CN109929799B - Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof - Google Patents
Human umbilical cord mesenchymal stem cell exosome and preparation method and application thereof Download PDFInfo
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Abstract
The preparation method of the human umbilical cord mesenchymal stem cell exosome comprises the following steps: collecting human umbilical cord tissues and extracting umbilical cord mesenchymal stem cells; and centrifuging the obtained umbilical cord mesenchymal stem cells to extract exosomes. The HucMSC exosome can activate primordial follicles and promote the development of follicles of newborn mice, and the quality of mature oocytes obtained after the follicles are developed and matured is not influenced. The HucMSC exosome can improve the reproductive function of the aged mice, and the litter size of the mice in the experimental group is obviously higher than that of the control group after the aged mice are mated with the mice after the aged mice are injected with the exosome in the ovary cyst.
Description
Technical Field
The invention belongs to the field of exosomes, and particularly relates to an umbilical cord mesenchymal stem cell-derived exosome, a preparation method thereof and application thereof in-vitro activation of primordial follicles.
Background
Exosomes are the most extensively studied subset of extracellular vesicles, first discovered by Pan and Johnstone in 1983. Exosomes are a class of extracellular vesicles derived from early endosomes, with membrane structures, whose diameters range from 50 to 150 nm. Several studies have shown that exosomes are secreted by most cells and can be detected in most body fluids, such as blood, urine, cerebrospinal fluid, ascites, follicular fluid, amniotic fluid, synovial fluid, etc. The exosome contains a large amount of protein, lipid, nucleic acid and other substances on the surface and inside, and the components inside the exosome from different cells are different. The exosome derived from the Mesenchymal Stem Cells (MSC) has similar characteristics to the derived cells, so the exosome has rich potential treatment capability in the aspects of wound repair, tissue reconstruction, cardiovascular diseases, brain injury, inflammation and the like, and has wide application prospect. As an emerging cell-free (cell-free) treatment mode, the exosome derived from the mesenchymal stem cells has low immunogenicity, does not have potential tumorigenicity, has a short half-life in vivo compared with the source cells, and can be better taken up by receptor cells. In addition, the exosome can be frozen for standby after being separated, the original biological activity of the exosome is not reduced within a long time, and the exosome can be used as a carrier for coating a medicament and delivered to a specific position in a targeted manner after being processed by means of modification and the like. Therefore, the exosome has higher safety in clinical application, low ethical risk and higher application potential and value.
In female mammals, the follicle is the basic functional unit that constitutes the ovary, consisting of a centrally located ovum and one or more layers of granulosa cells on the periphery. According to the different stages of the growth and development of the oocyte in the follicle, the follicle can be divided into primordial follicle, primary follicle, secondary follicle, luminal follicle and preovulatory follicle. Primordial follicles are a pool of follicles in the ovary in a quiescent state, formed either prenatally or postnatally in mammals, and are fixed in number, approximately 400,000 primordial follicles in each ovary in humans. During follicular growth and development, primordial follicles in the primordial follicle bank are activated into the growth phase, a process called initial recruitment. As the initial recruitment continues, the number of primordial follicles decreases, and when the number of primordial follicles in the human ovary is below 1000, the follicles will not develop and the woman will go through menopause. Menopause in women is around 50 years of age, however, under some pathological conditions premature ovarian failure is initiated by premature menopause (before age 40) in women due to insufficient primordial follicular reserve in the ovaries. Premature ovarian failure is a very common infertility disorder, with an incidence of approximately 1-2% in women of reproductive age. Currently, there is no very effective treatment for premature ovarian failure, and most patients achieve artificial menstrual cycles by hormone replacement therapy, requiring egg supply to meet fertility requirements. Primordial follicle in vitro activation technology (IVA), an emerging technology for treating ovarian tissues in vitro to activate primordial follicles in the ovarian tissues, is of great significance for treating premature ovarian failure patients, and is the first therapeutic technology proposed for primordial follicles in the world. In 2011, the first worldwide example of the IVA baby is born in Japan, and in 2015, the first national example of the IVA baby is born, which marks the clinical application success of the technology. The prior IVA technology mainly activates a PI3K/mTOR signal pathway in an oocyte through a signal pathway activator to realize the purpose of activating the primordial follicle. Whether exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC) have the effect of activating primordial follicles is not reported, however, the research is of great significance for the treatment of premature ovarian failure.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides a human umbilical cord mesenchymal stem cell exosome, and a preparation method and application thereof.
