Use of umbilical cord mesenchymal stem cell exosomes in preparation of a drug for repairing blood-testis barrier damage caused by cryptorchidism

Abstract

This invention discloses the application of umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for repairing blood-testis barrier damage caused by cryptorchidism. This invention is the first to discover that umbilical cord mesenchymal stem cells (UMSCs) can reside within testicular tissue and be absorbed by seminiferous tubule cells, repairing blood-testis barrier damage in a rat model of cryptorchidism after testicular descent surgery, promoting germ cell proliferation, and improving spermatogenesis. This invention provides a novel therapeutic approach for blood-testis barrier damage and has significant clinical and translational value in the treatment of reproductive system diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for repairing blood-testis barrier damage caused by cryptorchidism. Background Technology

[0002] The blood-testis barrier (BTB) is a highly specialized structure in the male reproductive system, located within the seminiferous tubules of the testis. Its primary function is to protect developing sperm cells from the immune system while maintaining a stable microenvironment within the testes, ensuring normal spermatogenesis. The BTB is mainly composed of Sertoli cells (SCs) and their tight junctions, the basement membrane, and vascular endothelial cells. SCs are the core of the BTB, interconnected by tight junctions to form a selectively permeable physical barrier. The basement membrane, composed of the extracellular matrix, provides structural support for the SCs and spermatogenic cells. Furthermore, the vascular endothelial cells within the testes also function as a barrier, restricting the entry of certain substances into the testicular tissue. These structures work together to ensure that the BTB protects developing sperm cells from immune attack and maintains a stable microenvironment within the testes.

[0003] Cryptorchidism refers to the failure of the testis to descend into the scrotum in men, including incomplete testicular descent and ectopic testis. Normally, the scrotum temperature is 2°C to 4°C lower than the abdominal cavity. Prolonged exposure to a high-temperature environment (abdominal or inguinal) can damage Sertoli cells and spermatogenic cells in undescended testes. This environment not only triggers oxidative stress and severe inflammation but also directly disrupts the tight junction proteins between Sertoli cells, thus causing basal testicular tract (BTB). Currently, treatment for BTB caused by cryptorchidism primarily relies on early surgery (orchiopexy), and effective medications are limited. Therefore, developing drugs to repair BTB caused by cryptorchidism is a crucial issue that urgently needs to be addressed in the field of reproductive diseases. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the purpose of this invention is to provide the application of umbilical cord mesenchymal stem cell exosomes in the preparation of drugs for repairing blood-testis barrier damage caused by cryptorchidism.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides the use of mesenchymal stem cell exosomes in the preparation of drugs for repairing blood-testis barrier damage.

[0006] In some implementations, the mesenchymal stem cells, also known as mesenchymal stem cells or mesenchymal stem cells, refer to a group of pluripotent stromal cells with certain differentiation potential, capable of differentiating into multiple cell types and possessing tissue repair functions. In addition to being found in the "bone marrow", they include pluripotent cells widely derived from other "non-bone marrow" tissues, such as: placenta, umbilical cord, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, and dental pulp of deciduous teeth.

[0007] Furthermore, the mesenchymal stem cells include umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, and placental mesenchymal stem cells.

[0008] Furthermore, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0009] Furthermore, the umbilical cord mesenchymal stem cells expressed positive CD105, CD73, and CD90, and negative CD11b, CD19, CD134, HLA-DR, and CD45, demonstrating trilineage differentiation capability.

[0010] Furthermore, the exosomes showed positive expression of HSP70, TSG101, and CD9, but negative expression of CALNEXIN.

