Composition for treating male erectile dysfunction through combination of human prepuce mesenchymal stem cell exosome and human placenta mesenchymal stem cells and application

Abstract

The invention provides a composition for treating male erectile dysfunction by combining human prepuce mesenchymal stem cell exosome with human placenta mesenchymal stem cells and application, and relates to the technical field of biological medicine. The composition comprises human prepuce mesenchymal stem cell exosome particles and human placenta mesenchymal stem cells. The human prepuce mesenchymal stem cell exosome particles and the human placenta mesenchymal stem cells are core components and have the repairing effects of revascularization, nerve repairing, fibrosis resistance and the like. The composition also comprises glutathione, L-arginine, icariin, ginseng stem and leaf total saponins, mecobalamin and human serum albumin, can provide functional support and antioxidant protection, ensures the instant effect, and creates an environment for restoration. According to the invention, multi-target and fundamental treatment of male erectile dysfunction is realized through an action mode of quick effect taking, synergistic repair and long-acting reconstruction, and symptoms of male erectile dysfunction are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a composition and application of human foreskin mesenchymal stem cell exosomes combined with human placental mesenchymal stem cells for the treatment of male erectile dysfunction. Background Technology

[0002] Erectile dysfunction (ED) is the persistent inability to achieve and / or maintain an erection sufficient for satisfactory sexual intercourse. It is a common men's health problem that seriously affects the quality of life, mental health, and family harmony of patients and their partners. With the aging of the global population, increased life stress, and rising incidence of cardiovascular diseases and diabetes, the prevalence of ED is showing a year-on-year increasing trend, and the patient population is becoming increasingly younger.

[0003] The causes of erectile dysfunction (ED) are complex and can be mainly divided into psychogenic, organic, and mixed types. Among them, organic ED is closely related to factors such as vascular dysfunction, nerve damage, and endocrine disorders, especially vascular endothelial dysfunction, which is considered one of the core pathological links. Common pathogenic factors include diabetes, hypertension, hyperlipidemia, cardiovascular disease, and neurovascular damage after pelvic surgery (such as radical prostatectomy).

[0004] Currently, first-line clinical treatments for erectile dysfunction (ED) mainly include oral medications, local treatments, and surgical treatments. Oral medications, such as phosphodiesterase-5 inhibitors, are effective for most patients, but their efficacy is temporary and symptom-dependent, and they cannot fundamentally repair damaged blood vessels and nerves. Local treatments, such as vacuum erection devices and intraurethral medications, carry risks such as inconvenience, pain, priapism, and fibrosis, resulting in poor patient compliance. Surgical treatment is an invasive end-stage treatment option, carrying risks such as surgical complications, infection, and mechanical malfunction, and once implanted, the patient will lose natural erectile function.

[0005] In summary, current therapies focus on symptom relief rather than addressing the underlying causes, lacking revolutionary treatment strategies that can fundamentally promote tissue regeneration and repair damaged blood vessels and nerves. Therefore, developing a novel therapy that targets the core pathological aspects of erectile dysfunction (ED) to address the root cause has significant clinical need and market value. Summary of the Invention

[0006] The purpose of this invention is to provide a composition and application of human foreskin mesenchymal stem cell exosomes combined with human placental mesenchymal stem cells for the treatment of male erectile dysfunction. The composition provided by this invention can significantly improve male erectile dysfunction.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a composition for treating male erectile dysfunction, comprising human foreskin mesenchymal stem cell exosomes and human placental mesenchymal stem cells; the concentration of the human placental mesenchymal stem cells is (3-7)×10⁻⁶. 6 The particle concentration of human foreskin mesenchymal stem cell exosomes was (2-3)×10⁻³ / mL. 11 Particles / mL.

[0009] Preferably, the particle size of the exosomes of the human foreskin mesenchymal stem cells is 50-120 nm.

[0010] Preferably, the human foreskin mesenchymal stem cell exosomes express CD9. + CD63 + and CD81 + The particle content is >90%.

[0011] Preferably, the human placental mesenchymal stem cells express CD29. + CD44 + CD73 + CD90 + CD105 + The cell count was ≥95%, and the expression of CD34 was ≥95%. + CD45 + HLA-DR + The cell count is ≤2%.

[0012] Preferably, it also includes glutathione, L-arginine, icariin, total saponins from ginseng stems and leaves, methylcobalamin, and human serum albumin.

[0013] More preferably, the concentration of glutathione is 1-3 mg / mL, the concentration of L-arginine is 0.5-1.5 mg / mL, the concentration of icariin is 0.1-0.2 mg / mL, the concentration of total saponins from ginseng stems and leaves is 0.2-0.5 mg / mL, the concentration of methylcobalamin is 0.5-1 mg / mL, and the concentration of human serum albumin is 5-10 mg / mL.

[0014] The present invention also provides a method for preparing the above composition, comprising: first mixing human serum albumin and physiological saline to obtain a human serum albumin solution, then sequentially adding glutathione, L-arginine, methylcobalamin, icariin and total saponins from ginseng stems and leaves to obtain a matrix solution; and mixing human foreskin mesenchymal stem cell exosome particles and human placental mesenchymal stem cells with the matrix solution to obtain the composition.

[0015] The present invention also provides the use of the above composition in the preparation of a medicament for treating male erectile dysfunction.

[0016] Preferably, the erectile dysfunction is diabetic or neurogenic.

