Endometrial repair formulations, methods of making and using the same

Abstract

The application provides an endometrial repair preparation and a preparation method and application thereof. The endometrial repair preparation comprises a conditioned culture solution of endometrial-derived cells, the endometrial-derived cells have precursor cell characteristics, and positively express stage-specific embryonic antigens, and help to construct an in-vitro co-culture system which has a positive effect on endometrial injury repair, and are of great significance for studying the pathophysiological repair of endometrium and treating intrauterine adhesion (IUA).

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to endometrial repair agents, their preparation methods, and applications. Background Technology

[0002] Human uterine tissue contains endometrial epithelial stem cells (eESCs) and endometrial mesenchymal stem cells (eMSCs). The proliferation and differentiation of adult stem cells play a crucial role in the repair of damaged tissues and organs. Endometrial repair also requires the participation of both epithelial and mesenchymal stem cells. Therefore, constructing in vitro culture systems for endometrial epithelial and mesenchymal stem cells is of great significance for studying the pathophysiological repair of the endometrium and the treatment of intrauterine adhesions (IUA).

[0003] Endometrial mesenchymal stem cells (eMSCs) have been shown to have the ability to differentiate into multiple lineages, while endometrial epithelial stem cells (eESCs) are difficult to culture in vitro for extended periods using traditional two-dimensional culture methods, thus limiting their functional assays in in vitro studies.

[0004] It is evident that the successful in vitro culture of eESCs is of great significance, not only helping to further understand the origin and repair mechanism of endometrial epithelial cells, but more importantly, helping to expand the cell source of endometrial stem cells. Summary of the Invention

[0005] The purpose of this invention is to provide an endometrial repair agent, its preparation method, and its application. Based on the successful in vitro culture of endometrial epithelial precursor cells and the further acquisition of conditioned culture medium for the endometrial epithelial precursor cells, an in vitro co-culture system with a positive effect on endometrial injury repair was constructed. This is of great significance for studying the pathophysiological repair of the endometrium and the treatment of intrauterine adhesions (IUA).

[0006] To achieve the above objectives, the endometrial repair preparation of the present invention comprises a conditioned culture medium of endometrial-derived cells, which possess precursor cell characteristics and positively express stage-specific embryonic antigens. Its beneficial effects are: the conditioned culture medium derived from endometrial-derived cells with precursor cell characteristics and positive expression of stage-specific embryonic antigens helps to construct an in vitro co-culture system with a positive effect on endometrial damage repair, which is of great significance for studying the pathophysiological repair of the endometrium and the treatment of intrauterine adhesions (IUA).

[0007] Preferably, the stage-specific embryonic antigen is SSEA-1.

[0008] Preferably, the endometrial-derived cells are endometrial epithelial precursor cells or endometrial epithelial precursor-like cells.

[0009] The method for preparing the endometrial repair agent of the present invention includes: providing the endometrial-derived cells, conditioned the endometrial-derived cells using a conditioned medium, and after the conditioned culture is completed, obtaining the conditioned culture supernatant of the endometrial-derived cells from the obtained culture product.

[0010] Preferably, the conditioned medium includes a basal medium and serum-like substances.

[0011] Preferably, the content of the serum-like substances does not exceed 20% based on the volume of the basal culture medium.

[0012] Preferably, the basal culture medium is any one of Hep-X basal medium, DMEM-high glucose medium, DMEM-low glucose medium, DMEM / F12 medium, MEM medium, William's Medium E medium, Ham's F-10 medium, Ham's F-12 medium, IMDM medium, McCoy'5A medium, RPMI-1640 medium, BME medium, M-199 Medium medium, Leibovitz Medium medium, CMRL1066 medium, Neurobasal medium, and Fischers medium.

[0013] Preferably, the endometrial-derived cells are obtained by in vitro culture of human primary endometrial cells expressing positive epithelial cell-related markers in endometrial precursor cell culture medium.

[0014] Preferably, the epithelial cell-related marker is any one of EpCAM, E-Cadherin, CD9, CK19, CD24, and CD44.

[0015] Preferably, the endometrial precursor cell culture medium includes the basal culture medium, growth factors, TGF-β signaling pathway inhibitors, Wnt signaling pathway agonists, ROCK kinase inhibitors, and serum-like substances.

[0016] More preferably, the intima precursor cell culture medium further includes at least one of N-acetyl-L-cysteine ​​and ascorbic acid.

[0017] Preferably, based on the volume of the basal culture medium, the content of sodium pyruvate is 5-20 μg / mL, the content of the growth factor is 5-50 ng / mL, the content of the Rock kinase inhibitor is 0.5-50 μmol / L, the content of the WNT signaling pathway agonist is 0.5-50 μmol / L, the content of the TGF-β signaling pathway inhibitor is 0.5-50 μmol / L, and the content of serum-like substances does not exceed 20%.

[0018] The application of the endometrial repair agent of the present invention in the preparation of drugs for repairing endometrial damage includes co-culturing the endometrial repair agent with endometrial stromal cells or vascular endothelial cells. Attached Figure Description

[0019] Figure 1 This is the flow cytometry analysis result of verifying the sorting efficiency of magnetic beads in the centrifuge tube cell suspension in Example 1 of the present invention;

[0020] Figure 2 This is the flow cytometry analysis result of verifying the sorting efficiency of magnetic beads in the cell suspension to be tested in the sorting tube in Example 1 of the present invention;

[0021] Figure 3 EpCAM of Embodiment 2 of the present invention + A comparison of the expression of EpCAM and SSEA-1 in P0, P1 and P2 cells obtained by flow cytometry detection of surface antibodies;

[0022] Figure 4 EpCAM amplification culture on day 4 using ECM in Example 2 of this invention + Cell light micrograph;

