Ovarian organ-derived exosome as well as preparation method and application thereof

Abstract

The invention provides an exosome derived from an ovary organ. The invention also provides an extraction method and application of the exosome. According to the method, the differential centrifugation-filtration combined technology is optimized, the in-vitro extraction and identification of the exosome from the human ovary organoid are realized for the first time, and the exosome with high purity and uniform particle size is obtained. The exosome provided by the invention can significantly inhibit abnormal apoptosis of human ovarian granular cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and in particular, the present application relates to an ovarian organoid-derived exosome and a preparation method and use thereof. BACKGROUND

[0002] The ovary is a core organ that regulates female reproductive function and endocrine homeostasis. Its dysfunction is directly related to many major diseases such as polycystic ovary syndrome (PCOS), premature ovarian insufficiency (POI), ovarian cancer, and infertility. Therefore, it is urgent to develop precise treatment strategies and new research tools. Existing ovarian research models rely on two-dimensional cell culture or animal experiments. However, the former cannot simulate the process of ovarian follicle development, hormone secretion, and dynamic periodic regulation due to the lack of three-dimensional tissue structure and cell-stromal interaction network. The latter is difficult to meet the needs of high-throughput drug screening and personalized diagnosis and treatment due to species differences, long experimental period, and ethical restrictions. In recent years, ovarian organoid technology uses pluripotent stem cells or ovarian tissue-derived cells to construct a three-dimensional functional model that can reproduce key functions such as follicle formation and steroid hormone synthesis. However, the study of exosomes, a key signal carrier secreted by the organoid, is still in its infancy.

[0003] As a key mediator of cell-to-cell communication, exosomes carry active molecules such as nucleic acids, proteins, and lipids, and have great potential in tissue regeneration, disease progression, and targeted therapy. However, current research on ovarian-related exosomes is mostly based on the separation of body fluids such as peripheral blood and follicular fluid, which has limitations such as mixed sources, low abundance, and poor targeting. Moreover, it is difficult to distinguish the specific secretory cell sources (such as granulosa cells, oocytes, or stromal cells), which limits their value in mechanism research and clinical application. In addition, the exosomes secreted by traditional ovarian models (such as immortalized cell lines) lack the spatiotemporal specificity of natural tissues, so they cannot truly reflect the physiological cascade reactions of disease microenvironment dynamics or drug intervention.

[0004] Ovarian organoid-derived exosomes may be enriched with ovarian-specific molecular markers closer to the physiological state or have unique functions such as regulating follicle activation and inhibiting granulosa cell apoptosis due to their three-dimensional tissue structure and directional differentiation characteristics of functional cells. However, current research on ovarian organoid exosomes has the following shortcomings: (1) Lack of standardized and high-efficiency organoid exosome separation and purification technology, existing ultracentrifugation or kit methods are prone to contamination of other vesicle components; (2) The molecular characteristics and functional atlas of exosomes have not been clearly defined, especially the dynamic change rules of exosome contents at different developmental stages or pathological states; (3) The translational potential of exosomes in disease model construction, targeted drug delivery, or ovarian function repair has not been explored.

[0005] Therefore, it is urgent to fill these technical gaps at present, to develop ovarian organoid exosome products with clear molecular markers and targeted effects by constructing a high-simulation ovarian organoid platform, combining specific exosome extraction and functional verification system, and providing innovative tools for the precise diagnosis and treatment of ovarian diseases. SUMMARY

[0006] In view of the problems in the prior art, the inventors have, through long-term technical practice and exploration, for the first time realized the in vitro extraction and identification of ovarian organoid-derived exosomes by optimizing differential centrifugation-filtering combined technology, and on this basis, verified the significant inhibitory effect of ovarian organoid-derived exosomes on granulosa cell apoptosis.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions. In a first aspect, the present application provides an exosome, which is an ovarian organoid-derived exosome.

[0008] According to some embodiments of the present application, the ovarian organoid is a human ovarian organoid.

[0009] Preferably, the human ovarian organoid is a human ovarian surface epithelial organoid.

[0010] According to some embodiments of the present application, the particle size distribution of the exosome is 30-150 nm.

[0011] Preferably, the particle size distribution of the exosome is 100-150 nm.

[0012] According to some embodiments of the present application, the exosome has markers Alix, TSG101 and CD9; and the exosome does not have marker GM130.

[0013] In a second aspect, the present application provides a preparation method of the exosome according to the first aspect of the present application, comprising the following steps: (1) in vitro isolation of ovarian primary cells from ovarian tissue; (2) adding the ovarian primary cells into Matrigel and mixing uniformly to obtain a mixture, adding the mixture into a culture medium, and culturing to obtain an ovarian organoid; (3) differential centrifugation treatment of the culture medium containing the ovarian organoid obtained in step (2), collecting the supernatant, filtering to obtain a filtrate; (4) high-speed centrifugation treatment of the filtrate, and collecting the precipitate; (5) washing the precipitate.

