Preparation method and application of stem cell preparation for repairing male sexual dysfunction

Abstract

The invention provides a preparation method and application of a stem cell preparation for repairing male sexual dysfunction. The preparation method comprises the following steps: extraction and primary culture of mesenchymal stem cells; regulating and controlling secretion signals of the mesenchymal stem cells; and preparing the preparation. According to the preparation method of the stem cell preparation for repairing the male sexual dysfunction, the mitochondrial repair signal inducer is added in the mesenchymal stem cell culture stage, and the mitochondrial function repair promoting component is added into the preparation, so that the obtained stem cell preparation can repair the mitochondrial energy metabolism function; the mitochondrial quality control is promoted; the cavernous body tissue microenvironment is improved. The preparation method provided by the invention can achieve the beneficial effects of performing targeted repair on the focus of male sexual dysfunction and improving the treatment and repair efficiency.

Description

Technical Field

[0001] This application belongs to the field of cell preparation technology, and more specifically, relates to a method for preparing and applying a stem cell preparation for repairing male sexual dysfunction. Background Technology

[0002] Male sexual dysfunction (MSD) is a common male reproductive system disorder with a significantly increased incidence rate with age. Globally, the prevalence among men over 40 years of age reaches 40%-50%, severely impacting patients' quality of life and partner relationships. Currently identified male sexual dysfunctions mainly include erectile dysfunction (ED), premature ejaculation, decreased libido, and ejaculatory disorders. Male sexual dysfunction is the result of the interaction of multiple factors, including physiological, psychological, lifestyle, and underlying medical conditions.

[0003] Current clinical treatments have significant limitations. Oral medications, such as PDE5 inhibitors (Phosphodiesterase type 5 inhibitors), can only relieve symptoms and cannot repair vascular damage. Long-term use can easily lead to drug resistance, and they are not effective for severe symptoms. Physical therapy usually uses physical methods such as vacuum erection devices and low-energy shockwaves, which can only play an auxiliary role by improving local microcirculation or nerve sensitivity. However, patient compliance is poor, and it may also cause damage to the corpus cavernosum tissue, resulting in secondary injury. Surgical treatment, such as penile prosthesis implantation, is highly invasive and has many complications, and is only suitable for end-stage patients.

[0004] Mesenchymal stem cells (MSCs) are considered a new direction for the treatment of male sexual dysfunction due to their self-renewal, multi-lineage differentiation potential, and paracrine effects. However, as an emerging field, the current technological system still has many shortcomings and areas that need improvement. For example, it does not target the repair of lesions, resulting in low treatment efficiency. Technologists need to provide more technical solutions to meet clinical needs. Summary of the Invention

[0005] The purpose of this application is to provide a method for preparing and applying a stem cell preparation for repairing male sexual dysfunction, so as to solve the technical problem in the prior art that the lesions are not specifically repaired, resulting in low treatment and repair efficiency.

[0006] To achieve the above objectives, the technical solution adopted in this application is: a method for preparing a stem cell preparation for repairing male sexual dysfunction, comprising the following steps: Mesenchymal stem cell extraction and initial culture: Healthy human tissues were selected, refined, and then digested using an enzymatic digestion method; after digestion, the mesenchymal stem cells were collected by filtration and centrifugation and cultured to the second generation; Regulation of secretory signals by mesenchymal stem cells: Mitochondrial repair signal inducers were added to the culture medium. After culturing mesenchymal stem cells for 36-48 hours, cell supernatant was collected to verify the mitochondrial repair function of secretory signals by mesenchymal stem cells. Mesenchymal stem cells were collected for formulation preparation. The mitochondrial repair signal inducers included 5-10 ng / mL transforming growth factor-β1 and 5-10 ng / mL hepatocyte growth factor. Preparation of the formulation: Using physiological saline containing 0.05%-0.2% trehalose, 1%-4% human serum albumin, and 0.5%-2% hydroxypropyl methylcellulose as the base solution, a mitochondrial function repair component was added to the base solution, followed by mesenchymal stem cells, and the mixture was suspended to obtain the stem cell formulation; the mitochondrial function repair component included 0.05%-0.2% vitamin E, 0.2-0.5 μmol / L coenzyme Q10, 0.5-1 mmol / L N-acetylcysteine, 3-7 μg / mL vascular endothelial growth factor, and 1-3 μmol / L MitoQ.

[0007] In one embodiment, in the mesenchymal stem cell extraction and initial culture step, the enzymatic digestion method employs a complex enzymatic digestion system, including collagenase IV at a mass fraction of 0.2%-0.5% and hyaluronidase at a mass fraction of 0.05%-0.1%.

[0008] In one embodiment, the pH value of the complex enzymatic hydrolysis system is 7.2-7.4.

[0009] In one embodiment, in the step of regulating the secretory signal of mesenchymal stem cells, the method for verifying the mitochondrial repair function of the secretory signal of mesenchymal stem cells is as follows: the cell supernatant of mesenchymal stem cells is co-incubated with damaged vascular endothelial cells and smooth muscle cells of the corpus cavernosum of the penis. After 24 hours, the expression levels of perkinin, peroxisome proliferator-activated receptor γ coactivator 1α, and the amount of adenine triphosphate produced in the damaged cells are detected.

[0010] In one embodiment, in the formulation preparation step, the amount of mesenchymal stem cells added is based on a final cell concentration of 2 × 10⁻⁶. 6 -3×10 6 per mL.

[0011] In one embodiment, the mitochondrial function repair component further includes 0.25-0.7 mmol / L creatine in the formulation preparation step.

[0012] In one embodiment, during the mesenchymal stem cell extraction and initial culture steps, when the mesenchymal stem cells are cultured to the second generation, the cell fusion rate is controlled at 70%-80%, so that the cells are in the logarithmic growth phase.

