Exosome preparation for treating premature ovarian failure

Abstract

The invention discloses an exosome preparation for treating premature ovarian failure, and belongs to the technical field of biology. And adding leonurine and estradiol into the umbilical cord mesenchymal stem cell culture solution, and co-culturing to prepare the exosome preparation for treating premature ovarian failure. The conventional exosome and the leonurine and estradiol co-cultured exosome with the same dosage can weaken the inhibition effect of the 4-OHCP on the KGN cell activity, and the leonurine and estradiol co-cultured exosome has a better human ovarian granular cell damage repair effect than the conventional exosome. When the leonurine and estradiol co-cultured exosome is used for treating a rat with premature ovarian failure, inflammatory cell infiltration in ovarian tissue interstitial substances is improved, follicle granular cell layers are increased, follicles in the growing period are increased, and mature follicles develop. Compared with a premature ovarian failure model group, the leonurine and estradiol co-cultured exosome group has the advantages that the serum FSH and LH levels of rats are reduced, the AMH level is increased, and the treatment effect on the premature ovarian failure rats is superior to that of a conventional exosome group.

Description

Technical Field

[0001] This invention belongs to the field of biological technology, and in particular relates to an exosome preparation for treating premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF) is a gynecological disease characterized by premature decline in ovarian function. It commonly affects women around 40 years of age, with menstrual irregularities, infertility, and abnormal sex hormone secretion being its main clinical manifestations, severely impacting women's reproductive function and quality of life. POF can cause perimenopausal symptoms such as amenorrhea or oligomenorrhea, sexual dysfunction, vaginal dryness, hot flashes, night sweats, and mood changes. Furthermore, it leads to a decrease in the number and / or quality of eggs, resulting in ovulation disorders, reduced pregnancy rates, and even infertility. Currently, the etiology of POF is not fully understood, but it may be related to genetics, immunity, and the inflammatory microenvironment. The number of older women becoming pregnant is on the rise, highlighting the urgent need to address the impact of aging on fertility, especially pregnancy difficulties caused by POF in older women, to reduce the incidence of infertility.

[0003] Exosomes possess a nanoscale bilayer membrane structure, containing lipids, microRNAs, and proteins that play crucial roles in intercellular communication. Due to their low immunogenicity and tumorigenicity, as well as their ease of storage and management, exosomes have emerged as an emerging biotherapy. Recent studies have found that exosomes and their contents, such as miRNAs, can act as important regulators of intercellular communication, playing a vital role in follicle development processes such as ovarian granulosa cell proliferation and differentiation, oocyte maturation, and oogenesis, thereby influencing the pathogenesis of ovarian-related diseases. Therefore, the development of highly effective exosome formulations holds promise for unlocking new avenues for the treatment of premature ovarian failure. Summary of the Invention

[0004] The purpose of this invention is to provide an exosome preparation for treating premature ovarian failure, which belongs to the field of biological technology.

[0005] To achieve the above objectives, the present invention provides the following technical solution: First, the present invention provides an exosome preparation for treating premature ovarian failure, wherein the exosomes are exosomes produced by adding leonurine and estradiol to umbilical cord mesenchymal stem cell culture medium.

[0006] Furthermore, the final concentration of leonurine in the culture medium for exosome production is 100 μM / L, and the final concentration of estradiol is 400 pg / mL.

[0007] Furthermore, the method for preparing the exosomes includes the following steps: (1) Isolation and culture of umbilical cord mesenchymal stem cells; (2) The passage density of umbilical cord mesenchymal stem cells is 1×10⁻⁶. 6 / mL, cultured in a carbon dioxide incubator for 24 hours until the cell density reaches more than 80%; (3) Add 100 μM / L leonurine and 400 pg / mL estradiol to the culture medium and continue culturing for 24 h. Centrifuge to separate the cell culture supernatant. (4) Extract exosomes with exosome extraction reagent and dissolve the exosomes with 1 / 10 volume of PBS of the original culture medium.

[0008] Secondly, the present invention provides an exosome for the repair of ovarian granulosa cell damage, wherein the exosome is an exosome produced by adding leonurine and estradiol to the umbilical cord mesenchymal stem cell culture medium.

[0009] Furthermore, the final concentration of leonurine in the culture medium for exosome production is 100 μM / L, and the final concentration of estradiol is 400 pg / mL.

