WO2021103817A1 - 蜕膜nk细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途 - Google Patents

蜕膜nk细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途 Download PDF

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WO2021103817A1
WO2021103817A1 PCT/CN2020/119327 CN2020119327W WO2021103817A1 WO 2021103817 A1 WO2021103817 A1 WO 2021103817A1 CN 2020119327 W CN2020119327 W CN 2020119327W WO 2021103817 A1 WO2021103817 A1 WO 2021103817A1
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exosomes
decidual
cells
cell
infertility
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French (fr)
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胡适
丁敏
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沣潮医药科技（上海）有限公司
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Publication of WO2021103817A1 publication Critical patent/WO2021103817A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention belongs to the field of biomedicine, and relates to the use of decidual NK cells and exosomes derived from cell subgroups in the preparation of infertility-related disease drugs and adjuvant therapeutic agents, a preparation method of the exosomes, and a method for containing the exosomes Body pharmaceutical composition.
  • Endometrial injury is mainly the injury of the basal layer of the endometrium.
  • the main reason is related to curettage during pregnancy. Compared with the normal endometrium, the basal layer of the endometrium during pregnancy is loose and more susceptible to injury. Based on the current situation of my country's national conditions and the increasing rate of induced abortion, the occurrence of endometrial damage cannot be ignored. Damage to the basal layer of the endometrium may result in damage or loss of endometrial stem cells; at the same time, infection and aseptic inflammation in the damaged endometrium will destroy the niche microenvironment of stem cells and cause epithelial and mesenchymal cell regeneration Obstacles to repair occur, blood vessel formation is blocked, and dense fibrous tissue is formed.
  • the uterine cavity covers only a small amount of intima, or even no intima, glands atrophy, and the uterine cavity loses its normal shape and function. Furthermore, the immune disorder in the uterine cavity further mediates the risk of endometrial immune abortion.
  • NK cells are an innate immune cell that exists in the endometrium. In early pregnancy, NK cells account for 40-70% of the total number of lymphocytes in the decidua, which is a type of lymphocytes with a higher content in normal decidua. Modern reproductive medicine studies believe that human decidual NK (dNK) cells have a unique CD56 bright CD16 - KIR + CD9 + CD49a + phenotype, which is different from peripheral NK cells and has almost no cytotoxicity. Decidual NK cells participate in local decidual tissue to promote angiogenesis, tissue remodeling, immune regulation and placenta formation.
  • Exosomes are the reverse budding of endosomes to form multivesicular endosomes.
  • the multivesicular endosomes fuse with the cell membrane and move toward the cell.
  • the external release forms exosomes.
  • Almost all cells can secrete exosomes.
  • the characteristics of these exosomes are: the diameter is between 30-150nm; the density is between 1.13-1.19g/mL; they express specific proteins and carry important signal molecules of the parent cell, including proteins, lipids and RNA, etc. ; Maintain the life activity similar to the parental cells.
  • decidual NK cell population itself is extremely heterogeneous, that is , the NK cells in the decidual tissue with the CD56 bright CD16 - KIR + CD9 + CD49a + phenotype can also be based on the expression of their surface markers. Different groups are divided into groups with different functions.
  • the prior art does not disclose or suggest a technical solution for preparing products derived from exosomes derived from decidual NK cell subsets expressing specific markers for disease treatment.
  • the present invention is made to solve the above technical problems, and aims to provide a use, preparation method and pharmaceutical composition containing the exosomes derived from decidual NK cells.
  • the first aspect of the present invention is to provide the use of exosomes derived from decidual NK cells and their cell subgroups in the preparation of drugs for infertility-related diseases and adjuvant therapeutic agents.
  • the surface marker of the decidua NK cell of the present invention is CD56 bright CD16 - CD49a + , and the cell subgroup is one or more of the surface markers of CD39 + , CD27 + , CD160 + and TIGIT + Combination of decidual NK cells.
