Vagina repair gel containing endometrial stem cells as well as preparation method and application of vagina repair gel

Abstract

The invention discloses an anti-GSK-3beta monoclonal antibody, and endometrial stem cells are prepared on the basis of the anti-GSK-3beta monoclonal antibody. On one hand, endometrial stem cells are combined with a natural polymer matrix material, and the novel vagina repairing gel is prepared. Different from traditional vagina repair gel, the gel provided by the invention not only can provide physical support for damaged tissues, but also can promote tissue regeneration through the action of endometrial stem cells. The vagina repair gel containing the endometrial stem cells provided by the invention has an obvious repair effect, can effectively promote healing of vagina injury, and provides a novel and efficient treatment method for treatment of vagina injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and specifically to a vaginal repair gel containing endometrial stem cells, and its preparation method and application. Background Art

[0002] The vagina is one of the most important structures in the female reproductive system. It not only bears the function of sexual life, but also participates in various physiological functions such as childbirth and urination. However, in many physiological or pathological conditions, the vagina of women may be damaged. Common vaginal injuries include postpartum vaginal lacerations, vaginal atrophy, chronic vaginitis, vaginal repair after surgery, etc. These injuries not only affect the physiological functions of women, but may also bring psychological troubles and affect the quality of life. Therefore, how to quickly, safely and effectively promote the repair of vaginal injuries has become an important research topic in the field of gynecology. At present, the methods for treating vaginal injuries mainly include local medication, surgical treatment and hormone therapy, etc. Local medications are mostly antibiotics, painkillers, anti-inflammatory drugs, etc., which are mainly used to relieve inflammation and pain, but cannot effectively promote the regeneration of damaged tissues. Surgical treatment is usually used to deal with relatively severe vaginal lacerations or deformities, but the surgery itself has certain risks and traumas, and the recovery period is relatively long. Hormone therapy is mainly used to treat vaginal atrophy caused by the decline of estrogen level, but the long-term use of hormones may cause other side effects, such as the risks of breast cancer, endometrial cancer, etc. Therefore, there is an urgent need for a treatment method that can not only effectively promote the repair of vaginal tissues, but also avoid the side effects of traditional treatment methods.

[0003] With the progress of stem cell research, stem cell therapy has gradually become a frontier field of tissue repair and regeneration. Stem cells have the ability of self-renewal and multi-directional differentiation, and can play an important role in repairing damaged or defective tissues. The application scope of stem cells includes multiple fields such as cardiovascular, nerve, bone, etc. In recent years, the research on the repair of the reproductive system by stem cells has also been gradually carried out. Especially, endometrial stem cells (ESCs) have become a very promising therapeutic material due to their advantages such as convenient source, low immunogenicity and strong differentiation potential. Endometrial stem cells are a type of adult stem cells derived from the endometrial tissue of women, and have strong proliferation and differentiation abilities. Research shows that endometrial stem cells can differentiate into various types of cells in vitro, such as epithelial cells, smooth muscle cells, fibroblasts, etc. These characteristics make endometrial stem cells have the potential in vaginal tissue repair.

[0004] Most of the gel products for vaginal repair on the current market contain some common biopolymer materials, such as sodium hyaluronate (HA), gelatin, sodium alginate, etc. These gel matrices can provide a certain moist environment and promote local healing. However, most of these gels are only used to relieve vaginal dryness, pain, inflammation, etc., and do not play an active role in the repair process. Therefore, how to enhance the biological repair function of vaginal repair gels has become a current research hotspot.