The technical scheme is as follows: a preparation method of exosome comprises the following steps: collecting human umbilical cord tissues, and extracting to obtain umbilical cord mesenchymal stem cells; and centrifuging the obtained umbilical cord mesenchymal stem cells to extract exosomes.
The preparation method of the exosome comprises the following steps: cleaning umbilical cord with artery and vein removed, and cutting into 1mm 3 The tissue blocks were evenly spread in petri dishes, 2mL of medium was added to each dish, incubated at 37 ℃ and 5% CO 2 After 6 hours in the incubator, 5mL of the medium was added to the dish, and the medium was changed every 2 to 3 days, 0.25% was used when the cells grew to 70% to 80% confluenceEDTA-pancreatin digestion, 1:3 passage to P3 to P7 cell, getting exosome.
When the umbilical cord mesenchymal stem cells grow until the confluence degree reaches 60% -70%, sucking out original culture solution, washing with PBS buffer solution, adding 10mL conditioned medium, collecting conditioned culture supernatant after culturing for 48 hours, centrifuging the collected conditioned medium for 10 minutes at 4 ℃, centrifuging the collected conditioned medium for 20 minutes by using 500g to remove dead cells and cell fragments by using 2000g to remove large vesicles and apoptotic bodies, centrifuging for 30 minutes by using 10000g to remove large vesicles and apoptotic bodies, centrifuging for 90 minutes by using 120000g of relative centrifugal force, sucking out supernatant, re-suspending exosomes at the bottom of a centrifuge tube by using 1mL of PBS, mixing all liquids in the centrifuge tube into the same centrifuge tube, adding PBS to fill the centrifuge tube with the liquid, centrifuging for 90 minutes by using 120000g again to clean exosomes, sucking out supernatant PBS liquid, and re-suspending exosomes by using 150-200 mu L for subsequent experiments.
The human umbilical cord mesenchymal stem cell exosome prepared by the preparation method.
The use of said exosomes for in vitro activation of primordial follicles for non-diagnostic therapeutic purposes.
The application of the exosome in preparing a primordial follicle in-vitro activating reagent.
The primordial follicle in-vitro activating reagent comprises the effective component of the human umbilical cord mesenchymal stem cell exosome.
Has the advantages that: the HucMSC exosome can activate primordial follicles and promote the development of follicles of newborn mice, and the quality of mature oocytes obtained after the follicles are developed and matured is not influenced. The HucMSC exosome can improve the reproductive function of the aged mice, and the litter size of the mice in the experimental group is obviously higher than that of the control group after the aged mice are mated with the mice after the aged mice are injected with the exosome in the ovary cyst.
Drawings
Figure 1 is a figure for identifying morphology and induced differentiation of human umbilical cord mesenchymal stem cells. The morphological characteristics of P5HucMSC cells; b: calcium nodule alizarin red staining; c: dyeing with lipid drop oil red O; d: staining with cartilage acid mucopolysaccharide a Li Xinlan. Scale =50 μm.
Figure 2 flow cytometric identification chart of surface marker of human umbilical cord mesenchymal stem cell. A: hucMSC positive marker CD44, CD73, CD90, CD105 expression; b: expression of the HucMSC negative markers CD11b, CD19, CD34, CD45, HLA-DQ/DR.