[0011] In this invention, the "exosome" refers to nanoscale vesicles secreted by cells, typically with a diameter between 30 and 150 nanometers, and possessing a typical phospholipid bilayer membrane structure. Exosomes encapsulate various bioactive molecules, including proteins, lipids, and nucleic acids (such as DNA, mRNA, and microRNA), whose contents reflect the type and physiological / pathological state of their originating cells. Exosomes play a crucial role in intercellular communication, participating in various physiological and pathological processes such as immune responses, tissue repair, and disease occurrence and development by transmitting biological information. Exosomes are widely present in body fluids, such as blood, saliva, urine, breast milk, and cerebrospinal fluid; almost all cell types can secrete exosomes under normal or pathological conditions. Specifically, the exosomes used in this invention are serum exosomes.

[0012] Furthermore, the causes of the blood-testis barrier injury include reproductive system infections, varicocele, cryptorchidism, and exposure to heavy metals or microplastics.

[0013] Furthermore, the blood-testis barrier damage is caused by cryptorchidism.

[0014] A second aspect of the present invention provides a drug for repairing damage to the blood-testis barrier, the drug comprising the mesenchymal stem cell exosomes described in the first aspect of the present invention.

[0015] A third aspect of the present invention provides a pharmaceutical preparation for repairing damage to the blood-testis barrier, the pharmaceutical preparation comprising the drug described in the second aspect of the present invention and pharmaceutically acceptable excipients.

[0016] In some embodiments, specific examples of the pharmaceutically acceptable excipients include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl chloride; benzalkonium chloride, benzyl chloride; phenol, butyl or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); proteins such as serum albumin, gelatin, or immunoglobulins; and hydrophilic substances. Polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEENTM, PLURONICS™, or polyethylene glycol (PEG).

[0017] Furthermore, the dosage form of the pharmaceutical preparation includes gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0018] In some embodiments, the dosage form of the pharmaceutical preparation of the present invention is a dosage form that is advantageous for administration, prepared by conventional methods, including but not limited to: gastrointestinal dosage forms and non-gastrointestinal dosage forms. Specific examples include but are not limited to: aqueous injections, powder injections, pills, powders, tablets, patches, suppositories, emulsions, creams, gels, granules, capsules, aerosols, sprays, powder inhalers, sustained-release agents, and controlled-release agents.

[0019] In some embodiments, the pharmaceutical preparations of the present invention are administered in a pharmaceutically effective amount. The effective amount refers to the amount of an active compound or pharmaceutical agent that elicits a biological or medical response sought by researchers, veterinarians, physicians, or other clinicians in a tissue, system, animal, or human. The therapeutic or pharmaceutically effective amount of the compound to be administered will be determined by such considerations and is the minimum amount necessary to improve, cure, or treat a disease or condition or one or more symptoms thereof. The pharmaceutical composition of the present invention will be formulated, administered, and applied in a manner consistent with good medical practice, i.e., the dosage, concentration, regimen, process, medium, and route of administration. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery, the method of administration, the administration regimen, and other factors known to a medical practitioner, such as the individual patient's age, weight, and response.

[0020] The fourth aspect of the present invention provides a pharmaceutical composition for repairing blood-testis barrier damage, the pharmaceutical composition comprising the drug described in the second aspect of the present invention or the pharmaceutical preparation described in the third aspect of the present invention, as well as other drugs that can be used to treat or adjuvantly treat blood-testis barrier damage.

[0021] In some implementations, the other drugs that can be used to treat or as adjunctive treatment for blood-testis barrier damage include, but are not limited to, cutting-edge targeted drugs (e.g., Apelin-APJ inhibitors), nonsteroidal anti-inflammatory drugs (e.g., ibuprofen extended-release, celecoxib) for relieving pain and inflammatory responses, antibiotics / antiviral drugs (e.g., cefixime, amoxicillin, ribavirin, oseltamivir phosphate) for controlling secondary infections, and circulation-promoting drugs.

[0022] In some embodiments, the other drugs that can be used to treat or assist in the treatment of blood-testis barrier damage are not limited to the specific drugs listed above in this invention, and any drug that may be used to treat or assist in the treatment of blood-testis barrier damage will fall within the protection scope of this invention.

