Application of breast milk-derived stem cells combined with umbilical cord mesenchymal stem cells in preparation of drugs for treating premature ovarian failure

Abstract

The application provides application of breast milk-derived stem cells combined with umbilical cord mesenchymal stem cells in preparation of a drug for treating premature ovarian failure, and belongs to the technical field of biological medicine. The application also provides a stem cell preparation for treating premature ovarian failure, which comprises breast milk-derived stem cells, umbilical cord mesenchymal stem cells, resveratrol glycoside, melatonin, Leonurus japonicus glycoside, vitamin C, human blood albumin and physiological saline. The breast milk-derived stem cells and the umbilical cord mesenchymal stem cells play a core role, promote follicle survival and regeneration, and improve the pathological state of ovarian ischemia and hypoxia; the Leonurus japonicus glycoside can assist in dredging and improving local microcirculation; the vitamin C, the melatonin and the resveratrol glycoside can remove excessive harmful free radicals in the ovary and the whole body, and curb the continuous damage of oxidative stress to the follicle. Animal experiments show that the stem cell preparation can significantly improve the serum hormone level of premature ovarian failure mice, increase the reserve and competence of follicles at all levels, and has a clear repairing effect on ovarian function.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of breast milk-derived stem cells combined with umbilical cord mesenchymal stem cells in the preparation of drugs for treating premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF), the terminal stage of early-onset ovarian insufficiency, is a complex endocrine disorder commonly seen in women under 40. POF severely impacts women's health, causing a significant drop in estrogen levels and triggering a range of perimenopausal symptoms such as hot flashes, sweating, insomnia, memory loss, vaginal dryness, and difficulty with intercourse. It also prevents the ovaries from ovulating normally, leading to infertility and causing immense physical and psychological distress for patients. Furthermore, POF increases the risk of osteoporosis, heart disease, and dementia; some patients may even experience psychological problems such as depression or anxiety.

[0003] Currently, traditional treatments for premature ovarian failure are very limited and their effectiveness is not ideal. While hormone replacement therapy can alleviate symptoms caused by estrogen deficiency to some extent, it cannot fundamentally restore ovarian function, and long-term use may lead to serious risks such as blood clots and breast cancer. Assisted reproductive technologies such as in-vitro fertilization also have extremely low success rates due to ovarian failure, a low number of follicles, or poor follicle quality, making it difficult to meet patients' fertility needs. Therefore, developing safe and effective new treatment methods has become a pressing challenge in the medical field. Summary of the Invention

[0004] The purpose of this invention is to provide the application of breast milk-derived stem cells combined with umbilical cord mesenchymal stem cells in the preparation of drugs for treating premature ovarian failure. This invention combines breast milk-derived stem cells with umbilical cord mesenchymal stem cells to alleviate premature ovarian failure.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides an application of breast milk-derived stem cells combined with umbilical cord mesenchymal stem cells in the preparation of drugs for treating premature ovarian failure.

[0007] Preferably, the drug is administered via intravenous injection.

[0008] Preferably, the preparation of the breast milk-derived stem cells includes: collecting breast milk, centrifuging the breast milk to remove fat, purifying it by Percoll gradient centrifugation and magnetic bead sorting, seeding and passaged, and obtaining breast milk-derived stem cells by passage to the 3rd-6th generation; the preparation of the umbilical cord mesenchymal stem cells includes: disinfecting and washing the umbilical cord, separating Wharton's jelly and cutting it into small pieces, attaching the tissue block to the wall, and passaged it with culture medium, obtaining umbilical cord mesenchymal stem cells by passage to the 3rd-6th generation.

[0009] The present invention also provides a stem cell preparation for treating premature ovarian failure, comprising: breast milk-derived stem cells, umbilical cord mesenchymal stem cells, resveratrol glycoside, melatonin, leonurin, vitamin C, human serum albumin, and physiological saline.

[0010] Preferably, the density of the breast milk-derived stem cells is (2-5)×10⁻⁶. 5 The density of umbilical cord mesenchymal stem cells was 6 × 10⁶ cells / mL. 5 -2×10 6 per mL.

[0011] Preferably, the concentration of resveratrol glycoside is 1-3 μM, the concentration of melatonin is 3-5 μM, the concentration of leonurin is 20-30 μM, the concentration of vitamin C is 200-300 μM, and the mass-volume percentage of human serum albumin is 2 w / v%-4 w / v.

[0012] Preferably, the osmotic pressure of the stem cell preparation is 305-317 mOsm / L.

[0013] Preferably, the pH value of the stem cell preparation is 7.2-7.6.

[0014] The present invention also provides a method for preparing the above-mentioned stem cell preparation, comprising: first mixing human serum albumin and physiological saline to obtain a human serum albumin solution, then adding vitamin C, resveratrol glycoside, melatonin, and leonurin to obtain a matrix solution; and resuspending breast milk stem cells and umbilical cord mesenchymal stem cells in the matrix solution to obtain the stem cell preparation.