[0014] In the present application, the culture medium for ovarian primary cell culture can refer to the related schemes in the prior art, which will not be described herein.

[0015] In the present application, the term "ovarian organoid" has the meaning commonly understood by a person skilled in the art, and specifically refers to a micro-model constructed in vitro using three-dimensional culture techniques, which has a structure and function similar to that of real ovarian tissue.

[0016] According to some embodiments of the present application, in step (1), the ovarian tissue is human ovarian tissue. Accordingly, the ovarian primary cells are human ovarian primary cells.

[0017] According to some embodiments of the present application, in step (2), the volume ratio of the ovarian primary cells to the Matrigel is 1: (2-4), preferably 1:3.

[0018] According to some embodiments of the present application, in step (3), the differential centrifugation treatment comprises the following steps in sequence: ① centrifugation at 300g to 500g for 5 to 10 minutes at 2°C to 8°C; ② centrifugation at 1500g to 2000g for 10 to 15 minutes at 2°C to 8°C; ③ centrifugation at 2000g to 3000g for 10 to 15 minutes at 2°C to 8°C; ④ centrifugation at 8000g to 10000g for 20 to 30 minutes at 2°C to 8°C.

[0019] According to some preferred embodiments of the present application, in step (3), the differential centrifugation treatment comprises the following steps in sequence: ① the culture medium containing the ovarian organoid obtained in step (2) is centrifuged at 300g for 5 minutes at 4°C to remove Matrigel in the culture medium; ② centrifugation at 2000g for 15 minutes at 4°C to remove dead cells in the culture medium; ③ centrifugation at 3000g for 15 minutes at 4°C to remove cell debris in the culture medium; ④ centrifugation at 10000g for 30 minutes at 4°C.

[0020] According to some embodiments of the present application, in step (3), the supernatant is subjected to filtration treatment by a filter screen.

[0021] Preferably, the filter screen is a 0.22-0.45µm filter screen.

[0022] More preferably, the filter screen is a 0.22µm filter screen.

[0023] According to some embodiments of the present application, in step (4), the high-speed centrifugation treatment is centrifugation at 110000g to 120000g for 70 to 90 minutes at 2°C to 8°C.

[0024] Preferably, the high-speed centrifugation is performed at 4°C at a speed of 120000g for 90 minutes.

[0025] According to some embodiments of the present application, in step (5), the washing comprises alternating PBS buffer resuspension of the pellet and high-speed centrifugation.

[0026] According to some preferred embodiments of the present application, in step (5), the washing comprises: first washing: resuspension of the pellet with PBS buffer to obtain a first suspension, centrifugation of the first suspension at 2°C to 8°C at a speed of 110000g to 120000g for 70 to 90 minutes, and then collection of the pellet; second washing: resuspension of the pellet collected after the first washing with PBS buffer to obtain a second suspension, centrifugation of the second suspension at 2°C to 8°C at a speed of 110000g to 120000g for 50 to 60 minutes.

[0027] Preferably, in step (5), the washing comprises: first washing: resuspension of the pellet with PBS buffer to obtain a first suspension, centrifugation of the first suspension at 4°C at a speed of 120000g for 70 minutes, and then collection of the pellet; second washing: resuspension of the pellet collected after the first washing with PBS buffer to obtain a second suspension, centrifugation of the second suspension at 4°C at a speed of 120000g for 60 minutes.

[0028] In a third aspect, the present application provides use of the exosome according to the first aspect of the present application in the preparation of a product for one or more of the following uses: (1) as a drug delivery carrier; (2) a medicament for preventing and / or treating ovarian damage and diseases related thereto.

[0029] Preferably, the disease related to ovarian damage is a disease associated with abnormal apoptosis of human ovarian granulosa cells.

[0030] More preferably, the disease associated with abnormal apoptosis of human ovarian granulosa cells is selected from one or more of premature ovarian failure, premature ovarian insufficiency, polycystic ovary syndrome, environmental toxicant-related ovarian damage, and endometriosis.

[0031] In a fourth aspect, the present application provides a pharmaceutical composition for preventing and / or treating ovarian damage and diseases related thereto, comprising an effective amount of the exosome according to the first aspect of the present application, and optionally a pharmaceutically acceptable adjuvant.

[0032] Preferably, the ovarian damage and its related diseases are diseases related to abnormal apoptosis of human ovarian granulosa cells.

[0033] More preferably, the diseases related to abnormal apoptosis of human ovarian granulosa cells are selected from one or more of premature ovarian failure, premature ovarian insufficiency, polycystic ovary syndrome, environmental toxicant-related ovarian damage and endometriosis.

