Endometrial stem cell preparation for cervical hypertrophy and repair of villi

Abstract

This invention discloses an endometrial stem cell preparation and its application for cervical hypertrophy and cuticle repair, belonging to the field of stem cell technology. The endometrial stem cell preparation includes a stem cell gel carrier, endometrial stem cells, auxiliary repair peptides, and a survival enhancer. In the endometrial stem cell preparation, the stem cell gel carrier, endometrial stem cells, auxiliary repair peptides, and survival enhancer work synergistically to improve the survival rate of endometrial stem cells in vivo. The auxiliary repair peptides can activate the MAPK / ERK pathway of endometrial stem cells, and the anti-inflammatory mimic peptides in the survival enhancer can inhibit the stress silencing of endometrial stem cells, thereby ensuring the secretory function of endometrial stem cells. Furthermore, the three-dimensional porous structure of the stem cell gel carrier enhances the targeted repair ability of endometrial stem cells and enhances the homing and colonization of endometrial stem cells to cervical lesions.

Description

Technical Field

[0001] This invention belongs to the field of stem cell technology, specifically relating to endometrial stem cell preparations and their applications for cervical hypertrophy and cuticle repair. Background Technology

[0002] Cervical hypertrophy is a common chronic cervical disease in women, usually caused by recurrent episodes of chronic cervicitis or persistent infection by pathogens. It manifests as proliferative changes in the glands and stroma of the cervical tissue, leading to an enlarged and hardened cervix. Long-term cervical hypertrophy not only affects women's reproductive health and increases the risk of infertility, but may also induce complications such as cervical erosion, cervical polyps, and even precancerous lesions of the cervix, seriously impacting patients' quality of life and mental health. Squamous cell lesions (referring to eversion of cervical columnar epithelium or erosion-like changes) are a pathological condition on the surface of the cervix, characterized by eversion or irregular proliferation of columnar epithelium at the external os of the cervix, often accompanied by contact bleeding, abnormal vaginal discharge, and other symptoms. Statistics show that the incidence of cervical hypertrophy and squamous cell lesions is high in women of reproductive age, seriously affecting their quality of life and reproductive health.

[0003] Currently, clinical treatments for cervical hypertrophy and cervical scaling mainly include medication, physical therapy, and surgery. However, medication often fails to completely eliminate the root cause of chronic inflammation and is prone to drug resistance; physical therapy can remove diseased tissue, but may damage the normal structure of the cervix, affecting fertility; while surgery is more invasive, has a slower recovery time, and carries the risk of recurrence.

[0004] In recent years, stem cell therapy, as an emerging biological treatment method, has shown great potential in tissue repair and regeneration. Among them, endometrial mechanicmal stem cells (EnSCs) are adult stem cells with multipotent differentiation potential and high self-renewal capacity, primarily derived from endometrial tissue. Endometrial stem cells have low immunogenicity, which can reduce immune rejection in allogeneic transplantation, improving the safety and efficacy of treatment. Simultaneously, endometrial stem cells possess strong tissue repair capabilities, secreting various growth factors and cytokines to promote tissue regeneration and repair. Furthermore, endometrial stem cells also have multiple functions, including inhibiting fibrosis, promoting angiogenesis, and regulating immune function. Although endometrial stem cells hold great potential in the treatment of cervical hypertrophy and squamous cell lesions, the survival rate and homing efficiency of endometrial stem cell preparations applied in vivo remain key factors limiting their efficacy. Low survival rate directly leads to a sharp reduction in the number of viable cells reaching the target site and functioning; low homing efficiency makes it difficult for the limited number of viable cells to effectively accumulate at the lesion site, failing to form a sufficient number and density of cell populations to secrete adequate bioactive substances or participate in tissue remodeling. The combined effect of low survival rate and low homing efficiency means that endometrial stem cell preparations require high cell doses to achieve barely visible effects in practical applications, thus increasing treatment costs and potential risks, and severely affecting the stability, reproducibility, and long-term prognosis of the treatment effect. To address these issues, we propose an endometrial stem cell preparation and its application for the repair of cervical hypertrophy and squamous cell lesions. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing endometrial stem cell preparations and their applications for cervical hypertrophy and cuticle repair.

[0006] This invention is achieved by providing an endometrial stem cell preparation for cervical hypertrophy and cuticle repair. The endometrial stem cell preparation includes a stem cell gel carrier, endometrial stem cells attached to the gel carrier, auxiliary repair peptides, and a survival enhancer. Endometrial stem cells can secrete anti-inflammatory cytokines, anti-inflammatory chemokines, and growth factors, promoting the proliferation and differentiation of cervical epithelial cells, repairing damaged cervical mucosal epithelium, and inhibiting abnormal expansion of cervical glands. Furthermore, endometrial stem cells possess multi-directional differentiation potential, and under specific microenvironments, they can differentiate into cervical epithelial cell-like cells or stromal repair cells, thereby supplementing the missing / damaged functional cells in the damaged cervical tissue, thus achieving cervical hypertrophy and cuticle repair. The stem cell gel carrier provides stable three-dimensional support for the endometrial stem cells and auxiliary repair peptides, maintaining their performance while the survival enhancer delays their retention time in the cervix / hair follicle. During cervical hypertrophy repair, high expression of fibronectin (FN) can promote cell migration and enhance the homing and colonization of endometrial stem cells to cervical lesions. Fibronectin (FN) binds to integrins on the surface of endometrial stem cells, activating the FAK / PI3K / AKT pathway, thereby enhancing the motility of endometrial stem cells and significantly increasing their enrichment efficiency at cervical lesions. Simultaneously, high expression of fibronectin (FN) can inhibit TGF-β1 fibrosis, reverse abnormal cervical stroma proliferation, and act as exosomes to directly target local cervical immune cells, promoting their M2 polarization and reducing the secretion of pro-inflammatory factors. High expression of hyaluronidase can regulate the cuticle microenvironment by degrading hyaluronic acid (HA) and synergistically crosslink keratin, which is crucial for maintaining tissue hydration and elasticity, contributing to cuticle repair and regeneration. Ultimately, endometrial stem cells, auxiliary repair peptides, and survival enhancers work synergistically. Endometrial stem cells provide the functional basis, auxiliary peptides enhance regulation, and survival enhancers ensure functional continuity. This achieves repair from the cellular level to the microenvironment level and then to the tissue level. It not only solves the bottleneck of low in vivo survival rate and poor homing efficiency of endometrial stem cells, but also promotes the repair of cervical hypertrophy and cuticles through a synergistic mechanism.

[0007] Furthermore, the present invention provides an endometrial stem cell preparation for cervical hypertrophy and squamous cell repair, the endometrial stem cell preparation comprising: a stem cell gel carrier, endometrial stem cells attached to the stem cell gel carrier, a repair-aiding polypeptide, and a survival enhancer, wherein the sequence of the repair-aiding polypeptide is shown in SEQ ID NO: 1, and the endometrial stem cell density in the endometrial stem cell preparation is 2 × 10⁻⁶. 6The concentration of the stem cell gel carrier was 25 mg / mL, the concentration of the auxiliary repair peptide was 15 mg / mL, the concentration of the survival enhancer was 8 mg / mL, and the concentration of the stem cell gel carrier was 25 mg / mL.