[0015] The present invention also provides an application of the above-mentioned stem cell preparation in the preparation of a drug for preventing and treating premature ovarian failure.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides the application of breast milk-derived stem cells combined with umbilical cord mesenchymal stem cells in the preparation of drugs for treating premature ovarian failure. This invention also provides a stem cell preparation for treating premature ovarian failure, comprising: breast milk-derived stem cells, umbilical cord mesenchymal stem cells, resveratrol glycoside, melatonin, leonurin, vitamin C, human serum albumin, and physiological saline.

[0018] In this invention, breast milk-derived stem cells and umbilical cord mesenchymal stem cells play a core role. Homecoming stem cells promote follicle survival and regeneration. Stem cells and their secreted active factors promote angiogenesis and improve the pathological state of ovarian ischemia and hypoxia. Leonurus japonicus glycosides help to unblock and improve local microcirculation, ensuring the smooth delivery of nutrients and repair factors. Vitamin C (extracellular), melatonin (intramitochondria), and resveratrol glycosides (paranuclear) constitute three three-dimensional antioxidant defense lines covering the whole body, cells, and organelles, which can eliminate excess harmful free radicals in the ovary and throughout the body, inhibiting the continuous damage of oxidative stress to follicles. Human serum albumin provides physical protection for stem cells during in vitro preparation and in vivo circulation, reducing cell death and providing necessary nutritional support for damaged ovarian tissue and transplanted stem cells. The technical solution of this invention achieves the effect of treating premature ovarian failure through multiple targets and a systematic approach. It is not just about passively replenishing cells, but about actively reshaping the micro and macro environment of the entire ovary. From eliminating pathogenic factors and protecting healthy tissues to ultimately rebuilding functional units, it forms a complete treatment system, thus providing a theoretical and technical basis for achieving substantial reversal of ovarian function.

[0019] Animal experiments have shown that the stem cell preparation of this invention can significantly improve serum hormone levels in mice with premature ovarian failure, increase the reserve and maturation capacity of follicles at all stages, and has a clear restorative effect on ovarian function. Detailed Implementation

[0020] This invention provides an application of breast milk-derived stem cells combined with umbilical cord mesenchymal stem cells in the preparation of drugs for treating premature ovarian failure.

[0021] The drug described in this invention is preferably administered via intravenous injection.

[0022] The preferred method for preparing breast milk-derived stem cells according to the present invention includes: collecting breast milk, centrifuging the breast milk to remove fat, purifying it by Percoll gradient centrifugation and magnetic bead sorting, seeding and passage culturing, and obtaining breast milk-derived stem cells by passage 3-6.

[0023] The preferred preparation method of the umbilical cord mesenchymal stem cells of the present invention includes: disinfecting and washing the umbilical cord, separating Wharton's jelly and cutting it into small pieces, attaching the tissue block to the wall, and then passaged it with culture medium until the 3rd to 6th generations to obtain umbilical cord mesenchymal stem cells.

[0024] The present invention also provides a stem cell preparation for treating premature ovarian failure, comprising: breast milk-derived stem cells, umbilical cord mesenchymal stem cells, resveratrol glycoside, melatonin, leonurin, vitamin C, human serum albumin, and physiological saline.

[0025] The preferred density of the breast milk-derived stem cells described in this invention is (2-5) × 10⁻⁶. 5 Cells / mL, more preferably 3×10⁻⁶ 5The density of umbilical cord mesenchymal stem cells was 6 × 10⁶ cells / mL. 5 -2×10 6 Cells / mL, more preferably 9 × 10⁻⁶ 5 The concentration of resveratrol glycosides in this invention is preferably 1-3 μM, more preferably 2 μM; the concentration of melatonin is preferably 3-5 μM, more preferably 4 μM; the concentration of leonurin is preferably 20-30 μM, more preferably 25 μM; the concentration of vitamin C is preferably 200-300 μM, more preferably 250 μM; and the mass-volume percentage of human serum albumin is preferably 2 w / v%-4 w / v%, more preferably 3 w / v.

[0026] The umbilical cord mesenchymal stem cells described in this invention can secrete factors such as TGF-β, PGE2, and IL-10, which can efficiently regulate immunity, inhibit excessive inflammation, and exert their immunomodulatory and anti-inflammatory effects more quickly, rapidly controlling local immune damage and the inflammatory environment of the ovary, paving the way for subsequent repair. Simultaneously, they secrete large amounts of VEGF and HGF, strongly promoting angiogenesis and improving ovarian blood supply. Breast milk-derived stem cells have extremely strong proliferative and self-renewal potential, secreting more factors related to tissue reconstruction and cell proliferation (such as growth factors and miRNAs). Combining the two can complement and enhance the secretory factor spectrum, enabling more effective long-term tissue reconstruction and cell replacement after inflammation is controlled by umbilical cord mesenchymal stem cells. After intravenous injection, breast milk-derived stem cells and umbilical cord mesenchymal stem cells can secrete large amounts of bioactive factors, exerting a paracrine effect. Furthermore, breast milk-derived stem cells and umbilical cord mesenchymal stem cells can migrate to damaged ovarian tissue (homing) and may differentiate into ovarian granulosa cells, follicular cells, etc., under the induction of the microenvironment, directly replenishing and repairing damaged cells.