[0023] In some embodiments, the two drugs in the pharmaceutical composition can be administered simultaneously, separately, or sequentially. Simultaneous administration means that the two drugs are administered concurrently. If not administered simultaneously, they are administered sequentially within a time frame so that both can be therapeutically effective within the same time frame. Therefore, sequential administration allows for the administration of one drug 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or several hours after administering one drug, provided that the circulating half-life of the first administered drug allows for a simultaneously therapeutically effective amount of both. The time delay between administrations of the components will vary depending on the exact nature of the components, their interactions, and their respective half-lives. This differs from simultaneous or sequential administration, which refers to a significant interval between the administration of one drug and another, meaning that when the second drug is administered, the first administered drug may no longer be present in the bloodstream at a therapeutically effective amount.

[0024] The fifth aspect of this invention provides the use of mesenchymal stem cell exosomes in the preparation of pharmaceutical formulations for repairing damage to the blood-testis barrier.

[0025] The sixth aspect of this invention provides the use of mesenchymal stem cell exosomes in the preparation of pharmaceutical compositions for repairing blood-testis barrier damage.

[0026] In some embodiments, the drug composition or drug formulation may be administered to the subject via injection, topical administration, or oral administration. For example, the method may include administering the drug composition or drug formulation to the subject three times a day, once a day, or every two days. In some embodiments, injection may include subcutaneous injection, intramuscular injection, intravenous injection, etc. In some embodiments, injection may include injecting the drug composition directly into or near the lesion. In some embodiments, topical administration may include rectal administration, nasal administration, ear administration, intramedullary administration, intra-articular administration, intrapleural administration, etc., or any combination thereof. In some embodiments, the drug composition or drug formulation may be administered to the subject via a combination of different administration methods.

[0027] Advantages and beneficial effects of the present invention: This invention is the first to discover that umbilical cord mesenchymal stem cells (UMSCs) can reside in testicular tissue and be absorbed by seminiferous tubule cells, repairing blood-testis barrier damage after testicular descent surgery in a rat model of cryptorchidism, promoting germ cell proliferation, and improving spermatogenesis. This invention provides a novel treatment approach for blood-testis barrier damage and has significant clinical and translational value in the treatment of reproductive system diseases. Attached Figure Description

[0028] Figure 1This study describes the isolation and identification of hUMSCs and their derived exosomes. A shows primary human umbilical cord mesenchymal stem cells (hUMSCs) cultured using the tissue block adherence method, with cells migrating from the umbilical cord tissue block (black arrows indicate migrating cells). B shows the cell morphology of primary hUMSCs. C shows the expression of surface markers on hUMSCs detected by flow cytometry. D shows Oil Red O staining results of hUMSCs after adipogenic induction. E shows Alizarin Red staining results of hUMSCs after osteogenic induction. F shows Alixin Blue staining results of hUMSCs after chondrogenic induction. G shows the exosome precipitate obtained after ultracentrifugation. H shows the particle size distribution of UMSC-derived exosomes (uExos) detected by nanoparticle tracking analysis (NTA). I ​​shows the morphology of uExos observed by transmission electron microscopy. J shows the expression of exosome-related markers detected by Western blot.

[0029] Figure 2 Intratesticular injection of uExos can improve spermatogenesis in cryptorchidistant rats after testicular fixation. A is a schematic diagram of the animal experiment; B shows the gross morphology of the testes in the three groups of rats; C shows the fluorescence imaging results at different time points after submucosal injection of DiR-labeled exosomes (DiR-exo); D shows frozen section images of testicular tissue after submucosal injection of DiO-labeled exosomes (DiO-exo), with white arrows indicating DiO-exo distributed within the testicular tissue (scale bar = 100 μm); E is testicular weight; F is testicular volume; G is epididymal weight; H is epididymal sperm count; I is epididymal sperm abnormality rate; J shows the hematoxylin-eosin (HE) staining results of testicular tissue, with asterisks indicating seminiferous tubules in the uExo group that showed more complete restoration of seminiferous epithelial structure (scale bar = 100 μm); K is the thickness of the seminiferous epithelium in the seminiferous tubules; L is the diameter of the seminiferous tubules; and M is the Johnsen score.