[0008] In this invention, the survival enhancer comprises the following components in weight percentage: anti-inflammatory mimic peptide: 15%; insulin-like growth factor-1: 20%; vitamin C: 10%; superoxide dismutase: 10%; curcumin: 5%; trehalose: 40%; the sequence of the anti-inflammatory mimic peptide is shown in SEQ ID NO: 2.

[0009] The method for preparing the anti-inflammatory mimic peptide includes:

[0010] Based on the target function of anti-inflammatory mimic peptides, a mimic peptide library was synthesized. The mimic peptide library was synthesized in solid phase using Rink Amide resin. During the synthesis, Rink Amide resin was placed in a reaction vessel, and an equal volume of DMF solution was added for swelling treatment for 1 hour. Then, it was washed three times with DCM solution, with each washing time being 30 seconds.

[0011] The amino acid sequences protected by Fmoc in the mimic peptide library were eluted with DMF solution containing 20% ​​piperidine to obtain amino acid sequences without Fmoc groups. The amino acid sequences after Fmoc removal were then coupled with Rink Amide resin using HBTU / DIEA coupling reagent to prepare anti-inflammatory mimic peptide-resin complexes.

[0012] The anti-inflammatory mimic peptide-resin complex was washed with methanol to obtain the washed anti-inflammatory mimic peptide-resin complex. The anti-inflammatory mimic peptide-resin complex was dried and then cleaved with a cleaving fluid. During the cleavage process, the stirring speed was 600 rpm and the stirring time was 50 min. After the cleavage process, a crude precipitate was precipitated and then filtered through a filter membrane. The precipitate was then frozen with liquid nitrogen to obtain the frozen anti-inflammatory mimic peptide.

[0013] The frozen anti-inflammatory mimic peptide was sterilized by gamma ray and then placed in physiological saline at pH 7 to obtain the anti-inflammatory mimic peptide.

[0014] The method for preparing the auxiliary repair peptide provided by this invention is similar to that for anti-inflammatory mimic peptides. Furthermore, the auxiliary repair peptide and the anti-inflammatory mimic peptide can be modified using conventional methods in the art. Such modifications can maintain the corresponding activity of the auxiliary repair peptide and the anti-inflammatory mimic peptide, or give them better cervical tissue repair properties.

[0015] The present invention also provides a method for preparing a survival rate enhancer, the method comprising:

[0016] Place trehalose powder in twice the volume of deionized water and stir for 10 minutes at a speed of 220-240 rpm. After stirring, let stand for 5 minutes to obtain a trehalose solution for later use.

[0017] Add vitamin C powder to an equal volume of PBS buffer and stir at 100-120 rpm. Then add superoxide dismutase lyophilized powder to the PBS buffer and stir at 50 rpm for 10 min. Finally, add the pre-prepared curcumin suspension to the PBS buffer and stir at 300 rpm to obtain an antioxidant mixture for later use.

[0018] Take the pre-prepared anti-inflammatory mimic peptide and insulin-like growth factor-1, dissolve the anti-inflammatory mimic peptide and insulin-like growth factor-1 in the same volume of PBS buffer as the mixture of anti-inflammatory mimic peptide and insulin-like growth factor-1, stir at 250 rpm for 20 min to obtain the active mixed component.

[0019] The antioxidant mixture and trehalose solution were placed in a reaction vessel and stirred for 15 minutes. Then, the pre-prepared active mixture was added along the wall and stirred at 150 rpm for 15 minutes to obtain a coarse mixture with enhanced survival rate.

[0020] A survival enhancer is prepared by filtering a coarse mixture containing a survival enhancer using a PES filter membrane. The purpose of the survival enhancer is to optimize the release rate or stabilize the drug. The survival enhancer may also include, for example, sugars, salts, amino acids, and bile salts. Specifically, sugars used herein include, for example, glucose, mannitol, lactose, trehalose, sucrose, erythritol, sorbitol, and xylitol; with glucose, mannitol, and lactose being preferred. Salts used herein include, for example, sodium chloride, potassium chloride, and calcium chloride; with sodium chloride being preferred. Amino acids used herein may include leucine, isoleucine, valine, proline, phenylalanine, methionine, tryptophan, serine, glutamine, threonine, cysteine, asparagine, tyrosine, aspartic acid, glutamic acid, lysine, arginine, and histidine; with lysine and arginine being preferred.

[0021] The present invention also provides a method for preparing curcumin, the method comprising:

[0022] Turmeric was harvested after 9-10 months of cultivation. The main rhizome of turmeric was cut to obtain turmeric root. The turmeric root was washed three times with deionized water and dried in a 60℃ oven for 2 hours. The dried turmeric root was then ground in a grinder and passed through an 80-mesh sieve to obtain turmeric powder.

[0023] Turmeric powder and turmeric extract were mixed at a material-to-liquid ratio of 1:10. The turmeric extract was an ethanol-deionized water mixture with a volume ratio of 3:2. The turmeric powder and turmeric extract were refluxed and extracted three times at 70°C. After extraction, the extracts were filtered and combined. The extracts were concentrated under reduced pressure at 40°C to obtain crude curcumin extract.

[0024] Take D101 macroporous resin, soak it in anhydrous ethanol solution, then pack the soaked D101 macroporous resin into a column using a wet method, rinse with deionized water until the effluent is clear and free of turbidity, soak the cleaned D101 macroporous resin in hydrochloric acid solution for 3 hours, and rinse with deionized water until neutral.

[0025] Curcumin crude extract was purified by adsorption using D101 macroporous resin, and the adsorbed D101 macroporous resin was eluted with 70% ethanol solution to prepare curcumin.

[0026] In this invention, the stem cell gel carrier comprises the following components by weight percentage: gelatin: 18%; glycerol: 8%; phosphate buffer: 4%; ferulic acid: 6%; polyglycerol sebate: 14%; chitosan: 13%; silk fibroin: 25%; trehalose: 12%.

[0027] Preferably, the method for preparing the stem cell gel carrier includes:

[0028] Gelatin and silk fibroin were placed in a reaction vessel, and then phosphate buffer was added. The mixture was stirred in a water bath at 65°C for 20 minutes at a stirring speed of 300-320 rpm to obtain the first intermediate carrier.

[0029] After mixing trehalose and ferulic acid, add 10 times the total volume of deionized water to the mixture. Vortex the mixture until the trehalose and ferulic acid are dissolved. Stir with a magnetic stirrer for 5 minutes and then sonicate for 10 minutes to obtain the second intermediate carrier.

[0030] Polyglycerol sebacic acid ester was dissolved in 3 times its volume of anhydrous ethanol and magnetically stirred at 45°C for 15 min to obtain a polyglycerol sebacic acid ester solution. The polyglycerol sebacic acid ester solution was added dropwise to the first intermediate carrier and sheared and emulsified at a speed of 2000 rpm during the addition. After the addition was completed, the mixture was stirred for 10 min to obtain the first emulsion mixture.

[0031] The second intermediate carrier is mixed with the first emulsion mixture and stirred at 200 rpm for 15 min to obtain the second emulsion mixture.