[0013] In one embodiment, the dosage form of the formulation is an injectable form.

[0014] This application also provides an application of a stem cell preparation for repairing male sexual dysfunction, wherein the stem cell preparation is obtained using the preparation method provided in this application, and the male sexual dysfunction is erectile dysfunction.

[0015] In one embodiment, erectile dysfunction is caused by one or both of insufficient arterial blood supply and venous closure dysfunction.

[0016] The method for preparing a stem cell preparation for repairing male sexual dysfunction provided in this application has the following beneficial effects: Compared with the prior art, the preparation method of this application, by adding mitochondrial repair signal inducers including transforming growth factor-β1 and hepatocyte growth factor during the mesenchymal stem cell culture stage, can induce mesenchymal stem cells to secrete extracellular vesicles containing mitochondrial protective factors, enhance the synthesis capacity of cellular antioxidant enzymes, and also enhance cell viability and promote the secretion of angiogenesis repair factors. The preparation method provided in this application also adds mitochondrial function repair components containing vitamin E, coenzyme Q10, N-acetylcysteine, vascular endothelial growth factor, and MitoQ to the preparation, which can efficiently scavenge reactive oxygen species in cells, reduce reactive oxygen species accumulation, activate the mitochondrial respiratory chain, repair mitochondrial energy metabolism function, promote mitochondrial quality control, and improve the microenvironment of the corpus cavernosum tissue. In summary, the preparation method provided in this application can achieve the beneficial effects of targeted repair of lesions in male sexual dysfunction and improve treatment and repair efficiency. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A statistical graph showing the relative expression levels of Parkin and PGC-1α and the amount of ATP produced in smooth muscle cells; Figure 2 A statistical graph showing the relative expression levels of Parkin and PGC-1α and the amount of ATP produced in vascular endothelial cells; Figure 3 This is a statistical chart showing the detection of erectile function indicators in a vascular erectile dysfunction model of SD rats. Detailed Implementation

[0019] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

[0020] It should be noted that when a component is referred to as being "fixed to" or "set on" another component, it can be directly on or indirectly on that other component. When a component is referred to as being "connected to" another component, it can be directly connected to or indirectly connected to that other component.

[0021] It should be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0022] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0023] Please refer to the following: Figure 1 , Figure 2 and Figure 3 The preparation method and application of the stem cell preparation for repairing male sexual dysfunction provided in the embodiments of this application will now be described.

[0024] This application provides a method for preparing a stem cell preparation for repairing male sexual dysfunction, comprising the following steps: Mesenchymal stem cell extraction and initial culture: Healthy human tissues were selected, refined, and then digested using an enzymatic digestion method; after digestion, the mesenchymal stem cells were collected by filtration and centrifugation and cultured to the second generation; Regulation of secretory signals by mesenchymal stem cells: Mitochondrial repair signal inducers were added to the culture medium. After culturing mesenchymal stem cells for 36-48 hours, cell supernatant was collected to verify the mitochondrial repair function of secretory signals by mesenchymal stem cells. Mesenchymal stem cells were collected for formulation preparation. The mitochondrial repair signal inducers included 5-10 ng / mL transforming growth factor-β1 and 5-10 ng / mL hepatocyte growth factor. Preparation of the formulation: Using physiological saline containing 0.05%-0.2% trehalose, 1%-4% human serum albumin, and 0.5%-2% hydroxypropyl methylcellulose as the base solution, a mitochondrial function repair component was added to the base solution, followed by mesenchymal stem cells, and the mixture was suspended to obtain the stem cell formulation; the mitochondrial function repair component included 0.05%-0.2% vitamin E, 0.2-0.5 μmol / L coenzyme Q10, 0.5-1 mmol / L N-acetylcysteine, 3-7 μg / mL vascular endothelial growth factor, and 1-3 μmol / L MitoQ.

[0025] The following is a detailed explanation of the steps for mesenchymal stem cell extraction and initial culture: For human tissues, placenta, umbilical cord, bone marrow, and adipose tissue can be selected. These tissues are commonly used for mesenchymal stem cell extraction, are ethically compliant, and yield cells with high viability and low immunogenicity. However, placenta and umbilical cord tissue are preferred because they are postpartum waste, have fewer ethical controversies, and yield mesenchymal stem cells with stronger proliferative capacity. Bone marrow aspiration is an invasive procedure, resulting in poor patient compliance and low cell yield; cells extracted from adipose tissue are easily affected by donor age and obesity, exhibiting significant differences in cell viability and secretory function, which is detrimental to the stability of formulation quality. For tissue refinement, the tissue is first rinsed three times with physiological saline, soaking for 5 minutes each time to remove surface impurities, and then cut into 1-2 mm pieces using sterile scissors. 3 Small tissue fragments are prepared to ensure thorough contact between the tissue and the enzymatic digestion process. The tissue suspension is then filtered and centrifuged using a 200-mesh sterile cell sieve to remove undigested tissue residue. Centrifugation should not be too high; 1000-1200 rpm for 8-10 minutes, with a centrifugal force of approximately 100-150 × g, is recommended. For mesenchymal stem cell culture, DMEM / F12 medium is used as a base, supplemented with human serum albumin (HSA), recombinant human albumin, or other nutrients that can replace bovine serum albumin (FBS) in conventional cell culture media. Mesenchymal stem cell culture is conducted under low oxygen conditions (1%-5% oxygen by volume), with other conditions including 37°C and 5% CO2. Culturing mesenchymal stem cells to the second generation helps stabilize the cells for subsequent steps.