[0010] Furthermore, the method for preparing the exosomes includes the following steps: (1) Isolation and culture of umbilical cord mesenchymal stem cells; (2) The passage density of umbilical cord mesenchymal stem cells is 1×10⁻⁶. 6 / mL, cultured in a carbon dioxide incubator for 24 hours until the cell density reaches more than 80%; (3) Add 100 μM / L leonurine and 400 pg / mL estradiol to the culture medium and continue culturing for 24 h. Centrifuge to separate the cell culture supernatant. (4) Extract exosomes with exosome extraction reagent and dissolve the exosomes with 1 / 10 volume of PBS of the original culture medium.

[0011] The present invention also provides a culture medium for umbilical cord mesenchymal stem cells, which can enhance the repair performance of umbilical cord mesenchymal stem cell exosomes on ovarian granulosa cell damage.

[0012] Furthermore, the culture medium is a serum-free culture medium for mesenchymal stem cells, to which leonurine at a final concentration of 100 μM / L and estradiol at a final concentration of 400 pg / mL are added.

[0013] The beneficial effects of this invention are as follows: This invention relates to the preparation of exosomes for treating premature ovarian failure by co-culturing umbilical cord mesenchymal stem cell (umbilical cord mesenchymal stem cell) culture medium with the addition of leonurine and estradiol. Compared with conventionally cultured umbilical cord mesenchymal stem cell exosomes, co-culturing with leonurine and estradiol increases the yield of umbilical cord mesenchymal stem cell exosomes. The gray value of the exosome marker protein CD63 was 1.84 times that of the group without leonurine and estradiol. Both conventional exosomes and exosomes co-cultured with leonurine and estradiol at the same dose attenuated the inhibitory effect of 4-OHCP on KGN cell viability. Exosomes co-cultured with leonurine and estradiol showed a stronger repair effect on human ovarian granulosa cell damage than conventional exosomes. Treatment trials of exosomes in rats with premature ovarian failure showed that the exosome group co-cultured with leonurine and estradiol exhibited improved inflammatory cell infiltration in the ovarian stroma, increased granulosa cell layer in follicles, increased number of follicles in the growth phase, and visible development of mature follicles, significantly superior to the conventional exosome group. Compared with the model group, the exosome group co-cultured with leonurine and estradiol showed decreased FSH and LH levels and increased AMH levels; the exosome group co-cultured with leonurine and estradiol had a better therapeutic effect on premature ovarian failure rats than the conventional exosome group. Attached Figure Description

[0014] Figure 1 The grayscale value of CD63, a marker protein of exosomes, was determined. Figure 2 The effect of exosomes on the repair of damaged human ovarian granulosa cells; Figure 3 Serum AMH levels in rats; Figure 4 Serum FSH levels in rats; Figure 5 The value represents the serum LH hormone level in rats. Detailed Implementation

[0015] The following is a more detailed description of the present invention, illustrated by examples. It should be understood that these examples are merely illustrative of the invention and are intended to explain the principles and functions of the invention, and are not intended to limit the scope of protection of the invention.

[0016] Example 1: Isolation and Culture of Umbilical Cord Mesenchymal Stem Cells (1) Obtain an appropriate amount of umbilical cord tissue from newborns delivered at full term using sterile instruments.

[0017] (2) The fresh umbilical cord tissue was transferred to a culture medium containing double antibodies, processed with sterile instruments, and then rinsed three times with PBS to remove blood and other foreign matter from the surface.

[0018] (3) After digesting with an appropriate amount of trypsin for about 1 hour, stop the digestion, transfer the digested cells to a centrifuge tube, add 15 times the volume of PBS and centrifuge, then discard the liquid after centrifugation.

[0019] (4) Add an appropriate amount of mesenchymal stem cell culture medium to resuspend the centrifuged cells and seed them in a cell culture flask. Transfer the flask to a carbon dioxide incubator with a carbon dioxide concentration of 5% and culture for about 2 hours. Check the cell growth status after 2 hours. Check the cell growth status and change the culture medium every 48 hours thereafter.

[0020] (5) When the cells have grown to 80%-90% of the bottom of the bottle, passage is started. When the cells are passaged to the second generation, they are cryopreserved. The stem cells used in this study are the third to fifth generation.