[0034] It should be noted that in the present application, "effective amount" refers to an amount of exosomes that is non-toxic but sufficient to provide the desired effect. Alternatively, "effective amount" refers to an amount of exosomes that is effective in providing the desired effect when used in combination with other ingredients. The "effective amount" will vary from subject to subject, for example, due to differences in age and general condition of the individual. Therefore, it is not always possible to refer to an exact "effective amount", however, the appropriate "effective amount" in any individual can be determined by a person of ordinary skill in the art using routine experimental methods.

[0035] It should be noted that in the present application, "pharmaceutically acceptable adjuvant" includes but is not limited to carriers, diluents, water-soluble fillers, pH adjusters, stabilizers, water for injection, osmotic pressure adjusters, etc.

[0036] Compared with the prior art, the technical scheme of the present application has the following beneficial effects: The present application optimizes the differential centrifugation-filter combination technology, and for the first time realizes the in vitro extraction and identification of exosomes derived from human ovarian organoids. The exosomes obtained by the extraction method provided by the present application have high purity and uniform particle size.

[0037] Through experimental verification, for the cyclophosphamide-induced granulosa cell apoptosis model (KGN cells), the ovarian organoid exosomes provided by the present application significantly inhibit apoptosis at a concentration threshold of 1x10 10 Thus, the anti-apoptosis function and mechanism of the ovarian organoid exosomes are clarified, and direct experimental evidence is provided for their use as ovarian damage repair agents or targeted drug carriers. BRIEF DESCRIPTION OF DRAWINGS

[0038] The embodiments of the present application will be described in detail below with reference to the accompanying drawings, in which: Figure 1 The construction process of the human ovarian surface epithelial organoid in Example 1 of the present application is shown.

[0039] Figure 2 The growth process of the human ovarian surface epithelial organoid in Example 1 of the present application at 1, 7, 14, 21 and 28 days after establishment is shown.

[0040] Figure 3The identification results of the human ovarian surface epithelial organoids in Example 1 of the present application are shown, wherein A shows the HE staining of the human ovarian organoids and the human ovarian tissue, B shows the expression of epithelial markers E-cadherin and N-cadherin in the human ovarian organoids and the human ovarian tissue, and C shows the expression of Ki67, ZO-1 and PAX8 related molecules in the human ovarian organoids.

[0041] Figure 4 The extraction process of the exosomes of the human ovarian surface epithelial organoids in Example 2 of the present application is shown.

[0042] Figure 5 The electron microscope images of the extracted exosomes of the human ovarian surface epithelial organoids in Example 2 of the present application are shown, wherein the right image is a magnified image of the exosomes in the box in the left image.

[0043] Figure 6 The NTA results of the extracted exosomes of the human ovarian surface epithelial organoids in Example 2 of the present application are shown.

[0044] Figure 7 The Western blot detection results of the markers GM130, Alix, TSG101 and CD9 of the extracted exosomes of the human ovarian surface epithelial organoids in Example 2 of the present application are shown.

[0045] Figure 8 The construction results of the in vitro cell apoptosis model in Example 3 of the present application are shown, wherein A is the flow cytometry detection results, and B is the corresponding quantitative statistical results of A. Indicates p <0.0001.

[0046] Figure 9 The EdU experiment results in Example 3 of the present application are shown, wherein A is the fluorescence staining results, and B is the corresponding quantitative statistical results of A. Indicates p <0.05.

[0047] Figure 10 The Incucyte detection results in Example 3 of the present application are shown.

[0048] Figure 11 The Ki67 staining results in Example 3 of the present application are shown, wherein A is the staining results, and B is the corresponding quantitative statistical results of A. Indicates p <0.001, Indicates p <0.01. DETAILED DESCRIPTION

[0049] The application will be further described in detail below with specific embodiments, and the examples given are only to illustrate the application, not to limit the scope of the application.

[0050] Example 1: Construction and identification of ovarian organoids

[0051] (I) Construction of human ovarian surface epithelial organoids

[0052] As shown in Figure 1 , the construction of human ovarian surface epithelial organoids includes the following steps: 1.1 Tissue processing: Place the ovarian tissue in PBS containing 2% PS (penicillin-streptomycin). Wash the ovarian tissue with PBS containing 2% PS three times to remove blood clots and other impurities. Then remove the ovarian medulla part (thinner, loose), retain the cortex part (thicker, white and transparent), cut the ovarian cortex into 1 cm pieces and further cut into a sticky state. Then, transfer the cut tissue to a 15 ml centrifuge tube containing Advanced DMEM / F12 (Thermo Fisher Scientific, 12634010), centrifuge (300g, 5 min, room temperature), and discard the supernatant.

[0053] 1.2 Digestion: Add 5 ml of Advanced DMEM / F12 to the centrifuge tube to resuspend the precipitate, then add 100 µl of collagenase (Sigma, C9407), and place the centrifuge tube in a 5% CO2, 37°C incubator for 15-30 minutes.