[0026] The following is a detailed explanation of the regulation of secretory signals in mesenchymal stem cells: From a cellular and molecular perspective, male sexual dysfunction is closely related to mitochondrial dysfunction in functional cells, such as penile cavernous vascular endothelial cells and smooth muscle cells. Mitochondria are important organelles within cells, primarily responsible for energy production and metabolic regulation, serving as the cell's power source and participating in cell life and death regulation. They can influence cellular metabolism and function by producing signaling molecules such as reactive oxygen species (ROS). Transforming growth factor-β1 (TGF-β1), by binding to TGF-β receptors I / II on the surface of mesenchymal stem cells, activates the Smad2 / 3 signaling pathway, promoting the secretion of extracellular vesicles (EVs) containing mitochondrial protective factors such as miR-133a and miR-378, while simultaneously enhancing the synthesis of cellular antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT). Hepatocyte growth factor (HGF) activates the PI3K / Akt signaling pathway by binding to c-Met receptors on the surface of mesenchymal stem cells. This enhances cell viability and specifically promotes the secretion of vascular repair factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Mitochondrial repair signaling inducers can also include components with similar functions, such as fibroblast growth factor-2 (FGF-2) and lipoic acid. The effects of mitochondrial repair signaling inducers require time; therefore, cells need to be treated for 36-48 hours.

[0027] The preparation of the formulation is described in detail below: In the base solution, trehalose, human serum albumin, and hydroxypropyl methylcellulose (HPMC) mainly serve to protect cells during cryopreservation. Trehalose, as a non-reducing disaccharide, can form hydrogen bonds with cell surface and mitochondrial membrane proteins, creating a protective film around the cells and reducing mechanical damage to cells from ice crystals during cryopreservation. It also stabilizes cell membrane structure and inhibits the aggregation of mesenchymal stem cells during suspension. Human serum albumin provides physiological colloid osmotic pressure, maintaining the stability of the formulation's osmotic pressure. As a carrier protein, it binds to and protects mitochondrial repair-promoting components, reducing their oxidative degradation. Hydroxypropyl methylcellulose, as a high-molecular-weight suspending agent, increases the formulation's viscosity, preventing the sedimentation of mesenchymal stem cells and repair components, ensuring the uniformity of the formulation. It also creates a viscous microenvironment, reducing direct collision damage between cells.

[0028] Among the components that promote mitochondrial function repair, vitamin E is a fat-soluble antioxidant that preferentially removes reactive oxygen species on the surface of cell membranes and mitochondrial membranes, protecting membrane lipids from oxidation and maintaining the structural integrity of mitochondrial membranes. Coenzyme Q10 (CoQ10), as an electron carrier in mitochondrial respiratory chain complexes II / III, directly participates in the ATP synthesis process, improving the efficiency of mitochondrial energy metabolism. It also has antioxidant functions, reducing ROS generated by the respiratory chain, and can synergize with endogenous coenzyme Q10. N-acetylcysteine ​​(NAC) is a water-soluble antioxidant that can penetrate cell membranes and enter cells. As a precursor to glutathione (GSH), it increases intracellular GSH levels and clears ROS from the cytoplasm and mitochondrial matrix. It can also chelate metal ions, inhibit the Fenton reaction, and reduce its damage to mitochondrial DNA (mtDNA) and mitochondrial membrane structure. Vascular endothelial growth factor (VEGF) can promote angiogenesis in the corpus cavernosum of the penis, improve local blood perfusion, provide oxygen and nutrients for mitochondrial repair, and activate the PI3K / Akt signaling pathway in damaged cells. It upregulates the expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) and promotes mitochondrial biosynthesis. MitoQ is a triphenylphosphine (TPP)-based mitochondrial-targeting antioxidant that can block hydrogen peroxide (H2O2)-induced intracellular ROS reactions and prevent oxidative damage. It targets and enriches itself in the mitochondrial matrix through its lipophilic cationic groups, selectively clearing ROS produced by mitochondria and protecting mtDNA from oxidative damage. MitoQ's targeting of mitochondria avoids non-specific effects on other parts of the cell. Furthermore, ingredients that promote mitochondrial function repair may also include nicotinamide mononucleotide (NMN), carnosine, and resveratrol.

[0029] The method for preparing a stem cell preparation for repairing male sexual dysfunction provided in this application, compared with existing technologies, induces the secretion of extracellular vesicles containing mitochondrial protective factors by adding mitochondrial repair signal inducers, including transforming growth factor-β1 and hepatocyte growth factor, during the mesenchymal stem cell culture stage. This enhances the cell's ability to synthesize antioxidant enzymes and improves cell viability, while also promoting the secretion of angiogenesis repair factors. Furthermore, the method further incorporates mitochondrial function repair components containing vitamin E, coenzyme Q10, N-acetylcysteine, vascular endothelial growth factor, and MitoQ into the preparation. This effectively scavenges reactive oxygen species in cells, reducing their accumulation; activates the mitochondrial respiratory chain, repairing mitochondrial energy metabolism; and promotes mitochondrial quality control and improves the microenvironment of the corpus cavernosum tissue. In summary, the preparation method provided in this application can achieve the beneficial effects of targeted repair of lesions in male sexual dysfunction, improving treatment and repair efficiency.

[0030] In one embodiment, in the mesenchymal stem cell extraction and initial culture step, the enzymatic digestion method employs a complex enzymatic digestion system, including collagenase IV at a mass fraction of 0.2%-0.5% and hyaluronidase at a mass fraction of 0.05%-0.1%.