[0027] In the stem cell preparation of this invention, the human serum albumin acts as a carrier, stabilizer, and protectant: it serves as a natural carrier for various small molecule substances (such as resveratrol glycosides and melatonin) in the preparation, improving their solubility and stability; it provides protection for stem cells in solution, reducing cell damage and apoptosis caused by shear force, changes in osmotic pressure, etc., and significantly improving cell survival rate after injection; it ensures that the osmotic pressure of the injection solution is within the physiological range, preventing abnormalities in blood volume or red blood cell morphology during intravenous administration.

[0028] The resveratrol glycosides described in this invention can exert a strong antioxidant effect and activate SIRT1, enhance cellular stress resistance, inhibit apoptosis, and improve mitochondrial function. This is crucial for protecting the remaining ovarian follicle reserve and can reduce oxidative stress and inflammatory damage to the ovary, paving the way for stem cell survival and function.

[0029] The melatonin described in this invention can easily enter the mitochondria, directly neutralize free radicals produced within the mitochondria, and protect cellular energy (oocytes are the cells with the most mitochondria in the human body), which is particularly important for improving follicle quality; it has antioxidant effects; it participates in regulating the hypothalamic-pituitary-gonadal axis (HPG axis), and has a positive effect on improving sleep and endocrine disorders in patients with premature ovarian failure.

[0030] The vitamin C described in this invention acts as the first line of defense, preferentially being oxidized during the formulation process, thereby protecting other more precious active ingredients (such as stem cells and resveratrol glycosides) from oxidative inactivation; as a cofactor for collagen synthesis, it can promote the synthesis of extracellular matrix and basement membrane, which helps in the repair of ovarian structure.

[0031] The leonurine described in this invention has a mild vasodilatory effect and improves microcirculation, thereby improving local blood supply to the ovary, providing more oxygen and nutrients to the delivered stem cells and nutrients, and facilitating the removal of metabolic waste. The improved hemodynamics may help stem cells home to the ovary more effectively.

[0032] In this invention, breast milk-derived stem cells and umbilical cord mesenchymal stem cells play a core role. Homing stem cells promote follicle survival and regeneration. Stem cells and their secreted active factors promote angiogenesis, improving the pathological state of ovarian ischemia and hypoxia. Leonurus japonicus glycosides help to unblock and improve local microcirculation, ensuring the smooth delivery of nutrients and repair factors. Vitamin C (extracellular), melatonin (intramitochondria), and resveratrol glycosides (paranuclear) constitute three three-dimensional antioxidant defense lines covering the whole body, cells, and organelles, which can eliminate excess harmful free radicals in the ovaries and the whole body, inhibiting the continuous damage of oxidative stress to follicles. Human serum albumin provides physical protection for stem cells during in vitro preparation and in vivo circulation, reducing cell death and providing necessary nutritional support for damaged ovarian tissue and transplanted stem cells. Melatonin ensures that cells have sufficient energy for repair work. The technical solution of this invention achieves the effect of treating premature ovarian failure through multiple targets and a systematic approach. It is not just about passively replenishing cells, but about actively reshaping the micro and macro environment of the entire ovary. From eliminating pathogenic factors and protecting healthy tissues to ultimately rebuilding functional units, it forms a complete treatment system, thus providing a theoretical and technical basis for achieving substantial reversal of ovarian function.

[0033] The osmotic pressure of the stem cell preparation described in this invention is 305-317 mOsm / L.

[0034] The pH value of the stem cell preparation described in this invention is preferably 7.2-7.6, more preferably 7.4. The pH value range can be adjusted by hydrochloric acid and sodium hydroxide. When preparing the stem cell preparation, vitamin C will make the solution system acidic, and sodium hydroxide solution is needed to adjust the system solution to slightly alkaline.

[0035] The present invention also provides a method for preparing the above-mentioned stem cell preparation, comprising: first mixing human serum albumin and physiological saline to obtain a human serum albumin solution, then adding vitamin C, resveratrol glycoside, melatonin, and leonurin to obtain a matrix solution; and resuspending breast milk stem cells and umbilical cord mesenchymal stem cells in the matrix solution to obtain the stem cell preparation.

[0036] The present invention also provides an application of the above-mentioned stem cell preparation in the preparation of a drug for preventing and treating premature ovarian failure.

[0037] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0038] Unless otherwise specified, the following embodiments are all conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1

[0041] Preparation of breast milk-derived stem cells

[0042] (1) Sample collection

[0043] 1) Donor selection: Select healthy breastfeeding women (excluding donors infected with (HIV, HBV, HCV, syphilis), taking immunosuppressants, or having metabolic diseases);

[0044] 2) Collection method: Before collection, the breast and breast pump should be cleaned. The breast pump needs to be sterilized. Use a sterile breast pump to collect breast milk electrically. Collect 100mL at a time. Immediately after collection, mark the donor information, collection time and postpartum days.