[0030] Figure 3To demonstrate that uExos restores the integrity of the blood-testis barrier and promotes spermatogenic cell proliferation, the following images are presented: A) Fluorescence image of biotin tracer in testicular tissue (scale bar = 100 μm); B) Transmission electron microscopy image of testicular tissue (black arrows indicate the basement membrane around the seminiferous tubules, white arrows indicate normal tight junction structures of the blood-testis barrier, and red arrows indicate areas of damaged tight junctions (scale bar = 1 μm); C) Western blot analysis of the expression of key blood-testis barrier proteins ZO-1, occludin, and claudin-11 in testicular tissue; D) Statistical analysis of biotin leakage rate in seminiferous tubules; E) Quantification of ZO-1 in C; F) Quantification of occludin in C; G) Quantification of claudin-11 in C; H) Immunofluorescence image of ZO-1 in testicular tissue of three groups of rats (scale bar = 100 μm); I) Immunofluorescence image of occludin (scale bar = 100 μm); J) Immunofluorescence image of claudin-11 (scale bar = 100 μm). μm; K represents the quantification of H; L represents the quantification of I; M represents the quantification of J; N represents the immunofluorescence image of SOX9, a Sertoli cell marker, in the testicular tissue of the three groups of rats, scale bar = 100 μm; O represents the immunofluorescence image of PCNA, a proliferation marker, in the testicular tissue of the three groups of rats, scale bar = 100 μm; P represents the immunofluorescence image of PLZF, a spermatogonial cell marker, in the testicular tissue of the three groups of rats, scale bar = 100 μm; Q represents the quantification of N; R represents the quantification of O; S represents the quantification of P; T represents the statistical analysis of plasma inhibin B levels in the three groups of rats. Detailed Implementation

[0031] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0032] Unless otherwise stated, all numbers used in this specification and claims representing volume, weight, temperature, time, density, parts by weight, technical effects and the like are in any case to be understood as modified by the term "about" or "approximately". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations. Unless otherwise defined, the terms used herein have the commonly understood meanings by those of ordinary skill in the art, and each numerical parameter shall be construed in accordance with the number of significant digits and conventional rounding methods, or in the manner understood by those skilled in the art.

[0033] Example 1 Study on the repair ability of umbilical cord mesenchymal stem cell exosomes on blood-testis barrier injury in rat cryptorchidism model 1. Experimental Methods 1. Experimental materials: 1.1 Experimental animals: The experimental animals selected in the present invention are healthy male SPF-grade Sprague-Dawley (SD) rats, 4 weeks old, approximately 80 g, purchased from Beijing Sibeifu Laboratory Animal Technology Co., Ltd., with the production license number SCXK (Beijing) 2019-0010. The rats are housed in the Animal Experiment Center of the Second Hospital of Hebei Medical University, with the experimental unit use license number SYXK (Hebei) 2021-003. The rats are fed with Sibeifu rat maintenance feed, with constant temperature and humidity controlled, and a 12 h light / dark cycle. This study complies with the principles of animal protection, animal welfare and ethics, and conforms to the relevant national regulations on laboratory animal welfare and ethics. The Research Ethics Committee of the Second Hospital of Hebei Medical University approved this research protocol, and the ethical review batch number is: 2024-AE001.

[0034] 1.2 Experimental reagents:

[0035] 2. Isolation and culture of umbilical cord mesenchymal stem cells (UMSCs): Under sterile conditions, the umbilical cord of a full-term cesarean section fetus (approximately 10-15 cm in length) is collected, immediately placed in PBS preservation solution containing double antibodies (penicillin 100 U / mL, streptomycin 100 μg / mL), and transported at a low temperature of 2-8°C to ensure treatment within 24 hours after isolation. In a super-clean workbench, the umbilical cord is repeatedly rinsed with PBS containing double antibodies to thoroughly remove blood clots and residual blood.

