Amniotic stem cell preparation and application thereof in treating erectile dysfunction

Abstract

The invention relates to the technical field of biological medicines, in particular to an amniotic stem cell preparation and application thereof in treating erectile dysfunction. The preparation method of the amniotic stem cell preparation comprises the following steps: adding amniotic stem cells, the active extract, butanediol, glycerol, trehalose, glucose, amino acid and vitamins into normal saline containing human umbilical cord blood plasma, uniformly mixing to obtain the amniotic stem cell preparation, and storing the amniotic stem cell preparation in a liquid nitrogen tank. The prepared amniotic stem cell can significantly increase the ICP / MAP ratio, improve the activity of NOS and GC, increase the content of NO and cGMP, effectively improve erectile dysfunction and repair damaged tissues.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and more specifically, to an amniotic membrane stem cell preparation and its application in the treatment of erectile dysfunction. Background Art

[0002] Erectile dysfunction is a common male sexual dysfunction that affects the quality of life of millions of people worldwide. Existing treatments mainly rely on oral medications, but these medications can cause headaches and facial flushing. A small number of patients may experience more serious side effects, such as vision problems and hearing loss. Long-term use may cause patients to become dependent on the drugs.

[0003] In recent years, with the continuous deepening of research on stem cell preparations, amniotic membrane stem cell preparations are expected to be an emerging method for the treatment of erectile dysfunction. They have multiple advantages such as pluripotency and immunomodulatory functions, and can effectively treat erectile dysfunction and restore damaged corpus cavernosum function by promoting angiogenesis and tissue repair. Although amniotic membrane stem cell preparations have shown certain potential in the treatment of erectile dysfunction, their effectiveness and safety still need further research and optimization. Therefore, there is an urgent need to provide a stem cell preparation with stable effects that can effectively improve the survival rate and differentiation ability of stem cells and enhance the therapeutic effect. Summary of the invention

[0004] In order to solve the technical problems mentioned in the background technology, the present application provides an amniotic stem cell preparation and its application in the treatment of erectile dysfunction.

[0005] An amniotic membrane stem cell preparation, wherein each 100 mL of the amniotic membrane stem cell preparation comprises 4×10 9 -10×10 9 , active extract 2.5-3g, butanediol 4-6g, glycerol 0.5-1g, trehalose 0.1-0.3g, glucose 0.3-1g, amino acid 0.1-0.3g, vitamin 0.01-0.02g, and the remainder is saline containing umbilical cord blood plasma.

[0006] The amino acids consist of histidine, threonine, isoleucine and phenylalanine in a mass ratio of 1-3:1-2:2-4:0.5-1.

[0007] The vitamins consist of vitamin D, vitamin E and vitamin B5 in a mass ratio of 10-12:1-3:1-2.

[0008] Preferably, the preparation steps of the active extract are as follows: (1) Drying Morinda officinalis and green tea, crushing and sieving, and then mixing, adding the mixed powder into an ethanol solution, ultrasonically treating at 60-70° C., centrifuging, taking the supernatant, filtering and sterilizing, rotary evaporating to remove the solvent, and drying to obtain a natural extract; (2) crushing epimedium and cistanche deserticola, adding them to physiological saline, stirring evenly, adding bacterial enzyme complex, controlling the stirring speed of the fermentation tank to 200-300 rpm, pH value to 6.5-7, temperature to 35-40°C, ventilation volume to 1-2 vvm, dissolved oxygen content to 30-60%, fermenting for 2-3 days to obtain a fermentation product; (3) centrifuging the fermentation product, collecting the supernatant, and sterilizing the supernatant with high-pressure steam to obtain an active fermentation product; (4) The active fermentation product and the natural extract are mixed in a mass ratio of 10-12:1-3 to obtain the active extract.

[0009] Preferably, the mixed powder in step (1) consists of Morinda officinalis and green tea in a mass ratio of 5-8:1-2.

[0010] Preferably, the fungus-enzyme complex in step (2) is composed of Trichoderma and complex enzyme in a mass ratio of 1-2:18-20.

[0011] Preferably, the concentration of live Trichoderma in step (2) is 1×10 9 -2×10 9 CFU / g.

[0012] Preferably, the complex enzyme in step (2) consists of α-amylase and papain in a mass ratio of 10-12:5-8.

[0013] Preferably, in step (2), the mass ratio of epimedium, Cistanche deserticola, physiological saline and bacterial enzyme complex is 20-30:5-8:40-50:0.5-1.

[0014] Preferably, the method for preparing the amniotic membrane stem cells is: Step S1, separating amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells from human amniotic membrane, and mixing them in a ratio of 10-12:1-2:2-3 to obtain mixed primary cells; Step S2, inoculating the mixed primary cells in a modified DMEM medium, culturing for 1-2 days under the conditions of a CO2 concentration of 5-6%, a humidity of 88-90%, and a temperature of 36-38°C, and digesting with trypsin when the cell confluence reaches 70-80% to obtain the first passage cells; Step S3, inoculating the first passage cells in a modified DMEM medium, culturing for 1-2 days under the conditions of a CO2 concentration of 5-6%, a humidity of 85-90%, and a temperature of 36-38°C, and when the cell confluence reaches 75-85%, digesting and passage with trypsin at a ratio of 1:2-3, repeating the passage step 3 times, and obtaining the fourth passage cells; Step S4: Disperse the first passage cells and the fourth passage cells in the modified DMEM medium respectively, count them using a cell counter, and adjust the final cell concentration of the first passage cells and the fourth passage cells to 2×10 6 -3×10 6 / mL, and mixed at a volume ratio of 2-3:0.5-1, and centrifuged to obtain amniotic stem cells.

[0015] Preferably, the seeding density of the mixed primary cells in step S2 is 1×10 6 -2×10 6 Pieces / mL.

[0016] Preferably, the preparation method of the improved DMEM culture medium in step S2 is: adding fetal bovine serum and penicillin-streptomycin solution to the basic DMEM culture medium, and shaking well.

[0017] Preferably, the volume ratio of the basic DMEM culture medium, fetal bovine serum and penicillin-streptomycin solution is 85-89:10-15:1-1.3; Preferably, the seeding density of the first passage cells in step S3 is 1.5×10 6 -2.5×10 6 Pieces / mL.

