Application of umbilical cord mesenchymal stem cell exosome in preparation of medicine for treating male infertility caused by hidden testis

Abstract

The invention relates to the technical field of biological medicines, in particular to an umbilical cord mesenchymal stem cell exosome and application of the umbilical cord mesenchymal stem cell exosome in preparation of a medicine for treating male infertility caused by hidden testis. The prepared high-concentration umbilical cord mesenchymal stem cell exosome suspension preparation is directly used for treating cultured spermatogonial cells and spermatoblasts in vitro, is injected into the testis of a mouse, and is used for improving the seminiferous tubule and spermatogenic microenvironment of testis tissue and promoting division and proliferation of the spermatogonial cells and the spermatoblasts. The method can effectively reverse oligospermia and azoospermia caused by the hidden testis, and compared with a model without intervention of treatment means, the method can effectively promote increase and even regeneration of sperms in the testis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to umbilical cord mesenchymal stem cell exosomes and their application in the preparation of a drug for male infertility caused by cryptorchidism. Background Art

[0002] Cryptorchidism is one of the most common urogenital anomalies in newborn males. It refers to the failure of one or both testicles to fully descend to the normal position at the bottom of the scrotum. Its prevalence in full-term infants is 1%-9%, with slightly different rates in different countries and regions. For the treatment of cryptorchidism, common interventions include hormone therapy and surgical treatment. The prognosis of these treatments varies greatly. Studies have shown that the prevalence of infertility in untreated bilateral cryptorchidism patients exceeds 90%, and approximately 9%-15% of unilateral cryptorchidism patients and 46% of bilateral cryptorchidism patients will still develop azoospermia after testicular descent surgery. The infertility caused by cryptorchidism is mainly manifested by a decrease in Sertoli cells in the seminiferous tubules of the testicles and a decrease in germ cells at all levels.

[0003] There is no proven treatment for male infertility caused by cryptorchidism. Clinically, therapeutic approaches are often used, such as the use of anti-oxidative and anti-inflammatory medications like coenzyme Q10 and L-carnitine. However, these treatments have limited success and fail to improve the inability to produce offspring caused by cryptorchidism. Patients with oligospermia due to cryptorchidism currently require assisted reproductive technologies, such as in vitro fertilization (IVF). However, these treatments are often ineffective and come with a significant financial burden. Summary of the Invention

[0004] The present invention aims to provide umbilical cord mesenchymal stem cell exosomes and their application in the preparation of a drug for male infertility caused by cryptorchidism.

[0005] To achieve the above-mentioned object, the technical solution adopted by the present invention is: use of umbilical cord mesenchymal stem cell exosomes in the preparation of a drug for male infertility caused by cryptorchidism.

[0006] A method for preparing umbilical cord mesenchymal stem cell exosomes, comprising the following steps: (1) Take out the human umbilical cord mesenchymal stem cell culture medium supernatant collected in advance from the -80 degree refrigerator and place it in cold water to melt completely; (2) Mix the thawed culture supernatant and transfer it to a high-speed centrifuge tube. Use an electronic balance to balance it and centrifuge it at 2000g for 20 minutes to remove dead cells and obtain supernatant ①. (2) After centrifugation, remove the precipitate and transfer the culture supernatant ① to a new tube, balance it, and centrifuge it at 10,000 g for 30 minutes to remove cell debris and obtain supernatant ②; (3) After discarding the precipitate, transfer supernatant ② to a new tube and use a 0.22 μm sterile filter to remove large vesicles and aggregated protein particles in the supernatant to obtain supernatant ③; (4) Balance the micrometer balance and centrifuge at 100,000 g in an ultracentrifuge at 4°C for 90 minutes; (5) After centrifugation, remove the supernatant and resuspend the pellet in each tube with a total volume of 1 mL of 1× PBS solution. Dilute to the corresponding volume with 1× PBS and balance the solution with a micrometer balance. Centrifuge again at 100,000 g at 4°C for 90 minutes. (6) After centrifugation, discard the supernatant again, resuspend the precipitate with 100uL 1×PBS solution, transfer the resuspension to a 1.5mL EP tube, and centrifuge at 10000g for 5 minutes at 4℃ to obtain the exosome suspension; In the present invention, to ensure subsequent treatment, the protein concentration of the exosome suspension was measured by Nanodrop, and the exosome suspension was repeatedly collected, added with a protective agent, and temporarily stored in a -80°C refrigerator. The collected exosomes were examined for exosome morphology using electron microscopy, particle size was measured using NTA, and the expression of key indicators CD9, CD81, and CD63 was identified by Western blotting.