[0017] Preferably, the drug is administered via intracavernosal injection.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention provides a composition for treating male erectile dysfunction, comprising human foreskin mesenchymal stem cell exosomes and human placental mesenchymal stem cells. The human foreskin mesenchymal stem cell exosomes and human placental mesenchymal stem cells are the core components, possessing fundamental repair functions such as angiogenesis, nerve repair, and anti-fibrosis. It also includes glutathione, L-arginine, icariin, total saponins from ginseng stems and leaves, methylcobalamin, and human serum albumin, which provide rapid functional support and antioxidant protection for the core components, ensuring immediate effects and creating an environment conducive to repair. This invention's composition achieves multi-target, fundamental treatment of male erectile dysfunction through a three-pronged approach of rapid onset, synergistic repair, and long-term reconstruction, not only improving symptoms but also comprehensively repairing and enhancing male erectile physiological function. Detailed Implementation

[0020] This invention provides a composition for treating male erectile dysfunction, comprising human foreskin mesenchymal stem cell exosomes and human placental mesenchymal stem cells; the concentration of the human placental mesenchymal stem cells is (3-7)×10⁻⁶. 6 The particle concentration of human foreskin mesenchymal stem cell exosomes was (2-3)×10⁻³ / mL. 11 Particles / mL.

[0021] The particle size of the human foreskin mesenchymal stem cell exosomes of the present invention is preferably 50-120 nm, and the human foreskin mesenchymal stem cell exosomes express CD9. + CD63 + and CD81 + The particle content is >90%. The exosomes of the foreskin mesenchymal stem cells of the inventors conform to the characteristics of exosomes.

[0022] The human placental mesenchymal stem cells described in this invention preferably express CD29. + CD44 + CD73 + CD90 + CD105 + The cell count was ≥95%, and the expression of CD34 was ≥95%. + CD45 + HLA-DR + The cell count is ≤2%.

[0023] The present invention preferably includes glutathione, L-arginine, icariin, total saponins from ginseng stems and leaves, methylcobalamin, and human serum albumin. The concentration of glutathione is preferably 1-3 mg / mL, more preferably 2 mg / mL; the concentration of L-arginine is 0.5-1.5 mg / mL, more preferably 1 mg / mL; the concentration of icariin is 0.1-0.2 mg / mL, more preferably 0.15 mg / mL; the concentration of total saponins from ginseng stems and leaves is 0.2-0.5 mg / mL, more preferably 0.3 mg / mL; the concentration of methylcobalamin is 0.5-1 mg / mL, more preferably 0.8 mg / mL; and the concentration of human serum albumin is 5-10 mg / mL, more preferably 8 mg / mL.

[0024] The core components of the composition of this invention are human preputial mesenchymal stem cell exosomes and human placental mesenchymal stem cells. Human preputial mesenchymal stem cell exosomes carry abundant miRNAs, cytokines, and growth factors, and can regulate target cell function without cell implantation. Their main functions are to promote vascular endothelial cell proliferation and migration, inhibit apoptosis, and resist fibrosis, thereby improving the structure and function of the cavernous body's vascular network. Human placental mesenchymal stem cells home to damaged cavernous body tissue and directly replace damaged cells through directed differentiation into vascular endothelial cells and smooth muscle cells. Simultaneously, they continuously secrete various nutritional factors and exosomes through paracrine function, providing long-term regulation of the local microenvironment and promoting tissue self-repair. Human foreskin mesenchymal stem cell exosomes can rapidly act on the corpus cavernosum, quickly inhibiting damage and promoting repair by releasing signaling molecules (miRNA, etc.), creating conditions for subsequent colonization and repair of human placental mesenchymal stem cells. Human placental mesenchymal stem cells directly reconstruct the damaged structure (differentiating into cells) on the one hand, and continuously secrete nutritional factors and exosomes on the other hand to maintain long-term stability. The combined use of the two achieves a synergistic effect of short-term symptom relief and long-term pathological reversal, together constituting a very promising regenerative treatment strategy for erectile dysfunction.

[0025] The glutathione described in this invention is one of the most important antioxidants in the body. It can eliminate excess reactive oxygen species generated during erection, reduce oxidative stress damage to vascular endothelial cells and nerves, protect NO activity, and prolong its duration of action. L-arginine is a direct substrate for the synthesis of nitric oxide (NO) in the body. Supplementing with L-arginine provides sufficient raw materials for NO synthesis, ensuring that penile vascular endothelial cells can produce enough NO to initiate the erection signaling pathway. Icariin is the main active ingredient of the traditional Chinese medicine Epimedium. Its effect is similar to that of natural phosphodiesterase 5 inhibitors (such as sildenafil). By inhibiting PDE5 activity, it reduces the degradation of cyclic guanosine monophosphate (cGMP), thereby enhancing and prolonging the action of NO. Ginseng stem and leaf total saponins have multiple effects, including vasodilation, microcirculation improvement, anti-fatigue, and neuroprotection. They can improve vascular endothelial function through multiple signaling pathways and may increase libido and overall energy, thus assisting in the treatment of male erectile dysfunction from both functional and psychological perspectives. Mecobalamin is particularly important for male erectile dysfunction caused by diabetic dysplasia or postoperative nerve damage. It can nourish and repair damaged peripheral nerves, including the cavernous nerves that innervate the penis, and restore nerve signal transmission. Human serum albumin, as a formulation excipient, mainly plays a role in stabilizing cells and exosomes, preventing their adhesion and aggregation, ensuring accurate dosage, and providing colloid osmotic pressure to reduce local irritation.

[0026] In this invention's composition, L-arginine, icariin, total saponins from ginseng stems and leaves, and glutathione provide rapid functional support and antioxidant protection, ensuring immediate effects and creating an environment conducive to repair. Human foreskin mesenchymal stem cell exosomes and human placental mesenchymal stem cells are the core components, possessing fundamental repair functions such as angiogenesis, nerve repair, and anti-fibrosis. This invention's composition, through a three-pronged approach of rapid onset, synergistic repair, and long-term reconstruction, achieves multi-target, fundamental treatment of male erectile dysfunction, not only improving symptoms but also comprehensively repairing and enhancing male erectile physiological function.

[0027] The present invention also provides a method for preparing the above composition, comprising: first mixing human serum albumin and physiological saline to obtain a human serum albumin solution, then sequentially adding glutathione, L-arginine, methylcobalamin, icariin and total saponins from ginseng stems and leaves to obtain a matrix solution; and mixing human foreskin mesenchymal stem cell exosome particles and human placental mesenchymal stem cells with the matrix solution to obtain the composition.