[0023] Figure 5 EpCAM cultured on day 2 using TEM-1 medium in Example 2 of this invention + Cell light micrograph;

[0024] Figure 6 and Figure 7 These are respectively 1-P3-SSEA-1 of Embodiment 3 of the present invention. + Cells and 2-P3-SSEA-1 + Graph showing the trend of OD value changes in cells;

[0025] Figure 8 1-P3-SSEA-1 of Embodiment 3 of the present invention + Light micrograph of clonal clumps formed by cells;

[0026] Figure 9 This is Example 4 of the present invention for 1-P3-SSEA-1 + A comparison of the expression levels of EpCAM, SUSD2, CD34, CD45, CD90, and CD105 obtained by flow cytometry analysis in cells;

[0027] Figure 10 This is Example 4 of the present invention for 1-P3-SSEA-1 + GO biological process clustering analysis diagram of the top 30 differentially expressed genes obtained from high-throughput transcriptome sequencing of cells.

[0028] Figure 11 This is Example 5 of the present invention using SSEA-1 + Comparative photographs of tubule formation observed under a light microscope at 6 and 12 hours after CM treatment of human vascular endothelial cells;

[0029] Figure 12 This is Example 5 of the present invention using SUSD2. + Comparative photographs of tubule formation observed under a light microscope at 6 and 12 hours after CM treatment of human vascular endothelial cells;

[0030] Figure 13 It is SSEA-1 of Embodiment 5 of the present invention. + Cells and SUSD2 + A comparison of the expression levels of VEGFA, TNF, IL-1A and IL-1B in cells;

[0031] Figure 14 This refers to the use of SSEA-1 in Embodiment 5 of the present invention. + -CM applies to SUSD2 + Comparative photographs of scratch healing observed under a light microscope at 0, 6 and 12 hours after the initiation of the scratch experiment;

[0032] Figure 15 This is SUSD2 in Embodiment 5 of the present invention. + -CM applies to SUSD2 + Comparative photographs of scratch healing observed under a light microscope at 0, 6 and 12 hours after the initiation of the scratch experiment;

[0033] Figure 16 It is SSEA-1 of Embodiment 5 of the present invention. + Cells and SUSD2+ A comparison of the expression levels of ITGB2, ITGB4, ITGB7, and NOTCH1 in cells. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art. The terms "comprising" and similar expressions used herein mean that the element or object preceding the word covers the element or object listed after the word and its equivalents, but does not exclude other elements or objects.

[0035] Human uterine tissue contains endometrial epithelial stem cells (eESCs) and endometrial mesenchymal stem cells (eMSCs). The proliferation and differentiation of adult stem cells play a crucial role in the repair of damaged tissues and organs. Endometrial repair also requires the participation of both epithelial and mesenchymal stem cells. Therefore, constructing in vitro culture systems for endometrial epithelial and mesenchymal stem cells is of great significance for studying the pathophysiological repair of the endometrium and the treatment of intrauterine adhesions (IUA).

[0036] Studies have found that eMSCs possess clonogenic and multilineage differentiation capabilities, and can be induced in vitro to differentiate into adipocytes, osteoblasts, chondrocytes, smooth muscle cells, and skeletal muscle cells using different induction media. However, due to the difficulty in obtaining and culturing endometrial epithelial precursor cells in vitro, related research has been scarce to date.

[0037] Therefore, the stable in vitro culture of human endometrial epithelial stem cells provides a cell model for studying the pathophysiological repair of the endometrium, which is of great scientific significance for the regenerative medicine treatment and exploration of IUA.

[0038] In this embodiment of the invention, human endometrial cells were used as seed cells for in vitro culture. The resulting endometrial epithelial precursor-like cells conformed to the characteristics of human endometrial epithelial stem cells and achieved stable in vitro culture.

[0039] In this embodiment of the invention, the endometrial epithelial precursor cells express stage-specific embryonic antigens, and the expression rate of the stage-specific embryonic antigens is not less than 80%.

[0040] In some specific embodiments, the stage-specific embryonic antigen is SSEA-1.

[0041] The method for preparing endometrial epithelial precursor cells according to embodiments of the present invention includes:

[0042] S1: Human primary endometrial cells that express positive epithelial cell-related markers;

[0043] S2: Human primary endometrial cells expressing the positive epithelial cell-related markers were cultured in vitro using endometrial precursor cell culture medium.

[0044] In step S1 of some embodiments of the present invention, the epithelial cell-related marker is any one of epithelial cell markers and epithelial precursor cell markers, and the expression rate is not less than 70%.

[0045] In step S1 of some specific embodiments, the epithelial cell marker is any one of EpCAM, E-Cadherin, CD9, and CK19.

[0046] In step S1 of some specific embodiments, the epithelial precursor cell marker is either CD24 or CD44.

[0047] In some specific embodiments, the stage-specific embryonic antigen is SSEA-1.

[0048] In step S1 of some embodiments of the present invention, the step of obtaining human primary endometrial cells expressing positive epithelial cell-related markers includes: sorting the primary cell suspension containing human endometrial cells by sieve, removing red blood cells by lysis, and sorting the cells to obtain the human primary endometrial cells expressing positive epithelial cell-related markers.

[0049] In some specific embodiments, the cell sorting is performed using immunomagnetic beads.

[0050] In some specific embodiments, the cell sorting is flow cytometry sorting.

[0051] In step S2 of some embodiments of the present invention, the step of culturing human primary endometrial cells expressing positive epithelial cell-related markers in vitro using endometrial precursor cell culture medium includes: using the endometrial precursor cell culture medium at 0.3 × 10⁻⁶... 4 -1×10 5Human primary endometrial cells expressing the positive epithelial cell-related markers were cultured at an inoculation density of cells / cm² until cell confluence was not less than 80%, then digested and passaged using the same endometrial precursor cell culture medium.