[0005] Currently, some studies have attempted to apply stem cells to the gel system and utilize the regenerative ability of stem cells to promote tissue repair. For example, some scholars have studied the use of bone marrow mesenchymal stem cells (MSC) and adipose stem cells (ADSC) for local injection or in vitro culture to promote tissue healing. However, as a new source of stem cells, endometrial stem cells have become the focus of research in recent years because they are closer to the physiological environment of the female reproductive system. Endometrial stem cells can secrete a variety of cytokines and have strong regenerative ability. Therefore, applying them to vaginal repair gels is expected to bring more significant repair effects. Summary of the Invention

[0006] The object of the present invention is to provide a vaginal repair gel containing endometrial stem cells and a preparation method thereof. The gel has good biocompatibility and can promote the self-repair of vaginal tissue through the action of endometrial stem cells. The stem cells in the gel not only help repair damaged vaginal tissue but also promote the regeneration of local tissue by secreting various cytokines.

[0007] Therefore, on the one hand, the present invention discloses an anti-GSK-3β monoclonal antibody, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-GSK-3β monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0008] On the one hand, the present invention also discloses a culture method for endometrial stem cells, and the method includes the following steps:

[0009] (1) Collection and transportation of endometrial tissue;

[0010] (2) Tissue dissociation and cell separation: Wash the collected endometrial tissue with sterile PBS to remove blood and impurities; Cut the endometrial tissue into 1-2 mm using sterile scissors and forceps 3Small pieces; put the cut tissue pieces into a digestive solution containing 2 - 5 mg / mL trypsin, gently shake the container, place it in a 37°C water bath, and digest for 30 - 60 minutes; add DMEM / F12 medium containing 10% fetal bovine serum to the digestive solution to terminate the digestion reaction, and centrifuge the container to remove trypsin; use microfluidic chip technology to efficiently screen and enrich the digested cells.

[0011] (3) Endometrial stem cell culture: Inoculate into a sterile culture flask at a density of 1×10 6 cells / mL, and use DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% penicillin - streptomycin, 1% glutamine, and 5 μg / mL anti - GSK - 3β monoclonal antibody; inoculate the cells into the culture flask, place it in an incubator at 37°C and 5% CO 2 , and culture for 2 - 3 days; change the medium every 2 - 3 days, and observe the cell growth status and morphological changes; check the cell growth and morphology through a microscope to ensure no contamination and cell death; when the cells grow to 70% - 80% confluence, digest the cells with 0.25% trypsin and perform a sub - culture at a ratio of 1:3 to 1:4.

[0012] In one aspect, the present invention also discloses a vaginal repair gel containing endometrial stem cells, and the formula of the gel is as follows:

[0013] (1) Endometrial stem cells: Each milliliter of the gel contains 1×10 6 to 1×10 7 viable cells;

[0014] (2) Gel matrix components

[0015] 1) Sodium hyaluronate: The concentration in the gel is 10 - 20 mg / mL;

[0016] 2) Gelatin: The concentration in the gel is 30 - 50 mg / mL;

[0017] 3) Sodium alginate: The concentration in the gel is 5 - 20 mg / mL;

[0018] (3) Cytokines and growth factors

[0019] 1) Epidermal growth factor: The concentration in the gel is 10 - 50 ng / mL;

[0020] 2) Transforming growth factor β: The concentration in the gel is 1 - 5 ng / mL;

[0021] 3) Platelet - derived growth factor: The concentration in the gel is 10 - 50 ng / mL;

[0022] 4) Matrix metalloproteinase: The concentration in the gel is 1 - 10 ng / mL;

[0023] (4) Other excipients

[0024] 1) Glycerol: The volume ratio in the gel is 3 - 5%;

[0025] 2) Phosphate buffer solution: Used to adjust the pH value of the gel and maintain the physiological stability of the gel. The concentration in the gel is appropriate to ensure that the pH value of the gel remains between 6.8 - 7.4.