[0028] The present invention also provides the use of the above composition in the preparation of a drug for treating erectile dysfunction in men, wherein the erectile dysfunction is preferably diabetic or nerve damage type; and the drug is preferably administered via intracavernosal injection.

[0029] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0030] Unless otherwise specified, the following embodiments are all conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Glutathione was purchased from Shandong Jincheng Biopharmaceutical Co., Ltd.

[0033] L-arginine was purchased from Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd., Baiyunshan Chemical Pharmaceutical Factory.

[0034] Epimedium glycoside was purchased from Beijing Bailingwei Technology Co., Ltd.

[0035] Total saponins from ginseng stems and leaves were purchased from Panjin Kaili Pharmaceutical Co., Ltd.

[0036] Methylcobalamin was purchased from Tianjin Tianyao Pharmaceutical Co., Ltd.

[0037] Human serum albumin was purchased from Shanghai Emerging Pharmaceuticals Co., Ltd.

[0038] The rat vascular endothelial growth factor (VEGF) ELISA kit was purchased from Beijing Bio-Lab Technology Co., Ltd.

[0039] Example 1

[0040] Preparation of human foreskin mesenchymal stem cell exosomes

[0041] 1. Preparation of human foreskin mesenchymal stem cells

[0042] (1) Sample collection

[0043] Donor selection: Male children requiring circumcision, excluding donors who are infected (HIV, HBV, HCV, syphilis), taking immunosuppressants, or have metabolic diseases;

[0044] (2) Washing and removing impurities

[0045] The foreskin tissue removed during circumcision was rinsed three times with PBS (pH 7.2, containing penicillin 100U / mL + streptomycin 100μg / mL) to remove blood stains, subcutaneous fat and connective tissue, and the dermis was preserved.

[0046] Use sterile scissors to cut the dermis into 1-2mm pieces. 3Add 0.25% trypsin-EDTA digestion solution to the small pieces and digest at 37°C for 30 minutes, gently shaking once every 10 minutes during the process;

[0047] Add an equal volume of DMEM / F12 medium containing 10% FBS to the 0.25% trypsin-EDTA digestion solution to terminate digestion, filter, and remove tissue blocks;

[0048] Centrifuge the filtered tissue suspension at 4°C and 1000×g for 5 min, discard the supernatant, resuspend the cells in DMEM / F12 medium, count them, and then seed them into T75 culture flasks.

[0049] Incubate in a CO2 incubator, change the medium after 48 hours (to remove non-adherent cells), and then change the medium every 2 days until the cell confluence reaches 85%.

[0050] (3) Transmission

[0051] When the cell confluence reached 85%, the culture medium was discarded, the cells were washed three times with PBS, 3 mL of 0.25% trypsin-EDTA digestion solution was added, and the cells were incubated at 37°C until they detached. Digestion was terminated by adding DME M / F12 medium containing 10% FBS, centrifuged at 1500×g for 5 min, and passaged to a new culture flask at a ratio of 1:3. After passage to the 3rd generation, P3 human foreskin mesenchymal stem cells were obtained.

[0052] P3 human foreskin mesenchymal stem cells were stained with PBS (containing 2% bovine serum albumin) to a concentration of 1×10⁻⁶. 6 P3 human foreskin mesenchymal stem cell suspension was obtained by averaging cells / mL.

[0053] (4) Identification

[0054] Flow cytometry analysis: 100 μL of P3 human preputial mesenchymal stem cells were added to flow cytometry tubes, and the expression of positive antibodies (CD29, CD44, CD73, CD90, CD105) and negative antibodies (CD34, CD45, HLA-DR) in the P3 human preputial mesenchymal stem cell suspension was detected by flow cytometry.

[0055] Experimental results showed that the positive expression rates of positive markers (CD29, CD44, CD73, CD90, CD105) of the P3 human foreskin mesenchymal stem cells of the present invention were all ≥95%, and the positive expression rates of negative markers (CD34, CD45, HLA-DR) were all ≤2%. This indicates that the human foreskin mesenchymal stem cells prepared by the present invention have high purity and their expression has highly similar phenotypic characteristics to mesenchymal stem cells, making them a type of cell with mesenchymal stem cell characteristics.

[0056] Multidirectional differentiation ability:

[0057] Osteogenic differentiation: P3 human foreskin mesenchymal stem cells were seeded in osteogenic induction medium (basal medium + 10 mmol / L sodium β-glycerophosphate + 50 μg / mL ascorbic acid + 100 nmol / L dexamethasone). After 2 weeks of culture, red calcium nodules were visible after Alizarin Red staining.

[0058] Adipogenic differentiation: P3 human preputial mesenchymal stem cells were cultured for 2 weeks in adipogenic induction medium (basal medium + 0.5 mmol / L IBMX + 1 μmol / L dexamethasone + 10 μmol / L insulin). Intracellular red lipid droplets were visible after Oil Red O staining.

[0059] Chondrogenic differentiation: P3 human prepuce mesenchymal stem cells were cultured for 3 weeks in chondrogenic induction medium (basal medium + 10 ng / mL TGF-β1 + 50 μg / mL ascorbic acid) using the microsphere culture method. Alcian blue staining revealed the cartilage matrix (glycosaminoglycans).

[0060] This demonstrates that the P3 human foreskin mesenchymal stem cells prepared by this invention have multi-directional differentiation potential, which conforms to the core definition of stem cells.

[0061] 2. Preparation of human foreskin mesenchymal stem cell exosomes

[0062] (1) When P3 human foreskin mesenchymal stem cells were cultured to a cell confluence of 85%, the culture medium was discarded, and the cells were washed three times with PBS. Then, they were cultured in serum-free DMEM / F12 medium containing 10 ng / mL LTGF-β1 and 30 ng / mL EGF for 24 h. After that, they were cultured in a hypoxic environment (3% O2, 5% CO2, balanced N2) for 36 h. The culture supernatant containing exosomes was collected.