[0018] Preferably, the preparation of the amniotic stem cell preparation comprises the following steps: adding amniotic stem cells, active extracts, butanediol, glycerol, trehalose, glucose, amino acids, and vitamins to physiological saline containing human umbilical cord blood plasma, mixing evenly to obtain the amniotic stem cell preparation, which is then stored in a liquid nitrogen tank.

[0019] Preferably, the volume fraction of human cord blood plasma in the physiological saline containing human cord blood plasma is 3-5%.

[0020] Preferably, the human umbilical cord blood plasma and the amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells are from the same provider.

[0021] Application of an amniotic membrane stem cell preparation prepared by the above method in the treatment of erectile dysfunction.

[0022] In summary, this application has the following beneficial effects: In the process of preparing active extracts, the present application adopts ultrasonic extraction for Morinda officinalis and green tea, which can better retain the original effective ingredients (such as Morinda officinalis polyphenol compounds, catechins in green tea), etc., and avoid the destruction of heat-sensitive and easily oxidized effective ingredients during high-temperature and long-term fermentation. In addition, the present application adopts fermentation for Epimedium and Cistanche deserticola, and utilizes bacterial enzyme complex to transform components such as icariin in Epimedium and phenylethanol glycosides in Cistanche deserticola, so that they are more easily absorbed by the human body and their bioavailability is improved. By utilizing the combined action of Trichoderma and complex enzyme, Trichoderma converts flavonoid compounds in Epimedium into a more absorbable form during the fermentation process, such as Icaritin, the latter has stronger biological activity and can promote the secretion of sex hormones. Trichoderma can also produce some secondary metabolites during the fermentation process, such as terpenoids, phenolic compounds, etc. These substances have antioxidant and anti-inflammatory effects, can reduce oxidative stress and inflammatory reactions, protect vascular endothelial cells, improve blood circulation, and thus help promote the secretion of sex hormones. Under the joint action of α-amylase and papain, the extraction rate of effective ingredients in Epimedium and Cistanche deserticola is significantly improved. The peptides and amino acids generated by enzymatic hydrolysis can provide nitrogen sources for Trichoderma, promote the metabolic activity of Trichoderma in the fermentation system, and ensure the normal progress of the fermentation process. The fermentation products and Morinda officinalis itself have the function of regulating the hypothalamus-pituitary-gonad axis, and work together on the endocrine system to stimulate the pituitary to secrete gonadotropin, promote the secretion of testosterone and other male hormones, enhance the sensitivity of androgen receptors, improve vascular endothelial function, promote the relaxation of cavernous smooth muscles, and increase the blood perfusion of the cavernous body of the penis, thereby improving erectile function. In addition, the polysaccharides in the fermentation products can also regulate the immune system, reduce immune rejection reactions, and increase the survival rate of stem cells. The natural extracts and the angiogenic factors or immunomodulatory substances transformed by microorganisms in the active fermentation products produce a synergistic effect, enhancing the overall therapeutic effect.

[0023] In the present application, the mixed culture of amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells is conducive to establishing mutual connections between cells. In the in vivo physiological environment, amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells are interdependent and work in collaboration. Mixed culture can better simulate this in vivo environment, so that the signal transduction pathways and interactions between cells can be established at an early stage (for example, amniotic vascular stem cells can receive the stimulation of growth factors such as vascular endothelial growth factor (VEGF) secreted by amniotic mesenchymal stem cells at an early stage, thereby better initiating angiogenesis-related gene expression programs and promoting their differentiation into vascular endothelial cells and the like). However, the amniotic vascular stem cells cultured alone may lack certain regulatory factors secreted by amniotic mesenchymal stem cells, and their angiogenesis ability is not fully stimulated during the single culture process. It will take a certain amount of time to restore and adjust this ability during subsequent mixing. In addition, the function of amniotic mesenchymal stem cells cultured alone may be disordered in secreting growth factors due to the lack of synergistic stimulation of amniotic vascular stem cells and amniotic stromal stem cells, and their differentiation direction may deviate from normal tissue repair needs. When these dysfunctional cells are mixed, they need time to rebalance and coordinate their functions, so they cannot fully recover to the optimal synergistic state, resulting in the inability to effectively exert the synergistic advantages of the three cells in tissue repair applications. By compounding the mixed first-generation cells and the mixed fourth-generation cells, the high vitality and proliferation ability of the first-generation cells and the differentiation potential and stability of the fourth-generation cells can be combined to achieve a synergistic effect, promote angiogenesis, improve blood circulation, enhance tissue repair ability, and improve the overall therapeutic effect of the amniotic stem cell preparation. DETAILED DESCRIPTION

[0024] The present application is further described in detail below with reference to the embodiments.

[0025] The herb Epimedium used in the examples and comparative examples of the present application was purchased from Nanxingyuan Chinese Herbal Medicine; Cistanche and Morinda officinalis were purchased from Zhongyaodu Medicinal Materials Store; green tea (article number: B53) was purchased from Haikou Longhua Lihuo Trading Company; basic DMEM culture medium (article number: D0822), trypsin (article number: T4049), fetal bovine serum (article number: F8687), penicillin-streptomycin solution (article number: TMS-AB2) were purchased from Merck Millipore; Trichoderma sp. (product name: Trichoderma sp., product number: BMZ073080) was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.; α-amylase (enzyme activity: 10,000-800,000 u / g) and papain (enzyme activity: 10,000-800,000 u / g) were purchased from Chengdu Wanxiang Hongrun Biotechnology Co., Ltd.; nitric oxide (NO) detection kit (article number: CB10963-Ra), nitric oxide synthase (nitric oxide synthase) synthase (NOS) detection kit (catalog number CB11462-Ra), cyclic guanosine monophosphate (cGMP) detection kit (catalog number: CB10438-Ra), and guanylate cyclase (GC) detection kit (catalog number: CB12301-Ra) were purchased from Shanghai Ke Ai Bo Biotechnology Co., Ltd.

[0026] Examples 1-3 and Comparative Examples 1-8 provide an amniotic membrane stem cell preparation and a preparation method thereof.

[0027] Example 1 An amniotic membrane stem cell preparation, each 100 mL of the amniotic membrane stem cell preparation includes 4×10 9 , 2.5g of active extract, 4g of butanediol, 0.5g of glycerol, 0.1g of trehalose, 0.3g of glucose, 0.1g of amino acids, 0.01g of vitamins, and the remainder is normal saline containing human umbilical cord blood plasma, wherein the amino acids are composed of histidine, threonine, isoleucine and phenylalanine in a mass ratio of 1:1:2:0.5; the vitamins are composed of vitamin D, vitamin E and vitamin B5 in a mass ratio of 10:1:1.