FIG. 3 characterization of exosomes. A: wester blotting detects positive protein Alix, tsg101, CD9, negative protein Gm130 and internal reference protein Gapdh of an exosome; b: observing the form of the exosome by a transmission electron microscope; c: NTA measures exosome particle diameter distribution.
FIG. 4 is a schematic representation of the results of treatment of neonatal mouse ovarian-activated primordial follicles by HucMSC exosomes. A: foxo3a immunohistochemical staining after 24h ovarian culture; b: the nucleus-yielding proportion of Foxo3a is increased after exosome treatment; c: p-rps6 immunohistochemical staining was performed 24h after ovarian culture. PBS, PBS treated control group, exo, exosome treated group. Scale =50 μm.
Fig. 5 is a graph of the development of neonatal mouse ovaries after 14 days post-subintimal transplantation of the kidneys after treatment with HucMSC exosomes. A: after the ovaries are cultured in pairs, the ovaries are transplanted, and after 14 days, the volume of a treatment group is larger than that of a control group; b: the development state of the treated group is better by the HE staining of the ovary morphology; c: counting and statistically analyzing follicles at all levels; d: ovarian PCNA staining shows that the ovarian and granular cell proliferation of the treated group is better. Scale of a =1mm, panel b, scale of D =50 μm.
FIG. 6 is a graph showing that HUCMSC exosomes improve geriatric mouse fertility. A: counting the number of the young mice and the non-young mice of the aged mice of the control group and the experimental group; b: mating experimental curves of aged mice; c: and (5) counting the number of eggs taken by the aged mice.
Detailed Description
The present invention will be described in detail below with reference to examples, but the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.
Example 1: isolation and culture of HucMSC
After removing about 15cm of umbilical cord, fresh umbilical cord tissue is placed in PBS buffer containing 1% penicillin/streptomycin double antibody, transported to a biosafety cabinet on ice, repeatedly washed with the above solution until there is no significant residual blood, mechanically stripped of umbilical cord arteries and veins with conventional surgical instruments,the umbilical cord tissue was again repeatedly rinsed. Carefully cutting the cleaned umbilical cord into 1mm with ophthalmic scissors 3 Small tissue blocks of about size, uniformly spread in 10cm diameter petri dishes, 2mL of medium per dish, left for culturing at 37 deg.C, 5% CO 2 After 6 hours in the incubator of (1), 5mL of the medium was added to the dish, and the solution was changed every 2-3 days, and when the cells were grown to a confluency of 70% -80%, they were passaged with 0.25% of EDTA-trypsin digestion, 1:3. P3 to P7 passage cells were used for the experiment (fig. 1:A).
Cell culture medium formula (50 mL)
| Composition (I) | Amount of the composition |
| DME/F12 | 44.25mL |
| FBS | 5mL |
| Double antibody | 750μL |
Example 2: multidirectional induced differentiation of HucMSC
When the cells of the P5 generation were cultured until the confluency reached 70% to 80%, they were digested with 0.25% EDTA-pancreatin, and then subjected to osteogenic and adipogenic induced differentiation experiments, the cells were seeded in a 6-well plate, and when chondrogenic induced differentiation was performed, the cells were cultured in the bottom of a 15mL centrifuge tube in the form of suspension spheres.
Osteogenic induced differentiation experiment: when the cells grew to reach 70% -80% confluence, the original medium was aspirated, the osteogenic differentiation medium was changed, induction was performed according to the instructions, the medium was changed every 2-3 days, after 21 days the cells were fixed with 4% pfa, and observed by staining with alizarin red stain (fig. 1:B);
adipogenic induced differentiation experiment: when the cell growth confluence reached 100%, the original medium was aspirated, the medium was changed to a adipogenic differentiation medium, induction was performed according to the instructions, the medium was changed every 2 to 3 days, and after 21 days, the cells were fixed with 4% pfa, and then stained with oil red O stain for observation (fig. 1:C);
chondrogenic induced differentiation experiment: when the confluency of cell growth reached 90% to 100%, after digestion with 0.25% EDTA-trypsin, the cells were placed in the bottom of a 15mL centrifuge tube in a suspension ball format, the original medium was aspirated, the medium was changed to a cartilage-inducing differentiation medium, the medium was changed every 2 days according to the instructions, the cell balls were fixed after 28 days, and after dehydration and embedding, the sections were stained with Alisine blue stain for observation (FIG. 1:D).