[0032] Take chitosan and glycerol, then prepare a 2% chitosan solution using glacial acetic acid as solvent, add glycerol to the chitosan solution, stir at 85℃ for 15 min, and then filter the insoluble matter using a filter membrane to obtain a chitosan-glycerol mixture;

[0033] The chitosan-glycerol mixture and the second emulsion mixture were placed in a reaction vessel and stirred at 200 rpm for 20 min to obtain a stem cell gel carrier. The stem cell gel carrier was then placed in a -80℃ low temperature freezer for 4 h to obtain a solidified stem cell gel carrier.

[0034] On the other hand, the present invention also provides a method for preparing an endometrial stem cell preparation, the method comprising:

[0035] Take the solidified stem cell gel carrier, add sterile PBS solution to the stem cell gel carrier, vortex for 10 min to obtain stem cell gel carrier solution, place the stem cell gel carrier solution in a 37℃ water bath for 15 min to obtain activated stem cell gel carrier solution.

[0036] The auxiliary repair peptide and the survival enhancer were mixed at a volume ratio of 1:1 and stirred at room temperature for 10 min to obtain the auxiliary repair peptide-survival enhancer mixture. The auxiliary repair peptide-survival enhancer mixture was added to the stem cell gel carrier solution and stirred magnetically for 10 min at a stirring speed of 40 rpm to obtain the stem cell gel carrier loaded with the auxiliary repair peptide.

[0037] Endometrial stem cells were treated at a constant temperature of 37°C for 10 minutes, and then resuspended in physiological saline. The endometrial stem cells were seeded into a stem cell gel carrier loaded with helper repair peptides, mixed by pipetting, and then allowed to stand at 37°C for 15 minutes to obtain the endometrial stem cell preparation.

[0038] Furthermore, the present invention also provides the use of endometrial stem cell preparations for cervical hypertrophy and cuticle repair in the preparation of drugs for cervical hypertrophy repair.

[0039] Furthermore, the present invention also provides the use of endometrial stem cell preparations for cervical hypertrophy and cuticle repair in the preparation of cuticle repair drugs.

[0040] Furthermore, the present invention provides cervical hypertrophy repair drugs and cuticle repair drugs that also contain pharmaceutically acceptable carriers.

[0041] Furthermore, the cervical hypertrophy repair drugs and cuticle repair drugs are in the form of tablets, pills, pellets, capsules, etc. The tablets, pills, pellets, and capsules may also contain binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, colorants, flavoring agents, etc.; preservatives, solvents, stabilizers, etc. for injectable formulations; and bases, diluents, lubricants, preservatives, etc. for topical formulations. In addition to the above types of substances, it may also contain a liquid carrier, such as vegetable oil or polyethylene glycol. Various other substances may exist as coatings or further modify the physical form of the solid unit dosage form.

[0042] Those skilled in the art can determine the appropriate dosage and range of the pro-repair peptides and anti-inflammatory mimics used, for example, based on in vitro and / or in vivo testing and / or other knowledge of compound dosage. These factors are well known to those skilled in the art and can be resolved simply through routine experiments. In some embodiments, a maximum dose is used, i.e., the highest safe dose based on reasonable medical judgment.

[0043] Compared with the prior art, the embodiments of this application have the following main advantages:

[0044] This invention provides an endometrial stem cell preparation and its application for cervical hypertrophy and cuticle repair. The endometrial stem cell preparation includes a stem cell gel carrier, endometrial stem cells attached to the stem cell gel carrier, auxiliary repair peptides, and a survival enhancer. Endometrial stem cells can secrete anti-inflammatory cytokines, anti-inflammatory chemokines, and growth factors, promoting the proliferation and differentiation of cervical epithelial cells, repairing damaged cervical mucosal epithelium, and inhibiting abnormal expansion of cervical glands. Endometrial stem cells also have multi-directional differentiation potential and can differentiate into cervical epithelial cell-like cells or stromal repair cells under specific microenvironments, thereby supplementing the missing / damaged functional cells in damaged cervical tissue and achieving cervical hypertrophy and cuticle repair. The stem cell gel carrier can provide stable three-dimensional support for endometrial stem cells and auxiliary repair peptides, maintaining the performance of endometrial stem cells and auxiliary repair peptides while delaying the retention time of endometrial stem cells and auxiliary repair peptides in the cervix / hair follicle through the survival enhancer. During cervical hypertrophy repair, high expression of fibronectin (FN) can promote cell migration and enhance the homing and colonization of endometrial stem cells to cervical lesions.

[0045] In this embodiment of the invention, the survival rate enhancer is composed of anti-inflammatory mimic peptides, insulin-like growth factor-1, vitamin C, superoxide dismutase, curcumin, and trehalose. The antioxidant components, namely vitamin C, superoxide dismutase, and curcumin, can eliminate reactive oxygen species generated during the preparation and storage of endometrial stem cell preparations, thereby avoiding the problem of low survival rate of endometrial stem cells due to oxidative stress. The anti-inflammatory mimic peptides can inhibit the inflammatory stress response of endometrial stem cells themselves, thereby avoiding the problem of endometrial stem cells ceasing to secrete functional molecules due to stress silencing. Furthermore, the survival rate enhancer can directly form a uniform cell-carrier-active ingredient complex system with endometrial stem cells and stem cell gel carriers, improving the uniformity and delivery efficiency of the preparation.

[0046] In this embodiment of the invention, curcumin can inhibit the secretion of TNF-α and IL-1β by local macrophages in the cervix, thereby reducing the production of ROS at the source. At the same time, curcumin can also directly remove the ROS that have been generated, alleviate the oxidative stress of the inflammatory microenvironment, improve the survival rate of endometrial stem cells, and ensure the secretory function of endometrial stem cells, thereby inhibiting cervical hypertrophy caused by fibrosis. In addition, it can work synergistically with hyaluronidase during cuticle repair to improve the cuticle microenvironment.

[0047] In this embodiment of the invention, the stem cell gel carrier in the endometrial stem cell preparation works synergistically with endometrial stem cells, auxiliary repair peptides, and survival enhancers to improve the survival rate of endometrial stem cells in vivo. The auxiliary repair peptides can activate the MAPK / ERK pathway of endometrial stem cells, and the anti-inflammatory mimic peptides in the survival enhancers can inhibit the stress silencing of endometrial stem cells, thereby ensuring the secretory function of endometrial stem cells. Furthermore, the three-dimensional porous structure of the stem cell gel carrier enhances the targeted repair ability of endometrial stem cells. Attached Figure Description

[0048] Figure 1 The figure shows the results of testing the relative expression levels of fibronectin FN in endometrial stem cells by different types of peptides.

[0049] Figure 2 The graph shows the results of the relative expression levels of hyaluronidase for different types of peptides.

[0050] Figure 3 The results of the tests on endometrial stem cell loading rate, auxiliary repair peptide loading rate, and endometrial stem cell survival rate in the stem cell gel carrier are shown in the figure.

[0051] Figure 4 The diagram shows the test results of the transverse diameter of the cervix in tests 1-4 in an embodiment of the present invention.

[0052] Figure 5 The diagram shows the test results of cervical appearance score and cervical squamous epithelium appearance score for tests 1-4 in an embodiment of the present invention.