[0026] Preferably, the formulation of the gel of the present invention is as follows:

[0027] (1) Endometrial stem cells: Each milliliter of the gel contains 1×10 6 to 1×10 7 viable cells;

[0028] (2) Gel matrix components

[0029] 1) Sodium hyaluronate: The concentration in the gel is 15 mg / mL;

[0030] 2) Gelatin: The concentration in the gel is 40 mg / mL;

[0031] 3) Sodium alginate: The concentration in the gel is 10 mg / mL;

[0032] (3) Cytokines and growth factors

[0033] 1) Epidermal growth factor: The concentration in the gel is 30 ng / mL;

[0034] 2) Transforming growth factor β: The concentration in the gel is 3 ng / mL;

[0035] 3) Platelet-derived growth factor: The concentration in the gel is 25 ng / mL;

[0036] 4) Matrix metalloproteinase: The concentration in the gel is 5 ng / mL;

[0037] (4) Other excipients

[0038] 1) Glycerol: The volume ratio in the gel is 4%;

[0039] 2) Phosphate buffer solution: Adjust the pH value appropriately to ensure that the pH value of the gel remains between 7.0 - 7.2.

[0040] In one aspect, the present invention also discloses the application of the anti-GSK-3β monoclonal antibody in the culture of endometrial stem cells.

[0041] The present invention combines endometrial stem cells with natural polymer matrix materials to prepare a novel vaginal repair gel. Different from traditional vaginal repair gels, the gel of the present invention can not only provide physical support for damaged tissues, but also promote tissue regeneration through the action of endometrial stem cells. This gel can achieve the repair of vaginal injuries through local application and has the following significant advantages:

[0042] (1) Strong biological repair ability: Endometrial stem cells have strong differentiation potential and self-repair ability, and can play an important role in tissue regeneration in damaged tissues.

[0043] (2) Auxiliary role of cytokines and growth factors: The cytokines (such as EGF, TGF-β, PDGF, etc.) added in the gel can promote local cell proliferation, migration and neovascularization, thus accelerating the repair process.

[0044] (3) Good biocompatibility: The matrix components of the gel include natural polymer materials such as sodium hyaluronate, gelatin, sodium alginate, etc., which have good biocompatibility and degradability and are non-irritating to tissues.

[0045] (4) Local treatment: This gel can play a therapeutic role through local vaginal application, avoiding the side effects and traumas of traditional treatment methods.

[0046] Therefore, the vaginal repair gel provided by the present invention has broad application prospects in the treatment of postpartum vaginal injuries, senile vaginal atrophy, postoperative vaginal repair, etc., and can provide a safer and more effective repair solution for women. Brief Description of the Drawings

[0047] Figure 1 Detection results of SDS-PAGE of anti-GSK-3β monoclonal antibody, where 1 is anti-GSK-3β monoclonal antibody. Detailed Embodiments

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0049] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0050] Example 1: Extraction and culture of endometrial stem cells

[0051] 1. Materials and Equipment

[0052] 1.1 Materials: DMEM / F12, basic component supplement, fetal bovine serum (FBS), penicillin-streptomycin, insulin, transferrin, selenium, etc.; cytokines (such as EGF, bFGF); flow cytometry marker antibodies such as CD73, CD90, CD105, CD34, CD45, etc.

[0053] 1.2 Equipment: Microfluidic chip (used for efficient separation and enrichment of endometrial stem cells, improving sorting efficiency and purity); CO 2 Incubator; microscope; biological safety cabinet; centrifuge; cell counter.

[0054] 2. Operation steps

[0055] 2.1 Collection and transportation of endometrial tissue

[0056] (1) Patient selection and consent signing: Endometrial tissue samples were collected from eligible female patients, and the patients were ensured to have signed an informed consent form explaining the sampling process, risks, and privacy protection.

[0057] (2) Aseptic collection: Aseptically obtain endometrial tissue samples through curettage, hysteroscopy, etc. The collected tissue should be immediately transferred to a sterile culture dish containing cold PBS to maintain tissue activity.

[0058] (3) Transportation conditions: Samples should be kept away from air for long periods of time and the temperature should be kept at 4°C during transportation to avoid tissue drying and cell death.