[0052] In some embodiments, the subculture ratio is not less than 1:3.

[0053] Furthermore, during the in vitro culture process, the endometrial precursor cell culture medium is replaced every 24-48 hours.

[0054] In this embodiment of the invention, the endometrial epithelial precursor cells obtained through step S2 positively express stage-specific embryonic antigens with an expression rate of not less than 80%.

[0055] In some embodiments, after step S2 is completed, the endometrial epithelial precursor cells expressing the positive stage-specific embryonic antigen are further expanded and cultured using the endometrial precursor cell culture medium.

[0056] In step S2 of some embodiments of the present invention, the endometrial precursor cell culture medium includes basal culture medium, growth factors, TGF-β signaling pathway inhibitors, Wnt signaling pathway agonists, ROCK kinase inhibitors, and serum-like substances.

[0057] Specifically, a reprogramming substance composed of growth factors, Rock signaling pathway inhibitors, WNT signaling pathway agonists, and TGF-β signaling pathway inhibitors efficiently induced endometrial cells to transform into endometrial epithelial precursor-like cells that positively expressed stage-specific embryonic antigens through reprogramming, thereby successfully achieving in vitro culture of endometrial epithelial precursor-like cells.

[0058] In some embodiments, the content of the growth factor is 5-50 nanograms / mL, the content of the Rock kinase inhibitor is 0.5-50 micromoles / L, the content of the WNT signaling pathway agonist is 0.5-50 micromoles / L, the content of the TGF-β signaling pathway inhibitor is 0.5-50 micromoles / L, and the content of the serum-like substances does not exceed 20%, based on the volume of the basal culture medium.

[0059] In some embodiments, the endometrial precursor cell culture medium further includes at least one of N-acetyl-L-cysteine ​​and ascorbic acid.

[0060] In some embodiments, the basal culture medium is any one of MEM, DMEM, BME, DMEM / F12, RPMI1640, CMRL1066, William E, Neurobasal, or Fischers medium.

[0061] In some embodiments, the basal medium is any one of Hep-X basal medium, DMEM-high glucose medium, DMEM-low glucose medium, Ham's F-10 medium, Ham's F-12 medium, IMDM medium, McCoy'5A medium, M-199 Medium, Leibovitz Medium, CMRL1066 medium, Neurobasal medium, and Fischers medium.

[0062] In some embodiments, the growth factor component is at least one of epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, interleukin-6, and tumor suppressor.

[0063] In some embodiments, the Rock signaling pathway inhibitor is at least one of Y27632, Fasudil, Thiazovivin, and SB-772077-B.

[0064] In some embodiments, the WNT signaling pathway agonist is at least one of recombinant WNT protein, recombinant R-spondin protein, BIO, CHIR99021, and TWS119.

[0065] In some embodiments, the TGF-β signaling pathway inhibitor is at least one of A8301, RepSox, and SB431542.

[0066] This invention also provides the application of the endometrial epithelial precursor-like cells in the preparation of drugs related to endometrial injury.

[0067] This invention also provides an endometrial repair preparation comprising a conditioned culture medium of endometrial-derived cells, wherein the endometrial-derived cells have precursor cell characteristics and positively express stage-specific embryonic antigens.

[0068] In some embodiments, the stage-specific embryonic antigen is SSEA-1.

[0069] In some embodiments, the endometrial-derived cells are endometrial epithelial precursor cells or endometrial epithelial precursor-like cells.

[0070] The method for preparing the endometrial repair agent according to the embodiments of the present invention includes: conditioned culture of the endometrial-derived cells using a conditioned culture medium; after the conditioned culture is completed, obtaining the conditioned culture supernatant of the endometrial-derived cells from the obtained culture product; the conditioned culture medium includes a basal culture medium and serum-like substances.

[0071] In some embodiments, the content of the serum-like substance does not exceed 20% of the volume of the basal culture medium.

[0072] In some embodiments, the serum-like substance is animal-derived serum.

[0073] In some embodiments, the animal-derived serum is fetal bovine serum.

[0074] In some embodiments, the animal-derived serum in the in vitro culture medium and the conditioned medium can be replaced with serum substitutes.

[0075] In some embodiments, the serum substitute is platelets and their derivatives that are free of animal-derived components.

[0076] In some embodiments, the serum substitute is sphingosine monophosphate and indoleacetic acid.

[0077] In all embodiments of the present invention, unless otherwise specified, cell culture is carried out in a cell culture incubator at 37 degrees Celsius and a carbon dioxide concentration of 5%.

[0078] In all embodiments of this invention, data involving statistical analysis were performed in at least three replicates per experiment. The experimental results were analyzed using GraphPad Prism 8.0 software. A two-tailed unpaired t-test was used to calculate statistical differences between two groups, and ANOVA was used to calculate statistical differences among multiple groups. A p-value < 0.05 was considered statistically significant.

[0079] Example 1

[0080] Example 1 of this invention provides a method for preparing primary endometrial cells that positively express the epithelial cell marker EpCAM.

[0081] In step S1 of Example 1, human primary endometrial cells expressing EpCAM were obtained using human endometrial tissue as the starting material.

[0082] Specifically, pathological examination of the human endometrial tissue showed that all of them were simple proliferative endometrium, specifically late proliferative endometrium (days 11-12 of menstruation).

[0083] Specifically, the endometrial tissue was obtained from surgical samples taken from female patients no older than 45 years of age. Medical examinations revealed no malignant endometrium, malignant tumors, or malignant lesions of the endometrium. The patients had not used any corticosteroids within three months prior to the surgery. The patients were fully informed about the purpose of obtaining the surgical sample and signed informed consent forms before the surgery.