[0028] The steps for preparing the active extract are as follows: (1) Dry Morinda officinalis and green tea, grind them through a 200-mesh sieve and mix them, add the mixed powder into a 60% ethanol solution by volume, perform ultrasonic treatment at 60°C, ultrasonic power 120W, ultrasonic frequency 40KHz, ultrasonic time 0.5h, centrifuge for 20min, centrifuge speed 5000rpm, take the supernatant, filter and sterilize it with a 0.22μm filter membrane, remove the solvent by rotary evaporation, and dry to obtain a natural extract, wherein the mixed powder is composed of Morinda officinalis and green tea in a mass ratio of 5:1, and the mass ratio of the mixed powder to the ethanol solution is 1:4; (2) The herb Epimedium and Cistanche deserticola were crushed, passed through a 200-mesh sieve, added to physiological saline, stirred evenly, and then the bacterial enzyme complex was added. The fermentation tank was stirred at a speed of 200 rpm, the pH value was 6.5, the temperature was 35°C, the ventilation volume was 1 vvm, and the dissolved oxygen content was 30%. The fermentation was carried out for 2 days to obtain a fermentation product, wherein the mass ratio of the herb Epimedium, Cistanche deserticola, physiological saline and bacterial enzyme complex was 20:5:40:0.5, the bacterial enzyme complex was composed of Trichoderma and complex enzyme in a mass ratio of 1:18, and the concentration of live Trichoderma was 1×10 9 CFU / g, the complex enzyme is composed of α-amylase and papain in a mass ratio of 2:1; (3) The fermentation product was centrifuged at a speed of 80,000 rpm for 15 min, the supernatant was collected, and the supernatant was sterilized by high-pressure steam at a sterilization temperature of 121°C, a sterilization pressure of 103.4 kPa, and a sterilization time of 30 min to obtain an active fermentation product; (4) The active fermentation product and the natural extract are mixed in a mass ratio of 10:1 to obtain the active extract.

[0029] Wherein, the preparation method of amniotic stem cells is: Step S1, separating amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells from human amniotic membrane, and mixing them in a ratio of 10:1:2 to obtain mixed primary cells; Step S2: The mixed primary cells were seeded at a density of 1×10 6 100 μg / mL was inoculated in a modified DMEM medium, and cultured for 1 day under the conditions of 5% CO2 concentration, 88% humidity, and 36°C. When the cell confluence reached 70%, the cells were digested with trypsin to obtain the first passage cells. The modified DMEM medium was prepared as follows: fetal bovine serum and penicillin-streptomycin solution were added to the basic DMEM medium, and the mixture was shaken well; the volume ratio of the basic DMEM medium, fetal bovine serum, and penicillin-streptomycin solution was 85:10:1; Step S3: The first passage cells were seeded at a density of 1.5×10 6Cells were inoculated into modified DMEM medium at 5% CO2, 85% humidity and 36°C for 1 day. When the cell confluence reached 75%, they were digested and subcultured with trypsin at a ratio of 1:2. The subculture steps were repeated 3 times to obtain the fourth subculture cells. Step S4: Disperse the first passage cells and the fourth passage cells in the modified DMEM medium respectively, count them using a cell counter, and adjust the final cell concentration of the first passage cells and the fourth passage cells to 2×10 6 / mL, and mixed at a volume ratio of 4:1, and centrifuged at 4°C at a speed of 1200 rpm for 5 minutes to obtain amniotic membrane stem cells.

[0030] Among them, the preparation of amniotic stem cell preparation includes the following steps: adding amniotic stem cells, active extracts, butanediol, glycerol, trehalose, glucose, amino acids, and vitamins to physiological saline containing human umbilical cord blood plasma, mixing them evenly, to obtain the amniotic stem cell preparation, which is stored in a liquid nitrogen tank. The volume fraction of human umbilical cord blood plasma in the physiological saline containing human umbilical cord blood plasma is 3%.

[0031] Example 2 An amniotic membrane stem cell preparation, each 100 mL of the amniotic membrane stem cell preparation includes 7×10 9 , 2.8g of active extract, 5g of butylene glycol, 0.8g of glycerol, 0.2g of trehalose, 0.5g of glucose, 0.2g of amino acid, 0.015g of vitamin, and the remainder is normal saline containing human umbilical cord blood plasma, among which the amino acid is composed of histidine, threonine, isoleucine and phenylalanine in a mass ratio of 2:1.5:3:0.8; the vitamin is composed of vitamin D, vitamin E and vitamin B5 in a mass ratio of 11:2:1.5.

[0032] (1) Dry Morinda officinalis and green tea, grind them through a 250-mesh sieve and mix them, add the mixed powder into a 70% ethanol solution by volume, perform ultrasonic treatment at 65°C, ultrasonic power 130W, ultrasonic frequency 50KHz, ultrasonic time 0.8h, centrifuge for 25min, centrifuge speed 6000rpm, take the supernatant, filter and sterilize with a 0.22μm filter membrane, remove the solvent by rotary evaporation, dry, and sterilize with high-pressure steam at a sterilization temperature of 121°C, a sterilization pressure of 103.4KPa, and a sterilization time of 15min to obtain a natural extract, wherein the mixed powder is composed of Morinda officinalis and green tea in a mass ratio of 4:1, and the mass ratio of the mixed powder to the ethanol solution is 6:25; (2) The herb Epimedium and the herb Cistanche deserticola were crushed, passed through a 250-mesh sieve, mixed, added to physiological saline, stirred evenly, and then the bacterial enzyme complex was added. The fermentation tank was stirred at a speed of 250 rpm, the pH value was 6.8, the temperature was 38°C, the ventilation volume was 1.5 vvm, and the dissolved oxygen content was 45%. The fermentation was carried out for 2.5 days to obtain a fermentation product, wherein the mass ratio of the herb Epimedium, the herb Cistanche deserticola, the physiological saline and the bacterial enzyme complex was 25:6:45:0.8, the bacterial enzyme complex was composed of Trichoderma and the complex enzyme in a mass ratio of 1.5:19, and the concentration of the live Trichoderma was 1.5×10 9 CFU / g, the complex enzyme is composed of α-amylase and papain in a mass ratio of 11:6; (3) The fermentation product was centrifuged at a speed of 9000 rpm for 18 min, the supernatant was collected, and the supernatant was sterilized by high-pressure steam at a sterilization temperature of 121°C, a sterilization pressure of 103.4 KPa, and a sterilization time of 35 min to obtain an active fermentation product; (4) The active fermentation product and the natural extract are mixed in a mass ratio of 11:2 to obtain the active extract.