[0059] 2.2 Tissue dissociation and cell separation

[0060] (1) Wash the collected endometrial tissue with sterile PBS to remove blood and impurities; use sterile scissors and forceps to cut the endometrial tissue into 1-2 mm 3 The cut tissue blocks were placed in a digestion solution containing 2-5 mg / mL trypsin, the container was gently shaken, and placed in a 37°C water bath for digestion for 30-60 minutes; DMEM / F12 culture medium containing 10% fetal bovine serum was added to the digestion solution to terminate the digestion reaction, and the container was centrifuged to remove the trypsin. The cell counting results are shown in Table 1.

[0061] (2) Use microfluidic chip technology to efficiently screen and enrich the above-digested cells. Through the microfluidic chip, endometrial stem cells can be precisely sorted according to cell surface markers (CD73, CD90, CD105) to obtain high-purity cells. The sorted cells are centrifuged (1000 rpm, 5 minutes) to remove the supernatant, and then the cells are resuspended with sterile PBS or medium. The cell counting results are shown in Table 1.

[0062] Table 1 Summary of Monitoring Results during the Extraction Process of Endometrial Stem Cells

[0063]

[0064] After the above process, especially the sorting by the microfluidic chip, this study achieved the efficient and specific separation and extraction of endometrial stem cells. Therefore, the application of microfluidic chip technology can improve the purity and yield of endometrial stem cells and avoid non-specific impurities in traditional methods.

[0065] 2.3 Culture of Endometrial Stem Cells

[0066] Inoculate at a density of 1×10 6 cells / mL into a sterile culture flask, and use DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine, and 5 μg / mL anti-GSK-3β monoclonal antibody (as described in Example 2); inoculate the cells into the culture flask and place it in an incubator at 37°C and 5% CO 2 for 2 - 3 days; change the medium every 2 - 3 days and observe the cell growth status and morphological changes; check the cell growth and morphology under a microscope to ensure no contamination and cell death; when the cells grow to 70% - 80% confluence, digest the cells with 0.25% trypsin and perform a subculture operation at a ratio of 1:3 to 1:4. After subculture, use for research or cryopreserve (store in liquid nitrogen for later use).

[0067] 2.4 Cell Identification and Quality Control

[0068] (1) Detection of cell surface markers: Use flow cytometry (FACS) to detect surface markers such as CD73, CD90, CD105, CD34, CD45, etc. to confirm the purity and phenotype of endometrial stem cells. The results are shown in Table 2, and the purity and specificity of endometrial stem cells are very strong.

[0069] Table 2 Summary of Detection Results of Cell Surface Markers

[0070]

[0071] (2) Detection of multi-directional differentiation ability: Induce cells to differentiate into adipose, bone, and cartilage directions, and identify them by Oil Red O staining, alkaline phosphatase staining, and chondroitin sulfate staining. The results are shown in Table 3. The endometrial stem cells prepared by the present invention have good differentiation ability.

[0072] Table 3 Summary of detection results of multi-directional differentiation ability

[0073]

[0074] 2.5 It should be particularly noted that this study compared the culture of endometrial stem cells with and without anti-GSK-3β monoclonal antibody. The results are shown in Table 4. Anti-GSK-3β antibody can be used as an effective regulator in the culture of endometrial stem cells, enhancing the cell proliferation ability and multi-directional differentiation potential, while maintaining its self-renewal ability.

[0075] Table 4 Summary of comparative experimental results of the culture of endometrial stem cells with or without anti-GSK-3β monoclonal antibody

[0076]

[0077] Example 2: Preparation and testing of anti-GSK-3β monoclonal antibody

[0078] GSK-3β (glycogen synthase kinase-3β) is an important kinase that participates in regulating many cellular functions, including cell proliferation, differentiation, and growth, etc. Anti-GSK-3β monoclonal antibody is widely used in the study of GSK-3β-related signaling pathways, especially in stem cell research, cancer research, and neurodegenerative diseases.