[0084] First, 1 cubic millimeter volume of endometrial tissue was washed and sterilized using sterile PBS buffer. Then, the endometrial tissue was digested for 60 minutes at 37°C using a 3 mL cell digestion solution consisting of type I collagenase, sterile PBS buffer, and trypsin digestion solution to obtain a primary cell suspension containing human endometrial cells. The sterile PBS buffer and trypsin digestion solution were of equal volume, and the type I collagenase constituted 1% of the modified buffer volume.

[0085] Then, the primary cell suspension was screened using a 100-mesh sterile sieve with the aid of sterile PBS buffer. The filtrate was collected and mucus and undigested tissue were removed to complete the screening process.

[0086] Then, the filtrate is centrifuged and the supernatant is removed. The resulting precipitate is resuspended in erythrocyte lysis equilibration solution and centrifuged again. This process is repeated until no erythrocytes are observed in the cell pellet after the second centrifugation, thus completing the erythrocyte lysis and removal. Specifically, each centrifugation is performed at a rate of 1000g for 3 minutes.

[0087] Finally, the cell pellet obtained after erythrocyte lysis was resuspended in 100 μL of staining buffer, followed by 10 μL of EpCAM primary antibody labeled with magnetic beads, and incubated for 20 minutes at 4°C. The incubation mixture formed after the first incubation was then mixed with 7 μL of EpCAM secondary antibody labeled with magnetic beads and incubated for 15 minutes at 4°C. The suspension formed after the second incubation was sorted using the EasySep magnetic bead sorting poles. Specifically, the sorting tube containing the suspension was placed in the EasySep magnetic bead sorting poles, 2.5 mL of staining buffer was added, and the mixture was allowed to stand at room temperature for 5 minutes. Then, the suspension in the sorting tube was quickly poured into a centrifuge tube, resulting in the centrifuge tube suspension and the remaining sorting tube suspension, thus completing the immunomagnetic bead sorting.

[0088] In Example 1, the cell suspensions obtained from immunomagnetic bead sorting in centrifuge tubes and sorting tubes were centrifuged separately. 100 μL of staining buffer and 5 μL of EpCAM flow cytometry antibody were added to the resulting cell pellets for resuspending, and the pellets were incubated at 4°C for 30 minutes. The supernatant was then discarded, and 600 μL of staining buffer was added to the resulting cell pellets for resuspending in flow cytometry tubes, yielding the cell suspensions for centrifuge tubes and sorting tubes. The EpCAM expression rate of cells in both cell suspensions was analyzed to verify the efficiency of the magnetic bead sorting. The corresponding Isotpye flow cytometry antibody was used as a control in all flow cytometry analyses.

[0089] Figure 1The results are flow cytometry analysis results used in Example 1 to verify the sorting efficiency of magnetic beads in the cell suspension tested in centrifuge tubes. Figure 2 The results are flow cytometry analysis results used in Example 1 to verify the sorting efficiency of magnetic beads in the cell suspension to be tested in the sorting tube.

[0090] Reference Figure 1 and Figure 2 , Figure 1 The flow cytometry results showed that the EpCAM expression rate of the cells in the centrifuge tube was less than 2%, which can be considered as almost no expression of EpCAM. Figure 2 The flow cytometry results showed that the EpCAM positive expression rate of cells in the cell suspension tested in the sorting tube exceeded 97%.

[0091] Therefore, it can be seen that the primary endometrial cells in the centrifuge tube suspension obtained after the sieve sorting, erythrocyte lysis removal, and immunomagnetic bead sorting in Example 1 negatively express EpCAM (abbreviated as EpCAM). - Primary endometrial cells in sorting tube suspension positively expressed EpCAM (abbreviated as EpCAM). + (cells), and the expression rate exceeds 97%.

[0092] Example 2

[0093] Example 2 used an endometrial progenitor cell culture medium (abbreviated as TEM-1 medium) to process the EpCAM cells from Example 1. + Cells were cultured in vitro to obtain endometrial epithelial precursor-like cells that positively expressed SSEA-1 (abbreviated as 1-SSEA-1). + This method successfully achieved stable in vitro culture of endometrial epithelial precursor-like cells.

[0094] The TEM-1 medium used in Example 2 consisted of DMEM / F12 medium, ascorbic acid, sodium pyruvate, HGF, EGF, Y27632, CHIR99021, A8301, S1P and LPA.

[0095] Based on the volume of TEM-1 medium: ascorbic acid content is 10 μg / mL; sodium pyruvate content is 1 mmol / L; HGF content is 20 ng / mL; EGF content is 20 ng / mL; Y27632 content is 10 μmol / L; CHIR99021 content is 3 μmol / L; A8301 content is 1 μmol / L; S1P content is 1 μmol / L; LPA content is 5 μmol / L.

[0096] In Example 2, the steps for in vitro culture using TEM-1 medium are as follows:

[0097] EpCAM+ Cells at 1×10 5 Inoculate cells per square centimeter into a 12-well plate, add 2 ml of TEM-1 medium to each well, and amplify the cells until the confluence is not less than 80%. Then, digest the cells with trypsin for 1-5 minutes, and continue to passage the cells to the third generation using TEM-1 medium.

[0098] To illustrate the effect of TEM-1 medium on EpCAM + The effect of EpCAM and SSEA-1 expression on cells during in vitro culture, as shown in Example 2, on EpCAM obtained after magnetic bead sorting. + Cells, EpCAM + Primary cells (P0 cells) were obtained by expanding and culturing cells in TEM-1 medium until the confluence was not less than 80%. The P0 cells were further cultured in TEM-1 medium for the first passage to obtain the first generation cells (P1 cells), and the second generation cells (P2 cells) were obtained by the second passage. The expression of EpCAM and SSEA-1 in different cells was investigated by flow cytometry.