[0036] Three blood vessels (two umbilical arteries and one umbilical vein) and the outer membrane of the umbilical cord are carefully removed from the umbilical cord with sterile ophthalmic scissors and tweezers, and the remaining part is the translucent Wharton's jelly. The Wharton's jelly is placed in a sterile petri dish and rinsed 1-2 times with PBS containing double antibodies.

[0037] Wharton's glue was repeatedly cut into pieces of approximately 1-3 mm using sterile surgical scissors. 3 Prepare tissue blocks of a specific size. Use a Pasteur tube to evenly inoculate the tissue blocks into a 10 cm culture dish, maintaining a spacing of approximately 0.3-0.5 cm between the tissue blocks. Cover the dish and invert it in a 37°C, 5% CO2 incubator for 1 hour to allow the tissue blocks to adhere fully to the incubator wall.

[0038] Once the tissue blocks have adhered firmly to the culture dish, gently invert the dish upright and slowly add an appropriate amount of specialized mesenchymal stem cell culture medium along the wall, taking care not to dislodge the tissue blocks and ensuring the medium completely submerges them. Place the culture flask in an incubator at 37°C, 5% CO2, and saturated humidity for static incubation. Do not move the culture flask during the first 5-7 days to avoid affecting cell migration.

[0039] The first medium change should be done 5-7 days after inoculation, followed by a half-volume medium change every 3 days. Observe cell migration around the tissue block daily under an inverted phase-contrast microscope. Cell migration is usually observed 10-14 days after inoculation; as culture time increases, the number of cells gradually increases and they grow in colony-like patterns around the tissue block. When the primary cells reach 80%-90% confluence (approximately 18-21 days of culture), passage or cryopreservation is performed.

[0040] 3. Extraction and identification of UMSCs exosomes: Collect the supernatant from UMSC cultured in a dedicated medium for 48 h, and centrifuge at low speed (300×g for 10 min to remove residual cells) and then at 2000×g for 10 min to remove cell debris. After low-speed centrifugation, the supernatant can be temporarily stored at -80℃. After collecting sufficient supernatant, proceed with subsequent ultracentrifugation. Thaw the frozen cell supernatant overnight at 4℃ the day before ultracentrifugation. Transfer the supernatant to ultracentrifuge tubes, centrifuge at 15,000×g for 30 min to remove apoptotic bodies and large vesicles, and then ultracentrifuge at 110,000×g for 70 min. Discard the supernatant, resuspend the exosome pellet in pre-chilled PBS, combine the pellets in one tube, and wash once more under the same conditions. Finally, resuspend the exosome pellet in a small amount of PBS, filter sterilize through a 0.22 μm filter, aliquot, and store at -80℃ for later use. All centrifugation steps were performed at 4℃ to maintain exosome viability. For BCA quantification, lysis is not required. Simply take the resuspended exosomes, add BCA reagent, incubate at 37°C for 30 min, measure the absorbance, and calculate the exosome concentration.

[0041] The NTA assay of exosomes was performed by Shanghai Yumeibo Biotechnology Co., Ltd. The procedure was as follows: Exosome samples were diluted with PBS to ensure a particle distribution of 10 to 100 particles per image. Video was recorded under the following conditions: camera level 10, camera shutter speed 20 ms, and camera gain 600. Each video segment was then analyzed to obtain the average particle size and particle concentration.

[0042] The exosome suspension obtained after ultracentrifugation was dropped onto a copper grid covered with a Formvar membrane. After adsorption at room temperature, excess liquid was absorbed with filter paper. Subsequently, negative staining was performed with phosphotungstic acid solution, followed by incubation at room temperature, removal of the staining solution, and air drying. After sample preparation, the morphology of the exosomes was observed and photographed under a transmission electron microscope.