[0079] 1. Preparation of anti-GSK-3β monoclonal antibody

[0080] 1.1 Immunize mice: Select GSK-3β recombinant protein (MCE, catalog number: HY-P74114). Mice are immunized once every two weeks for a total of 3 times, with an interval of 14 days. Each immunization injection contains 50 - 100 μg of GSK-3β protein. Use Freund's complete adjuvant (first immunization) and Freund's incomplete adjuvant (subsequent immunizations).

[0081] 1.2 Isolation of spleen cells and fusion with myeloma cells: Spleens were taken from immunized mice, single-cell suspensions were prepared, and washed with RPMI-1640 medium. Cells were counted and red blood cells were removed using red blood cell lysis buffer. The SP2 / 0 myeloma cell line was used, aseptically processed, and the cells were cultured to the logarithmic growth phase. Spleen cells and SP2 / 0 myeloma cells were mixed at a ratio of 5:1. Polyethylene glycol (PEG, molecular weight 1500 - 2000) was added for cell fusion. The fused cells were transferred to HAT selection medium for culture and screening.

[0082] 1.3 Screening and culture of hybridoma cells: Hybridoma cells capable of producing anti-GSK-3β monoclonal antibodies were screened by ELISA. After positive clones were selected, they were individually amplified, and the antibody titers were gradually detected. The amplified hybridoma cells were continuously cultured in RPMI-1640 medium, and the antibody yields were regularly detected.

[0083] 1.4 Antibody purification: The antibody was purified using a Protein A / G column to remove impurities in the culture medium, filtered through a 220 nm filter membrane to sterilize, and aliquoted and stored at -80 °C for later use. The concentration of the purified antibody was determined by the BCA method, and the concentration of this monoclonal antibody was 3.67 mg / mL.

[0084] 2. Examination of anti-GSK-3β monoclonal antibody

[0085] 2.1 SDS-PAGE detection: The purified monoclonal antibody was subjected to SDS-PAGE detection, and the results were as Figure 1 shown. The purity of this monoclonal antibody was very high, reaching over 90%.

[0086] 2.2 Sequence determination: mRNA was extracted from hybridoma cells, specifically amplified, and then sequenced. Through analysis, the amino acid sequences of the heavy chain variable region and light chain variable region of this monoclonal antibody were as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0087] 2.3 ELISA detection: 0.5 μg / mL of recombinant GSK-3β protein was coated on a 96-well plate and incubated overnight at 4 °C. 5% skim milk PBS solution was added and blocked at room temperature for 1 hour. Different concentrations of anti-GSK-3β monoclonal antibody (10 μg / mL, 5 μg / mL, 1 μg / mL, 0.1 μg / mL) were added and incubated at 37 °C for 1 hour. HRP-labeled secondary antibody was added and incubated at room temperature for 30 minutes. TMB substrate was added for a 10-minute color reaction. 2M H 2 SO 4The reaction was terminated. The results showed (Table 5) that the antibody of the present invention exhibited a higher OD value at a lower concentration, indicating its higher affinity.

[0088] Table 5 ELISA Detection Results of Monoclonal Antibodies

[0089]

[0090] Example 3: Vaginal Repair Gel Containing Endometrial Stem Cells

[0091] 1. Endometrial stem cells (stem cells prepared in Example 1): Each milliliter of the gel contains 1×10 6 to 1×10 7 viable cells, specifically adjusted according to needs.

[0092] 2. Gel matrix components

[0093] (1) Sodium hyaluronate (HA): It has good hydration, increases the gel wetness, and promotes cell adhesion and migration. The concentration in the gel is 10 - 20 mg / mL.

[0094] (2) Gelatin: It provides good gelation performance and enhances the flexibility of the matrix. The concentration in the gel is 30 - 50 mg / mL.

[0095] (3) Sodium alginate: A natural polysaccharide with biodegradability, providing physical support and helping tissue repair. The concentration in the gel is 5 - 20 mg / mL.

[0096] 3. Cytokines and growth factors

[0097] (1) Epidermal growth factor (EGF): Promotes the proliferation and migration of epithelial cells, and helps repair the damaged vaginal epithelium. The concentration in the gel is 10 - 50 ng / mL.