[0045] 3) Preservation: Fresh samples should be refrigerated at 4°C and delivered to the laboratory within 24 hours.

[0046] (2) Sample preprocessing

[0047] 1) Defatting by centrifugation: Transfer breast milk to a 50mL sterile centrifuge tube and centrifuge at 1500×g for 10min at 4℃. The sample will separate into three layers: an upper fat layer, a middle whey layer, and a lower cell pellet layer. Carefully aspirate the upper fat and middle whey layers, retaining the bottom cell pellet.

[0048] 2) Washing and removing impurities: Add an equal volume of PBS (pH 7.2, containing penicillin 100 U / mL + streptomycin 100 μg / mL) to the cell pellet, mix well by pipetting, centrifuge at 1000×g for 5 min at 4℃, repeat 3 times to remove residual whey protein and fat particles, collect the cells (the pellet), add 5 mL of PBS (pH 7.2, containing penicillin 100 U / mL + streptomycin 100 μg / mL) to the pellet, and gently pipette the pellet 5 times to obtain the cell suspension.

[0049] (3) Stem cell isolation and purification

[0050] 1) Density gradient centrifugation: Slowly add 5 mL of 1.07 g / mL Percoll solution to a 15 mL centrifuge tube, then spread the cell suspension on top of the Percoll solution. Centrifuge at 1500 × g for 20 min at 20°C. After centrifugation, the solution separates into four layers: the upper layer contains cell debris and impurities, the middle layer contains low-density cells (including stem cells), the lower layer contains high-density cells (such as granulocytes), and the bottom layer contains density media. Use a pipette to aspirate the cells from the middle layer.

[0051] 2) Immunomagnetic bead sorting: The mesolayer cells were incubated with fluorescently labeled stem cell marker antibodies (anti-CD29-PE, anti-CD44-FITC) at 4°C in the dark for 30 min. Then, magnetic bead-labeled secondary antibodies were added and incubated for 20 min. The cells were then placed in a magnetic field to adsorb positive cells (stem cells). Unbound negative cells were discarded. Cells bound to the magnetic beads were washed with PBS to obtain high-purity breast milk stem cells.

[0052] (4) Primary generation cultivation and propagation

[0053] 1) Culture medium: Add 12% fetal bovine serum (FBS), 1% penicillin 100U / mL + streptomycin 100μg / mL, and growth factors (EGF 10ng / mL, bFGF 5ng / mL) to DMEM / F12 (1:1) basal culture medium.

[0054] 2) Inoculation: High-purity breast milk-derived stem cells were inoculated at a rate of 3 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / mL in culture dishes coated with 0.01% gelatin and cultured at 37°C, 5% CO2, and saturated humidity for 24 hours. The culture medium was then replaced and non-adherent cells were removed. The culture medium was then replaced every 2 days, and cell morphology was observed (fibrillated, spindle-shaped, and arranged in whorls).

[0055] 3) Passaging: When the cell confluence reaches 80%, discard the old culture medium, wash twice with PBS, add 0.25% trypsin-EDTA solution, and incubate at 37℃ and 5% CO2 for 2-5 minutes. During this period, observe under a microscope every 1 minute. When most cells become round and the edges detach from the bottom of the culture dish, add culture medium to stop digestion. Centrifuge at 800×g for 5 minutes, collect the precipitated cells, and seed them in a new culture dish at a ratio of 1:5. Passage to the 5th generation to obtain maternal milk stem cells.

[0056] (5) Stem cell identification

[0057] 1) Flow cytometry

[0058] The prepared breast milk-derived stem cells were adjusted to 1×10⁻⁶ using staining buffer (PBS containing 2% bovine serum albumin). 6 A suspension of breast milk-derived stem cells was obtained by measuring cells / mL.

[0059] 100 μL of breast milk-derived stem cell suspension was added to flow cytometry tubes, and the expression of positive antibodies (CD29, CD44, CD73, CD90, CD105) and negative antibodies (CD31, CD34, CD45, EpCAM) in the breast milk-derived stem cell suspension was detected by flow cytometry.

[0060] Experimental results showed that the positive expression rates of positive markers (CD29, CD44, CD73, CD90, CD105) of the breast milk-derived stem cells of the present invention were all ≥95%, and the positive expression rates of negative markers (CD31, CD34, CD45, EpCAM) were all ≤2%. This indicates that the breast milk-derived stem cells prepared by the present invention have high purity and their expression has highly similar phenotypic characteristics to mesenchymal stem cells, making them a type of cell with mesenchymal stem cell characteristics.

[0061] 2) Identification of multi-directional differentiation potential

[0062] Osteogenic differentiation: Breast milk-derived stem cells were seeded into osteogenic induction medium (basal medium + 10 mmol / L sodium β-glycerophosphate + 50 μg / mL ascorbic acid + 100 nmol / L dexamethasone). After 2 weeks of culture, red calcium nodules were visible after Alizarin Red staining.

