An exosome preparation for treating thin endometrium and its preparation method
By combining collagen gel with sheep placenta powder extract and exosome preparations, the problem of poor vascular development in thin endometrium was solved, endometrial regeneration was promoted, treatment side effects were reduced, and effective endometrial repair was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANHUI KEMEN BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-09-11
- Publication Date
- 2026-05-26
AI Technical Summary
The formation of thin endometrium is related to poor endometrial vascular development leading to reduced blood supply, which affects endometrial regeneration. Existing treatment methods have side effects and adverse reactions.
Collagen gel was used as a sustained-release matrix, combined with growth factors and exosomes from sheep placenta powder extract to promote angiogenesis. Soy isoflavones were used to balance estrogen receptor expression, and exosome preparations were prepared to promote endometrial regeneration.
It effectively promotes endometrial regeneration, reduces adverse reactions, improves blood supply, avoids the side effects of single hormone therapy, provides three-dimensional structural support, and enhances bioavailability.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to an exosome preparation for treating thin endometrium and its preparation method. Background Technology
[0002] The endometrial tissue consists of the compact layer, the spongy layer, and the basal layer; the compact and spongy layers together are called the functional layers. In accordance with the regulation of hormone levels, the endometrium undergoes a complete cycle of growth, differentiation, and shedding throughout a woman's normal reproductive process. After menstruation, under the influence of estrogen, the remaining endometrial epithelial cells in the basal layer undergo re-epithelialization, and the newly formed stroma, glands, and blood vessels gradually mature, leading to the reformation of the functional layer and restoration of the normal endometrial morphology.
[0003] Thin endometrium typically refers to an endometrial thickness of <7mm as measured by ultrasound during the mid-luteal phase. Its formation is primarily related to damage to the basal layer, leading to impaired endometrial regeneration. The pathogenic mechanism involves poor endometrial vascular development and reduced blood supply, thereby inhibiting endometrial regeneration. Studies have found that the main causes of thin endometrium include age, a history of adverse intrauterine procedures, long-term medication use, and a history of severe intrauterine infection. In the field of reproductive medicine, thin endometrium is a significant factor inducing infertility. Endometrial thickness is not only a clinical indicator of endometrial receptivity but also a prognostic factor affecting pregnancy outcomes after embryo transfer. Recent studies have found that mesenchymal stem cells can migrate and differentiate into the endometrium, promoting the regeneration and repair of damaged endometrium.
[0004] Mesenchymal stem cells (MSCs) are a type of adult stem cell derived from the mesoderm. They can differentiate into various cell types, such as osteoblasts, cardiomyocytes, epithelial cells, epidermal cells, vascular endothelial cells, nerve cells, chondrocytes, and myofibroblasts, and are therefore pluripotent stem cells. MSCs can migrate to damaged tissues and organs and, under different induction conditions, differentiate into specific cell types to replace damaged cells and play a role in tissue repair. Exosomes are actively released by cells in a specific sequence of "invasion-fusion-expulsion." Exosomes possess biological functions similar to their source cells and carry a large number of signaling molecules such as lipids, proteins, and RNAs. They can act as transport mediators, interacting with recipient cells and regulating intercellular communication, thereby affecting the function of recipient cells. In recent years, MSCs and exosomes have been widely used in the repair and treatment of tissues and organs. Summary of the Invention
[0005] The purpose of this invention is to provide an exosome preparation for treating thin endometrium and its preparation method. Collagen gel is used as a sustained-release matrix to provide three-dimensional structural support for the active substances, thereby slowing down the degradation of the active substances. At the same time, the growth factors in sheep placenta powder extract work together with the exosomes to activate angiogenesis, improve blood supply, and promote the regeneration of endometrium.
[0006] To achieve the above objectives, the present invention provides a method for preparing an exosome preparation for treating thin endometrium, comprising the following steps:
[0007] Step 1, culture of mesenchymal stem cells: The frozen bone marrow mesenchymal stem cells (BSMCs) are thawed and cultured. P3-P5 generation mesenchymal stem cells are selected for expansion culture, and the supernatant is collected and stored at 4°C for later use.
[0008] Step 2, Exosome extraction: Collect the culture supernatant of bone marrow mesenchymal stem cells (BSMCs) into centrifuge tubes and purify to obtain exosomes;
[0009] Step 3, preparation of collagen solution: Dissolve bovine bone collagen powder in PBS solution and stir magnetically until completely dissolved. Then mix the collagen solution with NaHCO3 solution under ice bath conditions and store at 4°C for later use.