[0098] (2) Transforming growth factor β (TGF-β): Promotes the proliferation of fibroblasts and collagen synthesis, and avoids excessive fibrosis. The concentration in the gel is 1 - 5 ng / mL.

[0099] (3) Platelet-derived growth factor (PDGF): Promotes angiogenesis and the migration of fibroblasts, and helps tissue repair. The concentration in the gel is 10 - 50 ng / mL.

[0100] (4) Matrix metalloproteinases (MMPs): Participate in the degradation and remodeling of the extracellular matrix, and promote tissue repair. The concentration in the gel is 1 - 10 ng / mL.

[0101] 4. Other excipients

[0102] (1) Glycerol: It increases the moisture retention of the gel and keeps the local area moist. The volume ratio in the gel is 3 - 5%.

[0103] (2) Phosphate Buffered Saline (PBS): It is used to adjust the pH value of the gel and maintain the physiological stability of the gel. The concentration in the gel is appropriate to ensure that the pH value of the gel remains between 6.8 - 7.4.

[0104] 5. According to the above results, the preferred gel formulation is shown in Table 6.

[0105] Table 6 Vaginal repair gel formulation containing endometrial stem cells

[0106]

[0107] Example 4: Preparation of vaginal repair gel containing endometrial stem cells

[0108] 1. Dissolution of sodium hyaluronate, gelatin, and sodium alginate: Dissolve sodium hyaluronate, gelatin, and sodium alginate separately in PBS buffer, and gently heat to 60 °C until completely dissolved to form a viscous solution.

[0109] 2. Addition of glycerol: Add 3 - 5% glycerol or propylene glycol to the matrix solution and mix well to increase the moisture retention of the gel.

[0110] 3. Addition of growth factors and cytokines: Add EGF, TGF-β, PDGF, and MMPs to the matrix solution and adjust their concentrations according to specific requirements. Ensure that the solution temperature does not exceed 37 °C to avoid affecting the activity of growth factors due to high temperature.

[0111] 4. Addition of endometrial stem cells: Add the endometrial stem cell suspension (concentration of 1×10 6 to 1×10 7 cells / mL) prepared in Example 1 to the above matrix solution and mix evenly.

[0112] 5. Adjustment of pH value: Use PBS to adjust the pH value of the gel solution to the range of 6.8 - 7.4 to ensure its physiological compatibility during local vaginal use.

[0113] 6. Cooling and gelation: Place the final mixed gel solution at room temperature until gelation, and the gel can be adjusted to a solid or semi-solid form as needed.

[0114] 7. Sterile filtration: Filter the gel solution using a 0.22 μm sterile filter to remove possible bacteria or particles and ensure the sterility of the gel.

[0115] 8. Packaging: The prepared gel is filled into sterile packages to ensure its sterility before use. The gel can be stored at 4°C to avoid the influence of high temperature on the activity of stem cells and the stability of growth factors.

[0116] Example 5: Application of a vaginal repair gel containing endometrial stem cells

[0117] 1. Experimental grouping

[0118] (1) Blank group: No treatment, only simulating injury.

[0119] (2) Traditional gel group: Treated with a traditional repair gel (Fuyanjie, feminine antibacterial gel for gentle sterilization, itching relief, and deep cleaning of private parts for maintenance).

[0120] (3) Endometrial stem cell gel group: Treated with a gel containing endometrial stem cells and growth factors (prepared in Example 4).

[0121] (4) Endometrial stem cell gel without factors group: Treated with a gel containing only endometrial stem cells (prepared with the formula of Example 4 but without cytokines), without any growth factors.

[0122] 2. Experimental animals

[0123] (1) Animal species: 20 healthy female SD rats, weighing 180 - 220 g.

[0124] (2) Breeding conditions: Standard breeding conditions, temperature 22 ± 2°C, humidity 50 ± 10%, 12-hour light cycle.