[0099] Specifically, after discarding the TEM-1 medium, the cells were rinsed with sterile PBS buffer, then digested with trypsin digestion solution, and centrifuged to collect the precipitate. 100 μL of staining buffer was added to the precipitate to resuspend the cells in flow cytometry tubes. Then, 5 μL of the target flow cytometry antibody was added to each tube, and the cells were incubated for 20 minutes. After resuspending the cells in each tube with 6 μL of staining buffer, surface antibody flow cytometry detection was performed. The specific surface antibody flow cytometry detection method is a standard technique for those skilled in the art and will not be elaborated upon here.

[0100] Figure 3 For EpCAM + A comparison of the expression of EpCAM and SSEA-1 in P0, P1 and P2 cells obtained by flow cytometry detection of surface antibodies.

[0101] Reference Figure 3 EpCAM obtained after magnetic bead sorting +In TEM-1 culture, the proportion of cells positively expressing EpCAM reached over 97%, while only about 8% of cells positively expressed SSEA-1. In P0 cells obtained after expansion culture in TEM-1 medium, the proportion of cells positively expressing EpCAM decreased to approximately 50%, while the proportion of cells positively expressing SSEA-1 increased to approximately 50%. In P1 and P2 cells formed after passage, the characteristic marker EpCAM of endometrial epithelial cells was almost not expressed, while the proportion of cells positively expressing SSEA-1 further increased, with the proportion of P2 cells positively expressing SSEA-1 exceeding 80%. Therefore, the TEM-1 culture system not only successfully achieved in vitro culture of human endometrial epithelial cells but also influenced the expression of EpCAM and SSEA-1 in the cells, which is beneficial for obtaining SSEA-1. + cell.

[0102] To illustrate the effect of reprogramming substances in TEM-1 culture medium on EpCAM + Cells to SSEA-1 + The importance of cell transformation is further illustrated in Example 2, which provides a standard culture medium (ECM) consisting of DMEM F / 12, antibiotics, and fetal bovine serum for EpCAM. + Technical protocols for in vitro cell culture were investigated to examine the effect of ECM on EpCAM. + The effects on cells.

[0103] Specifically, the ECM consisted of 445 ml of DMEM F / 12, 5 ml of penicillin antibody, and 50 ml of fetal bovine serum. For specific amplification and culture methods, please refer to the section on in vitro culture of TEM-1 mentioned above.

[0104] On the second day of ECM culture, only about 10-20% of the cells adhered. On the 4th-5th day of ECM culture, a large number of floating cells were observed, indicating that a large number of cells had undergone apoptosis.

[0105] Figure 4 EpCAM amplification culture on day 4 using ECM + Cell light micrograph. Figure 5 EpCAM cultured on day two using TEM-1 medium + Cell light micrograph. See also Figure 4 and Figure 5 Compared to TEM-1 medium, EpCAM + Cell expansion culture under ECM culture showed significantly poor cell growth. Epithelial cell colony morphology was not distinct. EpCAM, on the other hand, showed poor cell growth. +Under TEM culture, the cells are in a vigorous growth state, and typical nest-shaped or rose-shaped epithelial cell colonies can be seen. The cells are large, oval, with large nuclei and prominent nucleoli. In addition, typical stromal cells can be seen adhering between the epithelial cell colonies, which are spindle-shaped or fibrous, and are slightly wider in the middle and pointed at both ends.

[0106] Therefore, it can be seen that the reprogramming material provided in the embodiments of the present invention is effective for EpCAM. + Cell proliferation and SSEA-1 + This is crucial for cell transformation.

[0107] Example 3

[0108] Example 3 used a different endometrial progenitor cell culture medium (abbreviated as TEM-2) for the EpCAM cells from Example 1. + In vitro culture of the cells also yielded endometrial epithelial precursor-like cells positively expressing SSEA-1 (abbreviated as 2-SSEA-1). + cell).

[0109] The TEM-2 medium used in Example 3 consisted of DMEM / F12 medium, ascorbic acid, sodium pyruvate, HGF, EGF, Thiazovivin, BIO, SB431542, S1P and LPA.

[0110] Based on the volume of TEM-1 medium: ascorbic acid content is 10 μg / mL; sodium pyruvate content is 1 mmol / L; HGF content is 20 ng / mL; EGF content is 20 ng / mL; Thiazovivin content is 1 μmol / L; BIO content is 2 μmol / L; SB431542 content is 10 μmol / L; S1P content is 1 μmol / L; LPA content is 5 μmol / L.

[0111] The in vitro culture process using TEM-2 medium in Example 3 differs from that using TEM-1 medium in Example 2 in that: the in vitro culture process described in Example 2 is performed using TEM-2 medium, and the obtained third-generation cells are cultured in TEM-2 medium until the confluence is not less than 80%, and then flow cytometry sorted to obtain 2-SSEA-1. + cell.

[0112] Specifically, the digestion, centrifugation, resuspending, and incubation processes before sorting are detailed in Example 1, outlining the steps before surface antibody flow cytometry detection. SSEA-1 was obtained through flow cytometry sorting using the MoFlo XDP flow cytometry system. + Cells. The specific process of flow cytometry sorting is a conventional technique for those skilled in the art and will not be described in detail here.

[0113] 1-SSEA-1 for Examples 2 and 3 + Cells and 2-SSEA-1 + Cells were passaged to the third generation using TEM-1 and TEM-2 media, respectively, yielding 1-P3-SSEA-1 cells. + Cells and 2-P3-SSEA-1 + Cells. The subculture procedure is the same as in Example 2.