[0053] Figure 6 A schematic diagram showing the collagen percentage test results of Test 1-Test 4 in an embodiment of the present invention is shown.

[0054] Figure 7 The diagram shows the test results of the relative expression levels of fibronectin FN and hyaluronidase in Tests 1-4 of the present invention. Detailed Implementation

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0056] Example 1

[0057] Preparation of anti-inflammatory mimic peptides: In this embodiment of the invention, the method for preparing the anti-inflammatory mimic peptides includes:

[0058] S101, based on the target function of anti-inflammatory mimic peptides, a mimic peptide library was synthesized. The mimic peptide library was synthesized in a solid phase using Rink Amide resin. During the synthesis, the Rink Amide resin was placed in a reaction vessel, and an equal volume of DMF solution was added for swelling treatment for 1 hour. Then, it was washed three times with DCM solution, with each washing time being 30 seconds. DMF, as a polar aprotic solvent, can fully expand the polymer skeleton of the resin, thereby exposing the internal amino reaction sites. The core of Rink Amide resin is the amide bond that connects the Fmoc-protected amino group. Only after swelling can the reagents penetrate evenly. If the swelling is insufficient, the subsequent coupling reaction will be incomplete, resulting in a decrease in product yield.

[0059] It should be noted that the simulated peptide library in this embodiment refers to an artificially designed short peptide library that can replace natural molecules to perform specific functions. Its essence is a functional modular tool, and by screening a short peptide in the library, an anti-inflammatory simulated peptide that can act precisely on the target can be obtained.

[0060] S102, the amino acid sequence protected by Fmoc in the simulated peptide library was eluted with DMF solution containing 20% ​​piperidine to obtain the amino acid sequence after the Fmoc group was removed. Then, the amino acid sequence after the Fmoc group was removed was coupled with Rink Amide resin using HBTU / DIEA coupling reagent to prepare the anti-inflammatory simulated peptide-resin complex.

[0061] S103, the anti-inflammatory mimic peptide-resin complex was washed with methanol to obtain the washed anti-inflammatory mimic peptide-resin complex. The anti-inflammatory mimic peptide-resin complex was dried and then cleaved using a cleaving fluid, which could be a mixture of deionized water, triisopropylsilane, and trifluoroacetic acid. During cleavage, the stirring speed was 600 rpm and the stirring time was 50 min. After cleavage, a crude precipitate formed, which was then filtered through a filter membrane and frozen with liquid nitrogen to obtain the frozen anti-inflammatory mimic peptide. Note that the TFA in the cleavage fluid is volatile and highly corrosive; adding cold diethyl ether precipitates the anti-inflammatory mimic peptide. After filtration, the crude peptide is obtained. Liquid nitrogen freezing can rapidly terminate the peptide chain degradation reaction and facilitates subsequent pulverization.

[0062] S104 involves sterilizing the frozen anti-inflammatory mimic peptide with gamma rays, then placing the anti-inflammatory mimic peptide in physiological saline at pH 7 to obtain the anti-inflammatory mimic peptide. The physiological saline at pH 7 has the same osmotic pressure as human body fluids, which can maintain the conformational stability of the anti-inflammatory mimic peptide.

[0063] In this embodiment, after the anti-inflammatory mimic peptide is prepared, it is subjected to primary screening, secondary screening and final screening. Anti-inflammatory mimic peptides that highly express fibronectin FN, associated mimic peptide 1, associated mimic peptide 2, associated mimic peptide 3 and invalid mimic peptides can be found from the mimic peptide library. The sequence of the anti-inflammatory mimic peptide finally obtained is shown in SEQ ID NO: 2.

[0064] Example 2

[0065] The preparation of auxiliary repair peptides can be carried out using the method for preparing anti-inflammatory mimic peptides as described in Example 1. In this embodiment, the method for preparing auxiliary repair peptides includes constructing an auxiliary repair peptide library, which can also be performed using Rink Amide resin for solid-phase synthesis. The constructed auxiliary repair peptide library is an artificially designed collection of short peptides capable of specific repair functions. It is a modular functional tool, and through screening, auxiliary repair peptides that precisely target repair sites can be obtained. After the auxiliary repair peptides are prepared, they undergo primary screening, secondary screening, and final screening to identify auxiliary repair peptides that highly express hyaluronidase, associated repair peptide 1, associated repair peptide 2, associated repair peptide 3, and ineffective repair peptides. The final sequence of the selected auxiliary repair peptides is shown in SEQ ID NO: 1.

[0066] Example 3

[0067] In this embodiment of the invention, the survival rate enhancer comprises the following components in weight percentage: anti-inflammatory mimic peptide: 15%; insulin-like growth factor-1: 20%; vitamin C: 10%; superoxide dismutase: 10%; curcumin: 5%; trehalose: 40%; the sequence of the anti-inflammatory mimic peptide is shown in SEQ ID NO: 2.

[0068] The method for preparing the survival enhancer includes:

[0069] S201. Take trehalose powder and place it in twice the volume of deionized water and stir for 10 minutes at a speed of 220-240 rpm. After stirring, let it stand for 5 minutes to obtain a trehalose solution. Trehalose has good chemical stability and can maintain its activity and function in subsequent preparation processes.

[0070] S202: Add vitamin C powder to an equal volume of PBS buffer and stir at 100-120 rpm. Then add superoxide dismutase lyophilized powder to the PBS buffer and stir at 50 rpm for 10 min. Finally, add the pre-prepared curcumin suspension to the PBS buffer and stir at 300 rpm to obtain an antioxidant mixture. Vitamin C, superoxide dismutase (SOD) and curcumin have synergistic antioxidant effects, which can effectively scavenge free radicals and reduce oxidative stress.

[0071] S203, take the pre-prepared anti-inflammatory mimic peptide and insulin-like growth factor-1, dissolve the anti-inflammatory mimic peptide and insulin-like growth factor-1 in the same volume of PBS buffer as the mixture of anti-inflammatory mimic peptide and insulin-like growth factor-1, stir at 250 rpm for 20 min to obtain the active mixed component. The combination of anti-inflammatory mimic peptide and insulin-like growth factor-1 can play a synergistic role, enhancing the anti-inflammatory and cell proliferation-promoting effects. The anti-inflammatory mimic peptide can reduce the apoptosis of endometrial stem cells by inhibiting TGF-β1 fibrosis signaling, while insulin-like growth factor-1 promotes the proliferation of endometrial stem cells by activating the PI3K / AKT pathway. The two work together to achieve dual protection of endometrial stem cells against apoptosis and promote proliferation.

[0072] S204, take the antioxidant mixture and trehalose solution and put them into the reaction vessel and stir for 15 min. Then add the pre-prepared active mixture to the wall and stir at 150 rpm for 15 min to obtain the survival rate enhanced coarse mixture.

[0073] S205 is prepared by filtering a coarse mixture with a survival rate enhancement agent using a PES filter membrane.