[0033] Wherein, the preparation method of amniotic stem cells is: Step S1, separating amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells from human amniotic membrane, and mixing them in a ratio of 11:1.5:2.5 to obtain mixed primary cells; Step S2: The mixed primary cells were seeded at a density of 1.5×10 6 The cells were inoculated with 100 μg / mL of modified DMEM medium, and cultured for 1.5 days under the conditions of CO2 concentration of 5.5%, humidity of 89%, and temperature of 37°C. When the cell confluence reached 75%, the cells were digested with trypsin to obtain the first passage cells. The modified DMEM medium was prepared as follows: fetal bovine serum and penicillin-streptomycin solution were added to the basic DMEM medium, and the mixture was shaken well; the volume ratio of the basic DMEM medium, fetal bovine serum, and penicillin-streptomycin solution was 87:12:1.2; Step S3: The first passage cells were seeded at a density of 2×10 6 The cells were inoculated into modified DMEM medium at 5.5% CO2, 88% humidity and 37°C for 1.5 days. When the cell confluence reached 80%, they were digested and subcultured with trypsin at a ratio of 1:2. The subculture steps were repeated 3 times to obtain the fourth subculture cells. Step S4: Disperse the first passage cells and the fourth passage cells in the modified DMEM medium respectively, count them using a cell counter, and adjust the final cell concentration of the first passage cells and the fourth passage cells to 2.5×10 6 / mL, and mixed at a volume ratio of 2.5:0.8, and centrifuged at 4°C with a centrifugal speed of 1300 rpm for 80 minutes to obtain amniotic membrane stem cells.

[0034] Among them, the preparation of amniotic stem cell preparation includes the following steps: adding amniotic stem cells, active extracts, butanediol, glycerol, trehalose, glucose, amino acids, and vitamins to physiological saline containing human umbilical cord blood plasma, mixing evenly, to obtain the amniotic stem cell preparation, which is stored in a liquid nitrogen tank, and the volume fraction of human umbilical cord blood plasma in the physiological saline containing human umbilical cord blood plasma is 4%.

[0035] Example 3 An amniotic membrane stem cell preparation, wherein each 100 mL of the amniotic membrane stem cell preparation comprises 1×10 10 , 3g active extract, 6g butanediol, 1g glycerol, 0.3g trehalose, 1g glucose, 0.3g amino acid, 0.02g vitamin, and the remainder is normal saline containing human umbilical cord blood plasma, wherein the amino acid is composed of histidine, threonine, isoleucine and phenylalanine in a mass ratio of 3:2:4:1; the vitamin is composed of vitamin D, vitamin E and vitamin B5 in a mass ratio of 12:3:2.

[0036] The steps for preparing the active extract are as follows: (1) Dry Morinda officinalis and green tea, grind them through a 300-mesh sieve, and mix them. Add the mixed powder into an ethanol solution with a volume fraction of 80%, perform ultrasonic treatment at 70°C, with an ultrasonic power of 150 W, an ultrasonic frequency of 60 kHz, and an ultrasonic time of 1 h. Centrifuge for 30 min and a centrifugal speed of 8000 rpm. Take the supernatant, filter and sterilize it with a 0.22 μm filter membrane, remove the solvent by rotary evaporation, and dry to obtain a natural extract, wherein the mixed powder is composed of Morinda officinalis and green tea in a mass ratio of 4:1, and the mass ratio of the mixed powder to the ethanol solution is 4:15; (2) The herb Epimedium and Cistanche deserticola were crushed, passed through a 300-mesh sieve, added to physiological saline, stirred evenly, and then the bacterial enzyme complex was added. The fermentation tank was stirred at a speed of 300 rpm, the pH value was 7, the temperature was 40°C, the ventilation volume was 2 vvm, and the dissolved oxygen content was 60%. The fermentation was carried out for 3 days to obtain a fermentation product, wherein the mass ratio of the herb Epimedium, Cistanche deserticola, physiological saline and bacterial enzyme complex was 30:8:50:1, the bacterial enzyme complex was composed of Trichoderma and complex enzyme in a mass ratio of 1:10, and the concentration of live Trichoderma was 2×10 9 CFU / g, the complex enzyme is composed of α-amylase and papain in a mass ratio of 3:2; (3) The fermentation product was centrifuged at a speed of 10,000 rpm for 20 min, the supernatant was collected, and the supernatant was sterilized by high-pressure steam at a temperature of 121°C, a pressure of 103.4 kPa, and a time of 40 min to obtain an active fermentation product; (4) The active fermentation product and the natural extract are mixed in a mass ratio of 4:1 to obtain the active extract.

[0037] Wherein, the preparation method of amniotic stem cells is: Step S1, separating amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells from human amniotic membrane, and mixing them in a ratio of 12:2:3 to obtain mixed primary cells; Step S2: The mixed primary cells were seeded at a density of 2×10 6 The cells were inoculated with 100 μg / mL of modified DMEM medium, and cultured for 2 days under the conditions of 6% CO2 concentration, 90% humidity, and 38°C. When the cell confluence reached 80%, the cells were digested with trypsin to obtain the first passage cells. The modified DMEM medium was prepared as follows: fetal bovine serum and penicillin-streptomycin solution were added to the basic DMEM medium and shaken well; the volume ratio of the basic DMEM medium, fetal bovine serum, and penicillin-streptomycin solution was 89:15:1.3; Step S3: The first passage cells were seeded at a density of 2.5×10 6 The cells were inoculated into modified DMEM medium at 1:1 ratio at 1:3 and cultured for 2 days at a CO2 concentration of 6%, a humidity of 90% and a temperature of 38°C. When the cell confluence reached 85%, the cells were digested and passaged with trypsin at a ratio of 1:3. The passage steps were repeated 3 times to obtain the fourth passage cells. Step S4: Disperse the first passage cells and the fourth passage cells in the modified DMEM medium respectively, count them using a cell counter, and adjust the final cell concentration of the first passage cells and the fourth passage cells to 3×10 6 / mL, and mixed at a volume ratio of 3:1, and centrifuged at 4°C at a centrifugal speed of 1500rpm for 10 minutes to obtain amniotic membrane stem cells.