[0010] Step 4, Preparation of Chinese medicine components: Pass sheep placenta powder through an 80-100 mesh sieve, then mix the powder with distilled water, soak and extract 3 times, 2 hours each time, combine the soaking liquids, filter to remove residue, and obtain sheep placenta powder extract.
[0011] Step 5: First, stir the collagen solution and sheep placenta powder extract evenly at 4°C. Then, add the exosome solution and mix evenly. Next, add the soy isoflavone solution and excipients. After the reaction is complete, the exosome gel is obtained and stored at 4°C.
[0012] Further, in step 1, P3 or P4 generation mesenchymal stem cells are selected for expansion culture; the P3 or P4 generation mesenchymal stem cells are cultured at a rate of 2 × 10⁻⁶. 6 The cells were seeded at a density of 1000 g / cm³ in cell culture flasks, and then DMEM / F12 complete medium was added. The cells were cultured at 37°C in a 5% CO2 incubator, with the medium changed periodically until the cell confluence reached 70-80%. Then, the medium was replaced with serum-free medium and cultured for another 48 hours. The supernatant of the medium was collected and stored at 4°C for later use.
[0013] Further, in step 2, the purification process is as follows: First, centrifuge at 2000g for 30 min at 4°C to remove dead cells and cell debris, then transfer to a new centrifuge tube and centrifuge at 10000g for 45 min at 4°C to remove organelles and particles. Filter using a 0.45μm filter membrane. Next, transfer the filtrate to a sterile ultracentrifuge tube and ultracentrifuge at 100000g for 70 min at 4°C. Discard the supernatant. The sediment at the bottom of the centrifuge tube contains exosomes containing impurities. Add pre-cooled PBS solution and mix by pipetting. Centrifuge again at 100000g for 70 min at 4°C and discard the supernatant to obtain purified exosomes. Finally, resuspend in pre-cooled PBS solution and store at -80°C for later use.
[0014] Furthermore, in step 3, the concentration of the collagen solution is 3%, and the collagen solution is mixed with...
[0015] The volume ratio of the NaHCO3 solution is 9:1, and the concentration of the NaHCO3 solution is 20mM.
[0016] Furthermore, in step 4, the mixing ratio of powder to distilled water is 1g:5mL.
[0017] Furthermore, in step 5, the concentration of the exosome solution is 10-25 μg / mL, and the concentration of the soy isoflavone solution is 5 μg / mL.
[0018] Further, in step 5, the volume ratio of collagen solution, sheep placenta powder extract, exosome solution, and soy isoflavone solution is 5-6:1:2-3:0.5-1; the excipients include one or more of water-soluble chitosan, poloxamer 407, and carbomer 934, and the excipients account for 3% of the total mass.
[0019] Further, in step 5, the preparation method of the soybean isoflavone solution is as follows: take water-soluble chitosan HCS solution, add soybean isoflavone, adjust the pH value to 5, and slowly add TPP aqueous solution under magnetic stirring at 300-600 r / min until the solution appears opalescent.
[0020] The present invention also provides an exosome preparation for treating thin endometrium, which is prepared by the above-described preparation method.
[0021] The present invention also provides the use of an exosome preparation in the preparation of a drug for treating thin endometrium, wherein the exosome preparation is one of the above-mentioned exosome preparations for treating thin endometrium.
[0022] The advantages and positive effects of the exosome preparation for treating thin endometrium and its preparation method described in this invention are as follows:
[0023] 1. In this invention, collagen gel is used as a sustained-release matrix to provide three-dimensional structural support for active substances (such as growth factors EGF, IGF-1, exosomes, etc. in sheep placenta powder extract), thereby slowing down the degradation of active substances. At the same time, it serves as a cell communication carrier to enhance their bioavailability.
[0024] 2. In this invention, the growth factors (such as VEGF, PDGF, etc.) in the sheep placenta powder extract work together with exosomes to activate angiogenesis, improve blood supply, and promote the regeneration of the endometrium.
[0025] 3. In this invention, soy isoflavones and exosomes are combined to balance estrogen receptor expression, thus avoiding the side effects of single hormone therapy.
[0026] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0027] Figure 1 This is a CCK-8 cytotoxicity experiment in an embodiment of the present invention, wherein A is the cell viability assay and B is the cell apoptosis detection result by flow cytometry;
[0028] Figure 2 This is a statistical chart of endometrial thickness after drug administration in an embodiment of the present invention. Detailed Implementation
[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0030] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0031] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.
[0032] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.