  • the exosomes derived from the decidual NK cells have the following characteristics: a diameter of about 30-150nm, a lipid membrane, enveloping genetic material such as protein, mRNA, and microRNA, isolated decidual NK cells, and expression specificity
  • the markers of decidual NK cell subpopulations are further enriched in the medium outside.
  • Infertility-related diseases include endometrial growth disorders and maternal-fetal immune tolerance disorders.
  • Endometrial growth disorders related diseases include endometrial injury, premature ovarian failure, sex hormone disorders, polycystic ovary syndrome, pelvic inflammatory disease, decreased endometrial receptivity, endometritis, endometrial polyps, intrauterine adhesions , Endometrial gland reduction, endometrial fibrosis, amenorrhea, abnormal uterine bleeding, adenomyosis and endometriosis, reproductive system infection, uterine fibroids, etc.; diseases related to maternal-fetal immune tolerance disorders Including recurrent spontaneous abortion, threatened abortion or failure of assisted reproductive technology treatment.
  • the drugs for treating infertility-related diseases mainly include drugs for treating endometrial growth disorders or drugs for treating diseases related to maternal-fetal immune tolerance disorders.
  • the drugs for the treatment of endometrial growth disorders are drugs that promote the increase of endometrial thickness, drugs that enhance endometrial cell viability, drugs that reduce endometrial cell damage, drugs that promote VEGF expression, and endometrial maintenance. Any one or more combinations of stromal cell stemness and proliferation-stimulating drugs.
  • the drugs for treating diseases related to maternal-fetal immune tolerance disorders are any one or a combination of drugs that exert immune tolerance, drugs for the treatment of spontaneous abortion, and drugs that increase the level of T-helper lymphocytes.
  • the adjuvant therapeutic agent for infertility-related diseases is a reagent composition that improves the normal development rate of fertilized eggs.
  • the composition may be an agent that promotes the development and maturation of fertilized eggs, blastocysts or blastocysts, or may be a fertilized agent containing the exosomes. Egg medium or medium for in vitro fertilization.
  • the reagent for promoting the development and maturation of fertilized eggs, blastocysts or blastocysts is preferably used in the medium of the fertilized eggs to increase the development rate of the fertilized eggs.
  • the reagent is administered orally, sublingually, subcutaneously, intravenously, intramuscularly, nasally, follicularly, vaginally to human or non-human animal mothers that are the source of eggs.
  • the composition for increasing the development rate of the present invention is not used, when the composition of the present invention is used, the normal development rate of the fertilized egg is significantly improved, and the conception rate is also improved. improve.
  • the composition can also be added to a medium for in vitro maturation of egg retrieval, an egg cryopreservation agent, and the like.
  • the animal fertilized egg culture medium or in vitro fertilization culture medium containing the exosomes is not particularly limited, as long as it is a culture medium capable of cultivating mammalian fertilized eggs, for example, HTF medium, m-HTF medium, Ham medium, Ham F-10 medium, MEM medium, 199 medium, BME medium, CMRL1066 medium, McCoy-5A medium, Weymouth medium, Trowell T-8 medium, Leibovitz L-15 medium, NCTC medium, William-E medium, Kane and Foote medium, Brinster medium, m-Tyrode medium, BWW medium, WK Whitten medium, TYH medium, Hoppes&Pitts medium, m-KRB medium, BO Medium, T6 medium, GPM medium, KSOM, HECM medium, and modified medium of these mediums.
  • Commercially available special media for in vitro fertilization such as CARD MEDIUM mouse in vitro fertilization medium and medium for porcine embryo development and culture (PZM-5), can also be
  • the second aspect of the present invention is to provide a method for preparing exosomes derived from decidual NK cells, which are prepared by a method including the following steps:
  • Lymphocyte acquisition Percoll (GE Healthcare) density gradient centrifugation.