[0054] 1.3 Stop digestion: Rinse the Pasteur tube with 2% FBS (fetal bovine serum), then add 100 µl of FBS to stop the digestion after repeatedly blowing the tissue fragments with the Pasteur tube until a small amount of cells are visible under a microscope. Repeat the blowing with the Pasteur tube several times after stopping the digestion.

[0055] 1.4 Sieving: Filter the digested tissue through a 100 µm filter screen, and rinse the residual cells on the screen. Centrifuge again (300g, 5 min, room temperature), and discard the supernatant.

[0056] 1.5 Red cell lysis: Add 1-2 ml of red cell lysis solution (Solarbio, R1010), and let it stand at room temperature for 5-10 min (observe whether there are red impurities in the cells during this step, which can be omitted). Centrifuge again (300g, 5 min, room temperature), and discard the supernatant.

[0057] 1.6 Washing: Resuspend the cells by adding 10 ml of Advanced DMEM / F12, and wash the cells again. Centrifuge (300g, 5 min, room temperature), discard the supernatant, and collect the cells to obtain human ovarian surface epithelial cells (OSE).

[0058] 1.7 Construction: Add human ovarian primary cells into Matrigel (Corning, 354230) (volume ratio of cells and Matrigel is 1:3), mix well by pipetting, and obtain the mixture of cells and Matrigel. Then inoculate the mixture of cells and Matrigel into a 24-well plate according to 40 μl mixture per well. Place the 24-well plate in a 5% CO2, 37°C incubator, and after standing for 15 min, add the OSE special medium (formula as shown in Table 1). Then replace the medium every 2 days.

[0059] Table 1: Human OSE special medium (50 ml system)

[0060] (B) Identification of human ovarian surface epithelial organoids

[0061] 2.1 Morphological identification

[0062] From the 7th day of the above 1.7 step, observe the growth of the organoids under a microscope, and the results are shown in Figure 2 As can be seen from Figure 2 , at about the 7th day, the morphology of the obvious OSE organoids (ovarian surface epithelial organoids) can be seen under a light microscope.

[0063] 2.2 Identification by HE (hematoxylin-eosin) staining and immunofluorescence staining

[0064] Determine whether some specific molecular markers such as Ki-67, E-cadherin, N-cadherin, etc. are expressed on human OSE organoids by HE staining and immunofluorescence staining.

[0065] 2.2.1 HE staining method

[0066] 1. Paraffin embedding: The organoids cultured for 14 days are scraped with 1 mL gun head with medium, collected into an EP tube, centrifuged at room temperature (600 g, 5 min), and the supernatant is discarded. Slowly add 4% paraformaldehyde (PFA), and fix at room temperature for 30 min.

[0067] 2. Centrifugation: centrifuge at room temperature at a speed of 500-600 g for 5 min, and then wash twice with PBS.

[0068] 3. Dehydration: dehydrate according to the concentration gradient of 70% ethanol→85% ethanol→95% ethanol→100% ethanol, 10 minutes for each gradient. After 85% ethanol treatment, stain with eosin for 2 minutes to facilitate subsequent embedding.

[0069] 4. Transparency: Place the organoids in xylene for 5 minutes to transparentize them, then replace the xylene once and continue processing for another 5 minutes.

[0070] 5. Infiltration with paraffin: Place the dehydrated organoid pieces in the middle of a folded lens cleaning paper, fold it four times, place it in the middle of the embedding cassette, close it tightly, and immerse it in the paraffin wax for 2 hours.

[0071] 6. Embedding and sectioning: The embedded wax block was cut into 5µm thick sections using paraffin sectioning and dried overnight at 37℃.

[0072] 7. Dewaxing and rehydration: The sections were treated sequentially with xylene for 10 min × 2 times → 100% ethanol for 8 min × 2 times → 95% ethanol for 5 min → 85% ethanol for 5 min → 70% ethanol for 5 min, and finally placed in distilled water.

[0073] 8. Staining: After rehydration, the organoid tissue was stained with hematoxylin for 1 min, placed in tap water for 3 min to reflect blue, then treated with eosin for 1 min, and then dehydrated by sequentially passing through a gradient of 95% ethanol for 1 min → 100% ethanol for 1 min → xylene for 2 min.

[0074] 9. After mounting with neutral resin, air dry and observe under a microscope.

[0075] 2.2.2 Immunofluorescence staining method

[0076] 1. Sample Collection: Add 300µl of 2% fetal bovine serum (FBS and PBS mixture) to human ovarian organoids cultured for approximately 28 days. Resuspend the gel and transfer it to a 15ml centrifuge tube. Add 200µl of 2% fetal bovine serum (FBS and PBS mixture) to each well for washing, and transfer the washings to the same centrifuge tube. Freeze the centrifuge tubes on ice for 10 minutes. Centrifuge at 400g for 5 minutes at 4℃. Discard the cell recovery solution.