It can be seen that the isolated HucMSC has good osteogenic, adipogenic, chondrogenic differentiation capacity.
Example 3: flow cytometric detection of HucMSC
P5 generation cells, when the cells growth to reach 70% -80% confluency, using 0.25% EDTA-pancreatin digestion, then using PBS to wash 2 times of centrifugation and resuspend cells, according to the antibody specification of the dosage, adding FITC labeled CD19, CD34, CD44, CD45, HLA-DQ/DR antibody and PE labeled CD11b, CD73, CD90 antibody, APC labeled CD105 antibody light-shielding incubation, 15 minutes later adding PI co-staining to mark dead cells, after co-staining for 15 minutes using 400g,4 ℃ centrifugation for 5 minutes to remove unbound antibody, using 200L PBS cells heavy suspension in the flow detection tube, machine detection. As shown in fig. 2: the P5 generation cells shown in A expressed the HucMSC positive markers CD44, CD73, CD90, CD105, and did not express the HucMSC negative markers CD11b, CD19, CD34, CD45, HLA-DQ/DR (FIG. 2:B).
Example 4: extraction and identification of HucMSC exosome
When the cells grew to reach 60% -70% confluence, the original culture solution was aspirated, the cells were carefully washed 3 times with PBS buffer, 10mL of conditioned medium was added, and conditioned culture supernatant was collected after 48 hours of culture. The collected conditioned medium was then centrifuged at 500g for 10 minutes to remove cells, 2000g for 20 minutes to remove dead cells and cell debris, 10000g for 30 minutes to remove large vesicles, apoptotic bodies and the like, and the resulting culture supernatant was filtered through a 0.22 μm sterile filter and stored temporarily at 4 ℃. During ultracentrifugation, the treated conditioned medium is carefully transferred into an ultracentrifuge tube, after balancing, the ultracentrifuge tube is centrifuged for 90 minutes by using 120000g of relative centrifugal force, supernatant is sucked, 1mL of PBS is used for resuspending the bottom sediment of the ultracentrifuge tube, liquid in all centrifuge tubes is mixed into the same centrifuge tube, PBS is added until the centrifuge tube is filled with the liquid, the centrifuge tube is centrifuged for 90 minutes again by using 120000g to clean exosomes, supernatant liquid is sucked, 150-200 muL of PBS is used for resuspending the exosomes for subsequent experiments, and the centrifugation in the previous steps is carried out under the environment of 4 ℃.
Conditioned cell culture medium formulation (50 mL)
| Composition (A) | Dosage of |
| DME/F12 | 44.25mL |
| FBS | 5mL |
| Double antibody | 750μL |
Example 5: detection of exosome nano-particle size (Nanosight)
The method adopts a nanoparticle tracking analysis technology, uses a nanoparticle tracking analyzer, operates according to a machine specification, dilutes the exosome sample to be detected by 50-100 times, and then operates on a machine to detect the particle size distribution range and the particle number of the sample. As shown in fig. 3: c, the detected particle diameter is 62.7-146.1nm, the diameter range of the exosome is met, the average particle concentration is 2.7X 1010particles/mL, and the protein is 0.79mg/mL.