[0033] Bovine bone collagen was purchased from Shandong Pingju Biotechnology Co., Ltd., with a content of ≥90%.
[0034] DMEM / F12 complete culture medium was purchased from Wuhan Punosei Life Science Technology Co., Ltd.
[0035] The sheep placenta powder was purchased from Baoji Fangcheng Biological Development Co., Ltd., with a content of ≥70%, product number FS-YTP.
[0036] Extraction of exosomes:
[0037] Culture of mesenchymal stem cells: Frozen bone marrow mesenchymal stem cells (BSMCs) were thawed and cultured. P3 generation BSMCs were selected for expansion culture at a rate of 2 × 10⁻⁶. 6 The cells were seeded at a density of 1000 mg / L in cell culture flasks, and then DMEM / F12 complete medium was added. The cells were cultured at 37°C in a 5% CO2 incubator, and the medium was changed periodically until the cell growth confluence reached 70-80%. Then, the medium was changed to serum-free medium and cultured for another 48 hours. The supernatant of the medium was collected and stored at 4°C for later use.
[0038] Exosome extraction: The culture supernatant of bone marrow mesenchymal stem cells (BSMCs) was collected into centrifuge tubes. First, the supernatant was centrifuged at 2000g for 30 min at 4°C to remove dead cells and cell debris. Then, it was transferred to a new centrifuge tube and centrifuged at 10000g for 45 min at 4°C to remove organelles and particles. The supernatant was filtered through a 0.45μm filter membrane. The filtrate was then transferred to a sterile ultracentrifuge tube and centrifuged at 100000g for 70 min at 4°C. The supernatant was discarded. The sediment at the bottom of the centrifuge tube contained exosomes with impurities. Pre-chilled PBS solution was added and the mixture was mixed by pipetting. The mixture was then centrifuged again at 100000g for 70 min at 4°C. The supernatant was discarded to obtain purified exosomes. Finally, the mixture was resuspended in pre-chilled PBS solution and stored at -80°C for later use.
[0039] Preparation of collagen solution:
[0040] Bovine bone collagen powder was dissolved in PBS solution and magnetically stirred until completely dissolved to obtain a 3% collagen solution. Then, the collagen solution was mixed with a 20mM NaHCO3 solution at a volume ratio of 9:1 under ice bath conditions, and then stored at 4°C for later use.
[0041] Preparation of Chinese medicine components:
[0042] The sheep placenta powder is passed through an 80-100 mesh sieve, and then the powder is mixed with distilled water (mixing ratio of 1g:5mL), and soaked and extracted 3 times, 2 hours each time. The soaking liquids are combined, filtered to remove the residue, and the sheep placenta powder extract is obtained.
[0043] Preparation of soybean isoflavone solution:
[0044] Take a water-soluble chitosan HCS solution, add soy isoflavones, adjust the pH to 5, and slowly add TPP aqueous solution dropwise under magnetic stirring at 300-600 r / min until the solution becomes opalescent.
[0045] Example 1: Preparation of exosome preparations
[0046] Collagen solution and sheep placenta powder extract were stirred evenly at 4°C, then exosome solution was added and mixed evenly. Soy isoflavone solution and excipients were then added and allowed to react fully to obtain exosome gel, which was then stored at 4°C.
[0047] The exosome solution concentration was 10 μg / mL, the soy isoflavone solution concentration was 5 μg / mL, and the volume ratio of collagen solution, sheep placenta extract, exosome solution, and soy isoflavone solution was 5:1:3:1; the excipients were water-soluble chitosan and poloxamer 407, and the excipients accounted for 3% of the total mass.
[0048] Example 2: Preparation of exosome formulation
[0049] Collagen solution and sheep placenta powder extract were stirred evenly at 4°C, then exosome solution was added and mixed evenly. Soy isoflavone solution and excipients were then added and allowed to react fully to obtain exosome gel, which was then stored at 4°C.
[0050] The exosome solution concentration was 10 μg / mL, the soy isoflavone solution concentration was 5 μg / mL, and the volume ratio of collagen solution, sheep placenta extract, exosome solution, and soy isoflavone solution was 6:1:3:1. The excipients were water-soluble chitosan, poloxamer 407, and carbomer 934, accounting for 3% of the total mass.
[0051] Example 3: Preparation of exosome formulation
[0052] Collagen solution and sheep placenta powder extract were stirred evenly at 4°C, then exosome solution was added and mixed evenly. Soy isoflavone solution and excipients were then added and allowed to react fully to obtain exosome gel, which was then stored at 4°C.