[0077] 2. Fixation: Add 1 ml of 4% paraformaldehyde and transfer the liquid to a 1.5 ml centrifuge tube. Place on ice for 15 minutes, then centrifuge at 700 g for 5 minutes.

[0078] 3. Discard the supernatant, add 1 ml of 2% fetal bovine serum (FBS and PBS mixture) to resuspend the cells, and store at 4°C for staining.

[0079] 4. Permeabilization: Permeabilize in 0.5% TX100 in PBS for 10 minutes, wash with 2% FBS / PBS for 5 minutes, and repeat twice.

[0080] 5. Blocking: Block with 3% BSA / PBS at room temperature for 1 hour.

[0081] 6. Incubate overnight at 4°C with primary antibodies Ki67 (Abcam, ab16667), ZO-1 (Abcam, ab96587), PAX8 (Abcam, ab13611), E-cadherin (Abcam, ab231303), and N-cadherin (Abcam, ab76057), then wash with 2% FBS / PBS for 5 minutes each time, for a total of 4 times.

[0082] 7. Incubate with 488 fluorescent rabbit secondary antibody (Beyotime, A0408) and 647 fluorescent mouse secondary antibody (Beyotime, A0468) at room temperature for 2 hours, then wash with 2% FBS / PBS for 5 minutes, a total of 4 times. Incubate with DAPI (Abcam, ab285390) and phalloidin (Abcam, ab176753) at room temperature for 1 hour, then wash with PBS for 10 minutes. Transfer the organoids to 1.5 mL EP tubes.

[0083] 8. Mounting: Centrifuge at 300g for 5 minutes to precipitate the organoids, then add approximately 40µl of mounting medium and mix with the organoids. Fix the organoids onto glass slides and secure with nail polish.

[0084] 2.2.3 Identification Results

[0085] The results are as follows Figure 3 As shown. Figure 3 The results showed that, through immunofluorescence staining of specific markers such as Ki-67, E-cadherin, and N-cadherin, organoids cultured from human ovarian epithelial tissue not only expressed epithelial markers such as E-cadherin and N-cadherin but also expressed Ki-67, demonstrating their strong proliferative capacity. In addition, HE staining also showed that their morphology conformed to the characteristics of ovarian organoids.

[0086] Example 2: Extraction and identification of ovarian organoid exosomes

[0087] (a) Extraction of exosomes from human ovarian surface epithelial organoids

[0088] like Figure 4 As shown, the extraction of human ovarian surface epithelial organoid exosomes includes the following steps: The culture medium containing human ovarian surface epithelial organoids from Example 1 (ovarian organoid culture medium collected after 14 days, at which point the organoids had stabilized) was collected in 50 ml centrifuge tubes. Using a Beckman floor-standing ultracentrifuge, the tubes were first centrifuged at 300 g for 5 min at 4°C to remove matrix gel contamination; then, centrifuged at 2000 g for 15 min at 4°C to remove dead cells; then, centrifuged at 3000 g for 15 min at 4°C to remove cell debris; then, centrifuged at 10000 g for 30 min at 4°C, discarding the precipitate and collecting the supernatant, which was filtered through a 0.22 μm filter; finally, centrifuged at 120000 g for 90 min at 4°C, discarding the cell culture supernatant and collecting the precipitate.

[0089] Next, wash the precipitate twice. First, resuspend the precipitate with PBS and centrifuge at 120,000g for 70 min at 4°C, discarding the supernatant. Then, resuspend the precipitate with PBS and wash again, centrifuging at 120,000g for 60 min at 4°C, discarding the supernatant and collecting the precipitate to obtain the exosomes. During washing, the PBS should be filled to 2-3 mm below the opening of the centrifuge tube.

[0090] The precipitate was then resuspended in PBS and stored at -80°C.

[0091] (II) Identification of ovarian organoid exosomes

[0092] 2.1 Transmission electron microscopy (TEM) Detection of Exosome Morphology

[0093] 10 µl of exocrine secretions were suspended in PBS and dropped onto a 0.125% polyvinyl formal (Formvar) / carbon-coated TEM grid, and negatively stained with 2% uranyl acetate. The grid was dried and then examined using an HT7700 transmission electron microscope (TEM) (Hitachi, Japan) at 120 kV.

[0094] The results are as follows Figure 5 As shown in the figure. Morphological identification of exosomes by transmission electron microscopy revealed that exosomes typically present a cup-shaped or saucer-shaped appearance. Furthermore, it was observed that exosomes possess a complete lipid bilayer membrane structure, and the internal electron density of exosomes exhibits a heterogeneous state, which is related to their contents.

[0095] 2.2 Exosome particle size and particle concentration

[0096] 1. Sample preparation: Dilute the exosome suspension 2000 times with sterile PBS (to avoid excessive concentration that may cause adhesion).

[0097] 2. Instrument Calibration: Calibrate the laser and camera parameters of the instrument (Malvern NanoSight NS300) using standards with known particle sizes.