[0063] (2) Isolation and purification of exosomes

[0064] Centrifuge the culture supernatant at 3000×g and 4℃ for 15 min to remove cell pellet, filter it with a 0.22μm sterile filter membrane (PES material) to remove impurities larger than 0.22μm in diameter, and obtain the supernatant.

[0065] Place the supernatant in an ultracentrifuge tube and centrifuge at 100,000 × g and 4 ° C for 120 min. Discard the supernatant, resuspend the precipitate in PBS, and incubate at 4 ° C for 30 min to allow the precipitate to dissolve completely.

[0066] Prepare 20%, 30%, 40%, and 60% (w / v) sucrose solutions with PBS, and slowly add them to the ultracentrifuge tubes in sequence (decreasing density from bottom to top to avoid layering disorder);

[0067] The above exocrine resuspension was slowly spread on the top layer of the sucrose gradient solution. After equilibration, it was centrifuged at 100,000×g and 4℃ for 16h. 1 mL of gradient component was collected sequentially from the bottom to the top of the tube using a sterile pipette, for a total of 8 tubes.

[0068] The sucrose density of each tube was measured using a refractometer, and the fraction with a density of 1.13-1.18 g / mL (the main distribution range of exosomes) was collected.

[0069] The collected target components were transferred to dialysis bags (molecular weight cutoff 100 kDa) and dialyzed with PBS at 4°C for 24 h (PBS was changed every 6 h) to remove sucrose.

[0070] The dialysis fluid was centrifuged at 100,000×g and 4℃ for 60 min. The supernatant was discarded, and the precipitate was the purified human foreskin mesenchymal stem cell exosome particles.

[0071] (3) Identification

[0072] 1) Measuring particle size

[0073] Human foreskin mesenchymal stem cell exosome particles were resuspended in 80 μL of sterile PBS, and the particle size was detected using a nanoparticle tracking analyzer.

[0074] Tests showed that the particle size of human foreskin mesenchymal stem cell exosomes was between 50-120 nm.

[0075] 2) Flow cytometry (high sensitivity) detection

[0076] Flow cytometry analysis: Human foreskin mesenchymal stem cell exosome particles were resuspended in 100 μL of sterile PBS. 100 μL of human foreskin mesenchymal stem cell exosome suspension was added to flow cytometry tubes, and the expression of positive antibodies (CD9, CD63, CD81) in the human foreskin mesenchymal stem cell suspension was detected by flow cytometry.

[0077] Experimental results show that the positive expression rates of positive markers (CD9, CD63, CD81) in the P3 human foreskin mesenchymal stem cell exosome particles of the present invention are all >90%, indicating that the human foreskin mesenchymal stem cell exosomes prepared by the present invention conform to the characteristics of exosomes.

[0078] Example 2

[0079] Preparation of human placental mesenchymal stem cells

[0080] 1. Sample collection

[0081] Placental donors must be healthy, full-term mothers, excluding those infected with HIV, HBV, HCV, or syphilis, those taking immunosuppressants, or those with metabolic diseases.

[0082] The placenta is retrieved within 1-2 hours after the cesarean section.

[0083] 2. Processing and segmentation of placental tissue

[0084] (1) Initial placental cleaning

[0085] Place the fresh placenta in a sterile large culture dish and rinse it repeatedly 4 times with pre-cooled PBS (pH 7.2, containing penicillin 100U / mL + streptomycin 100μg / mL) at 4°C to remove blood, amniotic fluid and mucus from the surface until the rinsing solution is clear.

[0086] Use sterile scissors to cut away the decidual tissue (dark red, soft in texture) and umbilical cord (keep separately, as it can be used to isolate umbilical cord mesenchymal stem cells) from the edge of the placenta, leaving only the amnion and chorion.

[0087] (2) Tissue layering and cutting

[0088] Gently separate the amnion and chorion with sterile forceps, and process them separately;

[0089] Lay the amnion / chorion membrane flat in a sterile culture dish and cut it into 1-2mm pieces with sterile scissors. 3 Place the small pieces into a 50mL centrifuge tube, add 10mL PBS (pH 7.2, containing penicillin 100U / mL + streptomycin 100μg / mL), gently invert to mix, let stand for 5min, discard the supernatant (to remove residual impurities), and repeat twice.

[0090] 3. Enzymatic digestion of placental tissue

[0091] (1) Collagenase digestion

[0092] Add 20 mL of serum-free α-MEM medium containing type I collagenase (1 mg / mL) and DNase I (10 μg / mL) to a centrifuge tube containing tissue blocks, and gently pipette to suspend the tissue blocks;

[0093] Place the centrifuge tube in a 37°C water bath and shake to digest. During digestion, remove the centrifuge tube every 20 minutes and observe the digestion under an inverted microscope (stop digestion when the edges of the tissue block become blurred and a small number of single cells appear, usually 60-90 minutes).

[0094] Filter the digestion solution through a 100-mesh sterile cell sieve, collect the filtrate, and add 10 mL of collagenase solution to any remaining undigested tissue blocks. Repeat the digestion for 30 minutes.

[0095] (2) Trypsin digestion

[0096] Combine the collagenase digestion solutions filtered twice, centrifuge at 1200×g at room temperature for 5 min, and discard the supernatant.

[0097] Add 5 mL of 0.25% trypsin-EDTA digestion solution to the precipitate, gently resuspend it, and incubate it in a CO2 incubator at 37°C for 10 min.

[0098] Add an equal volume of α-MEM medium containing 10% FBS to stop digestion, and gently pipette to form a single-cell suspension.

[0099] (3) Cell filtration and washing

[0100] Filter the single-cell suspension using a 200-mesh sterile cell sieve and collect the filtrate into a new 50 mL centrifuge tube.

[0101] Centrifuge at 1000×g at room temperature for 5 min, discard the supernatant, resuspend the cells in α-MEM medium containing 10% FBS and 1% penicillin and antibiotics, and repeat centrifugation once (to thoroughly remove enzyme residues);

[0102] Finally, the cells were resuspended in culture medium to obtain a single-cell suspension.