[0031] Specifically, collagenase IV specifically degrades type I and type III collagen in the tissue matrix, disrupting the fibrous network structure of the tissue and dispersing cell clusters into smaller cell clusters. Compared to collagenase I / II, collagenase IV causes less damage to stem cell surface antigens and is gentler on the intercellular matrix, reducing apoptosis caused by over-digestion. Hyaluronic acid is a key substance for intercellular adhesion; hyaluronidase can specifically degrade hyaluronic acid in the matrix, breaking down intercellular adhesion and further dispersing cell clusters into individual cells. The enzyme concentration should not be too high, otherwise it will lead to increased osmotic pressure of the enzyme solution, causing cell dehydration and increasing the shedding rate of contaminating cells such as fibroblasts. During digestion, the digestion system can be placed in a 37°C constant temperature water bath for 1-2 hours. The synergistic effect of the enzymes accelerates tissue dispersion and reduces the exposure time of cells in the enzyme solution.

[0032] Of course, some components can be added to the compound enzymatic hydrolysis system according to tissue type, cell characteristics and experimental needs, such as collagenase, elastase and chondroitin sulfate.

[0033] In one embodiment, the pH value of the complex enzymatic hydrolysis system is 7.2-7.4.

[0034] Specifically, strictly controlling the pH value within the physiological range of 7.2-7.4 is crucial for ensuring enzyme activity, cell viability, and tissue digestion efficiency. Under physiological pH conditions, the enzyme solution penetrates the tissue more evenly, avoiding localized over-digestion that could lead to cell damage. The enzymatic hydrolysis system can use calcium- and magnesium-free phosphate buffer (PBS) as the base solvent.

[0035] In one embodiment, in the step of regulating the secretory signal of mesenchymal stem cells, the method for verifying the mitochondrial repair function of the secretory signal of mesenchymal stem cells is as follows: the cell supernatant of mesenchymal stem cells is co-incubated with damaged vascular endothelial cells and smooth muscle cells of the corpus cavernosum of the penis. After 24 hours, the expression levels of perkinin, peroxisome proliferator-activated receptor γ coactivator 1α, and the amount of adenine triphosphate produced in the damaged cells are detected.

[0036] Specifically, in the physiological mechanisms of male erectile function, vascular endothelial cells and smooth muscle cells of the corpus cavernosum are important functional cells. The corpus cavernosum is rich in a vascular network, with vascular endothelial cells covering all vascular lumens. Their core function is to release NO, initiating vasodilation signaling pathways and regulating vascular tone to ensure smooth blood flow into the corpus cavernosum. They also regulate the intensity and duration of vasodilation through various mechanisms, avoiding excessive or insufficient vasodilation, such as NO degradation regulation and vascular tone sensing and feedback. The corpus cavernosum is composed of numerous smooth muscle cells forming cavernous trabeculae, which connect to each other as capillary sinuses. The contraction and relaxation of smooth muscle cells directly determine the volume and pressure of these sinuses. In male sexual dysfunction, ischemia, hypoxia, and oxidative stress damage the mitochondria of these cells, leading to functional abnormalities. Therefore, when verifying the mitochondrial repair function of mesenchymal stem cell secretion signals, damaged vascular endothelial cells and smooth muscle cells of the corpus cavernosum were used as verification cell models.

[0037] Parkin is a key protein in the mitophagy pathway, and increased expression of it indicates enhanced clearance of abnormal mitochondria. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is a core regulator of mitochondrial biosynthesis, and increased activity and expression of PGC-1α promote healthy mitochondrial regeneration. Adenine triphosphate (ATP) is the end product of mitochondrial energy metabolism, and its content directly reflects the degree of mitochondrial functional recovery. Currently, various biological methods and kits are available for detection. For example, conventional methods such as reverse transcription quantitative polymerase chain reaction (RT-qPCR) can be used to detect the expression levels of Parkin and PGC-1α, while ATP detection kits can be used to measure ATP production.

[0038] In one embodiment, during the formulation preparation step, the amount of mesenchymal stem cells added is based on a final cell concentration of 2 × 10⁻⁶. 6 -3×10 6 per mL.

[0039] Specifically, the paracrine effect of mesenchymal stem cells exhibits a density-effect relationship. When the cell concentration is too low, the concentration of repair signals such as VEGF and EVs secreted by mesenchymal stem cells per unit volume is insufficient, which cannot effectively activate the mitochondrial autophagy and biosynthesis pathways of damaged corpus cavernosum cells, thus weakening their repair effect. When the cell concentration is too high, mesenchymal stem cells are prone to aggregation in the formulation. Aggregated cell clusters can lead to an abnormal increase in the viscosity of the formulation, affecting its clinical application performance.

[0040] In one embodiment, the mitochondrial function repair component further includes 0.25-0.7 mmol / L creatine in the formulation preparation step.

[0041] Specifically, creatine is a nitrogenous organic acid naturally found in muscle tissue. Intracellularly, it reacts with ATP to generate phosphocreatine (PCr), forming the "ATP-PCr energy buffer system," which is particularly important in high-energy-consuming cells such as smooth muscle cells and endothelial cells. During erection, penile cavernous body smooth muscle cells rapidly consume large amounts of ATP. When mitochondrial ATP production is insufficient, phosphocreatine, through creatine kinase (CK) catalysis, transfers high-energy phosphate bonds to adenosine diphosphate (ADP) to generate ATP, thus stabilizing intracellular ATP levels for a short period and preventing smooth muscle relaxation dysfunction caused by a sudden drop in energy. Creatine can also enter the mitochondrial matrix via the creatine transporter protein (SLC6A8) on the mitochondrial membrane, combining with mitochondrial-generated ATP to generate PCr. PCr more easily diffuses across the mitochondrial membrane into the cytoplasm, improving mitochondrial energy output efficiency. Furthermore, creatine can protect mitochondrial structural integrity and reduce oxidative damage by inhibiting the opening of the mitochondrial permeability transition pore (mPTP) and enhancing the activity of antioxidant enzymes.