[0007] The beneficial technical effects of the present invention are as follows: The present invention provides umbilical cord mesenchymal stem cell exosomes and their use in the preparation of a drug for male infertility caused by cryptorchidism. By enriching the exosomes secreted by cells during the culture process of umbilical cord mesenchymal stem cells, a high-concentration umbilical cord mesenchymal stem cell exosome suspension preparation is prepared, which is directly used to treat cultured spermatogonia and spermatocytes in vitro and injected into the mouse testis to improve the spermatogenic microenvironment of the seminiferous tubules of testicular tissue and promote the division and proliferation of spermatogonia and spermatocytes. The present invention can effectively reverse oligospermia and azoospermia caused by cryptorchidism, and can effectively promote an increase in the number of sperm in the testicles and even regeneration compared to models without treatment intervention. In in vitro experiments, mesenchymal stem cell exosomes can effectively promote the division and proliferation of mouse spermatogonia and spermatocytes in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0009] Figure 1 It is the technical roadmap of the present invention.

[0010] Figure 2 This is a graph showing the electron microscopy results of Example 1.

[0011] Figure 3 This is the NTA detection result of UCMSC-EXOs in Example 1.

[0012] Figure 4 This is the result of protein immunoblotting detection of surface markers of mesenchymal stem cell exosomes in Example 1.

[0013] Figure 5 This is a HE (hematoxylin-eosin) staining image of mouse epididymal tissue used for sperm storage in Example 2.

[0014] Figure 6 This is a HE staining image of the seminiferous tubules of the testes of different groups of mice in Example 2.

[0015] Figure 7 This is Example 3: Effects of different concentrations of UCMSC-EXOs on the proliferation of spermatogonia (GC1-spg) and spermatocytes (GC2-spg).

[0016] Figure 8 This is Example 3, showing the effect of UCMSC-EXOs on improving the senescence of spermatogonia (GC1-spg) and spermatocytes (GC2-spg). DETAILED DESCRIPTION

[0017] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0018] Example 1

[0019] A method for preparing umbilical cord mesenchymal stem cell exosomes comprises the following steps: (1) Take out the human umbilical cord mesenchymal stem cell culture medium supernatant collected in advance from the -80 degree refrigerator and place it in cold water to melt completely; (2) Mix the thawed culture supernatant and transfer it to a high-speed centrifuge tube. Use an electronic balance to balance it and centrifuge it at 2000g for 20 minutes to remove dead cells and obtain supernatant ①. (2) After centrifugation, remove the precipitate and transfer the culture supernatant ① to a new tube, balance it, and centrifuge it at 10,000 g for 30 minutes to remove cell debris and obtain supernatant ②; (3) After discarding the precipitate, transfer supernatant ② to a new tube and use a 0.22 μm sterile filter to remove large vesicles and aggregated protein particles in the supernatant to obtain supernatant ③; (4) Balance the micrometer balance and centrifuge at 100,000 g in an ultracentrifuge at 4°C for 90 minutes; (5) After centrifugation, remove the supernatant and resuspend the pellet in each tube with a total volume of 1 mL of 1× PBS solution. Dilute to the corresponding volume with 1× PBS and balance the solution with a micrometer balance. Centrifuge again at 100,000 g at 4°C for 90 minutes. (6) After centrifugation, discard the supernatant again, resuspend the precipitate with 100uL 1×PBS solution, transfer the resuspension to a 1.5mL EP tube, and centrifuge at 10000g for 5 minutes at 4℃ to obtain the exosome suspension.