[0125] 3. Experimental procedures

[0126] (1) Establishment of a mild vaginal injury model in rats: The rats were anesthetized with isoflurane (concentration of 3 - 5%). A sterile scalpel was used to make an incision about 1 cm in the vaginal vestibule of the rats to simulate mild injury. According to the random principle, the 20 rats were divided into 4 groups, with 5 rats in each group.

[0127] (2) Treatment operations

[0128] 1) Blank group: No treatment, only simulating injury.

[0129] 2) Traditional gel group: Once a week, the traditional repair gel was applied to the injured area.

[0130] 3) Endometrial stem cell gel group: Once a week, the gel containing endometrial stem cells and growth factors was applied to the injured area.

[0131] 4) Endometrial stem cell - free gel group: Once a week, apply the gel containing only endometrial stem cells to the damaged area.

[0132] 5) Treatment cycle: Treat once a week for 4 consecutive weeks.

[0133] 4. Sampling and processing: After the treatment (4 weeks), euthanize all rats. Take out the tissues from the vaginal damaged area of the rats. The tissues are immediately cryopreserved for subsequent RNA extraction, protein extraction and related analysis.

[0134] 5. PCR detection

[0135] (1) Perform PCR analysis on the tissues from the vaginal damaged area of the rats to quantitatively detect the expression levels of the following genes: TGF-β, EGF, PDGF, Col-I (marker genes related to tissue repair), MMP-9 (genes related to tissue remodeling and degradation).

[0136] (2) Use SYBR Green PCR kit for real-time quantitative PCR, and use GAPDH as the internal reference gene to quantitatively analyze the mRNA expression levels of the target genes. The results show (Table 7) that the expression of oTGF-β, EGF, PDGF, Col-I: In the endometrial stem cell gel group, the expression levels of TGF-β, EGF, PDGF and Col-I are significantly higher than those in other groups, indicating that the combined treatment of stem cells and growth factors in this group effectively promoted tissue repair. Although the expression level of MMP-9 in the endometrial stem cell gel group is slightly lower, it still remains at a medium level, indicating that the tissue remodeling in this group is relatively mild, avoiding excessive degradation and fibrosis.

[0137] Table 7 Summary of PCR detection results

[0138]

[0139] 6. ELISA detection

[0140] (1) The ELISA detection method is used to quantitatively analyze the growth factor levels in the tissues: TGF-β (transforming growth factor β), EGF (epidermal growth factor), PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor).

[0141] (2) The protein concentrations of growth factors such as TGF-β, EGF, PDGF, and VEGF were measured using an ELISA kit. The results showed (Table 8) that the levels of TGF-β, EGF, PDGF, and VEGF were significantly increased in the endometrial stem cell gel group, especially TGF-β, EGF, and PDGF, indicating that these factors played a promoting role in the tissue repair process. The relatively high level of VEGF suggested that angiogenesis played an important role in the repair process, especially in the endometrial stem cell gel group where angiogenesis was significantly enhanced.

[0142] Table 8 Summary of ELISA test results

[0143]

[0144] The data obtained by PCR and ELISA tests indicated that the repair effect of the endometrial stem cell gel group was the most significant, and the expression of growth factors was highly correlated with the tissue repair process. All data were analyzed using SPSS software. Compared with other treatment groups, the endometrial stem cell gel group showed significant statistical differences (p < 0.05). Through one-way analysis of variance (ANOVA), the differences in the expression of growth factors and related genes among groups were statistically significant (p < 0.01).

[0145] 7. Summary

[0146] The endometrial stem cell gel group showed significant advantages in promoting the repair of mild vaginal injuries. The results of PCR and ELISA both indicated that this group promoted tissue repair, remodeling, and angiogenesis by enhancing the expression of growth factors.

[0147] The repair effects of the traditional gel group and the endometrial stem cell gel without factors group were relatively poor. Although some injuries were repaired, the repair process was slow, and the expression levels of growth factors were lower than those in the endometrial stem cell gel group.