[0074] In this embodiment of the invention, the survival rate enhancer is composed of anti-inflammatory mimic peptides, insulin-like growth factor-1, vitamin C, superoxide dismutase, curcumin, and trehalose. The antioxidant components, namely vitamin C, superoxide dismutase, and curcumin, can eliminate reactive oxygen species generated during the preparation and storage of endometrial stem cell preparations, thereby avoiding the problem of low survival rate of endometrial stem cells due to oxidative stress. The anti-inflammatory mimic peptides can inhibit the inflammatory stress response of endometrial stem cells themselves, thereby avoiding the problem of endometrial stem cells ceasing to secrete functional molecules due to stress silencing. Furthermore, the survival rate enhancer can directly form a uniform cell-carrier-active ingredient complex system with endometrial stem cells and stem cell gel carriers, improving the uniformity and delivery efficiency of the preparation.

[0075] In this embodiment of the invention, the method for preparing curcumin includes:

[0076] S2021. Turmeric is harvested after 9-10 months of planting. The main rhizome of turmeric is cut to obtain turmeric root. The turmeric root is washed 3 times with deionized water and dried in a 60℃ oven for 2 hours. The dried turmeric root is then ground in a grinder and passed through an 80-mesh sieve to obtain turmeric powder.

[0077] S2022, turmeric powder and turmeric extract were mixed at a material-liquid ratio of 1:10, wherein the turmeric extract was an ethanol-deionized water mixture with a volume ratio of 3:2. The turmeric powder and turmeric extract were refluxed and extracted 3 times at 70℃. After the extraction was completed, the extracts were filtered and combined. The extracts were concentrated under reduced pressure at 40℃ to obtain crude curcumin extract.

[0078] S2023, take D101 type macroporous resin, soak D101 type macroporous resin in anhydrous ethanol solution, then pack the soaked D101 type macroporous resin into a column by wet method, rinse with deionized water until the effluent is clear and free of turbidity, soak the cleaned D101 type macroporous resin in hydrochloric acid solution for 3 hours, and rinse with deionized water until neutral. Among them, the impurities (polysaccharides, proteins) in the crude curcumin extract cannot enter the resin pores due to their large molecular weight and are eluted, while curcumin is adsorbed by the resin.

[0079] S2024, the crude curcumin extract was purified by adsorption using the treated D101 macroporous resin, and the adsorbed D101 macroporous resin was eluted with 70% ethanol solution to prepare curcumin.

[0080] In this embodiment of the invention, curcumin can inhibit the secretion of TNF-α and IL-1β by local macrophages in the cervix, thereby reducing the production of ROS at the source. At the same time, curcumin can also directly remove the ROS that have been generated, alleviate the oxidative stress of the inflammatory microenvironment, improve the survival rate of endometrial stem cells, and ensure the secretory function of endometrial stem cells, thereby inhibiting cervical hypertrophy caused by fibrosis. In addition, it can work synergistically with hyaluronidase during cuticle repair to improve the cuticle microenvironment.

[0081] Example 4

[0082] Endometrial stem cell culture: Adult magnetic rats were selected and fasted for 10 hours before the procedure. A longitudinal incision was made in the midline of the abdomen of the anesthetized rats using a sterilized scalpel under aseptic conditions to expose the uterus. A uterine horn sample was obtained from the cervix. Fatty tissue near the uterine horn was removed to expose the cervix. The cervical sample was washed and then minced using sterile ophthalmic scissors. The cervical sample was mixed with 0.3% trypsin, centrifuged, and the supernatant was discarded to obtain digested cervical tissue. The digested cervical tissue was then filtered through a membrane. The cells were filtered to obtain a cell dispersion. Physiological saline was added to the cell dispersion, and the mixture was centrifuged at 2500 rpm for 15 min. The supernatant was discarded, and physiological saline was added. The mixture was then centrifuged again at 3000 rpm for 20 min. The supernatant was discarded, and the centrifuged cell pellet was resuspended in endometrial stem cell culture medium and incubated at 37°C until the fourth generation of cells was obtained. Endometrial stem cells were then detected by flow cytometry, and the results showed that all results were negative. Endometrial stem cells were successfully isolated and cultured from the surface.

[0083] Endometrial stem cells were collected to prepare an endometrial stem cell suspension, which was then divided into experimental groups 1-10 and blank groups 1-2. The suspension was administered at a concentration of 2×10⁻⁶ cells / mL. 6Endometrial stem cell suspension was aliquoted into 96-well plates at a density of 200 μL per well. The plates were then aseptically treated and placed in a CO2 incubator at 37°C and 5% CO2 concentration. Anti-inflammatory mimic peptides, associated mimic peptide 1, associated mimic peptide 2, associated mimic peptide 3, and null mimic peptides prepared in Example 1 were added to the endometrial stem cell suspensions of experimental groups 1, 2, 3, 4, and 5 at a dosage of 12 μg / mL per well. Blank group 1 received no treatment. Endometrial stem cells were cultured in the plates for 4 days. Cells from each well were then collected into 4 mL LEP tubes and analyzed by Western spectroscopy. The relative expression level of fibronectin (FN) was detected by blot (with blank group 1 as a reference). Then, the auxiliary repair peptides, associated repair peptides 1, 2, 3, and ineffective repair peptides prepared in Example 2 were introduced into the endometrial stem cell suspensions of experimental groups 6, 7, 8, 9, and 10 at a dosage of 12 μg / mL per well. Blank group 2 was left untreated. The endometrial stem cells in the culture plate were cultured for 4 days, and the cells in each well were collected into 4 mL LEP tubes. The relative expression level of hyaluronidase was detected (with blank group 2 as a reference). Table 1 and Figure 1 Table 2 shows the results of testing the relative expression levels of fibronectin (FN) in endometrial stem cells by different types of peptides. Figure 2 The results of the relative expression levels of hyaluronidase for different types of peptides are shown.

[0084] Table 1

[0085]

[0086] As shown in Table 1, the relative expression level of fibronectin FN, the anti-inflammatory mimic peptide, was the highest in experimental group 1, and was significantly higher than that in experimental groups 2-5 and blank group 1. The key driving factor of cervical hypertrophy is the overactivation of the TGF-β1 / Smad pathway: TGF-β1 induces fibroblasts to transform into myofibroblasts and secrete a large amount of type I / III collagen, leading to cervical stromal fibrosis and tissue hardening. Fibronectin FN can bind to TGF-β1 or its receptor, thereby blocking TGF-β1-induced Smad2 / 3 phosphorylation and inhibiting myofibroblast differentiation. As a major component of endometrial stem cell exosomes, high expression of fibronectin FN enhances the paracrine repair effect of endometrial stem cells.

[0087] Table 2

[0088]

[0089] As shown in Table 2, the relative expression level of hyaluronidase in experimental group 6 was significantly higher than that in blank group 2, indicating that the auxiliary repair peptides can effectively promote the expression of hyaluronidase. Since hyaluronidase plays an important role in the remodeling of the extracellular matrix and cell migration, its high expression helps to improve the microenvironment, thereby promoting tissue repair. Moreover, the high expression of hyaluronidase can degrade hyaluronic acid in the extracellular matrix, reduce the viscosity of the matrix, improve the microenvironment, thereby helping cell migration and nutrient transport, and promoting cuticle repair.