[0021] Example 2: Preparation of exosomes from conventional umbilical cord mesenchymal stem cells (1) Umbilical cord mesenchymal stem cells were passaged into 75T cell culture flasks, each containing 30 mL of serum-free mesenchymal stem cell culture medium, with a cell density of 1 × 10⁻⁶ cells / mL. 6 The cells were cultured at a concentration of 5% carbon dioxide for 24 hours until the cell density reached over 80%. Three replicates were performed.

[0022] (2) Continue culturing for 24 hours, collect cell culture supernatant, and mix exosome extraction reagent and ultrafiltered cell culture supernatant in a 1:1 ratio according to the instructions of the exosome extraction kit. After standing for 16 hours, centrifuge at 12000 rpm for 1 hour to collect the precipitate, and dissolve the exosomes with 1 / 10 volume of PBS of the original culture medium.

[0023] Example 3: Preparation of exosomes from umbilical cord mesenchymal stem cells for the treatment of premature ovarian failure (1) Umbilical cord mesenchymal stem cells were passaged into 75T cell culture flasks, each containing 30 mL of serum-free mesenchymal stem cell culture medium, with a cell density of 1 × 10⁻⁶ cells / mL. 6 Cells were cultured for 24 hours in a CO2 incubator with a carbon dioxide concentration of 5% at a concentration of 100 μM / L for 24 hours until the cell density reached over 80%. Leonurine and estradiol were added to the culture medium in the cell flasks to a final concentration of 100 μM / L. A control group without leonurine and estradiol was also included, with three replicates per group. Cells were cultured for another 24 hours, and the cell culture supernatant was collected. Following the exosome extraction kit instructions, the exosome extraction reagent and ultrafiltered cell culture supernatant were mixed at a 1:1 ratio. After standing for 16 hours, the mixture was centrifuged at 12000 rpm for 1 hour to collect the precipitate. The exosomes were dissolved in 1 / 10 volume of PBS (the original culture medium).

[0024] (2) Mix the exosomes with an equal volume of RIPA lysis buffer and incubate on ice for 1 hour, shaking for 1-2 minutes every 10 minutes. Centrifuge at 12000 rpm for 15 minutes at 4°C and collect the supernatant. Mix the sample with 2× Loading buffer at a 1:1 volume ratio and incubate in a boiling water bath for 10 minutes. Perform SDS-PAGE electrophoresis on the prepared sample, transfer it to a PVDF membrane, block with 5% skim milk powder solution for 1 hour, incubate with rabbit anti-human CD63 and GAPDH antibodies at 37°C for 1 hour, and wash three times with PBS; dilute with horseradish peroxidase-labeled goat anti-rabbit secondary antibody at a 1:2000 ratio, incubate at 37°C for 1 hour, and wash three times with PBS; finally, develop and expose with ECL luminescent reagent. Analyze the protein grayscale value using Quantity One v462 software and compare it with the internal control protein band to obtain the relative grayscale ratio. The grayscale value of the target protein = the OD value of the target protein / the corresponding OD value of the internal control.

[0025] from Figure 1 As can be seen, the gray value of exosome marker protein CD63 produced by control cells without the addition of leonurine and estradiol was 0.758, while the gray value of exosome marker protein CD63 produced by umbilical cord mesenchymal stem cells cultured with a final concentration of 100 μM / L leonurine and 400 pg / mL estradiol was 1.395, which was 1.84 times that of the group without the addition.

[0026] Example 4: Effect of exosomes on the repair of damaged human ovarian granulosa cells (1) Adjust the cell density of the human ovarian granulosa cell KGN cell line to 1×10⁻⁶. 5 / wells were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. After that, normal cells were added to each of the remaining wells and 100 µM 4-OHCP was added to each well for 24 hours.

[0027] (2) The first group was the 4-OHCP-induced human ovarian granulosa cell KGN injury model group. 100µM of 4-OHCP was added, followed by 50µL of cell culture medium. The second group was the conventional exosome group. 50µL of conventional umbilical cord mesenchymal stem cell exosomes from Example 2 were added to each well. The third group was the exosome group co-cultured with leonurine and estradiol. The exosomes produced by the umbilical cord mesenchymal stem cells co-cultured with leonurine and estradiol in Example 3 were diluted with culture medium (diluted to the same gray value as the exosome marker protein CD63 as the exosomes in the second group) and 50µL of diluted exosomes were added to each well.