[0148] The high expression of growth factors (especially TGF-β, EGF, PDGF, and VEGF) was closely related to the good repair effect of tissues, indicating that the endometrial stem cell gel had the potential to promote repair.

[0149] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An anti-GSK-3β monoclonal antibody, characterized in that: The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-GSK-3β monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. A method for culturing endometrial stem cells, characterized in that: The method comprises the following steps: (1) Collection and transportation of endometrial tissue; (2) Tissue dissociation and cell separation: Use sterile PBS to wash the collected endometrial tissue to remove blood and impurities; use sterile scissors and forceps to cut the endometrial tissue into 1-2 mm 3 The cut tissue blocks are placed in a digestion solution containing 2-5 mg / mL trypsin, the container is gently shaken, and the container is placed in a 37°C water bath for digestion for 30-60 minutes; DMEM / F12 culture medium containing 10% fetal bovine serum is added to the digestion solution to terminate the digestion reaction, and the container is centrifuged to remove trypsin; the cells after the above digestion are efficiently screened and enriched using microfluidic chip technology; (3) Endometrial stem cell culture: 1×10 6 cells / mL in a sterile culture flask, and supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine and 5 μg / mL of the anti-GSK-3β monoclonal antibody according to claim 1 using DMEM / F12 medium; inoculating the cells in the culture flask, placing it in an incubator at 37° C., 5% CO2, and culturing it for 2-3 days; replacing the culture medium every 2-3 days, and observing the cell growth status and morphological changes; examining the cell growth and morphology under a microscope to ensure that there is no contamination and cell death; when the cells grow to 70%-80% confluence, digesting the cells with 0.25% trypsin, and performing a 1:3 to 1:4 passaging.

3. A vaginal repair gel containing endometrial stem cells, characterized in that: The formula of the gel is as follows: (1) Endometrial stem cells prepared according to claim 2: each ml of gel contains 1×10 6 Up to 1×10 7 living cells; (2) Gel matrix components 1) Sodium hyaluronate: concentration in gel is 10-20 mg / mL; 2) Gelatin: The concentration in the gel is 30-50 mg / mL; 3) Sodium alginate: concentration in gel is 5-20 mg / mL; (3) Cytokines and growth factors 1) Epidermal growth factor: The concentration in the gel is 10-50 ng / mL; 2) Transforming growth factor β: concentration in gel is 1-5 ng / mL; 3) Platelet-derived growth factor: concentration in gel is 10-50 ng / mL; 4) Matrix metalloproteinases: concentration in gel is 1-10 ng / mL; (4) Other auxiliary materials 1) Glycerol: 3-5% by volume in the gel; 2) Phosphate buffer: used to adjust the pH value of the gel and maintain the physiological stability of the gel. The concentration in the gel is appropriate to ensure that the pH value of the gel remains between 6.8 and 7.

4.

4. The gel according to claim 3, characterized in that The formula of the gel is as follows: (1) Endometrial stem cells prepared according to claim 2: each ml of gel contains 1×10 6 Up to 1×10 7 living cells; (2) Gel matrix components 1) Sodium hyaluronate: concentration in gel is 15 mg / mL; 2) Gelatin: The concentration in the gel is 40 mg / mL; 3) Sodium alginate: concentration in gel is 10 mg / mL; (3) Cytokines and growth factors 1) Epidermal growth factor: concentration in gel is 30 ng / mL; 2) Transforming growth factor β: concentration in gel is 3 ng / mL; 3) Platelet-derived growth factor: concentration in gel is 25 ng / mL; 4) Matrix metalloproteinases: concentration in gel was 5 ng / mL; (4) Other auxiliary materials 1) Glycerol: 4% by volume in the gel; 2) Phosphate buffer: Adjust the pH value appropriately to ensure that the pH value of the gel remains between 7.0-7.

2.

5. Use of the anti-GSK-3β monoclonal antibody according to claim 1 in culturing endometrial stem cells.

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