[0042] In one embodiment, during the mesenchymal stem cell extraction and initial culture steps, when the mesenchymal stem cells are cultured to the second generation, the cell fusion rate is controlled at 70%-80%, so that the cells are in the logarithmic growth phase.

[0043] Specifically, the cell growth cycle can be divided into three phases: the latent phase, the logarithmic growth phase, and the plateau phase. Mesenchymal stem cells in the logarithmic growth phase are highly proliferating, with the shortest cell doubling time; their cells are in good condition, exhibiting uniform morphology, plump cell bodies, few cytoplasmic granules, and a moderate nucleocytoplasmic ratio. Furthermore, mesenchymal stem cells in this phase are in a highly active functional state, secreting more mitochondrial repair-related signaling molecules than in other phases, thus contributing to improved therapeutic efficacy.

[0044] When cell confluence is <70%, although the cells are in the early logarithmic growth phase, the low cell density leads to insufficient contact-dependent signaling. When confluence is >80%, the cells gradually enter a plateau phase, and contact inhibition triggers a series of functional declines, such as decreased paracrine function. Cell confluence can be determined using an inverted microscope. The principle is to observe the percentage of cell coverage on the culture dish surface, combined with cell morphology characteristics, to determine the range of confluence. It should be noted that cells may be unevenly distributed in the culture dish due to edge effects, requiring observation of multiple fields of view using the "five-point sampling method."

[0045] In one embodiment, the dosage form of the formulation is an injectable form.

[0046] Specifically, injectable formulations offer clinical advantages, delivering the medication directly to the affected area via a precise delivery route. Since the core lesion site in male sexual dysfunction is the corpus cavernosum, intracavernosal injection allows for rapid diffusion of the medication through the cavernous tissue's sinus network. Mesenchymal stem cells can directly engraft around damaged vascular endothelium and smooth muscle cells, and mitochondrial repair-promoting components achieve effective local concentrations, avoiding the low bioavailability caused by gastrointestinal degradation and the first-pass effect in the liver associated with oral formulations. Injectable formulations also offer advantages such as ease of administration, standardization, and high patient acceptance. Compared to surgical treatment, injection therapy is less invasive, allows for faster recovery, and enables dosage adjustments based on treatment efficacy, reducing patient psychological burden.

[0047] This application also provides an application of a stem cell preparation for repairing male sexual dysfunction, wherein the stem cell preparation is obtained by the preparation method provided above, and the male sexual dysfunction is erectile dysfunction.

[0048] Specifically, erectile dysfunction (ED) is the most common type of male sexual dysfunction, with a global prevalence of 42% among men aged 40-70, of which 9.6% are severe ED. The main clinical manifestation is the inability to achieve or maintain a sufficiently rigid penile erection for more than three months, resulting in the inability to complete sexual intercourse. The core pathological mechanism of ED is the inability of the corpora cavernosa to achieve or maintain adequate blood filling, leading to insufficient erectile rigidity or short duration of erection. The etiological classification of ED commonly includes three types: organic, psychogenic, and mixed. Organic ED mainly includes vascular, neurological, anatomical, and endocrine factors; psychogenic ED is mainly related to anxiety, depression, and relationship conflicts; and mixed ED is caused by the interaction of multiple pathogenic factors, both organic and psychogenic. Some diseases can also lead to ED, such as diabetes, metabolic syndrome, cardiovascular disease, Peronis' disease, and pelvic floor nerve damage. This is because the vascular homeostasis of the corpora cavernosa is mainly regulated by the vascular endothelium. Most of these diseases are related to endothelial dysfunction, which is related to the severity of ED.

[0049] In one embodiment, erectile dysfunction is caused by one or both of insufficient arterial blood supply and venous closure dysfunction.

[0050] Specifically, insufficient arterial blood supply leads to erectile dysfunction (ED) due to a lack of water at the source. When the cavernous arteries and their branches become narrowed, blocked, or lose elasticity, the arterial dilation under sexual stimulation cannot meet the blood demand of the corpora cavernosa, resulting in insufficient engorgement. The most common cause of insufficient arterial blood supply is atherosclerosis caused by damage to the vascular endothelium. Venous closure dysfunction results in short erection duration due to leakage of stored blood. When the function of the corpora cavernosa sinusoids, venous valves, or tunica albuginea is abnormal, even with sufficient arterial blood supply, blood will leak abnormally through the veins, failing to maintain sufficient pressure within the corpora cavernosa. This manifests as an erection that can be initiated but quickly becomes flaccid, or a gradual decrease in rigidity during sexual activity. A common cause of venous closure dysfunction is damage to the corpora cavernosa sinusoids due to necrosis of the sinusoidal cells. The stem cell preparation provided in this application can treat these pathological mechanisms.

[0051] The present invention will be further illustrated below through specific embodiments: Example 1 In this embodiment, during the mesenchymal stem cell extraction and initial culture steps, the enzymatic digestion method employs a composite enzymatic digestion system, consisting of 0.5% collagenase IV and 0.07% hyaluronidase by mass, with a pH of 7.4.

[0052] In this embodiment, during the step of regulating mesenchymal stem cell secretion signals, the mitochondrial repair signal inducers include 10 ng / mL transforming growth factor-β1 and 10 ng / mL hepatocyte growth factor.

[0053] In this embodiment, during the preparation of the formulation, the mitochondrial function repair component consists of 0.2% by mass of vitamin E, 0.5 μmol / L coenzyme Q10, 0.5 mmol / L of N-acetylcysteine, 5 μg / mL of vascular endothelial growth factor, 2 μmol / L of MitoQ, and 0.5 mmol / L of creatine.