[0098] 3. Data acquisition: Inject the sample, adjust the camera focus and sensitivity, and perform the detection.

[0099] 4. Parameter analysis: The software will automatically track the Brownian motion of the particles, calculate the particle size distribution (30-150nm) and particle concentration, and output the final measurement results.

[0100] The results are as follows Figure 6 As shown, the particle size of the extracted exosomes was determined using nanoparticle tracking analysis (NTA) in this embodiment. The results show that the particle size of the exosomes is mainly concentrated in the range of 100-150 nm, which is consistent with the size characteristics of exosomes.

[0101] 2.3 Western blot detection of exosome markers

[0102] Cells (as a negative control) or exosomes were lysed using lysis buffer (formulated as 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% nonionic detergent NP-40, and 0.1% sodium dodecyl sulfate), with the protease inhibitor Cocktail Set I (Biotool, Jupiter, FL) and benzoyl sulfonyl fluoride (PMSF, Sigma-Aldrich) added to the buffer. Total protein content of the lysates was determined using a BCA protein quantification kit (Pierce, Rockford, IL). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder. Protein expression was quantified by Western blotting using antibodies against CD9 (Abcam, ab236630), Alix (Abcam, ab225555), GM130 (Abcam, ab52649), and TSG101 (Abcam, ab30871). The results were visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, USA).

[0103] The results are as follows Figure 7 As shown.

[0104] Regarding the expression of exosome marker proteins, this embodiment selected CD9, a transmembrane protein widely expressed in exosomes, as well as Alix and TSG101, which are involved in exosome formation. Alix primarily participates in the ESCRT pathway of the endocytosis sorting complex, mediating endosome invagination and exosome formation. TSG101 is a core component of the ESCRT-I complex, playing a crucial role in ubiquitinated protein sorting and exosome formation. These three molecules were used as positive references for exosome identification. GM130, a marker of the Golgi apparatus, was used as a negative reference for exosome identification. Figure 7 Western blot results showed that exosomes derived from ovarian organoids expressed CD9, Alix, and TSG101, but none expressed GM130.

[0105] Example 3: Functional study of ovarian organoid exosomes

[0106] 3.1 Construction of an in vitro apoptosis model

[0107] An in vitro apoptosis model was constructed by adding 5 µM cyclophosphamide to KGN (human ovarian granulosa cell tumor cell line, donated by Professor Zhao Yue of Peking University Third Hospital) for 24 h.

[0108] After treatment, the cells were stained for apoptosis, and the number of apoptotic cells was detected by flow cytometry to confirm the successful construction of the model. The specific flow cytometry detection method is as follows: 1. Sample Preparation: KGN cells were treated with 5 µM cyclophosphamide for 24 h, then digested with trypsin and collected. Cells were washed twice with pre-cooled PBS, centrifuged (300 g, 5 min, room temperature), and the density adjusted to 1 × 10⁻⁶ cells / mL. Cells / mL were used as the model group (CTX). Cells not treated with cyclophosphamide served as the control group (Con).

[0109] 2. Staining procedure: Resuspend the cells in 50µL of TUNEL staining solution, incubate at 37°C in the dark for 60 minutes, and then wash three times with PBS.

[0110] 3. Flow cytometry: Set up the flow cytometer and use the FITC channel (Ex 488nm, Em 530nm) to detect Tunel.

[0111] Test results as follows Figure 8 As shown. Figure 8 The results showed that adding 5 µM cyclophosphamide to KGN for 24 h successfully constructed an in vitro cell apoptosis model.

[0112] 3.2 Study on the anti-apoptotic effect of exosomes using EdU (5-ethynyl-2'-deoxyuridine) assay

[0113] (1) Cell culture and treatment: KGN cells were seeded at a ratio of 4000 cells per well in 96-well plates and incubated overnight at 37°C. After cell attachment, the cells were divided into 5 groups as follows: Control group (Con): No treatment was given; Modeling group (CTX): treated with 5µM cyclophosphamide for 24h; Low-concentration exosome group (Exo-l): treated with 5 µM cyclophosphamide for 24 h, then the exosomes extracted in Example 2 were used at 1 × 10⁻⁶. 5 Treatment at a particle / mL concentration for 48 hours; Exosome concentration group (Exo-m): treated with 5 µM cyclophosphamide for 24 h, then exosomes extracted in Example 2 were used at 1 × 10⁻⁶. 8 Treatment at a particle / mL concentration for 48 hours; High-concentration exosome group (Exo-h): treated with 5 µM cyclophosphamide for 24 h, then the exosomes extracted in Example 2 were used at 1 × 10⁻⁶. 10 The concentration of particles / mL was treated for 48 hours.

[0114] Then, perform the following steps (2) to (5) on the cells of the above 5 groups respectively.