[0028] (3) After adding exosomes to KGN cells, all four groups of cells were cultured in a 37℃, 5% CO2 incubator for 6 hours. OD450 values ​​were measured using CCK-8 assay, and cell viability was calculated as follows: Cell viability = (OD450 of experimental group - OD450 of blank control group) / (OD450 of control group - OD450 of blank control group). Data are expressed as the average of three independent experiments.

[0029] from Figure 2 The results showed that the cell viability of KGN cells decreased to 61.70% after the addition of 100 µM 4-OHCP; the cell viability of the conventional exosome group was 83.22%, while the cell viability of the exosome group co-cultured with leonurine and estradiol was 99.47%. This indicates that both conventional exosomes and exosomes co-cultured with leonurine and estradiol at the same dose can attenuate the inhibitory effect of 4-OHCP on KGN cell viability. Furthermore, exosomes co-cultured with leonurine and estradiol showed a stronger repair effect on human ovarian granulosa cell damage than conventional exosomes.

[0030] Example 5: Cyclophosphamide-induced establishment of a rat model of premature ovarian failure. (1) Thirty 8-week-old SPF-grade female SD rats, weighing 180±20g, were selected. The room temperature was controlled at 19-22℃ and the relative humidity was controlled at 40-60%. They had free access to water and food. After one week of acclimatization, the rats were randomly divided into a control group (6 rats) and a model group (24 rats). The model group was induced by cyclophosphamide to establish a premature ovarian failure model.

[0031] (2) Modeling method: Rats were given an initial loading dose of cyclophosphamide of 200 mg / kg via intraperitoneal injection, followed by intraperitoneal injection of 8 mg / kg once a day for 14 days, for a total of 15 days. The control group rats were injected with an equal volume of physiological saline via intraperitoneal injection.

[0032] (3) Observation of the estrous cycle in rats by vaginal smear: A small amount of physiological saline was dripped into the vaginal opening of the rat, a small amount of vaginal secretion was collected and smeared on a glass slide, fixed in ethanol for 30 min, stained with rapid Wright's stain, and observed under an optical microscope. If the rats showed signs of estrous cycle and hormone level disorder, and increased apoptosis biomarkers after modeling, the model was considered successful.

[0033] The results showed that the control group rats had a regular estrous cycle, with distinct proestrus, estrus, metestrus, and diaestrus phases. During proestrus, numerous nucleated epithelial cells were observed; during estrus, large areas of keratinized cells were seen; during metestrus, keratinized cells and leukocytes coexisted; and during diaestrus, leukocytes predominated. The model group rats lacked an estrus phase, had a prolonged diaestrus, and exhibited disordered estrous cycles.

[0034] Example 6: Treatment of premature ovarian failure rats with exosomes (1) Grouping and Treatment Selection: Eighteen rats with successfully established models were randomly divided into three groups: a premature ovarian failure (POF) model group, a conventional exosome treatment group, and a leonurine and estradiol co-cultured exosome treatment group, with six rats in each group. Intervention Method: On the third day after modeling, exosomes in the conventional exosome treatment group and the leonurine and estradiol co-cultured exosome treatment group were injected into the ovarian sac of rats under anesthesia using an in situ injection method to evaluate the protective effect of the two types of exosomes on ovarian function in rats with POF. The control group and the POF model group were injected with an equal volume of physiological saline. Ten days after the injection of exosomes, their effect on ovarian function was evaluated.

[0035] (2) Tissue collection and processing: Intraperitoneal anesthesia was administered, blood was collected from the abdominal aorta, and the bilateral ovaries, uterus, and other tissues were quickly removed. Excess fat was removed, and one ovary was fixed in 4% paraformaldehyde solution. The other ovary was placed in a cryopreservation tube and stored in a -80°C freezer.

[0036] (3) Morphological observation of ovarian tissue: The fixed ovaries were routinely embedded in paraffin, sectioned at a thickness of 4µm, dewaxed, hydrated, stained with HE, dehydrated, cleared, and mounted with neutral resin. Pathological changes in ovarian tissue and the number of follicles at each stage were observed.