[0038] Among them, the preparation of amniotic stem cell preparation includes the following steps: adding amniotic stem cells, active extracts, butanediol, glycerol, trehalose, glucose, amino acids, and vitamins to physiological saline containing human umbilical cord blood plasma, mixing evenly, to obtain the amniotic stem cell preparation, which is stored in a liquid nitrogen tank, and the volume fraction of human umbilical cord blood plasma in the physiological saline containing human umbilical cord blood plasma is 5%.

[0039] Comparative Example 1 This comparative example is the same as Example 1, except that the preparation method of the active extract is different, as follows: The steps for preparing the active extract are as follows: (1) Dry Morinda officinalis and green tea, grind them through a 200-mesh sieve and mix them, add the mixed powder into a 60% ethanol solution by volume, perform ultrasonic treatment at 60°C, ultrasonic power 120W, ultrasonic frequency 40KHz, ultrasonic time 0.5h, centrifuge for 20min, centrifuge speed 5000rpm, take the supernatant, filter and sterilize it with a 0.22μm filter membrane, remove the solvent by rotary evaporation, and dry to obtain natural extract 1, wherein the mixed powder is composed of Morinda officinalis and green tea in a mass ratio of 5:1, and the mass ratio of the mixed powder to the ethanol solution is 1:4; (2) Grind the epimedium and Cistanche deserticola, pass through a 200-mesh sieve, add them into an ethanol solution, and perform ultrasonic treatment at 60°C, with an ultrasonic power of 120 W, an ultrasonic frequency of 40 kHz, and an ultrasonic time of 0.5 h to obtain an intermediate product, wherein the mass ratio of epimedium, Cistanche deserticola, and ethanol solution is 20:5:40, and the volume fraction of the ethanol solution is 60%; (3) The intermediate product was centrifuged at a speed of 80,000 rpm for 15 min, the supernatant was collected, and the supernatant was sterilized by high-pressure steam at a sterilization temperature of 121° C., a sterilization pressure of 103.4 KPa, and a sterilization time of 30 min, thereby obtaining natural extract 2; (4) Natural extract 2 and natural extract 1 are mixed in a mass ratio of 10:1 to obtain an active extract.

[0040] Comparative Example 2 This comparative example is the same as Example 1, except that the preparation method of the active extract is different, as follows: The steps for preparing the active extract are as follows: The Morinda officinalis and green tea were dried, crushed and mixed after being passed through a 200-mesh sieve, and the mixed powder was added into physiological saline, and the bacterial enzyme complex was added. The fermentation tank was stirred at a speed of 200 rpm, the pH value was 6.5, the temperature was 35°C, the ventilation volume was 1 vvm, and the dissolved oxygen content was 30%. The fermentation was carried out for 2 days, and the mixture was centrifuged for 20 min at a speed of 5000 rpm. The supernatant was filtered and sterilized using a 0.22 μm filter membrane, the solvent was removed by rotary evaporation, and the mixture was dried to obtain a fermentation product 1, wherein the mass ratio of Morinda officinalis, green tea, physiological saline and bacterial enzyme complex was 25:5:120:3, the bacterial enzyme complex was composed of Trichoderma and composite enzyme at a mass ratio of 1:18, and the concentration of live Trichoderma was 1×10 9 CFU / g, the complex enzyme is composed of α-amylase and papain in a mass ratio of 2:1; (2) crushing epimedium and Cistanche deserticola, passing through a 200-mesh sieve, adding them to physiological saline, stirring evenly, adding the bacterial enzyme complex, controlling the fermentation tank stirring speed at 200 rpm, pH value at 6.5, temperature at 35°C, ventilation volume at 1 vvm, and dissolved oxygen content at 30%, fermenting for 2 days to obtain an intermediate product, wherein the mass ratio of epimedium, Cistanche deserticola, physiological saline and bacterial enzyme complex is 20:5:40:0.5, the bacterial enzyme complex is composed of Trichoderma and complex enzyme in a mass ratio of 1:18, and the concentration of live Trichoderma is 1×10 9 CFU / g, the complex enzyme is composed of α-amylase and papain in a mass ratio of 2:1; (3) The intermediate product was centrifuged at a speed of 80,000 rpm for 15 min, the supernatant was collected, and the supernatant was sterilized by high-pressure steam at a sterilization temperature of 121° C., a sterilization pressure of 103.4 KPa, and a sterilization time of 30 min, to obtain fermentation product 2; (4) The fermentation product 2 and the fermentation product 1 are mixed in a mass ratio of 10:1 to obtain an active extract.

[0041] Comparative Example 3 This comparative example is the same as Example 1, except that the preparation method of the active extract is different, as follows: The steps for preparing the active extract are as follows: (1) Dry Morinda officinalis and green tea, grind them through a 200-mesh sieve and mix them, add the mixed powder into a 60% ethanol solution by volume, perform ultrasonic treatment at 60°C, ultrasonic power 120W, ultrasonic frequency 40KHz, ultrasonic time 0.5h, centrifuge for 20min, centrifuge speed 5000rpm, take the supernatant, filter and sterilize it with a 0.22μm filter membrane, remove the solvent by rotary evaporation, and dry to obtain a natural extract, wherein the mixed powder is composed of Morinda officinalis and green tea in a mass ratio of 5:1, and the mass ratio of the mixed powder to the ethanol solution is 1:4; (2) crushing epimedium and cistanche deserticola, passing through a 200-mesh sieve, adding the mixture to physiological saline, stirring evenly, adding complex enzyme, controlling the stirring speed of the fermentation tank to 200 rpm, the pH value to 6.5, the temperature to 35°C, the ventilation volume to 1 vvm, and the dissolved oxygen content to 30%, fermenting for 2 days to obtain a fermentation product, wherein the mass ratio of epimedium, cistanche deserticola, physiological saline and complex enzyme is 20:5:40:0.5, and the complex enzyme is composed of α-amylase and papain in a mass ratio of 2:1; (3) The fermentation product was centrifuged at a speed of 80,000 rpm for 15 min, the supernatant was collected, and the supernatant was sterilized by high-pressure steam at a sterilization temperature of 121°C, a sterilization pressure of 103.4 kPa, and a sterilization time of 30 min to obtain an active fermentation product; (4) The active fermentation product and the natural extract are mixed in a mass ratio of 10:1 to obtain the active extract.