[0115] (2) EdU incorporation: Dilute EdU to 20µM, add 100µL of EdU working solution to each well to make the concentration of EdU staining working solution 10µM, and continue to incubate at 37℃ for 2h to ensure that EdU is fully incorporated into the newly synthesized DNA. Discard the culture medium and wash the cells twice with PBS.

[0116] (3) Cell fixation: Add 50µL of 4% paraformaldehyde to each well to fix cells for 30min.

[0117] (4) Permeation: Permeation was performed for 10 min at room temperature using PBS containing 0.5% Triton X-100.

[0118] (5) Staining reaction: Add 50µL of EdU staining reaction solution to each well and incubate at room temperature in the dark for 30 min to allow the fluorescent dye Azide Alexa Fluor 555 (Beyotime, C0075S) to fully bind with EdU. After staining the nuclei with DAPI for 5-10 min, observe and photograph under a microscope.

[0119] The results are as follows Figure 9 As shown in the figure, by comparing the anti-apoptotic effects of different concentrations of exosomes derived from human ovarian organoids, it was found that high concentrations of exosomes derived from ovarian organoids were more effective in alleviating cyclophosphamide-induced apoptosis compared to low and medium concentrations.

[0120] 3.3 Identification of exosome function

[0121] Immunofluorescence and Incucyte assays using Ki67 confirmed that exosomes derived from ovarian organoids possess significant anti-apoptotic functions.

[0122] The experimental groups for Incucyte and immunofluorescence assays are as follows: Control group (Con): No treatment was given; Modeling group (CTX): treated with 5µM cyclophosphamide for 24h; Exosome group (OSE or OSE-Exo): treated with 5 µM cyclophosphamide for 24 h, with an added concentration of 1×10 10 Exosomes were treated with particles / mL for 48 hours.

[0123] 3.3.1 Incucyte detection method

[0124] 1. KGN cells were stimulated with 5 µM cyclophosphamide for 24 hours. Subsequently, ovarian organoid exosomes extracted in Example 2 were added at a concentration of 1 × 10⁻⁶. 10 Particles / mL. This is the exosome assembly (OSE).

[0125] In addition, a control group (Con) and a model group (CTX) were set up according to the above experimental grouping method.

[0126] 2. The cells from the three groups were placed in an Incucyte incubator and cultured for 48 hours, during which cell proliferation was continuously monitored.

[0127] 3. After the experiment, the cell proliferation data were analyzed using Incucyte 2022B Rev2 software.

[0128] The results are as follows Figure 10 As shown in the figure. Cell proliferation was detected by Incucyte. The addition of exosomes derived from human ovarian organoids significantly alleviated cyclophosphamide-induced apoptosis in KGN and promoted cell proliferation.

[0129] 3.3.2 Immunofluorescence staining method

[0130] 1. Sample processing: KGN was seeded into 96-well plates at a ratio of 4000 cells per well and incubated overnight at 37°C. After cell attachment, 100 µl of cyclophosphamide-containing medium was added to the KGN-seedled 96-well plates and incubated at 37°C for 24 h.

[0131] 2. Add a culture medium containing exosomes of the ovarian organoids extracted in Example 2, with an exosome concentration of 1×10⁻⁶. 10Particles / mL, cultured for 48 h. This is the exosome genome (OSE-Exo).

[0132] In addition, a control group (Con) and a model group (CTX) were set up according to the above experimental grouping method.

[0133] 3. Fixation: After discarding the culture medium, add 100µl of 4% paraformaldehyde and fix for 30min.

[0134] 4. Permeabilization: Discard the fixative, wash 3 times with PBS, add 100µl of 0.01% Triton-100 and treat for 10 min, then wash 3 times with PBS.

[0135] 5. Blocking: Block with 3% BSA / PBS at room temperature for 1 hour.

[0136] 6. Incubate with primary antibody Ki67 (Abcam, ab16667) overnight at 4°C, then wash with PBS for 5 minutes each time, for a total of 3 times.

[0137] 7. Incubate with 488 fluorescent rabbit secondary antibody (Beyotime, A0408) at room temperature for 1 hour, then wash with PBS for 5 minutes, repeating 3 times. Incubate with DAPI at room temperature for 10 minutes to stain the nucleus, then wash with PBS for 5 minutes, repeating 3 times.

[0138] 8. Take photos: Observe and photograph the stained cells under a fluorescence microscope.

[0139] The results are as follows Figure 11 As shown in the figure, immunofluorescence staining of exosomes derived from human ovarian organoids revealed an increase in Ki67 positive signal after the addition of exosomes in the cyclophosphamide-induced apoptosis model. This indicates that exosomes derived from human ovarian organoids can alleviate cyclophosphamide-induced KGN apoptosis and promote cell proliferation.

[0140] In summary, the immunofluorescence staining results of Incucyte and Ki67 indicate that exosomes extracted from ovarian organoids have the functions of promoting cell proliferation and resisting apoptosis in an in vitro apoptosis model.