[0054] In this embodiment, the method for verifying the mitochondrial repair function of mesenchymal stem cell secretion signal in the step of mesenchymal stem cell secretion signal regulation is as follows: the cell supernatant of mesenchymal stem cells is co-incubated with damaged vascular endothelial cells and smooth muscle cells of the corpus cavernosum of the penis. After 24 hours, the expression levels of Parkin and PGC-1α in the damaged cells are detected by RT-qPCR, and the amount of ATP generated is detected by ATP detection kit.

[0055] 1. The damaged vascular endothelial cells and smooth muscle cells of the corpus cavernosum were derived from a vascular erectile dysfunction (ED) model in SD rats. Specifically, after sacrificing rats with carbon dioxide, the corpus cavernosum tissue was harvested, rinsed with physiological saline, and cut into 1mm pieces. 3Small pieces were digested with collagenase II for 1 hour. After digestion, the cells were filtered and centrifuged to obtain a mixed cell suspension. The suspension was then purified using immunomagnetic bead sorting: for vascular endothelial cells, CD31 antibody-conjugated magnetic beads were used for sorting; for smooth muscle cells, α-smooth muscle actin (α-SMA) antibody-conjugated magnetic beads were used. The purified cells were cultured in specific media: vascular endothelial cells were cultured in ECM medium containing 10% FBS; smooth muscle cells were cultured in SMGM medium containing 10% FBS.

[0056] 2. The specific methods for detecting the expression levels of Parkin and PGC-1α, and the amount of ATP produced are as follows: (1) Detection of expression levels of Parkin and PGC-1α S1. Total RNA Extraction from Cells: Remove the culture medium from the cells to be tested, wash the cells twice with PBS, and add 1 mL of Trizol reagent. Gently agitate the culture dish to cover all cells, and repeatedly pipette to ensure complete cell lysis. Incubate at room temperature for 5 minutes. Transfer the lysis buffer to a 1.5 mL RNase-free centrifuge tube, add 200 μL of chloroform, vortex vigorously for 15 seconds, incubate at room temperature for 3 minutes, and centrifuge at 12,000 × g for 15 minutes at 4°C. After centrifugation, the liquid separates into three layers: a colorless aqueous phase containing RNA in the upper layer, a white middle layer, and an organic phase in the lower layer. Carefully aspirate the upper aqueous phase into a new RNase-free centrifuge tube, avoiding the middle layer; add an equal volume (500 μL) of isopropanol, gently invert to mix, and incubate at room temperature for 10 minutes; centrifuge at 12,000 × g for 10 minutes at 4°C, discard the supernatant, and a white RNA precipitate will be visible at the bottom of the tube; add 1 mL of 75% ethanol prepared with DEPC water, and gently invert to wash the precipitate; centrifuge at 7,500 × g for 5 minutes at 4°C, and aspirate as much supernatant as possible; invert the precipitate at room temperature for 10 minutes to air dry, avoiding complete drying, otherwise the RNA will be difficult to dissolve. Add 40 μL of DEPC water, and gently pipette until the precipitate is completely dissolved; incubate at 55°C for 10 minutes to promote dissolution.

[0057] S2. RNA quality and concentration detection: Take 1 μL of RNA solution and use NanoDrop to detect the concentration and purity. An A260 / A280 ratio of 1.8-2.0 indicates that the RNA is relatively pure and has little contamination; a concentration ≥50 ng / μL is preferable.

[0058] S3. Reverse transcription to synthesize cDNA and real-time quantitative PCR (qPCR): The BeyoFast™ SYBR GreenOne-Step qRT-PCR Kit, purchased from Beyotime Biotechnology, catalog number D7268S, was used. The experimental procedures were performed according to the kit instructions and will not be detailed here. GAPDH was used as the internal reference gene, and the primer sequences for each gene are shown in the table below: S4. Data Analysis: The relative expression level was calculated using the 2^(-ΔΔCt) method: ΔCt = Ct value of target gene - Ct value of internal reference gene; ΔΔCt = ΔCt of experimental group - ΔCt of control group; relative expression level = 2^(-ΔΔCt).

[0059] (2) ATP production detection The ATP assay kit was purchased from Beyotime Biotechnology, catalog number S0026. Refer to the kit instructions; the specific steps are as follows: S1. Collect test samples: Aspirate the culture medium from the cells to be tested, add lysis buffer at a ratio of 200 μL per well of a 6-well plate, and lyse the cells. After lysis, centrifuge at 12000g for 5 minutes at 4°C, and collect the supernatant for subsequent assays.

[0060] S2. Preparation for standard curve determination: Melt the reagents to be used on an ice bath, and dilute the ATP standard solution with ATP detection lysis buffer to a concentration gradient of 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM.

[0061] S3. Preparation of ATP detection working solution: Prepare an appropriate amount of ATP detection working solution according to the ratio of 100 μL of ATP detection working solution required for each sample or standard. Thaw the reagent to be used in an ice bath. Take an appropriate amount of ATP detection reagent and dilute it with ATP detection reagent diluent at a ratio of 1:9.

[0062] S4. Determination of ATP Concentration: Take a 96-well plate and add 100 μL of ATP detection working solution to each well. Incubate at room temperature for 3-5 minutes to allow all background ATP to be consumed, thereby reducing the background level. Add 20 μL of sample or standard to each well, mix quickly with a micropipette, and after at least 2 seconds, measure the RLU value using a chemiluminescence analyzer. Then calculate the ATP concentration in the sample based on the standard curve.