[0063] Adipogenic differentiation: After culturing in adipogenic induction medium (basal medium + 0.5 mmol / L IBMX + 1 μmol / L dexamethasone + 10 μmol / L insulin) for 2 weeks, intracellular red lipid droplets were visible after Oil Red O staining.

[0064] Chondrogenic differentiation: The chondrogenic differentiation was carried out by microsphere culture in chondrogenic induction medium (basal medium + 10 ng / mL LTGF-β1 + 50 μg / mL ascorbic acid) for 3 weeks. Alcian blue staining revealed the cartilage matrix (glycosaminoglycans).

[0065] This demonstrates that the breast milk-derived stem cells prepared by this invention have multi-directional differentiation potential, which conforms to the core definition of stem cells.

[0066] Example 2

[0067] Preparation of umbilical cord mesenchymal stem cells

[0068] (1) Umbilical cord examination

[0069] Check whether the container for storing the umbilical cord is intact and sealed; whether the label is clear, complete and correct; whether the umbilical cord is deteriorated or rotten; after checking and confirming that it is qualified, record the umbilical cord information, wipe the storage bottle with 75% alcohol twice for disinfection, and move it into the biosafety cabinet.

[0070] (2) Separation of Wharton's glue

[0071] Remove the umbilical cord from the preservation bottle using hemostatic forceps, record the cord length, place the cord in a sterile petri dish, pour in 75% alcohol and soak for 1 minute, then use a solution containing 10... 5 Rinse five times with normal saline containing U / L penicillin and 100mg / L streptomycin to remove residual blood from the umbilical vein and artery.

[0072] Clamp the umbilical cord with hemostats, cut it into segments of about 3cm, and remove the umbilical vein, artery, and outer membrane; wash away the bloodstains with saline to obtain Wharton's jelly.

[0073] (3) Adherent culture

[0074] Cut the Wharton's glue into 2mm pieces. 3 For tissue blocks, place the tissue into a sterile culture dish, pour in physiological saline, rinse gently, and discard the rinsing solution with a pipette; use a sterile dropper to evenly distribute the tissue blocks into T75 culture flasks, and record the number of inoculation flasks.

[0075] Incubate the inoculated T75 culture flasks at 37°C in a 5% CO2 incubator for 15 minutes to allow the tissue blocks to adhere to the flask walls. Add 8 ml of MSC complete culture medium to each flask, just enough to cover the tissue blocks (add the medium carefully, drop by drop, and avoid disturbing the tissue blocks). Incubate at 37°C in a 5% CO2 incubator to begin culturing. On the third day of culturing, perform a complete medium change by transferring the culture supernatant to a 50 mL centrifuge tube and centrifuging at 500×g for 5 minutes. After centrifugation, collect the supernatant for microbial testing and discard the rest of the supernatant. Place the tissue back into the culture flask, add 8 ml of culture medium to each flask, and incubate at 37°C in a 5% CO2 incubator for 6 days.

[0076] (4) Transmission

[0077] After 6 days of tissue culture, transfer the culture medium and tissue blocks from the culture flask to a 50 mL centrifuge tube and centrifuge at 500×g for 5 min; then gently rinse the cells twice with physiological saline.

[0078] Add 5 mL of 0.05% trypsin to each flask of cells and incubate at 37°C in a 5% CO2 incubator for 2-5 minutes, observing under a microscope every minute during this period. When most cells become round and their edges detach from the bottom of the culture dish, add complete culture medium containing 12% fetal bovine serum to stop digestion. Centrifuge at 800×g for 5 minutes, collect the cell pellet, wash twice with physiological saline, centrifuge at 800×g for 5 minutes, collect the cell pellet, and seed them at a 1:5 ratio into new seeding flasks containing complete culture medium. Passage to the 4th generation to obtain umbilical cord mesenchymal stem cells.

[0079] (5) Verification

[0080] 1) Flow cytometry

[0081] The prepared umbilical cord mesenchymal stem cells were adjusted to 1×10⁻⁶ using staining buffer (PBS containing 2% bovine serum albumin). 6 The concentration of cells / mL was increased to obtain a suspension of umbilical cord mesenchymal stem cells.

[0082] 100 μL of umbilical cord mesenchymal stem cell suspension was added to flow cytometry tubes, and the expression of positive antibodies (CD73, CD90, CD105) and negative antibodies (CD45, CD34, CD11b, CD19 and HLA-DR) in the umbilical cord mesenchymal stem cell suspension was detected by flow cytometry.

[0083] Experimental results showed that the positive expression rates of positive markers (CD73, CD90, CD105) of the umbilical cord mesenchymal stem cells of the present invention were all ≥95%, and the positive expression rates of negative markers (CD45, CD34, CD11b, CD19 and HLA-DR) were all ≤2%. This indicates that the umbilical cord mesenchymal stem cells prepared by the present invention have high purity and their expression has highly similar phenotypic characteristics to mesenchymal stem cells. They are a type of cell with the characteristics of mesenchymal stem cells.