[0043] 4. Establishment of a rat model of bilateral cryptorchidism: Rats were anesthetized with isoflurane gas. Abdominal hair was removed using depilatory cream, and a 1.5 cm midline incision was made in the lower abdomen. The scrotum was gently pushed to push the testis into the abdominal cavity, and then the gubernaculum of the testis was severed. To simulate cryptorchidism, 4-0 silk sutures were passed through the tunica albuginea of ​​the upper pole of the right testis, and both testes were fixed to the ipsilateral abdominal wall muscles.

[0044] Orchiopexy: Hair is removed from the abdomen and scrotum using depilatory cream. A second abdominal incision, approximately 1.5 cm long, is made along the same incision used in the modeling procedure. The sutures securing the testes to the abdominal wall muscles are cut, completely freeing both testes. A longitudinal incision, approximately 3 mm long, is made on each side of the scrotum. Using ophthalmic forceps, the lower pole of the testis is grasped and pulled into the scrotum through the scrotal incision. To ensure the testis remains within the scrotum, the lower pole is sutured to the bottom of the scrotum using 4-0 silk sutures. For the sham surgery side, a scrotal incision is made, the sutures on the tunica albuginea are removed, and the lower pole of the testis is also secured to the corresponding scrotum.

[0045] After completing the main procedures of modeling or testicular fixation, the abdominal cavity is cleared of blood, and penicillin is injected intraperitoneally to prevent infection. Finally, the incision is sutured in layers using 4-0 silk sutures. The scrotal muscles are sutured with figure-of-eight sutures, the abdominal muscles with simple continuous sutures, and the scrotal and abdominal skin with interrupted sutures. No further antibiotics are administered postoperatively.

[0046] 5. Epididymal sperm count: Epididymal sperm counting was performed using a modified method described previously. After anesthetizing the rats, intact epididymal tissue was isolated and placed in 2 mL of PBS. The epididymal tissue was then minced using ophthalmic scissors for approximately 1 minute, until the tissue fragments were approximately 1 mm in size. 3The suspension was then incubated in a 37°C water bath for 30 minutes to allow the sperm to swim out. After gentle mixing, the supernatant was collected, and the number of sperm was counted using a hemocytometer to calculate the total number of sperm in one epididymis for statistical purposes.

[0047] 6. Sperm Papanicolaou staining and abnormality rate statistics: 10 μL of sperm suspension was spread onto a glass slide, allowed to air dry, and then stained using a rapid Papanicolaou staining kit. The simplified steps were as follows: the smear was fixed in 95% ethanol for 2 min, rinsed with distilled water for 30 s, stained with nuclear staining solution for 2 min, rinsed with tap water for about 1 min, then stained with cytoplasmic staining solution for 2 min. The slide was then rinsed in destaining solution for 10 s until no excess stain remained. After air drying, the slide was mounted with resin. Three sperm smears were prepared from each epididymis. 100 sperm were counted in random fields on each smear, and the number of abnormal sperm was recorded. The sum of the three smears was used as the final sperm abnormality rate for that epididymis.

[0048] 7. Biotin tracer experiment: The permeability of BTB was assessed using a biotinylation method. After anesthetizing rats, approximately 100 µL of 10 mg / mL EZ-link Sulfo-NHS-LC-biotin was injected subtunic tunica albuginea using an insulin syringe. After 40 min to allow for sufficient diffusion of biotin into the testicular tissue, the rats were sacrificed, and the testes were immediately fixed in 4% paraformaldehyde. Paraffin sections were then immersed in antigen retrieval solution and placed in an autoclave for high-temperature heat retrieval for 5-8 min after the autoclave reached pressure. After antigen retrieval, the sections were immersed in antigen retrieval solution and allowed to cool naturally to room temperature. The sections were then immersed in PBS for 5 min × 3 times. The sections were then immersed in 0.5% Triton solution for 5 min × 3 times, and in PBS for 5 min × 3 times. Alexa Fluor was used. TM Streptavidin conjugated with 488 (10 μg / mL) was incubated at 37°C for 1 h in the dark, and all subsequent operations were performed in the dark. Sections were soaked in PBS for 5 min × 3 times. DAPI was diluted 8-fold with PBS and mounted. After 10 min, the sections were observed and photographed under a fluorescence microscope. To determine the biotin positivity rate, 100 seminiferous tubules were randomly counted in each testis from a random field of view; seminiferous tubules with significant biotin leakage were considered positive.