[0103] Mix 10 μL of cell suspension with 10 μL of trypan blue staining solution, add the mixture to a cell counting chamber, and count the live cells under an inverted microscope (live cells are colorless and transparent, while dead cells are blue). The cell viability was found to be 90%.

[0104] 3. Primary culture and subculture amplification

[0105] (1) Primary culture

[0106] Based on the cell count results, the single-cell suspension was prepared at 5 × 10⁻⁶. 5 -1×10 6 Seeds were placed at a density of 10 cells / flask into T25 culture flasks and 5 mL of α-MEM medium containing 10% FBS and 1% penicillin antibiotics were added.

[0107] Gently shake the culture flask to distribute the cells evenly, and place it in an incubator at 37°C, 5% CO2, and 95% humidity for static culture.

[0108] After the first medium change and 36 hours of culture, some cells were observed to adhere to the wall and grow under an inverted microscope. At this time, the supernatant was discarded, and the cells were gently washed twice with sterile PBS. Fresh culture medium was added and cultured for a longer period.

[0109] The medium was changed every two days thereafter until the P0 cells reached 85% confluence.

[0110] (2) Subculture

[0111] When the P0 cells reach 85% confluence, discard the culture medium, wash twice with PBS, add 2 mL of 0.25% trypsin-EDTA digestion solution, and incubate in a 37°C incubator for 3 min.

[0112] Add 3 mL of culture medium containing 10% FBS to stop digestion. Gently pipette the culture flask wall and collect the cell suspension into a centrifuge tube. Centrifuge at 1000×g at room temperature for 5 min.

[0113] Discard the supernatant, resuspend the cells in fresh culture medium, count them, and passage them into new T75 culture flasks at a ratio of 1:5.

[0114] After passage, cell morphology was observed daily under an inverted microscope, and the medium was changed every 2 days. When the cell confluence reached 85%, the next passage could be performed. The cells were passaged to P4 to obtain human placental mesenchymal stem cells.

[0115] 4. Identification

[0116] 1) Flow cytometry

[0117] P4 human placental mesenchymal stem cells were adjusted to 1×10⁻⁶ using staining buffer (PBS containing 2% bovine serum albumin). 6 Human placental mesenchymal stem cell suspension was obtained by averaging cells / mL.

[0118] 100 μL of human placental mesenchymal stem cell suspension was added to flow cytometry tubes, and the expression of positive antibodies (CD29, CD44, CD73, CD90, CD105) and negative antibodies (CD34, CD45, HLA-DR) in the human placental mesenchymal stem cell suspension was detected by flow cytometry.

[0119] Experimental results show that the positive expression rates of positive markers (CD29, CD44, CD73, CD90, CD105) of the human placental mesenchymal stem cells of the present invention are all ≥95%, and the positive expression rates of negative markers (CD34, CD45, HLA-DR) are all ≤2%, indicating that the human placental mesenchymal stem cells prepared by the present invention have high purity and conform to the characteristics of mesenchymal stem cells.

[0120] 2) Identification of multi-directional differentiation potential

[0121] Osteogenic differentiation: Human placental mesenchymal stem cells were seeded in osteogenic induction medium (basal medium + 10 mmol / L sodium β-glycerophosphate + 50 μg / mL ascorbic acid + 100 nmol / L dexamethasone). After 2 weeks of culture, red calcium nodules were visible after Alizarin Red staining.

[0122] Adipogenic differentiation: After culturing in adipogenic induction medium (basal medium + 0.5 mmol / L IBMX + 1 μmol / L dexamethasone + 10 μmol / L insulin) for 2 weeks, intracellular red lipid droplets were visible after Oil Red O staining.

[0123] Chondrogenic differentiation: The chondrogenic differentiation was carried out by microsphere culture in chondrogenic induction medium (basal medium + 10 ng / mL LTGF-β1 + 50 μg / mL ascorbic acid) for 3 weeks. Alcian blue staining revealed the cartilage matrix (glycosaminoglycans).

[0124] This demonstrates that the human placental mesenchymal stem cells prepared by this invention have multi-directional differentiation potential, which conforms to the core definition of stem cells.

[0125] Example 3

[0126] Preparation of a composition for treating male erectile dysfunction

[0127] First, human serum albumin and physiological saline were mixed and dissolved to obtain a human serum albumin solution. Glutathione, L-arginine, and methylcobalamin were added sequentially, and the mixture was stirred at 150 rpm for 3 min. The pH was then adjusted to 7.3 with sodium hydroxide. Icariin and total saponins from ginseng stems and leaves were added sequentially, and the mixture was stirred at 150 rpm for 15 min. The mixture was then filtered through a 0.22 μm sterile filter membrane to obtain a matrix solution. Human foreskin mesenchymal stem cell exosomes from Example 1 and human placental mesenchymal stem cells from Example 2 were added sequentially to the matrix solution to obtain a composition.

[0128] The concentration of human placental mesenchymal stem cells in the composition is 5 × 10⁻⁶. 6 The particle concentration of human foreskin mesenchymal stem cell exosomes was 2.5 × 10⁶ / mL. 11 The concentrations of glutathione (particles / mL), L-arginine (2 mg / mL), icariin (0.15 mg / mL), total ginseng stem and leaf saponins (0.3 mg / mL), methylcobalamin (0.8 mg / mL), and human serum albumin (8 mg / mL) were also measured.

[0129] The osmotic pressure of the composition was measured to be 312 mOsm / L using a freezing point osmometer.