[0053] The exosome solution concentration was 15 μg / mL, the soy isoflavone solution concentration was 8 μg / mL, and the volume ratio of collagen solution, sheep placenta powder extract, exosome solution, and soy isoflavone solution was 5:1:2:0.5; the excipients were water-soluble chitosan and poloxamer 407, and the excipients accounted for 3% of the total mass.
[0054] Comparative Example 1
[0055] No collagen solution was added; otherwise, it was the same as in Example 1.
[0056] The concentration of the exosome solution was 10 μg / mL, the concentration of the soy isoflavone solution was 5 μg / mL, and the volume ratio of sheep placenta powder extract, exosome solution, and soy isoflavone solution was 1:3:1; the excipients were water-soluble chitosan and poloxamer 407, and the excipients accounted for 3% of the total mass.
[0057] Comparative Example 2
[0058] Replace collagen with hyaluronic acid, otherwise remain the same as in Example 1.
[0059] The concentration of the exosome solution was 10 μg / mL, the concentration of the soy isoflavone solution was 5 μg / mL, and the volume ratio of hyaluronic acid solution, sheep placenta powder extract, exosome solution, and soy isoflavone solution was 5:1:3:1; the excipients were water-soluble chitosan and poloxamer 407, and the excipients accounted for 3% of the total mass.
[0060] Comparative Example 3
[0061] Replace the sheep placenta powder extract with a soy isoflavone solution, and the rest is the same as in Example 1.
[0062] The exosome solution concentration was 10 μg / mL, the soy isoflavone solution concentration was 5 μg / mL, and the volume ratio of collagen solution, exosome solution, and soy isoflavone solution was 5:3:2; the excipients were water-soluble chitosan and poloxamer 407, and the excipients accounted for 3% of the total mass.
[0063] Comparative Example 4
[0064] Replace the soy isoflavone solution with sheep placenta extract, and the rest is the same as in Example 1.
[0065] The exosome solution concentration was 10 μg / mL, and the volume ratio of collagen solution, exosome solution, and sheep placenta extract was 5:3:2. The excipients were water-soluble chitosan and poloxamer 407, accounting for 3% of the total mass.
[0066] CCK-8 cytotoxicity assay:
[0067] Well-grown HepG2 cells were diluted with complete medium (DMEM supplemented with 10% FBS and 1% penicillin-streptomycin) and seeded into 96-well plates at 10 cells per well. 4 Cells were cultured at 37°C under a humidified atmosphere of 5% CO2 for 24 h, and then cultured for 24 h each in DMEM medium containing different concentrations of exosome preparations (Examples 1 and 2) (0, 12.5, 25, 50, 100, 200, 300, 400 μg / mL). Cell viability was determined using the CCK-8 assay according to the kit instructions.
[0068] HepG2 cells were seeded in 12-well plates, with 10 cells per well. 6 Cells were cultured in complete medium for 24 h, then treated in DMEM medium with 100 μg / mL exosome preparation (Examples 1 and 2) for 24 h. Cells were then collected, washed twice with cold PBS, resuspended in 500 μL PBS, stained with PI and FITC, and detected by flow cytometry.
[0069] The results are as follows Figure 1 As shown, cell viability did not decrease significantly with increasing exosome concentration, indicating that the exosome preparation did not have significant cytotoxicity. Figure 1 In the study (A), after treating HepG2 cells with 100 μg / mL exosome preparation for 24 h, no significant apoptosis induction was observed. Figure 1 (B)
[0070] Effect test:
[0071] The effects of the exosome hydrogels prepared in Examples 1-3 and Comparative Examples 1-4 were tested.
[0072] Establishment of a rat model of thin endometrium:
[0073] SPF-grade healthy female SD rats (with normal estrous cycles), 7-8 weeks old, weighing 200-230 grams. All rats had free access to food and water and were acclimatized for one week before the experiment. Rats were randomly divided into 9 groups: blank control group, model control group, Example 1-3 groups, and Comparative Example 1-4 groups.