[0090] Example 5

[0091] Preparation of endometrial stem cell preparation: In this embodiment of the invention, the endometrial stem cell preparation comprises: a stem cell gel carrier, endometrial stem cells attached to the stem cell gel carrier, a repair-enhancing peptide, and a survival enhancer. The sequence of the repair-enhancing peptide is shown in SEQ ID NO: 1. The endometrial stem cell density in the endometrial stem cell preparation is 2 × 10⁻⁶. 6 The concentration of the stem cell gel carrier was 25 mg / mL, the concentration of the auxiliary repair peptide was 15 mg / mL, the concentration of the survival enhancer was 8 mg / mL, and the concentration of the stem cell gel carrier was 25 mg / mL.

[0092] In this embodiment of the invention, the stem cell gel carrier comprises the following components by weight percentage: gelatin: 18%; glycerol: 8%; phosphate buffer: 4%; ferulic acid: 6%; polyglycerol sebate: 14%; chitosan: 13%; silk fibroin: 25%; trehalose: 12%.

[0093] The method for preparing the stem cell gel carrier includes:

[0094] S301, place gelatin and silk fibroin in a reaction vessel, then add phosphate buffer, and stir in a water bath at 65℃ for 20 minutes at a stirring speed of 300-320 rpm to obtain the first intermediate carrier.

[0095] S302, after mixing trehalose and ferulic acid, add 10 times the total volume of deionized water to the mixture, vortex until the trehalose and ferulic acid are dissolved, start the magnetic stirrer to stir for 5 minutes, and use ultrasonic treatment for 10 minutes to obtain the second intermediate carrier.

[0096] S303, dissolve polyglycerol sebacic acid in 3 times the volume of anhydrous ethanol, stir magnetically for 15 min at 45°C to obtain polyglycerol sebacic acid solution, add the polyglycerol sebacic acid solution dropwise to the first intermediate carrier, and shear emulsify at a speed of 2000 rpm during the dropwise addition, and continue stirring for 10 min after the dropwise addition is completed to obtain the first emulsion mixture;

[0097] S304, the second intermediate carrier is mixed with the first emulsion mixture and stirred at 200 rpm for 15 min to obtain the second emulsion mixture;

[0098] S305, take chitosan and glycerol, then prepare a 2% chitosan solution with glacial acetic acid as solvent, add glycerol to the chitosan solution, stir at 85℃ for 15 min, and then filter the insoluble matter with a filter membrane to obtain a chitosan-glycerol mixture;

[0099] S306, the chitosan-glycerol mixture and the second emulsion mixture are placed in a reaction vessel and stirred at 200 rpm for 20 min to obtain a stem cell gel carrier. The stem cell gel carrier is then placed in a -80℃ low temperature freezer for 4 h to obtain a solidified stem cell gel carrier.

[0100] In this embodiment of the invention, the gelatin in the stem cell gel carrier has good biocompatibility and cell adhesion, which can provide initial adhesion sites for endometrial stem cells. Silk fibroin can form a porous network, thereby maintaining the stable morphology of the stem cell gel carrier and avoiding the problem of rapid degradation after injection. Ferulic acid has antioxidant and anti-inflammatory activities, which can reduce oxidative stress damage to endometrial stem cells and enhance their ability to secrete IL-10. Polyglycerol sebacic acid is a biodegradable emulsifier that forms a stable emulsion structure through shear emulsification, which can slow down the degradation rate of the gel and ensure the continuous action of endometrial stem cells at the target site (cervix / hair follicle).

[0101] In this embodiment of the invention, the method for preparing the endometrial stem cell preparation includes:

[0102] S10, take the solidified stem cell gel carrier, add sterile PBS solution to the stem cell gel carrier, vortex for 10 min to obtain stem cell gel carrier solution, place the stem cell gel carrier solution in a 37℃ water bath for 15 min to obtain activated stem cell gel carrier solution.

[0103] S20, the auxiliary repair peptide and the survival enhancer are mixed at a volume ratio of 1:1 and stirred at room temperature for 10 min to obtain the auxiliary repair peptide-survival enhancer mixture. The auxiliary repair peptide-survival enhancer mixture is added to the stem cell gel carrier solution and magnetically stirred for 10 min at a stirring speed of 40 rpm to obtain the stem cell gel carrier loaded with the auxiliary repair peptide.

[0104] S30. Endometrial stem cells were treated at a constant temperature of 37°C for 10 min in a water bath. Then, the endometrial stem cells were resuspended in physiological saline. The endometrial stem cells were seeded into a stem cell gel carrier loaded with helper repair peptides. After mixing by pipetting, the mixture was allowed to stand at 37°C for 15 min to obtain the endometrial stem cell preparation.

[0105] In this embodiment of the invention, the stem cell gel carrier in the endometrial stem cell preparation works synergistically with endometrial stem cells, auxiliary repair peptides, and survival enhancers to improve the survival rate of endometrial stem cells in vivo. The auxiliary repair peptides can activate the MAPK / ERK pathway of endometrial stem cells, and the anti-inflammatory mimic peptides in the survival enhancers can inhibit the stress silencing of endometrial stem cells (avoiding inflammatory factors from inhibiting their secretory function), thereby ensuring the secretory function of endometrial stem cells. Furthermore, the three-dimensional porous structure of the stem cell gel carrier enhances the targeted repair ability of endometrial stem cells.

[0106] The stem cell gel carrier prepared in Example 5 of this invention was used, and the stem cell gel carrier was divided into gel carrier 1-gel carrier 5. Then, commercially available gel 1 was used as a control carrier. Gel carrier 1-gel carrier 5 and the control carrier were packaged into 96-well plates. The formulation and preparation method of gel carrier 1-gel carrier 5 were the same. 2×10⁻⁶ endometrial stem cell suspension was used. 6 The density of cells / well was determined, and endometrial stem cell suspension, auxiliary repair peptide, and survival enhancer were injected into the wells corresponding to gel carriers 1-5 and the control carrier, respectively. The culture plates were then aseptically treated and placed in a CO2 incubator at 37°C and 5% CO2 concentration. Endometrial stem cells were cultured for 7 days to obtain gel solutions loaded with endometrial stem cells and auxiliary repair peptides. The endometrial stem cell loading rate, auxiliary repair peptide loading rate, and endometrial stem cell survival rate were calculated using a cell counter and HPLC methods. (Table 3 and...) Figure 3 The results of tests on endometrial stem cell loading rate, auxiliary repair peptide loading rate, and endometrial stem cell survival rate in stem cell gel carriers are shown.

[0107] Table 3

[0108]

[0109] As can be seen from Table 3, the porous structure of the stem cell gel carrier prepared in the embodiments of the present invention provides sufficient adhesion sites for endometrial stem cells, improves the cell adhesion ability of endometrial stem cells, and enhances the colonization ability of endometrial stem cells. The interpenetrating network structure (gelatin-silk fibroin-chitosan) of the gel carrier can adsorb peptides through hydrogen bonds and electrostatic interactions, and the emulsification effect of polyglycerol sebate forms a stable emulsion, which delays the release of peptides and further ensures the long-term effect of the endometrial stem cell preparation.