[0130] Example 4

[0131] Preparation of a composition for treating male erectile dysfunction

[0132] First, human serum albumin and physiological saline were mixed and dissolved to obtain a human serum albumin solution. Glutathione, L-arginine, and methylcobalamin were added sequentially, and the mixture was stirred at 120 rpm for 5 min. The pH was then adjusted to 7.2 with sodium hydroxide. Icariin and total saponins from ginseng stems and leaves were added sequentially, and the mixture was stirred at 120 rpm for 18 min. The mixture was then filtered through a 0.22 μm sterile filter membrane to obtain a matrix solution. Human foreskin mesenchymal stem cell exosomes from Example 1 and human placental mesenchymal stem cells from Example 2 were added sequentially to the matrix solution to obtain a composition.

[0133] The concentration of human placental mesenchymal stem cells in the composition is 3 × 10⁻⁶. 6 The particle concentration of human foreskin mesenchymal stem cell exosomes was 3 × 10⁶ / mL. 11 The concentrations of glutathione, L-arginine, icariin, total saponins from ginseng stems and leaves, mecobalamin, and human serum albumin were 10 mg / mL.

[0134] The osmotic pressure of the composition was measured to be 315 mOsm / L using a freezing point osmometer.

[0135] Example 5

[0136] Preparation of a composition for treating male erectile dysfunction

[0137] First, human serum albumin and physiological saline were mixed and dissolved to obtain a human serum albumin solution. Glutathione, L-arginine, and methylcobalamin were added sequentially, and the mixture was stirred at 180 rpm for 2 min. The pH was then adjusted to 7.4 with sodium hydroxide. Icariin and total saponins from ginseng stems and leaves were added sequentially, and the mixture was stirred at 180 rpm for 12 min. The mixture was then filtered through a 0.22 μm sterile filter membrane to obtain a matrix solution. Human foreskin mesenchymal stem cell exosomes from Example 1 and human placental mesenchymal stem cells from Example 2 were added sequentially to the matrix solution to obtain a composition.

[0138] The concentration of human placental mesenchymal stem cells in the composition is 7 × 10⁻⁶. 6 The particle concentration of human foreskin mesenchymal stem cell exosomes was 2 × 10⁶ / mL. 11 The concentrations of glutathione, L-arginine, icariin, total ginseng stem and leaf saponins, mecobalamin, and human serum albumin were 5 mg / mL.

[0139] The osmotic pressure of the composition was measured to be 317 mOsm / L using a freezing point osmometer.

[0140] Comparative Example 1

[0141] The specific implementation method is the same as in Example 3, except that human placental mesenchymal stem cells are discarded and the concentration of human foreskin mesenchymal stem cell exosomes is adjusted to 5 × 10⁻⁶. 11 Particles / mL.

[0142] Comparative Example 2

[0143] The specific implementation method is the same as in Example 3, except that human foreskin mesenchymal stem cell exosomes are discarded, and the concentration of human placental mesenchymal stem cells is adjusted to 2×10⁻⁶. 7 per mL.

[0144] Comparative Example 3

[0145] The specific implementation method is the same as in Example 3, except that human foreskin mesenchymal stem cell exosome particles and human placental mesenchymal stem cells are discarded.

[0146] Comparative Example 4

[0147] The specific implementation method is the same as in Example 3, except that glutathione, L-arginine, methylcobalamin, icariin, and total saponins from ginseng stems and leaves are omitted.

[0148] Comparative Example 5

[0149] The specific implementation method is the same as in Example 3, except that glutathione, L-arginine and methylcobalamin are omitted.

[0150] Comparative Example 6

[0151] The specific implementation method is the same as in Example 3, except that icariin and total saponins from ginseng stems and leaves are omitted.

[0152] Experimental Example 1

[0153] Neurogenic erectile dysfunction

[0154] (1) Construction of a model of diabetic erectile dysfunction

[0155] Ten-week-old SPF-grade male SD rats were selected and acclimatized for one week. Ten rats were selected as the sham-operated group, while the other rats were used to establish a neurogenic erectile dysfunction model.

[0156] After fasting for 8 hours but not watering, 1% sodium pentobarbital was injected intraperitoneally at a dose of 50 mg / kg. After successful anesthesia, the rat was fixed in a supine position on the operating table, and the hair from the lower abdomen to the penis area was shaved off. The surgical area was disinfected three times alternately with povidone-iodine and 75% ethanol.

[0157] Make a longitudinal incision (about 2-3 cm long) along the midline of the lower abdomen, and cut through the skin, subcutaneous tissue and linea alba layer by layer. Gently separate the rectus abdominis muscle to expose the pubic symphysis and the pelvic region behind it. Wrap the intestine with gauze soaked in physiological saline and gently push it upward, or fix it with a self-made retractor to fully expose the bladder and prostate. Perform the operation under a surgical microscope. Clamp the bilateral cavernous nerves with microvascular forceps (clamp position at the level of the middle of the prostate) for 1 minute, and then remove the vascular forceps.

[0158] After confirming the absence of active bleeding, the linea alba and muscle layer were continuously sutured with absorbable sutures, and the skin incision was intermittently sutured with non-absorbable sutures. Iodine was applied to the wound surface for disinfection, and penicillin (30,000 units / rat) was injected subcutaneously to replenish body fluids and prevent infection. The rats were placed alone in a warm, clean cage for resuscitation, and were returned to the group cages after they were fully awake.

[0159] Rats in the sham-operated group underwent the exact same surgical procedure, including exposure of the bilateral cavernous nerves, but the nerves were only gently touched with microforceps without clamping or damaging them.

[0160] (2) Model Validation

[0161] Functional verification was performed 4 weeks post-surgery to ensure that the pathological changes following nerve injury had stabilized.

[0162] Both the rats used to construct the model and the sham-operated group were anesthetized. One common carotid artery was isolated, and a pressure sensor was inserted to monitor mean arterial pressure (MAP). A PE-50 catheter filled with heparinized saline was inserted into the corpus cavernosum of the penis, connected to another pressure sensor to monitor intracavernosal pressure (ICP). The pelvic nerve or cavernous nerve (on the contralateral side not measured) was stimulated using an electrical stimulator. Common stimulation parameters were: voltage 5-7.5V, frequency 15-16Hz, pulse width 5ms, duration 60s. The maximum ICP (mICP) and mean arterial pressure (MAP) generated during stimulation were recorded, and the mICP / MAP ratio was calculated. The mICP / MAP ratio is a core indicator for evaluating erectile function; a higher ratio indicates better erectile function.