[0074] Rats in the model control group, Examples 1-3, and Comparative Examples 1-4 were used to establish the rat model. The modeling method is as follows:
[0075] The rats were fasted for 12 hours before surgery, but not allowed to drink. After weighing, 10% chloral hydrate (0.3 ml / 100g) was slowly injected into the rats to anesthetize them. After the anesthesia took effect, the rats were placed abdomen-up, their limbs were fixed, and the skin was shaved and prepared on the lower abdomen. The skin around the incision was disinfected. About 2 cm above the urethral opening, the skin was cut open with sterilized tissue scissors, and the abdominal wall layers were separated in turn to expose the uterus. Then, 0.5 ml of 95% ethanol was slowly and evenly injected into the uterine cavity with a syringe and left for 5 minutes to keep the uterine cavity full, causing damage to the endometrium. The 95% ethanol was withdrawn from the syringe, and the uterine cavity was repeatedly flushed with sterile saline to ensure that any residual 95% ethanol was washed away and to avoid adhesion of the uterine tissue. The abdominal cavity of the rats was carefully flushed three times with sterile saline, the sterile saline was dried, the uterus was returned to its original position, the abdomen was closed layer by layer, and 1% povidone-iodine was applied around the incision. For three consecutive days after surgery, administer penicillin intramuscularly at 160,000 U / day and apply povidone-iodine to the incision to prevent postoperative infection. Observe and record the rats' mental state, diet, weight, and stool condition daily.
[0076] The experimental groupings and drug administration details are as follows:
[0077] Table 1 Experimental Groups and Drug Administration
[0078] Dosage Blank control group Normal rats were injected into the uterus with sterile saline (1 mL). Model control group Model rats were injected into the uterus with sterile saline (1 mL). Example 1 Model rats + exosome gel (1 mL) from Example 1 were injected into the uterus Example 2 Model rats + exosome gel (1 mL) from Example 2 were injected into the uterus Example 3 Model rats + exosome gel (1 mL) from Example 3 were injected into the uterus Comparative Example 1 Model rats + Comparative Example 1 exosome gel (1 mL) were injected into the uterus Comparative Example 2 Model rat + Comparative Example 2 exosome gel (1 mL) was injected into the uterus Comparative Example 3 Model rats + Comparative Example 3 exosome gel (1 mL) were injected into the uterus Comparative Example 4 Model rat + Comparative Example 4 exosome gel (1 mL) was injected into the uterus
[0079] The drug was administered every two days at 10:00 AM for 30 days. After administration, the rats were sacrificed, and the uterus was harvested for HE staining to observe endometrial thickness (the vertical distance from the junction of the endometrium and myometrium to the uterine cavity; five sites were randomly selected from each section, and the average value was taken as the endometrial thickness). Results are shown in Table 2 and... Figure 2 As shown:
[0080] Table 2 Endometrial Thickness
[0081]
[0082]
[0083] Depend on Figure 2 It can be seen that the endometrial thickness of the rats in the model control group was 373.40±82.21μm, which was significantly smaller than that of the rats in the blank control group (543.22±67.44μm), indicating that the model was successfully established.
[0084] Compared with the model control group, the endometrial thickness of rats in Examples 1-3 was significantly increased after injection of the exosome gel prepared in Examples 1-3, indicating that the exosome gel prepared in Examples 1-3 can promote endometrial growth and alleviate thin endometrium.
[0085] Compared to Example 1, the endometrial thickness of rats in Comparative Example 1 increased less, but increased compared to the model control group. Comparative Example 1 did not contain added collagen, and lacked a sustained-release matrix for active substances such as exosomes, resulting in rapid release of these substances. Although the endometrial thickness increased compared to the model control group, the rats experienced uterine bleeding as an adverse reaction during treatment. This indicates that collagen, as a sustained-release matrix for active substances such as exosomes, can reduce adverse reactions and improve therapeutic efficacy.
[0086] Compared to Example 1, Comparative Example 2 rats showed less increase in endometrial thickness, but compared to the model control group, endometrial thickness increased, and the effect was better than Comparative Example 1. Comparative Example 2 replaced collagen with hyaluronic acid. Hyaluronic acid has lower mechanical strength and poorer support for active substances such as exosomes, leading to faster release of these substances. This resulted in uterine bleeding as an adverse reaction in the rats during treatment. Therefore, this invention uses collagen as a matrix for the sustained release of active substances such as exosomes, which not only reduces adverse reactions but also improves the therapeutic effect.
[0087] Compared to Example 1, the increase in endometrial thickness in Comparative Example 3-4 rats was less. Comparative Example 3-4 rats were injected with either a single soy isoflavone solution or sheep placenta extract, i.e., a single hormone + exosome treatment regimen. Although the endometrial thickness increased compared to the model control group, the rats experienced adverse reactions such as uterine bleeding during treatment. This indicates that the treatment regimen of soy isoflavone + sheep placenta extract + exosomes used in Example 1 not only restored the endometrium to normal levels but also reduced the occurrence of adverse reactions.