[0020] (7) After centrifugation, discard the supernatant again, resuspend the precipitate with 100uL 1×PBS solution, transfer the resuspension to a 1.5mL EP tube, and centrifuge at 10000g for 5 minutes at 4℃ to obtain the exosome suspension; (7) Protein concentration measured by Nanodrop ≥10 μg / mL; (8) Repeatedly collect the exosome suspension, add a protective agent, and store it in a -80°C refrigerator; (9) The collected exosomes were examined for morphology using an electron microscope and confirmed to be shaped like a stalk. Figure 2 ; (10) NTA detection of particle size. The exosome particle size range is 40-150nm. The exosome particle size of mesenchymal stem cells is 113nm, accounting for more than 90%. See the test results. Figure 3 ; (11) Protein immunoblotting assay showed positive expression of key indicators CD9, CD81, and CD63. Figure 4 .

[0021] Example 2

[0022] (1) Surgical model construction: ① Use a No. 11 scalpel blade to open the mouse scrotum and cut off the gubernaculum of the mouse testis; ② Use a No. 11 scalpel blade to open the abdominal cavity, transfer the testicles after cutting the gubernaculum to the abdominal cavity, and use 3.0-specification non-absorbable surgical sutures to fix the testicles and vas deferens to the mouse's lower abdominal wall to prevent testicular torsion and necrosis; ③ Use 3.0-specification absorbable sutures to suture the lower abdominal and scrotal wounds, and use antibiotics to clean the abdominal cavity. After surgery, give antibiotics for 3 days to prevent infection; ④ After 7 or 14 days, use a No. 11 scalpel to open the abdomen again, untie the non-absorbable sutures, release the testicles, open the scrotum, transfer the testicles to the mouse scrotum again, clean the abdominal cavity with antibiotics, suture the wound after surgery, and give antibiotics for 3 days to prevent infection. ⑤ After two surgeries, the cryptorchidism model is completed after one week of recovery. This study established two models of infertility caused by cryptorchidism of varying degrees through preliminary experiments: a cryptorchidism model in which the testicles were placed in the abdominal cavity for 7 days (referred to as the 7-day cryptorchidism model) and a cryptorchidism model in which the testicles were placed in the abdominal cavity for 14 days (referred to as the 14-day cryptorchidism model). The patients recovered for one week after surgery. (2) After the model was established, 100 μL of 1.5×10 10 Particles / mL concentration of exosome solution (1.5×10 9 Particles / testis) for treatment; Injection details: The exosome solution was injected into the testicle using a 1mL syringe. After aspirating to remove all blood, the solution was injected into the testicles of the model mouse at four locations (25μL each at the upper, lower, left, and right points). After injection, the wound was gently pressed until no liquid flowed out. The injection was completed. During the treatment period, the mice maintained normal activities and diet. Treatment frequency: 3 days / time, for a total of 4 times.

[0023] (3) After exosome treatment, mice were allowed to resume their activities and diet for 5 weeks without restriction; (4) After recovery, the animals were killed to evaluate the therapeutic effect; (5) The results of the study showed that exosomes have an excellent effect in treating azoospermia or oligospermia caused by cryptorchidism; they can effectively reverse oligospermia and azoospermia caused by cryptorchidism, such as Figure 5 As shown in the figure, compared with the model without treatment intervention, umbilical cord mesenchymal stem cell exosomes can effectively promote the increase in the number of sperm in the testicle and even regeneration. The red arrows indicate sperm in the mouse epididymis, the purple ones in the lumen are free sperm, and the pink ones are phospholipid particles. Figure 6 As shown, the testicular tissue of normal mice treated with exosomes did not show obvious structural changes, but the testicular tissue of mouse models with cryptorchidism of different degrees showed significant improvement after treatment intervention with this method.

[0024] Example 3

[0025] (1) Mouse spermatogonial cell line GC1-spg (BNCC338398) and mouse spermatocyte GC2-spg (BNCC307026) purchased from Beibeina Biotechnology-Henan Industrial Microbial Engineering Technology Research Center were used in this experimental study.