[0114] Two P3-SSEA-1 assays were investigated using a cell viability assay kit (CCK-8). + Cell proliferation capacity. Specifically, this is observed in two types of P3-SSEA-1... + After digestion with trypsin solution, cells were counted. Following counting, the cells were digested again and then divided into 5 × 10⁻⁶ cells. 3 Each well was seeded into a 96-well plate, with 100 μL of the corresponding TEM-1 medium added to each well for incubation. Nine replicates were set up and grouped according to the number of days, with one plate per group for 7 days. Measurements were performed continuously for 7 days at fixed time points. For testing, 10 μL of CCK-8 assay solution was added to each well and mixed thoroughly. The plate was incubated at 37°C for 2 hours, and then the OD value at 450 nm was measured using a microplate reader and recorded. Figure 6 and Figure 7 The 1-P3-SSEA-1 shown respectively + Cells and 2-P3-SSEA-1 + A graph showing the trend of OD values ​​in cells. (Refer to...) Figure 6 and Figure 7 Two types of P3-SSEA-1 + The cells were still in the exponential growth phase after 7 days of TEM culture, demonstrating strong self-renewal and proliferative capabilities.

[0115] The 1-P3-SSEA-1 was examined. + Cellular clonogenic capacity. For 1-P3-SSEA-1 + Cells at 1×10 3 After culturing in 6-well plates at the appropriate seeding density in TEM-1 medium for 14 days, the medium was discarded, and the cell clusters were washed three times with PBS. The cells were then fixed with 4% paraformaldehyde at room temperature. After removing the paraformaldehyde and washing the cell clusters again with PBS, 0.1% crystal violet solution was added for staining at room temperature. Residual dye was rinsed off with PBS, and the clonal clusters were observed under a microscope, photographed, and counted. Figure 8 The 1-P3-SSEA-1 shown + Light micrograph of clonal clumps formed by cell formation. (See reference) Figure 8 1-P3-SSEA-1 +Cytogenetic cells are large, polygonal or tadpole-shaped single-cell clones, distributed in rose-like or nest-like patterns.

[0116] Clonal clumps with at least 50 cells were defined as large clonal clumps. Cell counting indicated that 1-P3-SSEA-1... + The cells were able to form 134±6 large clonal clumps, demonstrating good cloning ability.

[0117] Example 4

[0118] Example 4 examined P3-SSEA-1 + The phenotypic characteristics of the cells further confirm SSEA-1 + The cells are adult stem cells derived from human endometrial epithelium.

[0119] For 1-P3-SSEA-1 + Cells and 2-P3-SSEA-1 + All cells were subjected to flow cytometry detection of surface antibodies. For specific detection methods, please refer to Example 2. The results showed that both P3-SSEA-1 antibodies... + None of the cells expressed characteristic markers of human endometrial mesenchymal stem cells (eMSCs).

[0120] With 1-P3-SSEA-1 + For example, see cells. Figure 9 The 1-P3-SSEA-1 shown + A comparison of the expression levels of EpCAM, SUSD2, CD34, CD45, CD90, and CD105 in cells. SUSD2, CD34, CD45, CD90, and CD105 are characteristic markers of human endometrial mesenchymal stem cells (eMSCs). 1-P3-SSEA-1 + The cells do not express SUSD2, EpCAM, CD34, CD45, CD90, and CD105, indicating that SSEA-1... + The cells are not interstitial cells.

[0121] Additionally, P3-SSEA-1 + The cells did not express EpCAM, further demonstrating the effect of TEM culture on EpCAM. + Cells to SSEA-1 + The crucial role of cell transformation. More importantly, P3-SSEA-1 + The cells do not express the mature somatic marker EpCAM, indicating that P3-SSEA-1 + The cell is in a dedifferentiated state.

[0122] Example 4 for 1-P3-SSEA-1 +The cells underwent high-throughput transcriptome sequencing, yielding results such as... Figure 10 The diagram shows the GO biological process clustering analysis of the top 30 differentially expressed genes. From... Figure 10 As can be seen, SSEA-1 + Cells express more genes related to epithelial differentiation.

[0123] Specifically, the high-throughput transcriptome sequencing was performed by Shanghai Jikai Gene Chemical Technology Co., Ltd., which prepared three biological replicates of the sample, with three technical replicates for each.

[0124] Example 5

[0125] As is common knowledge in the field, angiogenesis and the migration of endometrial stromal cells play a very important role in the process of endometrial repair.

[0126] Example 5 describes the preparation of an endometrial repair agent, namely 1-P3-SSEA-1. + Conditioned culture medium for cells (abbreviated as SSEA-1) + -CM) and investigated its effects on human vascular endothelial cells and human endometrial stromal cells, proving that SSEA-1 + The cells have the ability to induce angiogenesis and promote the migration of endometrial stromal cells, showing therapeutic potential for endometrial regeneration and repair, and can be applied in the preparation of drugs related to endometrial injury.

[0127] SSEA-1 + The preparation method of -CM is as follows: 1-P3-SSEA-1 + Cells at 6×10 5 Inoculate 6-well plates at a density of 1 cell / well, and incubate each well with 2.5 mL of TEM-1 medium until 80% confluence. Then, replace the medium with ECM medium and incubate for 24 hours. Replace the ECM medium completely and collect the supernatant as SSEA-1. + -CM.

[0128] This embodiment also examined endometrial stromal cells (abbreviated as SUSD2). + Cells, derived from Shanghai Celive Biotechnology Co., Ltd., were cultured in conditioned medium (abbreviated as SUSD2). + Effects of -CM on human vascular endothelial cells and on the migration of endometrial stromal cells. SUSD2 + For the preparation method of -CM, please refer to the aforementioned SSEA-1. + Preparation method of -CM.