[0110] Example 6

[0111] Safety Testing of Auxiliary Repair Peptides and Anti-inflammatory Mimic Peptides: In this implementation case, 300 female SD rats were selected as experimental subjects and randomly assigned to 12 experimental groups, with 25 rats in each group. These 12 groups of SD rats were further subdivided into high-dose, medium-dose, and low-dose groups for the auxiliary repair peptides and anti-inflammatory mimic peptides. Simultaneously, 6 control groups using PBS solvent were included in this embodiment to comprehensively assess and compare abnormal toxic reactions in each group, including changes in body weight and overall health status. Twenty-four hours after administration, detailed dermatopathological examination, blood biochemical index measurements, and toxicity assessments of vital organs such as the liver, kidneys, and spleen were performed on the experimental animals. The results showed that with prolonged treatment time, the high-dose peptide group exhibited a more significant improvement in therapeutic efficacy, with significantly better efficacy than other dose groups and the control group. Further dermatopathological examination of the rats confirmed that no abnormal pathological changes such as edema or erythema were observed in the skin, and the squamous endothelial cell layer structure remained intact. Blood biochemical index results were all within the normal range, indicating that peptide treatment did not cause significant metabolic or physiological disorders. Meanwhile, histological examination of major organs revealed no obvious toxic damage or pathological changes, confirming the good safety of the repair-aiding peptides and anti-inflammatory mimic peptides under experimental conditions.

[0112] Example 7

[0113] The efficacy of endometrial stem cell preparations for cervical hypertrophy and cuticle repair was verified in a rat model. First, a rat model of cervical hypertrophy and cuticle damage was established using 600 naked female rats aged 10-12 weeks. The experimental temperature was room temperature. Before the experiment, the rats in the cervical hypertrophy and cuticle damage rat model group had free access to food and water. During the establishment of the cervical hypertrophy and cuticle damage rat model, the rats were intraperitoneally injected three times a week with phenol gel and lipopolysaccharide for four consecutive weeks to simulate a hormonal imbalance environment, inducing the proliferation of cervical stromal fibroblasts and collagen deposition, thus obtaining the cervical hypertrophy and cuticle damage rat model. Behavioral tests and histological examinations confirmed successful model establishment. The successfully modeled rats were then divided into the following groups for testing:

[0114] Test 1: Starting on the seventh day after surgery, endometrial stem cells were injected into the cervical region of the rat model group with cervical hypertrophy and cuticle damage. The injections were given every two days for a total of 4 weeks.

[0115] Test 2: Starting on the seventh day after surgery, endometrial stem cell preparations were injected into the cervical region of the rat model group with cervical hypertrophy and squamous cell damage. The injections were given every two days for a total of 4 weeks.

[0116] Test 3: Starting on the seventh day after surgery, PBS solution was injected into the cervical region of the rat model group with cervical hypertrophy and cuticle damage, once every 2 days for a total of 4 weeks.

[0117] Test 4: Starting on the seventh day after surgery, progesterone was injected into the cervical region of the rat model group with cervical hypertrophy and cuticle damage, once every 2 days for a total of 4 weeks.

[0118] Behavioral and morphological examinations were conducted on the rat models of cervical hypertrophy-squamous lesions tested in tests 1-4 of this embodiment. Behavioral examination showed that the rats in test 2 exhibited enhanced activity, indicating a good treatment effect. Morphological examination involved measuring the transverse diameter of the vaginal portion of the cervix with calipers and observing the cervical surface under a microscope. The cervical appearance was scored as follows: 0 points: smooth cervical surface without hyperplasia; 1 point: slight congestion of the cervical surface; 2 points: moderate erosion of the cervical surface; 3 points: severe hyperplasia with ulceration of the cervical surface. The integrity of the cervical squamous epithelium was also scored. For the cervical squamous epithelium integrity test, the vaginal portion of the cervix was observed under a stereomicroscope. Scores were: 0 points: intact vaginal epithelium without defects; 1 point: slight incomplete keratinization of the vaginal epithelium; 2 points: moderate erosion of the vaginal epithelium; 3 points: severe ulceration of the vaginal epithelium. Masson's trichrome staining method was used to quantitatively analyze collagen fiber deposition and calculate the collagen percentage. Western blot was used to detect the relative expression levels of fibronectin (FN) and hyaluronidase. Table 4 shows the results of tests 1-4 in this embodiment of the invention for cervical vaginal diameter, cervical appearance score, cervical squamous epithelium appearance score, collagen percentage, relative expression levels of fibronectin (FN), and relative expression levels of hyaluronidase. Figure 4 The diagram shows the test results of the transverse diameter of the cervix in tests 1-4 in an embodiment of the present invention. Figure 5 The diagram shows the test results of cervical appearance score and cervical squamous epithelium appearance score for tests 1-4 in an embodiment of the present invention. Figure 6 A schematic diagram showing the collagen percentage test results of Test 1-Test 4 in an embodiment of the present invention is shown. Figure 7 The diagram shows the test results of the relative expression levels of fibronectin FN and hyaluronidase in Tests 1-4 of the present invention.

[0119] Table 4

[0120]

[0121] As shown in Table 4, the cervical diameter of test group 2 was significantly smaller than that of test groups 3 and 4, and the cervical appearance score was significantly lower than that of test groups 3 and 4. This indicates that the endometrial stem cell preparation used for cervical hypertrophy and squamous cell repair can reduce cervical volume, alleviate erosion, and reduce inflammatory secretions. Meanwhile, the cervical appearance score and cervical squamous epithelial appearance score of test group 2 were significantly lower than those of test groups 3 and 4, and higher than those of test group 1. This indicates that the endometrial stem cell preparation prepared in the embodiments of the present invention can repair squamous epithelial defects, restore epithelial smoothness, inhibit excessive squamous epithelial proliferation, restore cell polarity, and has better effects than simple endometrial stem cells. Meanwhile, the results of collagen percentage test, relative expression of fibronectin FN, and relative expression of hyaluronidase showed that the endometrial stem cell preparation could upregulate the relative expression of hyaluronidase and fibronectin FN, repair HA metabolism and cell connections in cervical squamous epithelium, restore barrier function, reduce cervical volume, inhibit stromal hyperplasia and collagen deposition, and block the TGF-β1 fibrosis pathway. The endometrial stem cell preparation showed better effects on cervical hypertrophy and cuticle repair than endometrial stem cells alone, progesterone, and PBS solution.

[0122] In summary, this invention provides an endometrial stem cell preparation and its application for cervical hypertrophy and cuticle repair. The endometrial stem cell preparation includes a stem cell gel carrier, endometrial stem cells attached to the stem cell gel carrier, auxiliary repair peptides, and a survival enhancer. Endometrial stem cells can secrete anti-inflammatory cytokines, anti-inflammatory chemokines, and growth factors, promoting the proliferation and differentiation of cervical epithelial cells, repairing damaged cervical mucosal epithelium, and inhibiting abnormal expansion of cervical glands. Furthermore, endometrial stem cells possess multi-directional differentiation potential, and under specific microenvironments, they can differentiate into cervical epithelial cell-like cells or stromal repair cells, thereby supplementing the missing / damaged functional cells in damaged cervical tissue, thus achieving cervical hypertrophy and cuticle repair. The stem cell gel carrier provides stable three-dimensional support for endometrial stem cells and auxiliary repair peptides, maintaining their performance while the survival enhancer delays their retention time in the cervix / hair follicle. During cervical hypertrophy repair, high expression of fibronectin (FN) can promote cell migration and enhance the homing and colonization of endometrial stem cells to cervical lesions.