[0026] (2) Cell recovery: The two purchased cells were taken out of the liquid nitrogen tank and placed in a -80℃ ultra-low temperature freezer for 30 minutes to balance and release the pressure. Liquid nitrogen in the tube caused the cryopreservation tube to burst. After the pressure release is completed, the frozen cells are picked up with tweezers and placed in a 37℃ constant temperature water bath to completely melt into liquid. Then, 1mL of cell solution is transferred to a 1.5mL centrifuge tube using a pipette and a disposable sterile pipette in a biosafety cabinet and centrifuged at 1000g for 5 minutes. After the centrifugation is completed, the supernatant is discarded using a pipette and a disposable sterile pipette in a biosafety cabinet and the cells are resuspended in a medium containing 10% fetal bovine serum (Gibco: 16000-044), 1% double antibody (penicillin-streptomycin) (White Shark: BL505A), and 89% DMEM high glucose medium (GE: Hyclone SH30022.01), referred to as serum-containing medium. After resuspension, transfer the cell suspension to a 15 mL centrifuge tube using a pipette and add 3 mL of serum-containing culture medium to form a diluted cell suspension. Transfer the diluted cell suspension to a cell culture flask (Corning: 430639 T25) and place it horizontally in a cell culture incubator containing 5% CO2 to complete cell recovery.

[0027] (3) Cell passaging, culture and expansion: After 72 hours of culture after recovery, the GC1-spg and GC2-spg cell lines have a cell confluence of nearly 90%, and the cells need to be passaged at this time. First, take out the GC1-spg and GC2-spg cell lines that have reached 90% confluence in the cell culture incubator and place them in a biosafety cabinet. Use a pipette to discard the cell culture medium in the culture bottle and use 2mL of sterile PBS solution (phosphate buffer) to wash the serum-containing culture medium remaining in the cells. After washing, discard the PBS solution in the bottle and wash twice. After washing, use a pipette to add 1mL of 0.25% trypsin (without EDTA and phenol red white shark BL526A) to fully infiltrate the cell surface. Then place the cell culture bottle in the cell culture incubator for digestion for 3 minutes. After digestion, use an inverted microscope to observe the digestion effect. When all cells in the field of view become round and large pieces fall off from the bottom of the culture bottle, the digestion is complete. After digestion is complete, transfer the cell culture flask containing the cells to a biosafety cabinet and add 3 mL of serum-containing medium to terminate trypsin digestion. During this time, use a pipette to aspirate the cell mixture and gently pipette against the bottom of the cell culture flask to ensure that most cells fall off the bottom of the flask and enter the trypsin-serum-containing medium mixture (cell suspension). After digestion is complete, transfer the mixed suspension to a 15 mL centrifuge tube and centrifuge at 1000 g for 5 minutes. After centrifugation, use a pipette and a disposable sterile pipette tip to discard the supernatant and resuspend the cells in 1 mL of serum-containing medium to create a cell suspension. After resuspension, add 19 mL of serum-containing medium to prepare a new cell suspension and evenly transfer the suspension to five culture flasks to complete cell passage, culture, and expansion.

[0028] (4) Cell proliferation experiment: GC1-spg and GC2-spg cells were cultured in serum-containing medium and the cells could be used for this experiment after the confluence reached 30%.

[0029] Group design (at least 3 replicates per group): control group: cultured in normal culture medium; low-dose exosome group: MSC-Exosomes (concentration of 1×10 7 particles / ml); high-dose exosome group: MSC-Exosomes (concentration 1×10 8 GC1-spg cell culture: routine subculture to the logarithmic growth phase, and starve with serum-free medium for 12-24 h before inoculation to synchronize the cell cycle. Exosome concentration optimization: Cell suspension preparation: adjust the cell density to 5×10³~1×10 4 cells / mL.

[0030] Plating: Inoculate 1000 cells per well, add culture medium to 2 mL, and shake gently to mix evenly.

