and are shown in Table 2 below.

[0063] Table 2

[0064] Example 2 BMSCs-sec promotes physiological function of USMCs and tube formation of HUVECs in vitro: To further investigate the regulatory role of BMSCs-sec on the major structural cells of the uterus, USMCs, this part of the experiment observed the effects of BMSCs-sec on some physiological functions of USMCs (Figure 1).

[0065] (1) The effect of BMSCs-sec on the proliferation of USMCs was detected by CCK8 assay. The results showed that BMSCs-sec could promote the proliferation of USMCs, and the cell activity was proportional to the concentration of secretory group (Figure 2). (2) The proliferation of USMCs was further verified using the EDU cell proliferation assay, and the results were consistent with those of the CCK8 assay (see results). Figure 3 , Figure 4 ).

[0066] (3) To clarify whether BMSCs-sec can exert a chemotactic effect on USMCs, the effect of different concentrations of secretion groups on USMCs was observed using a Transwell assay to simulate the in vitro environment. The results showed that BMSCs-sec can chemotactically induce migration of USMCs, and the efficiency increases with increasing concentration (see [link to study]). Figure 5 , Figure 6 ).

[0067] Then, simulating an in vitro wound, the cells were "scratched" to observe the effect of BMSCs-sec on the migration ability of USMCs. The results suggested that BMSCs-sec can promote the migration of USMCs. Figure 7 , Figure 8 ).

[0068] (4) The results of the cell tube formation experiment showed that BMSCs-sec promoted HUVEC tube formation in vitro (Figure 9 and 10). Figure 10 ).

[0069] In summary, BMSCs-sec can significantly promote the proliferation, chemotaxis, and migration of USMCs, and promote angiogenesis.

[0070] Example 3 BMSCs-sec reduces necrosis and apoptosis of USMCs: In vitro cell experiments were conducted to simulate tissue and cell damage caused by the inflammatory microenvironment. First, the phenotypic changes of USMCs after LPS treatment were detected. The results showed that after LPS treatment, the expression of inflammation-related genes IL-1α, TNF-α and apoptosis marker Bax were all upregulated (A, B, C in Figure 11).

[0071] Then, by intervening with BMSCs-sec at different concentrations, Hoechst 33342 / PI double staining results suggested that BMSCs-sec can effectively reduce necrosis and apoptosis of damaged USMCs. Figure 12 , Figure 13 Furthermore, highly concentrated BMSCs-sec is more beneficial for the protection of USMCs.

[0072] Example 4 BMSCs-sec regulate macrophage polarization: (1) In order to demonstrate whether BMSCs-sec can affect macrophage phenotype, primary mononuclear cells were extracted from rat bone marrow and induced to differentiate into BMDMs after being induced in a medium containing M-CSF (Figure 14A).

[0073] Under a light microscope, the cells were spindle-shaped and gradually stretched out with prolonged culture time. Flow cytometry analysis showed that CD68-positive cells accounted for 87.3%. Figure 14 B, C).

[0074] In the ground state (GS), the CD86 positivity rate of BMDM was 10.9%. LPS treatment significantly increased the CD86 positivity rate of BMDM to 77.5%, but simultaneous BMSCs-sec treatment significantly reduced the CD86 positivity rate to 46.43%.

[0075] The positivity rate of CD163 in the ground state was 2.802%. LPS treatment had no significant effect on the expression level of CD163, but after BMSCs-sec treatment, the positivity rate of CD163 increased significantly to 39%. Figure 15 , Figure 16 ).

[0076] (2) The regulatory effect of BMSCs-sec on macrophages was observed in animals. First, 10× BMSCs-sec was combined with hydrogel (the hydrogel is commercially known as VitroGel 3D-RGD Hydrogel, a hydrogel product for biomedical research produced by The Well Bioscience, USA. The BMSC-sec / RGD Hydrogel complex helps BMSC-sec adhere to the wound surface). As an ion-crosslinked hydrogel, the mixing of BMSCs-sec and hydrogel (BMSCs-sec and hydrogel were mixed at a volume ratio of 3:1) can initiate the crosslinking process to form a semi-solid BMSCs-sec / Hydrogel soft gel. The BMSCs-sec / Hydrogel soft gel was then injected into the animal model. When the syringe was pushed, the BMSCs-sec / Hydrogel soft gel changed from a semi-solid to a liquid due to mechanical force. After 5 minutes, it could re-gel on the tissue wound, which facilitated drug delivery to the tissue wound.