Example 6: exosome observed by transmission electron microscope
The exosome pellet obtained by ultracentrifugation was resuspended in 100. Mu.L of 2.5% glutaraldehyde solution, fixed overnight at 4 ℃, 20. Mu.L of the sample was pipetted onto a clean copper mesh, the liquid was carefully blotted with a piece of dust-free paper, dried at room temperature for 1-2 minutes, then stained with 2% uranyl acetate at room temperature for 2 minutes, subsequently dried at room temperature, the copper mesh was carefully transferred to a drop of ultrapure water, after 2 minutes, the liquid was carefully blotted with a piece of dust-free paper, and the sample was mounted on a machine for observation after waiting for room temperature drying. As shown in fig. 3: b shows that the vesicles separated under the transmission electron microscope have a typical exosome round cup-shaped structure and the diameter of the vesicles is about 100 nm.
Example 7: exosome fluorescent labels
After ultracentrifugation, the supernatant medium was carefully aspirated and the exosome pellet was resuspended in 0.5mL _ of Diluent C using the PKH67 fluorescent labeling kit. Meanwhile, 4 μ L of the ph-67 dye solution was added to 0.5mL of solvent C, the two were mixed, incubated at room temperature for 4min in the dark, then 1mL of 1-mL bsa/PBS solution was rapidly added to stop the staining reaction, the sample was transferred to an ultracentrifuge tube, centrifuged at 120000g for 90 min at 4 ℃, the supernatant was carefully aspirated, the bottom exosome pellet was resuspended in 200 μ L of PBS, and stored at-20 ℃ in the dark for future use.
Example 8: immunohistochemical staining after in vitro exosome culture of newborn mouse ovary
15 new-born 3-day ICR female mice were sacrificed by freezing, sterilized by alcohol spray and placed in a 10cm diameter petri dish. In the cell culture room, a small opening was made in the dorsal intercostal angle of the suckling mice, the bilateral ovaries were removed, placed in a microdrop of L15 equilibration fluid, and the ovaries were carefully dissected under a stereoscope to expose ovarian tissue. After washing the free ovarian tissues in clean L15 balance solution for 2 times, randomly dividing the free ovarian tissues into 2 groups, randomly dividing each group into 6 groups, culturing the groups in pairs in built-in culture chambers in a 24-well plate, adding 400 mu L of ovarian tissue culture solution into each hole, and respectively adding basal culture solution, PBS and exosome into each group of holes. And collecting ovarian tissues after 24h of culture, and fixing the embedded sections.
As shown in fig. 4: a shows that marked by using a primordial follicle-activated marker molecule Foxo3a, obvious nuclei of Foxo3a can be observed in an ovary of an exosome-treated group. The follicle count results also indicated a significant increase in the number of activated primordial follicles (FIG. 4:B). In addition, increased proliferation in exosome-treated groups was also found using PCNA staining (fig. 4:C). It can be seen that exosome treatment was able to activate primordial follicles and promote proliferation of cells within the ovaries.
Example 9: transplantation of kidney capsule of newborn mouse ovary after exosome culture
Selecting 5-6 weeks old ICR female mice as recipient mice, injecting 240mg/kg of avermectin into abdominal cavity for anesthesia until the mice do not respond, shaving off hairs near the back waist side of the mice, disinfecting skin with alcohol, transversely placing on an operating table, confirming the position of the kidney with fingers, cutting a small opening with the length of 1cm, carefully exposing subcutaneous tissues, entering the abdominal cavity layer by layer, after finding the kidney, carefully pulling out the kidney, clamping the kidney at the position of the incision, tearing a small opening on a kidney capsule with a sharp forceps, carefully plugging the cultured ovary into the small opening and fixing the position to prevent the ovary from leaking. After the ovary transplantation is completed, finding out the self ovary tissue of a receptor mouse along the adipose tissue near the kidney, shearing off the ovary by using the ophthalmological scissors after high-temperature heating disinfection, using high-temperature coagulation to confirm that no bleeding exists, then carefully storing tissues exposed outside the body into the body according to the sequence, suturing wounds layer by layer, placing the mouse on a warm table for resuscitation after being disinfected by iodine. The animals were returned to the animal center for feeding, and 10IU FSH was intraperitoneally injected every 1 day, and wound recovery was observed. After 14 days of transplantation, a part of mice were taken back, sacrificed by carbon dioxide method, the abdominal cavity was exposed, the kidney was carefully cut off, the kidney capsule was peeled off, the transplanted ovary was exposed, after taking a picture under a stereoscope, the embedded section was fixed, the number of follicles at each stage was recorded, and the PCNA protein expression was detected by immunohistochemistry.