[0163] Tests showed that the mICP / MAP ratio of rats in the sham-operated group was >0.8, indicating normal erectile function. Rats with an mICP / MAP ratio <0.4 were considered to have successfully established the model.

[0164] (3) Group administration

[0165] Rats with an mICP / MAP ratio <0.4 were selected and randomly divided into 9 groups (model group, positive control group, Example 3 group, comparative example 1 group, comparative example 2 group, comparative example 3 group, comparative example 4 group, comparative example 5 group and comparative example 6 group), with 10 rats in each group.

[0166] The sham-operated group received an equal volume of saline solution, while the positive control group received 0.2 μg of prostaglandin E1 per corpus cavernosum via a micro-injection needle injected from one side of the base of the penis at a very small angle (almost parallel). Injections were administered once a week for four consecutive weeks.

[0167] (4) Detection

[0168] 1) On day 7 after the last injection, the mICP / MAP ratio of rats in each group was measured. The specific results are shown in Table 1.

[0169] Table 1. mICP / MAP ratio of rats in each group

[0170] Grouping mICP / MAP Grouping mICP / MAP Sham surgery group 0.85 Comparative Example 2 0.54 Model control group 0.32 Comparative Example 3 0.37 Positive control group 0.76 Comparative Example 4 0.60 Example 3 0.81 Comparative Example 5 0.62 Comparative Example 1 0.58 Comparative Example 6 0.65

[0171] Table 1 shows that the ratio of the control group to the sham-operated group was significantly lower, indicating severe impairment of erectile function in rats, thus demonstrating the successful establishment of the neural-induced erectile dysfunction model. The ratio in Example 3 was higher than that in the positive control group, indicating that the composition in Example 3 has excellent repair effects on neural-induced erectile dysfunction. Data from Example 3 and Comparative Examples 1-3 show that human foreskin mesenchymal stem cell exosome particles and human placental mesenchymal stem cells have a synergistic effect in treating neural-induced erectile dysfunction; using either alone, doubling the dosage of a single component, or omitting either component fails to achieve the desired repair effect. Data from Example 3 and Comparative Examples 4-6 show that glutathione, L-arginine, methylcobalamin, icariin, and total saponins from ginseng stems and leaves can assist human foreskin mesenchymal stem cell exosome particles and human placental mesenchymal stem cells in treating neural-induced erectile dysfunction.

[0172] 2) After detecting the mICP / MAP ratio, rats were euthanized by spinal dislocation, and the spongy tissue of the rats was taken and added to PBS solution at 4℃ at a ratio of 1:9. The mixture was homogenized to obtain spongy tissue homogenate. Vascular endothelial growth factor (VEGF) was detected using an ELISA kit. The specific results are shown in Table 2.

[0173] 3) The higher the VEGF content, the stronger the cavernous body's blood vessel repair ability.

[0174] Table 2. VEGF content in the corpus cavernosum of rats in each group

[0175] Grouping VEGF (pg / mL) Grouping VEGF (pg / mL) Sham surgery group 85.26 Comparative Example 2 52.64 Model control group 32.53 Comparative Example 3 44.21 Positive control group 75.80 Comparative Example 4 57.79 Example 3 80.18 Comparative Example 5 62.52 Comparative Example 1 55.35 Comparative Example 6 65.27

[0176] Table 2 shows that VEGF levels were significantly lower in the model control group compared to the sham surgery group, indicating impaired vascular function in the corpus cavernosum and successful establishment of the neurogenic erectile dysfunction model. The composition in Example 3 effectively restored VEGF levels in the corpus cavernosum and improved erectile function by promoting vascular repair, showing better results than the prostaglandin E1 in the positive control group. Comparison of data from Example 3 and Comparative Examples 1-3 shows that human foreskin mesenchymal stem cell exosomes and human placental mesenchymal stem cells synergistically promote VEGF secretion. Comparison of data from Example 3 and Comparative Examples 4-6 shows that glutathione, L-arginine, methylcobalamin, icariin, and total saponins from ginseng stems and leaves have positive effects on the treatment of neurogenic erectile dysfunction.

[0177] Experimental Example 2

[0178] Diabetic erectile dysfunction

[0179] Eight-week-old SPF-grade male SD rats were selected and acclimatized for one week. Ten of these rats were used as a blank control group, while the other rats were used to establish a diabetic male erectile dysfunction model.

[0180] (1) Construction of a model of erectile dysfunction in diabetic men

[0181] 1) Rats were fasted for 12 hours but allowed free access to water. Each rat was given a single intraperitoneal injection of streptozotocin at a dose of 65 mg / kg. Six hours after the injection, rats were provided with ample drinking water and food. 10% sucrose could be added to the drinking water for 48 hours.

[0182] Seventy-two hours after streptozotocin injection, fasting blood glucose was measured by taking blood from the tail vein using a blood glucose meter. If the blood glucose level was ≥16.7 mmol / L, the type 1 diabetes model was considered to have been successfully established.

[0183] 2) Six weeks after successful establishment of the type I diabetes model, the mICP / MAP ratio of all rats was verified according to the model verification method in (2) of Experiment 1.

[0184] Tests showed that the mICP / MAP ratio in the blank control group was >0.8, indicating normal erectile function, while rats with an mICP / MAP ratio <0.4 were considered to have successfully established the model.

[0185] (2) Group administration

[0186] Rats with an mICP / MAP ratio <0.4 were selected and randomly divided into 9 groups (model group, positive control group, Example 3 group, comparative example 1 group, comparative example 2 group, comparative example 3 group, comparative example 4 group, comparative example 5 group and comparative example 6 group), with 10 rats in each group.

[0187] The sham-operated group received an equal volume of saline solution, while the positive control group received 10 μL of prostaglandin E1 at a concentration of 3 μg / kg per penis. Injections were administered once a week for four consecutive weeks.