[0063] The method for preparing the stem cell preparation for repairing male sexual dysfunction in this embodiment includes the following steps: Mesenchymal stem cell extraction and initial culture: Healthy human placental tissue was selected. During tissue refinement, the tissue was first rinsed three times with physiological saline, soaking for 5 minutes each time to remove surface impurities. Then, it was cut into 1-2 mm pieces using sterile scissors. 3Small tissue fragments were collected. A complex enzymatic digestion system was used to digest the tissue fragments, and the digestion system was placed in a 37°C water bath for 2 hours. After digestion, the digested tissue suspension was filtered through a 200-mesh sterile cell sieve to remove undigested tissue residue. Mesenchymal stem cells were collected by centrifugation at 1200 rpm for 8 minutes and cultured in culture dishes. The culture medium was DMEM / F12 based, supplemented with 10% human serum albumin (HSA), and the culture conditions were 37°C, 3% O2, and 5% CO2. When the mesenchymal stem cells reached the second generation, cell confluence was controlled at 70%-80% using an inverted microscope, indicating that the cells were in the logarithmic growth phase.

[0064] Regulation of secretory signals in mesenchymal stem cells: Mitochondrial repair signal inducers were added to the culture medium. After culturing mesenchymal stem cells for 48 hours, cell supernatant was collected to verify the mitochondrial repair function of secretory signals in mesenchymal stem cells. Mesenchymal stem cells were also collected for formulation preparation.

[0065] Formulation preparation: Using physiological saline containing 0.2% trehalose, 3% human serum albumin, and 1% hydroxypropyl methylcellulose as the base solution, a mitochondrial function repair-promoting component was added to the base solution, and then the final cell concentration was adjusted to 3 × 10⁻⁶. 6 Mesenchymal stem cells were added at a concentration of 1 / mL and suspended to obtain a stem cell preparation. The dosage form was for injection, and each vial contained 1 mL.

[0066] In this embodiment, the stem cell preparation is used to repair erectile dysfunction caused by insufficient arterial blood supply and venous closure dysfunction.

[0067] Example 2 Based on Example 1, the difference lies in the step of mesenchymal stem cell extraction and initial culture, where healthy adipose tissue is selected for the extraction of mesenchymal stem cells.

[0068] Example 3 Based on Example 1, the difference is that the mitochondrial function repair component does not include creatine in the preparation of the formulation.

[0069] Comparative Example 1 Based on Example 1, the difference is that no mitochondrial repair signal inducer was added to the culture medium in the step of regulating the secretory signal of mesenchymal stem cells.

[0070] Comparative Example 2 Based on Example 1, the difference is that no mitochondrial function repair components are added in the preparation of the formulation.

[0071] Comparative Example 3 Based on Example 1, the difference is that in the step of regulating the secretory signal of mesenchymal stem cells, no mitochondrial repair signal inducer was added to the culture medium; and in the step of preparation, no mitochondrial function repair components were added.

[0072] Experimental Example 1 The procedure was performed according to the method described in Example 1 for verifying the mitochondrial repair function of mesenchymal stem cell secretion signals. The expression levels of Parkin and PGC-1α, and the amount of ATP produced in damaged vascular endothelial cells and smooth muscle cells of the corpus cavernosum used in each example and comparative example were measured.

[0073] Group setup: A control group, experimental groups for Examples 1-3 and Comparative Example 1 were set up, and vascular endothelial cells and smooth muscle cells were detected, respectively. The cells in the control group were not incubated with the culture supernatant of mesenchymal stem cells, but were cultured in fresh culture medium for mesenchymal stem cells. It should be noted that the detection methods for Comparative Examples 1-3 were the same for this experimental example; therefore, only Comparative Example 1 was detected.

[0074] The results are analyzed as follows: Figure 1 A statistical graph showing the relative expression levels of Parkin and PGC-1α and the amount of ATP produced in smooth muscle cells; Figure 2 This is a statistical graph showing the relative expression levels of Parkin and PGC-1α and the amount of ATP produced in vascular endothelial cells. It should be noted that the asterisks (*) in the graph indicate statistical significance: * indicates p-value < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.001. Figure 1 and Figure 2 As shown, the expression levels of Parkin and PGC-1α, as well as the ATP production, in Examples 1-3 were all higher than those in Comparative Example 1, indicating that the stem cell preparation provided in this application can enhance the mitochondrial repair function of mesenchymal stem cell secretion signals in male sexual dysfunction cells. However, the effects of Examples 2 and 3 were not as good as those of Example 1, indicating that in this experiment, placental-derived mesenchymal stem cells were superior to adipose-derived mesenchymal stem cells; adding creatine to the mitochondrial repair-promoting components can further enhance mitochondrial repair function.

[0075] Experiment Example 2 The repair effect of stem cell preparations was verified using a SD rat model of vascular erectile dysfunction (ED). Healthy SPF-grade male SD rats, aged 8-10 weeks and weighing 280-320g, were used in this experiment. The animal model was established by bilateral internal iliac artery ligation. The specific steps are as follows: (1) Constructing an animal model S1. Combined anesthesia was performed using 1% sodium pentobarbital and 0.5% lidocaine. After confirming the anesthesia effect, a midline incision of approximately 2 cm was made in the rat's abdomen. The intestine was then pushed to the left to expose the retroperitoneal space. The internal iliac artery was located at the bifurcation of the abdominal aorta. The internal iliac artery was approximately 0.4 mm in diameter, with visible fine branches on its surface and a palpable pulsation. At a distance of 0.3-0.5 mm from the bifurcation of the internal iliac artery, the adipose tissue on the artery surface was first wiped with a 75% ethanol swab to clearly expose the vessel. A loose knot was then tied with 5-0 silk suture, which was slipped onto the artery. After confirming under a microscope that no nerve fibers were present, the knot was tightened. A second knot was tied at the same location for double ligation to ensure complete occlusion of blood flow. The disappearance of arterial pulsation after ligation was used as the criterion for judgment. After the surgery, the rat was gently returned to its cage and fed normally, with ample food, water, and space for movement.