[0042] Comparative Example 4 This comparative example is the same as Example 1, except that the preparation method of the active extract is different, as follows: The steps for preparing the active extract are as follows: (1) Dry Morinda officinalis and green tea, grind them through a 200-mesh sieve and mix them, add the mixed powder into a 60% ethanol solution by volume, perform ultrasonic treatment at 60°C, ultrasonic power 120W, ultrasonic frequency 40KHz, ultrasonic time 0.5h, centrifuge for 20min, centrifuge speed 5000rpm, take the supernatant, filter and sterilize it with a 0.22μm filter membrane, remove the solvent by rotary evaporation, and dry to obtain a natural extract, wherein the mixed powder is composed of Morinda officinalis and green tea in a mass ratio of 5:1, and the mass ratio of the mixed powder to the ethanol solution is 1:4; (2) The herb Epimedium and Cistanche deserticola were crushed, passed through a 200-mesh sieve, added to physiological saline, stirred evenly, and then the bacterial enzyme complex was added. The fermentation tank was stirred at a speed of 200 rpm, the pH value was 6.5, the temperature was 35°C, the ventilation volume was 1 vvm, and the dissolved oxygen content was 30%. The fermentation was carried out for 2 days to obtain a fermentation product, wherein the mass ratio of the herb Epimedium, Cistanche deserticola, physiological saline and bacterial enzyme complex was 20:5:40:0.5, the bacterial enzyme complex was composed of Trichoderma and α-amylase in a mass ratio of 1:18, and the concentration of live Trichoderma was 1×10 9 CFU / g; (3) The fermentation product was centrifuged at a speed of 80,000 rpm for 15 min, the supernatant was collected, and the supernatant was sterilized by high-pressure steam at a sterilization temperature of 121°C, a sterilization pressure of 103.4 kPa, and a sterilization time of 30 min to obtain an active fermentation product; (4) The active fermentation product and the natural extract are mixed in a mass ratio of 10:1 to obtain the active extract.

[0043] Comparative Example 5 This comparative example is the same as Example 1, except that the preparation steps of the active extract are different, as follows: The preparation steps of amniotic stem cells are as follows: (1) Dry Morinda officinalis and green tea, grind them through a 200-mesh sieve and mix them, add the mixed powder into a 60% ethanol solution by volume, perform ultrasonic treatment at 60°C, ultrasonic power 120W, ultrasonic frequency 40KHz, ultrasonic time 0.5h, centrifuge for 20min, centrifuge speed 5000rpm, take the supernatant, filter and sterilize it with a 0.22μm filter membrane, remove the solvent by rotary evaporation, and dry to obtain a natural extract, wherein the mixed powder is composed of Morinda officinalis and green tea in a mass ratio of 5:1, and the mass ratio of the mixed powder to the ethanol solution is 1:4; (2) The herb Epimedium and Cistanche deserticola were crushed, passed through a 200-mesh sieve, added to physiological saline, stirred evenly, and then the bacterial enzyme complex was added. The fermentation tank was stirred at a speed of 200 rpm, the pH value was 6.5, the temperature was 35°C, the ventilation volume was 1 vvm, and the dissolved oxygen content was 30%. The fermentation was carried out for 2 days to obtain a fermentation product, wherein the mass ratio of the herb Epimedium, Cistanche deserticola, physiological saline and bacterial enzyme complex was 20:5:40:0.5, the bacterial enzyme complex was composed of Trichoderma and papain in a mass ratio of 1:18, and the concentration of live Trichoderma was 1×10 9 CFU / g; (3) The fermentation product was centrifuged at a speed of 80,000 rpm for 15 min, the supernatant was collected, and the supernatant was sterilized by high-pressure steam at a sterilization temperature of 121°C, a sterilization pressure of 103.4 kPa, and a sterilization time of 30 min to obtain an active fermentation product; (4) The active fermentation product and the natural extract are mixed in a mass ratio of 10:1 to obtain the active extract.

[0044] Comparative Example 6 This comparative example is the same as Example 1, except that the preparation steps of amniotic stem cells are different, as follows: The preparation steps of amniotic stem cells are as follows: Step S1, separating amniotic vascular stem cells and amniotic stromal stem cells from human amniotic membrane, and mixing them in a ratio of 5:1 to obtain mixed primary cells; Step S2: The mixed primary cells were seeded at a density of 1×10 6 100 μg / mL was inoculated in a modified DMEM medium, and cultured for 1 day under the conditions of 5% CO2 concentration, 88% humidity, and 36°C. When the cell confluence reached 70%, the cells were digested with trypsin to obtain the first passage cells. The modified DMEM medium was prepared as follows: fetal bovine serum and penicillin-streptomycin solution were added to the basic DMEM medium, and the mixture was shaken well; the volume ratio of the basic DMEM medium, fetal bovine serum, and penicillin-streptomycin solution was 85:10:1; Step S3: The first passage cells were seeded at a density of 1.5×10 6Cells were inoculated into modified DMEM medium at 5% CO2, 85% humidity and 36°C for 1 day. When the cell confluence reached 75%, they were digested and subcultured with trypsin at a ratio of 1:2. The subculture steps were repeated 3 times to obtain the fourth subculture cells. Step S4: Disperse the first passage cells and the fourth passage cells in the modified DMEM medium respectively, count them using a cell counter, and adjust the final cell concentration of the first passage cells and the fourth passage cells to 2×10 6 / mL, and mixed at a volume ratio of 4:1, and centrifuged at 4°C at a speed of 1200 rpm for 5 minutes to obtain amniotic membrane stem cells.