[0188] (3) Detection

[0189] 1) On day 7 after the last injection, the mICP / MAP ratio of rats in each group was measured, and the results are shown in Table 3.

[0190] Table 3. mICP / MAP ratios of rats in each group

[0191] Grouping mICP / MAP Grouping mICP / MAP Blank control group 0.88 Comparative Example 2 0.50 Model control group 0.30 Comparative Example 3 0.40 Positive control group 0.65 Comparative Example 4 0.55 Example 3 0.73 Comparative Example 5 0.57 Comparative Example 1 0.52 Comparative Example 6 0.60

[0192] Table 3 shows that the mICP / MAP ratio in the model control group was significantly lower than that in the blank control group, and less than 0.4, indicating that the animal model of diabetic erectile dysfunction in men was successfully established. Example 3 still performed best in the diabetic model, and was higher than the positive control group, indicating that the composition of Example 3 can alleviate erectile dysfunction caused by diabetes. Data from Example 3 and Comparative Examples 1-3 show that there is a synergistic effect between human foreskin mesenchymal stem cell exosome particles and human placental mesenchymal stem cells; their combined use can significantly alleviate the symptoms of erectile dysfunction caused by diabetes. Data from Example 3 and Comparative Examples 4-6 show that glutathione, L-arginine, methylcobalamin, icariin, and total saponins from ginseng stems and leaves can assist human foreskin mesenchymal stem cell exosome particles and human placental mesenchymal stem cells in the treatment of diabetic erectile dysfunction.

[0193] 2) After detecting the mICP / MAP ratio, rats were euthanized by spinal dislocation, and the spongy tissue of the rats was taken and added to PBS solution at 4℃ at a ratio of 1:9. The mixture was homogenized to obtain a spongy tissue homogenate. Vascular endothelial growth factor (VEGF) was detected using an ELISA kit. The specific results are shown in Table 4.

[0194] Table 4. VEGF content in the corpus cavernosum of rats in each group

[0195] Grouping VEGF (pg / mL) Grouping VEGF (pg / mL) Blank control group 88.51 Comparative Example 2 50.23 Model control group 30.28 Comparative Example 3 40.85 Positive control group 62.47 Comparative Example 4 52.89 Example 3 78.62 Comparative Example 5 55.56 Comparative Example 1 51.74 Comparative Example 6 58.30

[0196] Table 4 shows that VEGF levels were significantly lower in the model control group compared to the sham-operated group, indicating the successful establishment of the diabetic erectile dysfunction rat model. The composition in Example 3 effectively restored VEGF levels in the corpus cavernosum, with better results than the prostaglandin E1 in the positive control group. Comparison of data from Example 3 and Comparative Examples 1-3 shows that human foreskin mesenchymal stem cell exosomes and human placental mesenchymal stem cells synergistically promote VEGF secretion. Comparison of data from Example 3 and Comparative Examples 4-6 shows that glutathione, L-arginine, methylcobalamin, icariin, and total saponins from ginseng stems and leaves have positive effects on the treatment of diabetic erectile dysfunction.

[0197] Overall, the composition of the present invention is more effective than that for erectile dysfunction in men with neurological damage than for men with diabetic erectile dysfunction, possibly because diabetic erectile dysfunction is often caused by mixed factors, including vascular lesions, neuropathy, and psychological factors.

[0198] In summary, the composition of the present invention is effective for two common types of male erectile dysfunction: nerve damage and diabetes. Moreover, it is superior to traditional drugs in the case of highly difficult diabetic erectile dysfunction, and has good potential for broad-spectrum application.

[0199] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition for the treatment of erectile dysfunction in men, characterized in that, The composition comprises human foreskin mesenchymal stem cell exosome particles and human placental mesenchymal stem cells. The concentration of the human placental mesenchymal stem cells is (3-7) x 10 6 The particle concentration of the human foreskin mesenchymal stem cell exosome is (2-3) x 10 11 particles / mL.

2. The composition of claim 1, wherein, The particle size of the human foreskin mesenchymal stem cell exosome particles is 50-120 nm.

3. The composition of claim 1, wherein, The human foreskin mesenchymal stem cell exosome expresses CD9 + , CD63 + , and CD81 + at a particle ratio of >90%.

4. The composition of claim 1, wherein, The number of cells expressing CD29 + , CD44 + , CD73 + , CD90 + , CD105 + in the human placental mesenchymal stem cells is ≥ 95%, and the number of cells expressing CD34 + , CD45 + , HLA-DR + is ≤ 2%.

5. The composition of claim 2, wherein The composition further comprises glutathione, L-arginine, icariin, panax notoginseng stem and leaf total saponins, methylcobalamin and human blood albumin.

6. The composition of claim 5, wherein, The concentration of the glutathione is 1-3 mg / mL, the concentration of the L-arginine is 0.5-1.5 mg / mL, the concentration of the icariin is 0.1-0.2 mg / mL, the concentration of the panax notoginseng stem and leaf total saponins is 0.2-0.5 mg / mL, the concentration of the methylcobalamin is 0.5-1 mg / mL, and the concentration of the human blood albumin is 5-10 mg / mL.

7. A process for the preparation of a composition according to claim 5, characterized in that, The composition comprises: The human blood albumin and normal saline are mixed first to obtain a human blood albumin solution, and then the glutathione, L-arginine, methylcobalamin, icariin and panax notoginseng stem and leaf total saponins are added in sequence to obtain a matrix solution; the human foreskin mesenchymal stem cell exosome particles and human placental mesenchymal stem cells are mixed with the matrix solution to obtain the composition.

8. Use of the composition according to any one of claims 1-6 in the preparation of a medicament for treating male erectile dysfunction.

9. Use according to claim 8, characterized in that, The male erectile dysfunction is of the diabetes type or the nerve injury type.

10. Use according to claim 9, characterized in that, The medicament is administered by intracavernous injection.