[0141] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through minor modifications or variations of the disclosed technical content without departing from the scope of the present invention fall within the scope of the present invention.

Claims

1. An exosome derived from ovarian organoids; Preferably, the ovarian organoid is a human ovarian organoid; More preferably, the human ovarian organoid is a human ovarian surface epithelial organoid.

2. The exosome according to claim 1, wherein, The exosomes have a particle size distribution of 30 nm to 150 nm, preferably 100 nm to 150 nm.

3. The exosome according to claim 1, wherein, The exosomes have the markers Alix, TSG101 and CD9; the exosomes do not have the marker GM130.

4. The method for preparing exosomes according to any one of claims 1 to 3, comprising the following steps: (1) Primary ovarian cells were isolated from ovarian tissue in vitro; (2) The primary ovarian cells are added to the matrix gel and mixed evenly to obtain a mixture. The mixture is then added to the culture medium and cultured to obtain ovarian organoids. (3) The culture medium containing the ovarian organoids obtained in step (2) is subjected to differential centrifugation, the supernatant is collected, filtered, and the filtrate is obtained. (4) The filtrate is subjected to high-speed centrifugation, and the precipitate is collected; (5) Wash the precipitate.

5. The preparation method according to claim 4, wherein, In step (1), the ovarian tissue is human ovarian tissue; Preferably, in step (2), the volume ratio of the primary ovarian cells to the matrix gel is 1:(2-4), more preferably 1:

3.

6. The preparation method according to claim 4, wherein, In step (3), the differential centrifugation process includes the following steps in sequence: ① Centrifuge at 300g to 500g for 5 to 10 minutes at 2℃ to 8℃; ② Centrifuge at 1500g to 2000g for 10 to 15 minutes at 2℃ to 8℃; ③ Centrifuge at 2000g to 3000g for 10 to 15 minutes at 2℃ to 8℃; ④ Centrifuge at 8000g to 10000g for 20 to 30 minutes at 2℃ to 8℃; Preferably, the differential centrifugation process includes the following steps in sequence: ① Centrifuge at 300g for 5 minutes at 4℃; ② Centrifuge at 2000g for 15 minutes at 4℃; ③ Centrifuge at 3000g for 15 minutes at 4℃; ④ Centrifuge at 10000g for 30 minutes at 4℃.

7. The preparation method according to claim 4, wherein, In step (3), the supernatant is filtered through a filter screen; preferably, the filter screen is a 0.22-0.45µm filter screen; more preferably, the filter screen is a 0.22µm filter screen; Preferably, in step (4), the high-speed centrifugation is performed at 2°C to 8°C at a speed of 110,000g to 120,000g for 70 to 90 minutes. More preferably, the high-speed centrifugation is performed at 4°C at a speed of 120,000g for 90 minutes.

8. The preparation method according to claim 4, wherein, In step (5), the washing includes alternating resuspension of the precipitate with PBS buffer and high-speed centrifugation; Preferably, the washing includes: First wash: The precipitate was resuspended in PBS buffer to obtain a first suspension, which was centrifuged at 110,000 g to 120,000 g for 70 to 90 minutes at 2 to 8 °C, and then the precipitate was collected. Second wash: The precipitate collected after the first wash was resuspended in PBS buffer to obtain a second suspension, which was then centrifuged at 110,000 g to 120,000 g for 50 to 60 minutes at 2 to 8 °C. More preferably, the washing includes: First wash: The precipitate was resuspended in PBS buffer to obtain a first suspension, which was centrifuged at 120,000 g for 70 minutes at 4°C, and then the precipitate was collected. Second wash: The precipitate collected after the first wash was resuspended in PBS buffer to obtain a second suspension, which was then centrifuged at 120,000 g for 60 minutes at 4°C.

9. Use of exosomes according to any one of claims 1 to 3 in the preparation of products for one or more of the following purposes: (1) As a drug delivery carrier; (2) Medications used to prevent and / or treat ovarian damage and related diseases; Preferably, the ovarian injury and related diseases are diseases related to abnormal apoptosis of human ovarian granulosa cells; More preferably, the disease associated with abnormal apoptosis of human ovarian granulosa cells is selected from one or more of premature ovarian failure, premature ovarian insufficiency, polycystic ovary syndrome, environmental toxin-related ovarian damage, and endometriosis.

10. A pharmaceutical composition for the prevention and / or treatment of ovarian injury and related diseases, comprising an effective amount of exosomes according to any one of claims 1 to 3, and optionally pharmaceutically acceptable excipients; Preferably, the ovarian injury and related diseases are diseases related to abnormal apoptosis of human ovarian granulosa cells; More preferably, the disease associated with abnormal apoptosis of human ovarian granulosa cells is selected from one or more of premature ovarian failure, premature ovarian insufficiency, polycystic ovary syndrome, environmental toxin-related ovarian damage, and endometriosis.

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