[0045] Comparative Example 7 This comparative example is the same as Example 1, except that the preparation steps of amniotic stem cells are different, as follows: The preparation steps of amniotic stem cells are as follows: Step S1, separating amniotic vascular stem cells and amniotic mesenchymal stem cells from human amniotic membrane, and mixing them at a ratio of 10:1 to obtain mixed primary cells; Step S2: The mixed primary cells were seeded at a density of 1×10 6 100 μg / mL was inoculated in a modified DMEM medium, and cultured for 1 day under the conditions of 5% CO2 concentration, 88% humidity, and 36°C. When the cell confluence reached 70%, the cells were digested with trypsin to obtain the first passage cells. The modified DMEM medium was prepared as follows: fetal bovine serum and penicillin-streptomycin solution were added to the basic DMEM medium, and the mixture was shaken well; the volume ratio of the basic DMEM medium, fetal bovine serum, and penicillin-streptomycin solution was 85:10:1; Step S3: The first passage cells were seeded at a density of 1.5×10 6 Cells were inoculated into modified DMEM medium at 5% CO2, 85% humidity and 36°C for 1 day. When the cell confluence reached 75%, they were digested and subcultured with trypsin at a ratio of 1:2. The subculture steps were repeated 3 times to obtain the fourth subculture cells. Step S4: Disperse the first passage cells and the fourth passage cells in the modified DMEM medium respectively, count them using a cell counter, and adjust the final cell concentration of the first passage cells and the fourth passage cells to 2×10 6 / mL, and mixed at a volume ratio of 4:1, and centrifuged at 4°C at a speed of 1200 rpm for 5 minutes to obtain amniotic membrane stem cells.

[0046] Comparative Example 8 This comparative example is the same as Example 1, except that the preparation steps of amniotic stem cells are different, as follows: The preparation steps of amniotic stem cells are as follows: Step S1, isolating amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells from human amniotic membrane; Step S2: amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells are seeded at a density of 1×10 6 The cells were inoculated with 100 μg / mL of modified DMEM medium, and cultured for 1 day under the conditions of 5% CO2 concentration, 88% humidity, and 36°C. When the cell confluence reached 70%, the cells were digested with trypsin to obtain three first passage cells. The modified DMEM medium was prepared as follows: fetal bovine serum and penicillin-streptomycin solution were added to the basic DMEM medium and shaken well; the volume ratio of the basic DMEM medium, fetal bovine serum, and penicillin-streptomycin solution was 85:10:1; Step S3: The three first passage cells were seeded at a density of 1.5×10 6 100 cells / mL were inoculated in modified DMEM medium and cultured for 1 day under the conditions of 5% CO2 concentration, 85% humidity and 36°C. When the cell confluence reached 75%, they were digested and passaged with trypsin at a ratio of 1:2. The passage steps were repeated 3 times to obtain three types of fourth passage cells. Step S4: Disperse the three first passage cells and the corresponding fourth passage cells in the modified DMEM medium, count them using a cell counter, and adjust the final cell concentration of the first passage cells and the fourth passage cells to 2×10 6 / mL, and mixed at a volume ratio of 4:1, and centrifuged at 1200 rpm at 4°C for 5 minutes to obtain three mixed amniotic stem cells; Step S5, mixing the three mixed amniotic stem cells to obtain amniotic stem cells; wherein the number ratio of the mixed amniotic vascular stem cells, the mixed amniotic mesenchymal stem cells and the mixed amniotic stromal stem cells in the amniotic stem cells is 10:1:2.

[0047] Performance Testing The comprehensive properties of the amniotic stem cell preparations prepared in Examples 1-3 and Comparative Examples 1-8 of the present application are as follows: Experimental animals: 78 SPF male SD rats, weighing 180-220 g, were raised in an environment with a room temperature of 20-25°C, a humidity of 45-55%, and a 12-h light-dark cycle.

[0048] Model establishment: 78 rats were randomly divided into a normal group (6 rats) and a modeling group (72 rats). They were adaptively fed for 1 week and fasted for 12 hours before modeling. The rats in the normal group were intraperitoneally injected with sodium citrate-citric acid buffer, and the rats in the modeling group were intraperitoneally injected with streptozotocin solution (35 mg / kg) prepared with an equal amount of sodium citrate-citric acid buffer. After continuous injection for 3 days, blood was collected by pricking the tail vein to measure the random blood glucose of each group of rats. If the random blood glucose was greater than 16.7 mmol / L, it was considered a diabetic (DM) rat model. After 8 weeks, diabetic erectile dysfunction (DMED) rats were screened out by apomorphine erection test, indicating that the animal model was successfully established.

[0049] Experimental grouping and administration: 72 DMED rats were randomly divided into 12 groups, with 6 rats in each group. 100 μL of the amniotic membrane stem cell preparations prepared in Examples 1-3 and Comparative Examples 1-8 were injected into the corpus cavernosum of the penis of 11 groups of rats (respectively referred to as Example 1-3 groups and Comparative Example 1-8 groups), 100 μL of normal saline was injected into one group (respectively referred to as the experimental control group), and 100 μL of normal saline was injected into the normal group (respectively referred to as the normal group). The test was performed 4 weeks after the injection; Intracavernosal pressure (ICP), intracarotid artery pressure (MAP) and ICP / MA detection: SD rats in each group were anesthetized with 3% sodium pentobarbital (30 mg / kg) by intraperitoneal injection. A longitudinal incision was made in the midline abdomen of the anesthetized rats to find the prostate of the rats and extend it to the dorsal side of its posterior lobe to fully expose the rat cavernous nerve. The PE-50 hose filled with sodium heparin saline was connected to the pressure sensor powerlab / 16sp. The stimulation duration was set to 60s, the parameters were 1.5mA and 20Hz, and it was inserted into the proximal end of the common carotid artery and the corpus cavernosum of the rats, respectively. The average values ​​of MAP and ICP were recorded. Finally, ICP / MAP was calculated to evaluate the erectile function of the rats. Determination of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) content, and determination of nitric oxide synthase (NOS) and guanylate cyclase (GC) activity: Penile tissue was obtained, minced, added with protein lysis buffer, and prepared into a homogenate using a tissue grinder. The mixture was lysed on ice for 30 min, centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was obtained. The NO and cGMP levels and NOS and GC activities in the penile tissue were determined according to the instructions of the kit. The specific test results are shown in Tables 1 and 2 below.