[0123] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions are also within the scope of protection of the present invention.

Claims

1. An endometrial stem cell preparation for cervical hypertrophy and squamous cell repair, characterized in that, The endometrial stem cell preparation comprises: a stem cell gel carrier, endometrial stem cells attached to the stem cell gel carrier, a repair-enhancing peptide, and a survival-enhancing agent. The sequence of the repair-enhancing peptide is shown in SEQ ID NO:

1. The endometrial stem cell density in the endometrial stem cell preparation is 2 × 10⁻⁶. 6 The concentration of the auxiliary repair peptide was 15 mg / mL, the concentration of the survival enhancer was 8 mg / mL, and the concentration of the stem cell gel carrier was 25 mg / mL. The survival enhancer comprises the following components in weight percentage: anti-inflammatory mimic peptide: 15%; insulin-like growth factor-1: 20%; vitamin C: 10%; superoxide dismutase: 10%; curcumin: 5%; trehalose: 40%; the sequence of the anti-inflammatory mimic peptide is shown in SEQ ID NO: 2; The method for preparing the survival enhancer includes: Place trehalose powder in twice the volume of deionized water and stir for 10 minutes at a speed of 220-240 rpm. After stirring, let stand for 5 minutes to obtain a trehalose solution for later use. Add vitamin C powder to an equal volume of PBS buffer and stir at 100-120 rpm. Then add superoxide dismutase lyophilized powder to the PBS buffer and stir at 50 rpm for 10 min. Finally, add the pre-prepared curcumin suspension to the PBS buffer and stir at 300 rpm to obtain an antioxidant mixture for later use. Take the pre-prepared anti-inflammatory mimic peptide and insulin-like growth factor-1, dissolve the anti-inflammatory mimic peptide and insulin-like growth factor-1 in the same volume of PBS buffer as the mixture of anti-inflammatory mimic peptide and insulin-like growth factor-1, stir at 250 rpm for 20 min to obtain the active mixed component. The antioxidant mixture and trehalose solution were placed in a reaction vessel and stirred for 15 minutes. Then, the pre-prepared active mixture was added along the wall and stirred at 150 rpm for 15 minutes to obtain a coarse mixture with enhanced survival rate. A survival rate enhancer was prepared by filtering a coarse mixture with a survival rate enhancement using a PES filter membrane. The stem cell gel carrier comprises the following components by weight percentage: gelatin: 18%; glycerol: 8%; phosphate buffer: 4%; ferulic acid: 6%; polyglycerol sebate: 14%; chitosan: 13%; silk fibroin: 25%; trehalose: 12%; The method for preparing the stem cell gel carrier includes: Gelatin and silk fibroin were placed in a reaction vessel, and then phosphate buffer was added. The mixture was stirred in a water bath at 65°C for 20 minutes at a stirring speed of 300-320 rpm to obtain the first intermediate carrier. After mixing trehalose and ferulic acid, add 10 times the total volume of deionized water to the mixture. Vortex the mixture until the trehalose and ferulic acid are dissolved. Stir with a magnetic stirrer for 5 minutes and then sonicate for 10 minutes to obtain the second intermediate carrier. Polyglycerol sebacic acid ester was dissolved in 3 times its volume of anhydrous ethanol and magnetically stirred at 45°C for 15 min to obtain a polyglycerol sebacic acid ester solution. The polyglycerol sebacic acid ester solution was added dropwise to the first intermediate carrier and sheared and emulsified at a speed of 2000 rpm during the addition. After the addition was completed, the mixture was stirred for 10 min to obtain the first emulsion mixture. The second intermediate carrier is mixed with the first emulsion mixture and stirred at 200 rpm for 15 min to obtain the second emulsion mixture. Take chitosan and glycerol, then prepare a 2% chitosan solution using glacial acetic acid as solvent, add glycerol to the chitosan solution, stir at 85℃ for 15 min, and then filter the insoluble matter using a filter membrane to obtain a chitosan-glycerol mixture; The chitosan-glycerol mixture and the second emulsion mixture were placed in a reaction vessel and stirred at 200 rpm for 20 min to obtain a stem cell gel carrier. The stem cell gel carrier was then placed in a -80℃ low temperature freezer for 4 h to obtain a solidified stem cell gel carrier.

2. The endometrial stem cell preparation for cervical hypertrophy and cuticle repair as described in claim 1, characterized in that: Methods for preparing curcumin include: Turmeric was harvested after 9-10 months of cultivation. The main rhizome of turmeric was cut to obtain turmeric root. The turmeric root was washed three times with deionized water and dried in a 60℃ oven for 2 hours. The dried turmeric root was then ground in a grinder and passed through an 80-mesh sieve to obtain turmeric powder. Turmeric powder and turmeric extract were mixed at a material-to-liquid ratio of 1:

10. The turmeric extract was an ethanol-deionized water mixture with a volume ratio of 3:

2. The turmeric powder and turmeric extract were refluxed and extracted three times at 70°C. After extraction, the extracts were filtered and combined. The extracts were concentrated under reduced pressure at 40°C to obtain crude curcumin extract. Take D101 macroporous resin, soak it in anhydrous ethanol solution, then pack the soaked D101 macroporous resin into a column using a wet method, rinse with deionized water until the effluent is clear and free of turbidity, soak the cleaned D101 macroporous resin in hydrochloric acid solution for 3 hours, and rinse with deionized water until neutral. Curcumin crude extract was purified by adsorption using D101 macroporous resin, and the adsorbed D101 macroporous resin was eluted with 70% ethanol solution to prepare curcumin.

3. The endometrial stem cell preparation for cervical hypertrophy and cuticle repair as described in claim 2, characterized in that: The method for preparing the endometrial stem cell preparation includes: Take the solidified stem cell gel carrier, add sterile PBS solution to the stem cell gel carrier, vortex for 10 min to obtain stem cell gel carrier solution, place the stem cell gel carrier solution in a 37℃ water bath for 15 min to obtain activated stem cell gel carrier solution. The auxiliary repair peptide and the survival enhancer were mixed at a volume ratio of 1:1 and stirred at room temperature for 10 min to obtain the auxiliary repair peptide-survival enhancer mixture. The auxiliary repair peptide-survival enhancer mixture was added to the stem cell gel carrier solution and stirred magnetically for 10 min at a stirring speed of 40 rpm to obtain the stem cell gel carrier loaded with the auxiliary repair peptide. Endometrial stem cells were treated at a constant temperature of 37°C for 10 minutes, and then resuspended in physiological saline. The endometrial stem cells were seeded into a stem cell gel carrier loaded with helper repair peptides, mixed by pipetting, and then allowed to stand at 37°C for 15 minutes to obtain the endometrial stem cell preparation.

4. The use of the endometrial stem cell preparation for cervical hypertrophy and cervical cuticle repair as described in claim 1 in the preparation of drugs for cervical hypertrophy and cervical cuticle damage repair.

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