As shown in fig. 5: A. b, C, the ovaries of the exosome-treated group were significantly enlarged, and the results of morphological analysis and follicle counting showed that follicular development increased and the number of antral follicles significantly increased. In addition, the PCNA markers were also seen to be favorable for granulosa cell proliferation in the exosome ovary-treated group. It can be seen that exosome treatment can promote follicular development.
Example 10: improvement of reproductive function of old mice by using HucMSC exosome
30 old mice aged 50 weeks are selected and randomly divided into 2 groups for intra-ovarian cyst injection. One group was injected with PBS, one group was injected with exosomes, and 3 weeks later were caged for mating experiments. We continuously monitored the birth status of 14 weeks of each group of mice and plotted the mating curve. The number of mice born in the experimental group was significantly increased compared to the control group (4 mice) (9 mice). By plotting the mating curves, it can be seen that the experimental group gave an average of 5.40 litters per mouse and the control group gave an average of 2.13 litters per mouse. Therefore, exosomes also play a role in improving reproductive function in aged mice (fig. 6).
Claims (2)
1. The application of the human umbilical cord mesenchymal stem cell exosome in the in-vitro activation of primordial follicles for non-diagnosis and treatment purposes comprises the following preparation steps: cleaning umbilical cord with artery and vein removed, and cutting into 1mm 3 Tissue mass, evenly spread in culture dishes, add 2mL of medium per dish, incubate at 37 ℃ 5% 2 After 6 hours in the incubator of (1), 5mL of the medium was added to the dish, and the medium was changed every 2 to 3 days, and when the cells grew to a confluence of 70 to 80%, they were passaged to P3 to P7 generation cells using 0.25% EDTA-pancreatin digestion, 1:3, to obtain exosomes.
2. The application of the exosome of the human umbilical cord mesenchymal stem cells in preparing the in-vitro primordial follicle activation reagent comprises the following steps: cleaning umbilical cord with artery and vein removed, and cutting into 1mm pieces 3 Tissue mass, evenly spread in culture dishes, add 2mL of medium per dish, incubate at 37 ℃ 5% 2 After 6 hours in the incubator of (1), 5mL of the medium was added to the dish, and the solution was changed every 2 to 3 days, and when the cells grew to reach 70% to 80% confluence, they were passaged to P3 to P7 generation cells using 0.25% of EDTA-trypsin digestion, 1:3, to obtain exosomes.
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| CN110478368A (en) * | 2019-08-21 | 2019-11-22 | 中国科学院动物研究所 | The purposes of umbilical cord mesenchymal stem cells conditioned medium |
| CN111394307A (en) * | 2020-02-17 | 2020-07-10 | 天津医科大学眼科医院 | Method for separating and purifying exosome from plasma and application |
| CN113244272B (en) * | 2021-06-18 | 2024-05-17 | 陕西中鸿科瑞再生医学研究院有限公司 | Composition for improving premature ovarian failure and preparation method and application thereof |
| WO2023040938A1 (en) * | 2021-09-15 | 2023-03-23 | 青岛卓云海智医疗科技有限公司 | Method for treating ilk signaling pathway related diseases using exosome derived from mesenchymal stem cells, and pharmaceutical composition |
| CN114134107B (en) * | 2021-11-29 | 2024-01-16 | 南京医科大学 | Artificial ovary with mesenchymal stem cells participating, and preparation method and application thereof |
| CN115463215A (en) * | 2022-07-26 | 2022-12-13 | 苏州大学 | New use of HDAC9 and inhibitors thereof |
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