[0049] 8. Small animal live imaging: Dir staining of exosomes: Dissolve 5 mg Dir powder in 1 mL DMSO, using sonication to aid dissolution, resulting in a 5 mM stock solution. Aliquot and store at -20°C. Add 60 μL of Dir stock solution to every 500 μL of exosomes and incubate at 37°C for 30 min. Transfer to a 10 kDa ultrafiltration tube and centrifuge at 1300×g for 5 min until the volume of the upper layer is reduced by half. Add 250 μL of PBS to the upper layer, mix thoroughly by pipetting, and centrifuge again at 1300×g for 5 min to remove excess dye. Collect the upper layer after ultrafiltration; this is the Dir-stained exosome (Dir-exo). Stain immediately before use, protect from light throughout the process, and store temporarily at 4°C for later use.

[0050] Three 6-week-old SD rats underwent bilateral surgical cryptorchidism modeling, maintained in the cryptorchidistant state for 4 weeks, and then underwent bilateral testicular descent and fixation. During the operation, 200 μL of Dir-exo was injected subtunically into the tunica albuginea. Subsequently, small animal in vivo imaging was performed at 0, 1, 3, 5, 7, and 14 days postoperatively to observe the distribution of exosomes.

[0051] 9. Exosome uptake experiment: Dio staining of exosomes: Dissolve 100 mg Dio powder in 1134 μL DMSO, using sonication to aid dissolution, resulting in a 10 mM stock solution. Aliquot and store at -20°C. Add 0.6 μL of Dio stock solution to every 200 μL of exosomes and incubate at 37°C for 30 min. Transfer to a 10 kDa ultrafiltration tube and centrifuge at 1300×g for 5 min until the volume of the upper layer is reduced by half. Add 250 μL of PBS to the upper layer, mix thoroughly by pipetting, and centrifuge again at 1300×g for 5 min to remove excess dye. Collect the upper layer after ultrafiltration; this is the Dio-stained exosome (Dio-exo). Stain immediately before use, protect from light throughout the process, and store temporarily at 4°C for later use.

[0052] Three 6-week-old SD rats underwent bilateral surgical cryptorchidism modeling, maintaining the cryptorchidism state for 4 weeks. Following this, bilateral testicular descent and fixation were performed, with 200 μL of Dio-exo injected subtunically during the procedure. Twenty-four hours after injection, the rats were sacrificed, and testicular tissue was collected and fixed in formalin at 4°C for 24 hours. The tissue was then dehydrated using 5% and 30% sucrose solutions until it settled. OCT was embedded and frozen sectioned to a thickness of 10 μm. Sections were incubated at room temperature for 1 hour to ensure close adhesion to the slide. OCT was removed by washing with PBS for 5 minutes. The sections were then fixed in ice-cold methanol at 4°C for 15 minutes, followed by three 5-minute washes with PBS to remove the methanol. Next, the sections were washed with 0.5% Triton for 5 minutes three times, followed by three 5-minute washes with PBS. DAPI was diluted 8-fold with PBS and mounted. After 10 minutes, the sections were observed and photographed under a fluorescence microscope.