[0129] SSEA-1 was investigated through tubule formation experiments. +The effect of CM on human vascular endothelial cells is specifically as follows: human vascular endothelial cells are processed at a concentration of 6 × 10⁻⁶. 5 Inoculate at a density of 1 cell / well in 6-well plates, and incubate each well with 2.5 μL of ECM-1 medium until 80% confluence. Then digest with trypsin digestion solution and SSEA-1. + -CM was resuspended in digestion solution and inoculated onto a matrix gel-coated 96-well plate to initiate the tubelet experiment. Tube formation was observed and photographed under a light microscope at 6 and 12 hours after the start of the tubelet experiment.

[0130] SUSD2 was investigated through tube formation experiments. + The steps regarding the effect of CM on human vascular endothelial cells are described in the previous section on investigating SSEA-1 through tubule formation experiments. + -Steps of the action of CM on human vascular endothelial cells.

[0131] Figure 11 Is it using SSEA-1? + Comparative photographs of tubule formation observed under a light microscope at 6 and 12 hours after CM treatment of human vascular endothelial cells. (Reference) Figure 11 SSEA-1 + -CM showed significant angiogenesis as early as 6 hours after application to human vascular endothelial cells, with even denser and more pronounced angiogenesis at 12 hours, indicating the presence of SSEA-1. + -CM can effectively promote tubule formation.

[0132] Figure 12 It uses SUSD2 + Comparative photographs of tubule formation observed under a light microscope at 6 and 12 hours after CM treatment of human vascular endothelial cells. (Reference) Figure 12 SUSD2 + Angiogenesis was observed 6 hours after CM was applied to human vascular endothelial cells, and the angiogenesis became more concentrated and significant at 12 hours.

[0133] Reference Figure 11 and Figure 12 Even SUSD2 + The density and significance of blood vessels 12 hours after CM treatment of human vascular endothelial cells were also significantly lower than those of... Figure 11 The SSEA-1 shown + - The density and significance of blood vessels observed 6 hours after CM treatment of human vascular endothelial cells.

[0134] Furthermore, statistical analysis of the average length of the tubes formed per unit area reveals the following:

[0135] SUSD2 +At 6 hours after CM treatment of human vascular endothelial cells, the average length of tubules formed per unit area was 2 mm, while SSEA-1... + The average tubule length of -CM applied to human vascular endothelial cells reached 18 mm 6 hours after treatment, showing a significant increase (P<0.05).

[0136] SUSD2 + At 12 hours after CM treatment of human vascular endothelial cells, the average length of tubules formed per unit area was 15 mm, while SSEA-1... + The average tubule length of -CM applied to human vascular endothelial cells reached 25 mm 12 hours after treatment, showing a significant increase (P<0.05).

[0137] Furthermore, this embodiment addresses SSEA-1. + Cells and SUSD2 + Cells were analyzed by qPCR to examine the expression of angiogenesis-related factors in the two cell types, and the results were obtained. Figure 13 The SSEA-1 shown + Cells and SUSD2 + A comparison of the expression levels of VEGFA, TNF, IL-1A, and IL-1B in cells. (Refer to...) Figure 13 Compared to SUSD2 + Cells, SSEA-1 + The relative expression levels of angiogenesis-related factors VEGFA, TNF, IL-1A and IL-1B in cells were significantly increased (P<0.001).

[0138] In conclusion, SSEA-1 + -CM has a stronger ability to promote tubule formation than SUSD2. + -CM.

[0139] SSEA-1 was examined through a scratch test. + -CM to SUSD2 + The role of cells. Specifically, SUSD2 + Cells at 1×10 6 Inoculate at a density of 1 cell / well on a 6-well plate, adding 2.5 μL of SSEA-1 to each well. + - Culture in CM for 24 hours until cell confluence is greater than 90%; use a 10 μL pipette tip to make 3 scratches per well in a 6-well plate, wash twice with PBS, and then use SSEA-1. + -CM culture was used to initiate the scratch assay. Scratch healing was observed and recorded under a light microscope at 0, 12, and 24 hours after the initiation of the scratch assay. Figure 14 The example shown uses SSEA-1 +-CM applies to SUSD2 + Comparative photographs of scratch healing observed under a light microscope at 0, 6 and 12 hours after the initiation of the scratch experiment.

[0140] SUSD2 was examined through a scratch test. + -CM to SUSD2 + The role of cells. For specific steps, please refer to the previous section on investigating SSEA-1 through scratch assay. + -CM to SUSD2 + The experimental steps for cell action were obtained. Figure 15 SUSD2 shown + -CM applies to SUSD2 + Comparative photographs of scratch healing observed under a light microscope at 0, 6 and 12 hours after the initiation of the scratch experiment.

[0141] Reference Figure 14 and Figure 15 Whether it is SSEA-1 + -CM applies to SUSD2 + Cells or SUSD2 + -CM applies to SUSD2 + In the cell initiation scratch assay, cell migration was clearly observed for 12 hours after initiation, indicating that SSEA-1... + -CM and SUSD2 + -CM can promote the migration of endometrial stromal cells. Among them:

[0142] SUSD2 + -CM applies to SUSD2 + In the cell-initiated scratch assay, the average percentage of scratch-healed area obtained at 6 hours after initiation was 25%, while SSEA-1 + -CM applies to SUSD2 + The percentage of scratch-healed area obtained by cell-initiated scratch assay at 6 hours after initiation was 62% on average, which was significantly increased (P<0.05).