[0084] 2) Identification of multi-directional differentiation potential

[0085] Osteogenic differentiation: Umbilical cord mesenchymal stem cells were seeded into osteogenic induction medium (basal medium + 10 mmol / L sodium β-glycerophosphate + 50 μg / mL ascorbic acid + 100 nmol / L dexamethasone). After 2 weeks of culture, red calcium nodules were visible after Alizarin Red staining.

[0086] Adipogenic differentiation: After culturing in adipogenic induction medium (basal medium + 0.5 mmol / L IBMX + 1 μmol / L dexamethasone + 10 μmol / L insulin) for 2 weeks, intracellular red lipid droplets were visible after Oil Red O staining.

[0087] Chondrogenic differentiation: The chondrogenic differentiation was carried out by microsphere culture in chondrogenic induction medium (basal medium + 10 ng / mL LTGF-β1 + 50 μg / mL ascorbic acid) for 3 weeks. Alcian blue staining revealed the cartilage matrix (glycosaminoglycans).

[0088] This demonstrates that the umbilical cord mesenchymal stem cells prepared by this invention have multi-directional differentiation potential and are mesenchymal stem cells with standard biological characteristics.

[0089] Example 3

[0090] Preparation of stem cell preparations for treating premature ovarian failure

[0091] (1) Composition and concentration of stem cell preparations

[0092] The stem cell preparation consists of the following components: breast milk-derived stem cells prepared in Example 1, umbilical cord mesenchymal stem cells prepared in Example 2, resveratrol glycoside, melatonin, leonurin, vitamin C, human serum albumin, and physiological saline, with sodium hydroxide to adjust the pH value.

[0093] Among them, the density of breast milk-derived stem cells was 3×10⁻⁶. 5 The density of umbilical cord mesenchymal stem cells was 9 × 10⁶ cells / mL. 5 The concentrations of resveratrol glycoside (2 μM), melatonin (4 μM), leonurin (25 μM), vitamin C (250 μM), and human serum albumin (3 w / v) were all present.

[0094] (2) Preparation of stem cell preparations

[0095] First, human serum albumin and physiological saline were allowed to stand for 30 minutes to obtain a human serum albumin solution. Then, vitamin C was added, and the pH was adjusted to 7.4 with sodium hydroxide. Next, resveratrol glycoside, leonurin, and melatonin were added sequentially. After each addition, the mixture was stirred at 120 rpm for 8 minutes to obtain a matrix solution. Breast milk-derived stem cells and umbilical cord mesenchymal stem cells were resuspended in the matrix solution, and the pH was adjusted to 7.4 with sodium hydroxide to obtain the stem cell preparation.

[0096] The osmotic pressure of the stem cell preparation was measured to be 310 mOsm / L using a freezing point osmometer.

[0097] Example 4

[0098] Preparation of stem cell preparations for treating premature ovarian failure

[0099] (1) Composition and concentration of stem cell preparations

[0100] The stem cell preparation consists of the following components: breast milk-derived stem cells prepared in Example 1, umbilical cord mesenchymal stem cells prepared in Example 2, resveratrol glycoside, melatonin, leonurin, vitamin C, human serum albumin, and physiological saline, with sodium hydroxide to adjust the pH value.

[0101] The density of breast milk-derived stem cells was 2 × 10⁻⁶. 5 The density of umbilical cord mesenchymal stem cells was 2 × 10⁶ cells / mL. 6 The concentrations were: 1 μM of resveratrol glycoside, 5 μM of melatonin, 20 μM of leonurin, 300 μM of vitamin C, and 2 w / v of human serum albumin.

[0102] (2) Preparation of stem cell preparations

[0103] First, human serum albumin and physiological saline were allowed to stand for 25 minutes to obtain a human serum albumin solution. Then, vitamin C was added, and the pH was adjusted to 7.2 with sodium hydroxide. Next, resveratrol glycoside, leonurin, and melatonin were added sequentially. After each addition, the mixture was stirred at 100 rpm for 10 minutes to obtain a matrix solution. Breast milk-derived stem cells and umbilical cord mesenchymal stem cells were resuspended in the matrix solution, and the pH was adjusted to 7.2 with sodium hydroxide to obtain the stem cell preparation.

[0104] The osmotic pressure of the stem cell preparation was measured to be 317 mOsm / L using a freezing point osmoremeter.

[0105] Example 5

[0106] Preparation of stem cell preparations for treating premature ovarian failure

[0107] (1) Composition and concentration of stem cell preparations

[0108] The stem cell preparation consists of the following components: breast milk-derived stem cells prepared in Example 1, umbilical cord mesenchymal stem cells prepared in Example 2, resveratrol glycoside, melatonin, leonurin, vitamin C, human serum albumin, and physiological saline, with sodium hydroxide to adjust the pH value.