[0031] Exosome intervention: After adhesion, the culture medium containing exosomes was replaced every 3 days and the culture was continued for 7-12 days.

[0032] Termination of clone formation and staining fixation: discard the culture medium, wash with PBS and then add 4% paraformaldehyde to fix at room temperature for 30 minutes.

[0033] Staining: Add 1 mL of 0.1% crystal violet staining solution to each well, stain for 30 minutes in the dark, and rinse gently with running water until no floating color remains.

[0034] (5) Cell senescence experiment: GC1-spg and GC2-spg cells were cultured in serum-containing medium until the confluence reached 70%. The experiment was then carried out. Group design (at least 3 replicates per group): control group: cultured in normal medium; D-galactose model group: treated with D-galactose (concentration of 30 mg / ml); exosome intervention group: treated with D-galactose and then added with MSC-Exosomes (concentration of 1×10 9 particles / ml); 2. D-galactose induced cell senescence; cells were seeded in 6-well plates (density: 1×10 5 cells / well). After adherence, replace the medium containing D-galactose. Treatment time: 48 hours. Observe cell morphological changes (e.g., increase in volume, flattening).

[0035] 3. Mesenchymal stem cell exosome intervention Exosome pretreatment: Exosomes were resuspended in PBS, filtered and sterilized, and then added to the culture medium of the model group.

[0036] Intervention duration: 24 hours.

[0037] β-Galactosidase (SA-β-gal) staining: Discard the culture medium, wash with PBS, and then add fixative (2% formaldehyde + 0.2% glutaraldehyde) for 15 minutes at room temperature. Staining: Prepare X-Gal staining solution according to the kit instructions. Add staining solution to each well to cover the cells and incubate at 37°C in the dark for 24 hours until a blue precipitate is observed.

[0038] Termination and observation: After washing with PBS, take pictures under a microscope (at least 5 random fields / well).

[0039] Determination of positive cells: blue particles appear in the cytoplasm.

[0040] By detecting the mouse spermatogonia GC1-spg and spermatocyte GC2-spg exposed to umbilical cord mesenchymal stem cell exosomes UCMSC-EXOs, it was found that UCMSC-EXOs significantly promoted the proliferation of spermatogonia and spermatocytes in vitro. Figure 7 In terms of anti-cell aging, our exosome therapy can treat and improve the aging of cells. Figure 8.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Use of umbilical cord mesenchymal stem cell exosomes in the preparation of a drug for male infertility caused by cryptorchidism.

2. The use according to claim 1, characterized in that The method for preparing umbilical cord mesenchymal stem cell exosomes comprises the following steps: (1) Take out the human umbilical cord mesenchymal stem cell culture medium supernatant collected in advance from the -80 degree refrigerator and place it in cold water to melt completely; (2) Mix the thawed culture supernatant and transfer it to a high-speed centrifuge tube. Use an electronic balance to balance it and centrifuge it at 2000g for 20 minutes to remove dead cells and obtain supernatant ①. (2) After centrifugation, remove the precipitate and transfer the culture supernatant ① to a new tube, balance it, and centrifuge it at 10,000g for 30 minutes to remove cell debris and obtain supernatant ②; (3) After discarding the precipitate, transfer supernatant ② to a new tube and use a 0.22 μm sterile filter to remove large vesicles and aggregated protein particles in the supernatant to obtain supernatant ③; (4) Balance the micrometer balance and centrifuge at 100,000 g for 90 minutes at 4°C. (5) After centrifugation, remove the supernatant and resuspend the pellet in each tube with a total volume of 1 mL of 1× PBS solution. Dilute to the corresponding volume with 1× PBS and balance the solution with a micrometer balance. Centrifuge again at 100,000 g at 4°C for 90 minutes. (6) After centrifugation, discard the supernatant again, resuspend the precipitate with 100uL 1×PBS solution, transfer the resuspension to a 1.5 mL EP tube, and centrifuge at 10000g for 5 minutes at 4℃ to obtain the exosome suspension.

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