[0088] Therefore, the present invention employs the above-mentioned exosome preparation for treating thin endometrium and its preparation method, using collagen gel as a sustained-release matrix to provide three-dimensional structural support for the active substances, thereby slowing down the degradation of the active substances. At the same time, the growth factors in sheep placenta powder extract work together with the exosomes to activate angiogenesis, improve blood supply, and promote the regeneration of endometrium.
[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A method for preparing an exosome preparation for treating thin endometrium, characterized in that, Includes the following steps: Step 1, culture of mesenchymal stem cells: The frozen bone marrow mesenchymal stem cells (BSMCs) are thawed and cultured. P3-P5 generation mesenchymal stem cells are selected for expansion culture, and the supernatant is collected and stored at 4°C for later use. Step 2, Exosome extraction: Collect the culture supernatant of bone marrow mesenchymal stem cells (BSMCs) into centrifuge tubes and purify to obtain exosomes; Step 3, preparation of collagen solution: Dissolve bovine bone collagen powder in PBS solution and stir magnetically until completely dissolved. Then mix the collagen solution with NaHCO3 solution under ice bath conditions and store at 4°C for later use. The collagen solution concentration was 3%, the volume ratio of collagen solution to NaHCO3 solution was 9:1, and the NaHCO3 solution concentration was 20mM. Step 4, Preparation of Chinese medicine components: Pass sheep placenta powder through an 80-100 mesh sieve, then mix the powder with distilled water, soak and extract 3 times, 2 hours each time, combine the soaking liquids, filter to remove residue, and obtain sheep placenta powder extract. The mixing ratio of powder to distilled water is 1g:5mL; Step 5: First, stir the collagen solution and sheep placenta powder extract evenly at 4°C, then add the exosome solution and mix evenly, then add the soy isoflavone solution and excipients. After the reaction is complete, the exosome gel is obtained and stored at 4°C. The concentration of the exosome solution was 10-25 μg / mL, and the concentration of the soy isoflavone solution was 5 μg / mL. The volume ratio of collagen solution, sheep placenta powder extract, exosome solution, and soy isoflavone solution is 5-6:1:2-3:0.5-1; the excipients include one or more of water-soluble chitosan, poloxamer 407, and carbomer 934, and the excipients account for 3% of the total mass.
2. The method for preparing an exosome preparation for treating thin endometrium according to claim 1, characterized in that: In step 1, P3 or P4 generation mesenchymal stem cells are selected for expansion culture; the P3 or P4 generation mesenchymal stem cells are cultured at a rate of 2 × 10⁻⁶. 6 The cells were seeded at a density of 1000 g / cm³ in cell culture flasks, and then DMEM / F12 complete medium was added. The cells were cultured at 37°C in a 5% CO2 incubator, with the medium changed periodically until the cell confluence reached 70-80%. Then, the medium was replaced with serum-free medium and cultured for another 48 hours. The supernatant of the medium was collected and stored at 4°C for later use.
3. The method for preparing an exosome preparation for treating thin endometrium according to claim 1, characterized in that, In step 2, the purification process is as follows: First, centrifuge at 2000g for 30 min at 4℃ to remove dead cells and cell debris. Then, transfer the filtrate to a new centrifuge tube and centrifuge at 10000g for 45 min at 4℃ to remove organelles and particles. Filter the filtrate using a 0.45μm filter membrane. Next, transfer the filtrate to a sterile ultracentrifuge tube and centrifuge at 100000g for 70 min at 4℃. Discard the supernatant. The sediment at the bottom of the centrifuge tube contains exosomes containing impurities. Add pre-chilled PBS solution and mix by pipetting. Centrifuge again at 100000g for 70 min at 4℃ and discard the supernatant to obtain purified exosomes. Finally, resuspend the exosomes in pre-chilled PBS solution and store them at -80℃ for later use.
4. The method for preparing an exosome preparation for treating thin endometrium according to claim 1, characterized in that, In step 5, the preparation method of the soy isoflavone solution is as follows: take water-soluble chitosan HCS solution, add soy isoflavone, adjust the pH value to 5, and slowly add TPP aqueous solution under magnetic stirring at 300-600 r / min until the solution appears opalescent.
5. An exosome preparation for treating thin endometrium, characterized in that, It is prepared by the preparation method according to any one of claims 1-4.
6. The application of exosome preparations in the preparation of drugs for treating thin endometrium, characterized in that, The exosome preparation is the exosome preparation for treating thin endometrium as described in claim 5.
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CN115054678A