[0109] Among them, the density of breast milk-derived stem cells was 5×10⁻⁶. 5 The density of umbilical cord mesenchymal stem cells was 6 × 10⁶ cells / mL. 5 The concentration of resveratrol glycoside was 3 μM, the concentration of melatonin was 3 μM, the concentration of leonurin was 30 μM, the concentration of vitamin C was preferably 200 μM, and the mass-volume percentage of human serum albumin was 4 w / v.

[0110] (2) Preparation of stem cell preparations

[0111] First, human serum albumin and physiological saline were allowed to stand for 35 minutes to obtain a human serum albumin solution. Then, vitamin C was added, and the pH was adjusted to 7.6 with sodium hydroxide. Next, resveratrol glycoside, leonurin, and melatonin were added sequentially. After each addition, the mixture was stirred at 150 rpm for 8 minutes to obtain a matrix solution. Breast milk-derived stem cells and umbilical cord mesenchymal stem cells were resuspended in the matrix solution, and the pH was adjusted to 7.6 with sodium hydroxide to obtain the stem cell preparation.

[0112] The osmotic pressure of the stem cell preparation was measured to be 305 mOsm / L using a freezing point osmometer.

[0113] Comparative Example 1

[0114] The specific implementation method is the same as in Example 3, except that the density of breast milk-derived stem cells in step (1) is 3×10 5 The density of umbilical cord mesenchymal stem cells was 9 × 10⁶ cells / mL. 5 Replace "number / mL" with "the density of breast milk-derived stem cells is 12 × 10⁻⁶". 5 "number / mL", and correspondingly delete the umbilical cord mesenchymal stem cells in step (2).

[0115] Comparative Example 2

[0116] The specific implementation method is the same as in Example 3, except that the density of breast milk-derived stem cells in step (1) is 3×10 5 The density of umbilical cord mesenchymal stem cells was 9 × 10⁶ cells / mL. 5 Replace "cells / mL" with "the density of umbilical cord mesenchymal stem cells is 12 × 10⁻⁶". 5 "number / mL", and correspondingly delete the breast milk stem cells in step (2).

[0117] Comparative Example 3

[0118] (1) Composition and concentration of stem cell preparations

[0119] The stem cell preparation consists of the following components: breast milk-derived stem cells prepared in Example 1, umbilical cord mesenchymal stem cells prepared in Example 2, leonurin, human serum albumin, and physiological saline, with sodium hydroxide to adjust the pH value.

[0120] Among them, the density of breast milk-derived stem cells was 3×10⁻⁶. 5 The density of umbilical cord mesenchymal stem cells was 9 × 10⁶ cells / mL. 5 The concentration of leonurin was 25 μM, and the mass-volume percentage of human serum albumin was 3 w / v.

[0121] (2) Preparation of stem cell preparations

[0122] First, human serum albumin and physiological saline were allowed to stand and mix for 30 minutes to obtain a human serum albumin solution. Then, leonurin was added and stirred at 120 rpm for 8 minutes to obtain a matrix solution. Breast milk-derived stem cells and umbilical cord mesenchymal stem cells were resuspended in the matrix solution, and the pH value was adjusted to 7.4 with sodium hydroxide to obtain the stem cell preparation.

[0123] Comparative Example 4

[0124] The specific implementation method is the same as in Example 3, except that the concentration of leonurin is adjusted to 15 μM.

[0125] Comparative Example 5

[0126] The specific implementation method is the same as in Example 3, except that the concentration of leonurin is adjusted to 40 μM.

[0127] Experimental Example 1

[0128] animal experiments

[0129] Healthy SPF-grade 8-week-old female ICR mice weighing 18-22g were selected. After one week of acclimatization feeding, the mice were randomly divided into 11 groups (normal control group, model control group, positive control group, Example 3-5 groups and Comparative Examples 1-5), with 10 mice in each group.

[0130] Except for the normal control group, mice in the other groups were injected intraperitoneally with cyclophosphamide (120 mg / kg) and busulfan (12 mg / kg) daily to establish a premature ovarian failure model. The normal control group was injected with an equal volume of physiological saline. The model was established for 7 days.

[0131] On day 8, groups 3-5 of Examples and groups 1-5 of Comparative Examples were injected with the corresponding stem cell preparation via the tail vein at a dose of 0.2 mL / animal. The positive control group was injected with physiological saline containing 0.02 mg / mL estradiol via the tail vein at a dose of 0.2 mL / animal. The normal control group and the model group were injected with physiological saline via the tail vein at a dose of 0.2 mL / animal. The injections were given once every 4 days for 4 consecutive weeks.

[0132] (1) Serum hormone levels

[0133] 24 hours after the last administration, blood was collected from the facial vein, incubated at 4°C for 2 hours, and centrifuged at 3000 rpm for 15 minutes to obtain serum. The levels of estradiol and follicle-stimulating hormone were detected by ELISA, and the average values ​​were taken. The specific results are shown in Table 1.

[0134] Table 1. Serum estradiol and follicle-stimulating hormone levels in mice of each group.