[0050] Table 1 Comparison of MAP, ICP and ICP / MAP in each group of rats

[0051] (Note: Compared with the normal group: ##P<0.01; compared with the experimental control group: *P<0.05, **P<0.01) Table 2 Comparison of NO, cGMP levels and NOS, GC activities in penile tissues of rats in each group

[0052] (Note: Compared with the normal group: ## P<0.01; compared with the experimental control group: *P<0.05, **P<0.01) As shown in Tables 1 and 2, the amniotic membrane stem cell preparation prepared in the present application can significantly increase the ICP / MAP ratio and improve erectile dysfunction. Compared with the normal group, the ICP and ICP / MAP of the rats in the experimental control group were significantly reduced (P < 0.01), and there was no statistically significant difference in MAP (P > 0.05). Compared with the groups of Examples 1-3 and Comparative Examples 1-8, the ICP and ICP / MAP of the rats in each drug-treated group were significantly increased (P < 0.01), and there was no statistically significant difference in MAP (P > 0.05). Compared with the normal group, the NO and cGMP levels and NOS and GC activities in the penile tissues of the rats in the experimental control group were The results showed that the NO and cGMP levels in the penile tissues of the rats in the experimental control group were significantly increased (P<0.05, 0.01), and the NOS activity and GC activity were significantly increased (P<0.05, 0.01). NO, as a vasodilator factor, can promote the formation of new blood vessels. By increasing the cGMP level, it can help cells resist oxidative stress and protect cells from free radical damage. NO and cGMP are involved in regulating various cell functions, such as cell proliferation and differentiation, which are crucial to the repair of damaged tissues, thereby improving the erectile function of DMEM rats.

[0053] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. An amniotic membrane stem cell preparation, characterized in that: Each 100 mL of the amniotic membrane stem cell preparation includes 4×10 9 -10×10 9 , active extract 2.5-3g, butanediol 4-6g, glycerol 0.5-1g, trehalose 0.1-0.3g, glucose 0.3-1g, amino acid 0.1-0.3g, vitamin 0.01-0.02g, and the remainder is normal saline containing human umbilical cord blood plasma.

2. The amniotic membrane stem cell preparation according to claim 1, characterized in that: The preparation steps of the active extract are as follows: (1) Drying Morinda officinalis and green tea, crushing and sieving, and then mixing, adding the mixed powder into an ethanol solution, ultrasonically treating at 60-70° C., centrifuging, taking the supernatant, filtering and sterilizing, rotary evaporating to remove the solvent, and drying to obtain a natural extract; (2) crushing epimedium and cistanche deserticola, sieving, adding into physiological saline, stirring evenly, adding bacterial enzyme complex, controlling the stirring speed of the fermentation tank to 200-300 rpm, pH value to 6.5-7, temperature to 35-40°C, ventilation volume to 1-2 vvm, dissolved oxygen content to 30-60%, fermenting for 2-3 days to obtain fermentation product; (3) centrifuging the fermentation product, collecting the supernatant, and sterilizing the supernatant with high-pressure steam to obtain an active fermentation product; (4) The active fermentation product and the natural extract are mixed in a mass ratio of 10-12:1-3 to obtain the active extract.

3. The amniotic membrane stem cell preparation according to claim 2, characterized in that: The mixed powder in step (1) is composed of Morinda officinalis and green tea in a mass ratio of 5-8:1-2.

4. The amniotic membrane stem cell preparation according to claim 2, characterized in that: The bacterial enzyme complex in step (2) is composed of Trichoderma and complex enzyme in a mass ratio of 1-2:18-20; the concentration of live Trichoderma is 1×10 9 -2×10 9 CFU / g; the complex enzyme consists of α-amylase and papain in a mass ratio of 10-12:5-8.

5. The amniotic membrane stem cell preparation according to claim 2, characterized in that: In step (2) The mass ratio of epimedium, Cistanche deserticola, normal saline and bacterial enzyme complex is 20-30:5-8:40-50:0.5-1.

6. The amniotic membrane stem cell preparation according to claim 1, characterized in that The preparation method of the amniotic membrane stem cells is as follows: Step S1, separating amniotic vascular stem cells, amniotic mesenchymal stem cells and amniotic stromal stem cells from human amniotic membrane, and mixing them in a ratio of 10-12:1-2:2-3 to obtain mixed primary cells; Step S2, inoculating the mixed primary cells in a modified DMEM medium, culturing for 1-2 days under the conditions of a CO2 concentration of 5-6%, a humidity of 88-90%, and a temperature of 36-38°C, and digesting with trypsin when the cell confluence reaches 70-80% to obtain the first passage cells; Step S3, inoculating the first passage cells in a modified DMEM medium, culturing for 1-2 days under the conditions of a CO2 concentration of 5-6%, a humidity of 85-90%, and a temperature of 36-38°C, and when the cell confluence reaches 75-85%, digesting and passage with trypsin at a ratio of 1:2-3, repeating the passage step 3 times, and obtaining the fourth passage cells; Step S4: Disperse the first passage cells and the fourth passage cells in the modified DMEM medium respectively, count them using a cell counter, and adjust the final cell concentration of the first passage cells and the fourth passage cells to 2×10 6 -3×10 6 / mL, and mixed at a volume ratio of 2-3:0.5-1, and centrifuged to obtain amniotic stem cells.

7. The amniotic membrane stem cell preparation according to claim 6, characterized in that: The seeding density of the mixed primary cells in step S2 is 1×10 6 -2×10 6 / mL; the preparation method of the modified DMEM medium is: adding fetal bovine serum and penicillin-streptomycin solution to the basic DMEM medium, and shaking well to obtain the medium; the volume ratio of the basic DMEM medium, fetal bovine serum and penicillin-streptomycin solution is 85-89:10-15:1-1.3; the seeding density of the first passage cells in step S3 is 1.5×10 6 -2.5×10 6 Pieces / mL.

8. The amniotic membrane stem cell preparation according to claim 1, characterized in that The preparation of the amniotic membrane stem cell preparation comprises the following steps: Add amniotic stem cells, active extracts, butanediol, glycerol, trehalose, glucose, amino acids, and vitamins to physiological saline containing human umbilical cord blood plasma, mix well, and then obtain an amniotic stem cell preparation, which is stored in a liquid nitrogen tank.

9. The amniotic membrane stem cell preparation according to claim 1, characterized in that: The volume fraction of human umbilical cord blood plasma in the physiological saline containing human umbilical cord blood plasma is 3-5%.

10. Use of the amniotic membrane stem cell preparation according to any one of claims 1 to 9 in treating erectile dysfunction.

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