[0077] Immunohistochemical results showed that in rat uterine wounds, the positive area of ​​CD86, a marker of M1 macrophages, in the BMSCs-sec group was significantly lower than that in the NR group, while the positive area of ​​CD163, a marker of M2 macrophages, was significantly higher in the BMSCs-sec group than in the NR group. Figure 17 and Figure 18 ).

[0078] Comparative Example 1 Effects of primary (P0) and P3 generation BMSCs secretory groups on the migration ability of USMCs: The secretory groups of P0 and P3 generation BMSCs were extracted separately (the specific steps are the same as in Example 1), concentrated 5 times, and then subjected to USMCs scratch test. See results Figure 19 and Figure 20 It can be seen that P3 BMSCs-sec can promote the migration of USMCs, while P0 BMSCs-sec has little impact on the migration capability of USMCs.

[0079] Application Example 1 The constructed BMSC-sec / RGD-hydrogel system was transplanted into a rat uterine injury model to observe the state of uterine wound scars.

[0080] First, a rat full-thickness uterine injury model was constructed ( Figure 21 A) Observing the characteristics of natural healing after uterine injury, it was found that the uterine wound was accompanied by a large number of inflammatory cells infiltrating, and the repair process of each tissue layer was different: the endometrium was repaired by complete and partial regeneration, the myometrium had weak regeneration ability, severe defects, hyalinization of fibrous connective tissue, and a large amount of collagen deposition. The uterine wound showed a pathological change of fibrosis accompanied by chronic inflammation. Figure 21 B).

[0081] The gross appearance of uterine specimens at different time points after injury was observed. It was found that the natural healing group and the gel group were prone to varying degrees of inflammatory reactions and adhesions, resulting in severe tissue damage at the injury site, difficulty in regeneration, and the formation of contracture scars. The gross uterine morphology of the BMSCs-sec / RGD-hydrogel group was closest to that of the normal group. Figure 21 C).

[0082] Table 3 describes the appearance of uterine wound scars in different treatment groups.

[0083] Table 3

[0084] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing a preparation BMSCs-sec for repairing scarred uterus, characterized in that: include, Well-grown primary bone marrow mesenchymal stem cells (BMSCs) were passaged to the third generation, and BMSCs were collected. BMSCs were seeded in culture flasks and cultured in complete culture medium. When the cell adhesion and confluence rate reached 80%, the supernatant was discarded, the cells were washed with PBS, DMEM / F12 was added for culture, the cell supernatant was collected, centrifuged, cell debris and dead cells were removed, filtered, and the filtrate was collected. The filtrate was transferred to an ultrafiltration tube, centrifuged, and concentrated cell supernatant was obtained to produce the formulation BMSCs-sec.

2. The preparation method according to claim 1, characterized in that: The primary BMSCs were derived from rat bone marrow.

3. The preparation method according to claim 1 or 2, characterized in that: The incubation time with DMEM / F12 is 24-48 hours.

4. The preparation method according to claim 3, characterized in that: The centrifugation process removes cell debris and dead cells, wherein the centrifugation speed is 1000×g and the centrifugation time is 15~20min.

5. The preparation method according to claim 4, characterized in that: The filter is used for filtration, wherein the filter particle size is 0.22µm.

6. The preparation method according to claim 1 or 5, characterized in that: The ultrafiltration tube has a specification of 3K.

7. The preparation method according to claim 6, characterized in that: The centrifugation process yields concentrated cell supernatant, wherein the centrifugation temperature is 4°C and the centrifugation speed is 5000×g.

8. The formulation BMSCs-sec prepared by any one of the preparation methods described in claims 1 to 7.

9. The use of the formulation BMSCs-sec according to claim 8 in the preparation of a medicament for repairing scarred uterus.

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