[0037] The results showed that in the control group, the ovarian cortex and medulla of rats were clearly distinguishable, with neatly arranged granulosa cells, and normally developing follicles in the growth phase were visible, along with mature follicles and fewer atretic follicles. Compared with the control group, the ovarian structure of the model group rats was unclear, with varying degrees of inflammatory cell infiltration in the stroma, a reduced granulosa cell layer in follicles, fewer follicles in the growth phase, and an increased number of atretic follicles. In the conventional exosome group, the inflammatory cell infiltration in the stroma of the ovarian tissue of rats was improved, the granulosa cell layer of follicles increased, and the number of follicles in the growth phase increased. In the exosome group co-cultured with leonurine and estradiol, the inflammatory cell infiltration in the stroma of the ovarian tissue of rats was improved, the granulosa cell layer of follicles increased, the number of follicles in the growth phase increased, and mature follicle development was visible, which was significantly better than that of the conventional exosome group.

[0038] (4) ELISA detection of rat serum AMH, FSH and LH hormone levels: After arterial blood was left to stand at room temperature for 1 hour, it was centrifuged at 3500 r / min for 15 min. The upper serum layer was collected and the rat serum AMH, FSH and LH hormone levels were detected according to the ELISA kit instructions.

[0039] Depend on Figure 3 , Figure 4 , Figure 5The results showed that, compared with the control group, the serum FSH and LH levels in the model group were significantly increased, while the AMH level was significantly decreased; compared with the model group, the FSH and LH levels in the conventional exosome group were decreased, while the AMH level was increased; compared with the model group, the FSH and LH levels in the exosome group co-cultured with leonurine and estradiol were decreased, while the AMH level was increased; the exosome group co-cultured with leonurine and estradiol showed better results than the conventional exosome group.

Claims

1. An exosome preparation for treating premature ovarian failure, characterized in that, The exosomes are exosomes produced by adding leonurine and estradiol to the culture medium of umbilical cord mesenchymal stem cells.

2. The exosome preparation according to claim 1, characterized in that, The final concentration of leonurine in the culture medium produced by the exosomes was 100 μM / L, and the final concentration of estradiol was 400 pg / mL.

3. The exosome preparation according to claim 1, characterized in that, The method for preparing the exosomes includes the following steps: (1) Isolation and culture of umbilical cord mesenchymal stem cells; (2) The passage density of umbilical cord mesenchymal stem cells is 1×10⁻⁶. 6 / mL, cultured in a carbon dioxide incubator for 24 hours until the cell density reaches more than 80%; (3) Add 100 μM / L leonurine and 400 pg / mL estradiol to the culture medium and continue culturing for 24 h. Centrifuge to separate the cell culture supernatant. (4) Extract exosomes with exosome extraction reagent and dissolve the exosomes with 1 / 10 volume of PBS of the original culture medium.

4. An exosome for repairing ovarian granulosa cell damage, characterized in that, The exosomes are exosomes produced by adding leonurine and estradiol to the culture medium of umbilical cord mesenchymal stem cells.

5. The exosome according to claim 4, characterized in that, The final concentration of leonurine in the culture medium produced by the exosomes was 100 μM / L, and the final concentration of estradiol was 400 pg / mL.

6. The exosome according to claim 4, characterized in that, The method for preparing the exosomes includes the following steps: (1) Isolation and culture of umbilical cord mesenchymal stem cells; (2) The passage density of umbilical cord mesenchymal stem cells is 1×10⁻⁶. 6 / mL, cultured in a carbon dioxide incubator for 24 hours until the cell density reaches more than 80%; (3) Add 100 μM / L leonurine and 400 pg / mL estradiol to the culture medium and continue culturing for 24 h. Centrifuge to separate the cell culture supernatant. (4) Extract exosomes with exosome extraction reagent and dissolve the exosomes with 1 / 10 volume of PBS of the original culture medium.

7. A culture medium for umbilical cord mesenchymal stem cells, characterized in that, The culture medium can enhance the repair performance of umbilical cord mesenchymal stem cell exosomes on ovarian granulosa cell damage. The culture medium is a serum-free culture medium for mesenchymal stem cells with added leonurine at a final concentration of 100 μM / L and estradiol at a final concentration of 400 pg / mL.

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