[0143] SUSD2 + -CM applies to SUSD2 + In cell-initiated scratch assays, the average percentage of scratch-healed area was 60% at 12 hours post-initiation, while SSEA-1... + -CM applies to SUSD2 + The percentage of scratch-healed area obtained by cell-initiated scratch assay at 12 hours after initiation was 90% on average, which was significantly increased (P<0.05).

[0144] Furthermore, this embodiment addresses SSEA-1. + Cells and SUSD2 + Cells were subjected to qPCR detection to examine the expression of migration-related factors in the two cell types, and the results were obtained. Figure 16 The SSEA-1 shown + Cells and SUSD2 + A comparison of the expression levels of ITGB2, ITGB4, ITGB7, and NOTCH1 in cells. (Refer to...) Figure 16 Compared to SUSD2 + Cells, SSEA-1 + The relative expression levels of cell migration-related factors ITGB2, ITGB4, ITGB7 and NOTCH1 were significantly increased (P<0.01).

[0145] In summary, compared to SUSD2 + -CM, SSEA-1 + -CM has a stronger promoting effect on the migration of endometrial stromal cells.

[0146] Using SSEA-1 + -CM to SUSD2 + Cells were cultured, and SSEA-1 was further investigated using qPCR. + -CM to SUSD2 + The effect of cellular fibrosis-related factors on SSEA-1 demonstrates + SUSD2 cultured in CM + The mRNA expression of TGF-β1, β-catenin, COL1A, and αSMA in cells was lower than that in SUSD2. + SSEA-1 was significantly downregulated at the cellular level (P<0.05). This indicates that SSEA-1... + Cells have the ability to inhibit SUSD2 + The ability to express cell fibrosis-related factors.

[0147] Specifically, SUSD2 + Cells at 6×10 5 Seeds were inoculated into 6-well plates, with 2 ml of TEM added to each well. When the confluence reached approximately 80%, the plates were divided into two groups: one group continued to be cultured with ECM, and the other group was cultured with SSEA-1. + Cultured in CM medium with complete daily medium changes, RNA was extracted after 7 days for qPCR testing. See Table 1 for the specific downregulation levels of fibrosis-related factors. This demonstrates SSEA-1... + SUSD2 cultured in CM + The mRNA expression of TGF-β1, β-catenin, COL1A, and αSMA in cells was lower than that in SUSD2.+ SSEA-1 was significantly downregulated at the cellular level. This indicates that SSEA-1... + Cells have the ability to inhibit SUSD2 + The ability to express cell fibrosis-related factors.

[0148] Table 1

[0149]

[0150] The internal standard used in the qPCR test in this embodiment is β-actin, and all primers are from Shanghai Sangon Biotech Co., Ltd.

[0151] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for preparing an endometrial repair agent, characterized in that, Includes the following steps: Step S1 involves sorting the primary cell suspension containing human endometrial cells using a sieve, removing red blood cells through lysis, and further sorting the cells to obtain human primary endometrial cells positively expressing epithelial cell-related markers, wherein the epithelial cell-related marker is EpCAM. Step S2, use endometrial precursor cell culture medium at 0.3 × 10⁻⁶. 4 -1×10 5 Human primary endometrial cells expressing the positive epithelial cell-related markers were cultured at an inoculation density of cells / cm² until cell confluence was not less than 80%, then digested and passaged in the same endometrial precursor cell culture medium at a passage ratio of not less than 1:3 to obtain endometrial epithelial precursor-like cells. These cells positively expressed stage-specific embryonic antigen (SSEA-1) at an expression rate of not less than 80%. Step S3: Conditioning the endometrial epithelial precursor-like cells with a conditioned medium. After the conditioned culture is completed, the conditioned culture supernatant of the endometrial epithelial precursor-like cells is obtained from the culture product. The conditioned medium includes basal medium and serum-like substances. The intima precursor cell culture medium is selected from TEM-1 medium or TEM-2 medium. The TEM-1 medium consisted of DMEM / F12 medium, ascorbic acid, sodium pyruvate, HGF, EGF, Y27632, CHIR99021, A8301, S1P, and LPA, with the following concentrations per unit volume of TEM-1 medium: ascorbic acid 10 μg / mL; sodium pyruvate 1 mmol / L; HGF 20 ng / mL; EGF 20 ng / mL; Y27632 10 μmol / L; CHIR99021 3 μmol / L; A8301 1 μmol / L; S1P 1 μmol / L; and LPA 5 μmol / L. The TEM-2 medium consists of DMEM / F12 medium, ascorbic acid, sodium pyruvate, HGF, EGF, Thiazovivin, BIO, SB431542, S1P, and LPA, with the following concentrations per unit volume of TEM-2 medium: ascorbic acid 10 μg / mL; sodium pyruvate 1 mmol / L; HGF 20 ng / mL; EGF 20 ng / mL; Thiazovivin 1 μmol / L; BIO 2 μmol / L; SB431542 10 μmol / L; S1P 1 μmol / L; and LPA 5 μmol / L.

2. The method for preparing the endometrial repair agent according to claim 1, characterized in that, The basal culture medium is any one of Hep-X basal medium, DMEM-high glucose medium, DMEM-low glucose medium, DMEM / F12 medium, MEM medium, William's Medium E medium, Ham's F-10 medium, Ham's F-12 medium, IMDM medium, McCoy'5A medium, RPMI-1640 medium, BME medium, M-199 Medium medium, Leibovitz Medium medium, CMRL1066 medium, Neurobasal medium, and Fischers medium.

3. An endometrial repair preparation, prepared by the method of the endometrial repair preparation according to claim 1 or 2.

4. The application of the endometrial repair agent as described in claim 3 in the preparation of drugs for repairing endometrial damage, characterized in that, This includes co-culturing the endometrial repair agent with endometrial stromal cells or vascular endothelial cells.

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