[0053] II. Experimental Results This invention successfully isolated UMSCs cells ( Figure 1 AB), and flow cytometry analysis revealed that UMSCs cells highly expressed CD105, CD73, and CD90, and lowly expressed CD11b, CD19, CD134, HLA-DR, and CD45 (AB). Figure 1 C), staining results showed that UMSCs cells possessed trilineal differentiation ability ( Figure 1 DF). The exosomes obtained by ultracentrifugation had a particle size of 100-200 nm. Figure 1 GH), TEM shows a typical disk-like structure ( Figure 1 I), Western blotting showed positive results for HSP70, TSG101, and CD9, but negative for CALNEXIN. Figure 1 J).

[0054] In vivo tracing showed that exosomes injected under the tunica albuginea could remain in the testes for at least 14 days. Figure 2 AC), and can be taken up by the seminiferous tubules ( Figure 2 D). Compared with the control group, the exo group showed significantly increased testicular volume, testicular mass, epididymal mass, and epididymal sperm count, and significantly decreased sperm abnormality rate. Figure 2 EI). HE staining showed that the exo group had fully recovered seminiferous tubules (EI). Figure 2 J), the seminiferous tubule diameter, seminiferous epithelial thickness, and Johnsen score were all significantly higher than those in the control group (J). Figure 2 KM).

[0055] Biotin tracer analysis showed that the blood-testis barrier leakage rate in the exo group (approximately 30%) was significantly lower than that in the control group (approximately 100%). Figure 3 A), Transmission electron microscopy shows that the exo group is tightly connected, continuous and intact. Figure 3 B), Western blotting and immunofluorescence showed that the expression of ZO-1, occludin, and claudin-11 in the exo group was significantly restored ( Figure 3 CM). Immunofluorescence showed no significant difference in the number of supporting cells among the three groups, but the number of proliferating cells (PCNA) was significantly increased in the exo group. Figure 3 NS). ELISA showed that plasma inhibin B levels in the exo group were higher than those in the control group ( Figure 3 T).

[0056] In summary, via subtunic injection, UMMSC exosomes can remain in testicular tissue and be absorbed by seminiferous tubule cells. UMSC exosomes repair blood-testis barrier damage following testicular descent in a rat model of cryptorchidism, promote germ cell proliferation, and improve spermatogenesis.

[0057] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of mesenchymal stem cell exosomes in the preparation of drugs for repairing blood-testis barrier damage.

2. The application according to claim 1, characterized in that, The mesenchymal stem cells include umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, and placental mesenchymal stem cells. Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells; Preferably, the umbilical cord mesenchymal stem cells are positive for CD105, CD73, and CD90, and negative for CD11b, CD19, CD134, HLA-DR, and CD45, and possess trilineage differentiation ability.

3. The application according to claim 1, characterized in that, The exosomes expressed HSP70, TSG101, and CD9 positively, and CALNEXIN negatively.

4. The application according to claim 1, characterized in that, The causes of blood-testis barrier damage include reproductive system infections, varicocele, cryptorchidism, and exposure to heavy metals or microplastics. Preferably, the blood-testis barrier damage is caused by cryptorchidism.

5. A drug for repairing damage to the blood-testis barrier, characterized in that, The drug comprises mesenchymal stem cell exosomes as described in any one of claims 1-3.

6. A pharmaceutical preparation for repairing damage to the blood-testis barrier, characterized in that, The pharmaceutical preparation comprises the drug of claim 5 and pharmaceutically acceptable excipients.

7. The pharmaceutical preparation according to claim 6, characterized in that, The dosage forms of the pharmaceutical preparations include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

8. A pharmaceutical composition for repairing damage to the blood-testis barrier, characterized in that, The pharmaceutical composition includes the drug of claim 5 or the pharmaceutical preparation of claim 6 or 7, as well as other drugs that can be used to treat or as adjunctive treatment for blood-testis barrier damage.

9. Application of mesenchymal stem cell exosomes in the preparation of drug formulations for repairing blood-testis barrier damage.

10. Application of mesenchymal stem cell exosomes in the preparation of pharmaceutical compositions for repairing blood-testis barrier damage.

Images