[0076] S2. Two weeks after model establishment, the peak systolic velocity (PSV) and the intracavernosal pressure (ICP / MAP) ratio during erection were measured in rats to verify the success of the model establishment. The judgment criteria are shown in the table below: (2) Experimental group setup: A control group, a model group, and control and comparative groups were set up. Rats that successfully developed the model were randomly assigned to each group except the control group, with 10 rats in each group. Ten normal rats were selected as the control group. Rats in each group were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and the perineal skin was disinfected. A 27G needle was inserted into the side of the base of the penis, 1 cm away from the glans, with the needle tip pointing towards the center of the corpus cavernosum. 1 mL of 0.9% saline or the stem cell preparation of each example and comparative group was slowly injected (0.9% saline was injected into the control and model groups). After the injection, the needle was removed, and the injection site was compressed with a sterile cotton ball for 5 minutes to stop the bleeding and prevent leakage or hematoma formation. The injection was given once a week for 2 consecutive weeks.

[0077] (3) Detect the peak systolic velocity (PSV) and the intracavernosal pressure / mean arterial pressure (ICP / MAP) ratio of the penile cavernous artery in rats.

[0078] (4) Results analysis: Figure 3 This is a statistical chart showing the erectile function indicators in a vascular erectile dysfunction model of SD rats. It should be noted that PSV during erection reflects the blood supply capacity of the penile cavernous arteries; a higher value indicates more sufficient arterial blood flow. Peak ICP reflects erectile rigidity; a higher value indicates stronger erectile function. The ICP / MAP ratio is used to eliminate individual differences in blood pressure and more objectively reflects the relative strength of the erection. (The chart is presented as follows...) Figure 3As shown, the indicators of Example 1 are closest to those of the normal control group, and the therapeutic effects of the stem cell preparations of Examples 1-3 are all superior to those of Comparative Examples 1-3, indicating that the stem cell preparations provided in this application can be used to repair vascular erectile dysfunction. Furthermore, the repair effect of the stem cell preparations in this application can be further enhanced by using placental-derived mesenchymal stem cells and adding creatine to the mitochondrial function repair components.

[0079] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a stem cell preparation for the treatment of male sexual dysfunction, characterized in that, The method comprises the following steps: Mesenchymal stem cell extraction and primary culture: healthy human tissues are selected, and after being finely processed, enzyme digestion is performed; after digestion, mesenchymal stem cells are collected by filtration and centrifugation, and are cultured to the second generation; Mesenchymal stem cell secretion signal regulation: a mitochondrial repair signal inducer is added to the culture medium, and after the mesenchymal stem cells are cultured for 36-48 hours, the cell supernatant is collected for verifying the mitochondrial repair function of the mesenchymal stem cell secretion signal, and the mesenchymal stem cells are collected for preparation of a preparation; the mitochondrial repair signal inducer comprises 5-10 ng / mL transforming growth factor-β1 and 5-10 ng / mL hepatocyte growth factor; Preparation of the preparation: physiological saline containing 0.05%-0.2% trehalose, 1%-4% human blood albumin and 0.5%-2% hydroxypropyl methyl cellulose is used as a base solution, a mitochondrial function repair promoting component is added to the base solution, and then the mesenchymal stem cells are added to obtain a stem cell preparation by suspension; the mitochondrial function repair promoting component comprises 0.05%-0.2% vitamin E, 0.2-0.5 μmol / L coenzyme Q10, 0.5-1 mmol / L N-acetylcysteine, 3-7 μg / mL vascular endothelial growth factor and 1-3 μmol / L MitoQ.

2. The production method according to claim 1, wherein In the mesenchymal stem cell extraction and primary culture step, a complex enzyme digestion system is used, which comprises 0.2%-0.5% collagenase IV and 0.05%-0.1% hyaluronidase.

3. The production method according to claim 2, wherein The pH value of the complex enzyme digestion system is 7.2-7.

4.

4. The production method according to claim 1, wherein In the mesenchymal stem cell secretion signal regulation step, the method for verifying the mitochondrial repair function of the mesenchymal stem cell secretion signal is as follows: the cell supernatant of the mesenchymal stem cells is co-incubated with penile corpus cavernosum damaged vascular endothelial cells and smooth muscle cells, and after 24 hours, the expression amount of parkin, peroxisome proliferator-activated receptor γ coactivator 1α and the amount of adenine nucleotide triphosphate of the damaged cells are detected.

5. The production method according to claim 1, wherein In the preparation step of the formulation, the amount of mesenchymal stem cells added is 2 x 10 6 -3 x 10 6 cells / mL as a final concentration of cells.

6. The production method according to claim 1, wherein In the preparation step, the mitochondrial function repair promoting component further comprises 0.25-0.7 mmol / L creatine.

7. The production method according to claim 1, wherein In the mesenchymal stem cell extraction and primary culture step, when the mesenchymal stem cells are cultured to the second generation, the cell fusion degree is controlled at 70%-80%, so that the cells are in the logarithmic growth phase.

8. The production method according to any one of claims 1 to 7, wherein The dosage form of the preparation is an injection type.

9. Use of a stem cell preparation for the treatment of male sexual dysfunction, said stem cell preparation being obtained by the preparation method according to any one of claims 1 to 8, characterized in that, The male sexual dysfunction is erectile dysfunction.

10. The use of a stem cell preparation according to claim 9, wherein the stem cell preparation is a stem cell preparation according to any one of claims 1 to 8. The erectile dysfunction is caused by one or both of insufficient arterial blood supply and venous occlusion dysfunction.

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