[0135]

[0136]

[0137] Table 1 shows that, compared with the normal control group, the model group had significantly lower estradiol and significantly higher follicle-stimulating hormone (FSH) levels, proving the successful establishment of the premature ovarian failure (POF) mouse model. In other groups, estradiol levels were higher than in the model group, and FSH levels were lower, demonstrating that each group had a positive effect on the treatment of POF. Specifically, the effects of Examples 3-5 were comparable to the positive control group, indicating that the stem cell preparation of this invention can effectively improve ovarian endocrine function, promote estradiol secretion, and inhibit excessive FSH levels. A comparison of the data from Example 3 and Comparative Examples 1-2 shows that breast milk-derived stem cells and umbilical cord mesenchymal stem cells have a synergistic effect, and their combination is more effective in treating POF. The efficacy of Comparative Example 3 was worse than that of Example 1, possibly because the lack of resveratrol glycosides, melatonin, and vitamin C reduced the antioxidant properties of the stem cell preparation, causing some of the active ingredients in the stem cells to be used against harmful free radicals in the ovary, thus reducing the efficacy of the stem cell preparation. A comparison of the data from Example 3 and Comparative Examples 4-5 shows that the concentration of leonurus japonicus glycosides can affect the therapeutic effect of the stem cell preparation on POF.

[0138] (2) Ovarian function

[0139] After collecting blood from the facial veins of mice, the mice were euthanized by cervical dislocation. Ovarian tissue was collected, stained with H&E, and the number of follicles at each stage (primitive, primary, secondary, and mature follicles) was observed and counted. The average value was taken, and the specific results are shown in Table 2.

[0140] Table 2. Number of primordial, primary, secondary, and mature follicles in each group of mice.

[0141]

[0142]

[0143] Table 2 shows that the number of follicles at all stages decreased sharply in the model group, especially mature follicles, which is consistent with the characteristics of premature ovarian failure, indicating that the mouse model of premature ovarian failure was successfully established. The number of follicles at all stages in Examples 3-5 was significantly higher than that in the model control group and close to that in the positive control group, indicating that the stem cell preparation of this invention can effectively promote ovarian tissue regeneration and increase follicle reserve and developmental capacity. A comparison of data from Comparative Examples 1-2 and Example 3 shows that the combined use of breast milk stem cells and umbilical cord mesenchymal stem cells is more conducive to follicle generation; a comparison of data from Comparative Example 3 and Example 3 shows that resveratrol glycosides, melatonin, and vitamin C can enhance the antioxidant properties of the stem cell preparation, improve the bioactivity of breast milk stem cells and umbilical cord mesenchymal stem cells, and thus promote follicle maturation; a comparison of data from Comparative Examples 4-5 and Example 3 shows that an appropriate concentration of leonurine can promote blood circulation in the ovarian region and improve the therapeutic effect of the stem cell preparation on premature ovarian failure.

[0144] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A stem cell preparation for treating premature ovarian failure, characterized in that, It consists of the following components: breast milk-derived stem cells, umbilical cord mesenchymal stem cells, resveratrol glycosides, melatonin, leonurin, vitamin C, human serum albumin, and physiological saline. The density of the breast milk-derived stem cells was (2-5)×10⁻⁶. 5 The density of umbilical cord mesenchymal stem cells was 6 × 10⁶ cells / mL. 5 -2×10 6 cells / mL; The concentrations of resveratrol glycosides, melatonin, leonurin, and vitamin C were 1-3 μM, 3-5 μM, 20-30 μM, and 200-300 μM, respectively. The mass-volume percentage of human serum albumin was 2 w / v%-4 w / v%. The preparation of the breast milk-derived stem cells includes: collecting breast milk, centrifuging the breast milk to remove fat, purifying it by Percoll gradient centrifugation and magnetic bead sorting, seeding and passage culture, and obtaining breast milk-derived stem cells by passage to the 3rd-6th generation. The preparation of the umbilical cord mesenchymal stem cells includes: disinfecting and washing the umbilical cord, separating Wharton's jelly and cutting it into small pieces, attaching the tissue block to the wall, adding culture medium for passage culture, and obtaining umbilical cord mesenchymal stem cells by passage to the 3rd-6th generation.

2. The stem cell preparation according to claim 1, characterized in that, The osmotic pressure of the stem cell preparation is 305-317 mOsm / L.

3. The stem cell preparation according to claim 1, characterized in that, The pH value of the stem cell preparation is 7.2-7.

6.

4. A method for preparing a stem cell preparation according to any one of claims 1-3, characterized in that, include: First, human serum albumin and physiological saline are mixed to obtain a human serum albumin solution. Then, vitamin C, resveratrol glycoside, melatonin, and leonurin are added to obtain a matrix solution. The stem cell preparation was obtained by resuspending breast milk-derived stem cells and umbilical cord mesenchymal stem cells in a matrix solution.

5. The use of a stem cell preparation according to any one of claims 1-3 in the preparation of a drug for preventing and treating premature ovarian failure.

6. The application according to claim 5, characterized in that, The drug is administered via intravenous injection.

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