  • decidual NK cells Use flow cytometry to separate decidual NK cells, first use general technology to separate some impurity cells, and then use anti-CD56 antibody, anti-CD16 antibody and anti-CD49a antibody to perform flow cytometry or magnetic bead method Separate decidual NK cells, the marker of decidual NK cells is CD56 bright CD16 - CD49a + .
  • the decidual NK cell subset marker is any one of CD39 + , CD27 + , CD160 + , and TIGIT + and/or a combination of at least two.
  • NK cells or cell subpopulations to CTS AIM-V medium without serum, 1640 medium or DMEM medium.
  • the culture time is 24-96 hours at 37°C and 5% CO 2.
  • the medium is generally not Add serum.
  • step B After filtering the liquid culture medium in step B with a 0.45 ⁇ m filter membrane, it was centrifuged at 4°C and 1000g for 10 minutes; 4°C and 2000g for 20 minutes; 4°C and 10000g for 30 minutes; after 110000g for 90 minutes, discard the supernatant and use phosphate Resuspend the pellet in buffer; centrifuge again at 110000g for 90 min, discard the supernatant, resuspend the pellet in a small amount of phosphate buffer, and filter with a 0.45 ⁇ m filter to obtain exosomes.
  • the third aspect of the present invention is to provide a pharmaceutical composition for infertility-related diseases, which is composed of decidual NK cell-derived exosomes and pharmaceutically acceptable excipients.
  • the exosomes are the main or even the only active ingredient.
  • the excipients help the exosomes to exert their curative effect more stably.
  • liquid formulations can be stored at 2°C-8°C for at least three months, and lyophilized formulations can be stored at -30°C for at least six months.
  • the pharmaceutical composition includes tablets, pills, powders, injections, tinctures, solutions, extracts, ointments, etc. commonly used in the pharmaceutical field; it also includes preparations for uterine mucosal administration, such as membranes, suppositories, and tablets.
  • Drugs, effervescent tablets, gels, and stents and other intrauterine drug delivery systems, etc.; can also include mucosal absorption enhancers, such as surfactants, chelating agents, fatty acids, fatty alcohols, fatty acid esters, cyclodextrin-derived Substances, protease inhibitors, etc.
  • the present invention also provides the use of the pharmaceutical composition in the preparation of products for the treatment and prevention of infertility-related diseases.
  • An effective amount of exosomes and/or compositions can be administered to subjects with infertility-related diseases (human or animal), and an effective amount of exosomes and/or compositions can also be administered prophylactically to healthy subjects at risk of infertility Exosomes and/or compositions.
  • the fourth aspect of the present invention provides an auxiliary therapeutic agent for infertility-related diseases, which is composed of decidual NK cell-derived exosomes and pharmaceutically acceptable excipients, or fertilized animals containing the exosomes Egg medium or medium for in vitro fertilization.
  • the mass-to-volume ratio of the exosomes is 0.02% (the mass of exosomes refers to the mass of protein contained in the exosomes), or other values, as long as the normal development of the fertilized egg can be obtained.
  • the effect of increasing the rate is sufficient, and it is not particularly limited.
  • the fifth aspect of the present invention provides a kit for in vitro or in vivo development in order to increase the normal development rate of fertilized eggs, as long as the exosomes are included as an active ingredient, and there are no particular restrictions on them, and they can be used.
  • the present invention provides the use of decidual NK cells and exosomes derived from cell subgroups in the preparation of drugs for infertility-related diseases and auxiliary therapeutic agents.
  • the exosomes treat the endometrium by promoting the increase of endometrial thickness, promoting endometrial cell viability, reducing endometrial cell damage, promoting VEGF expression, maintaining the stemness of endometrial stromal cells and stimulating proliferation.
  • Growth disorders increase the pregnancy success rate of endometrial injury model mice from 20% to 50-70%; treat maternal-fetal immunity by exerting immune tolerance, reducing the rate of spontaneous abortion and increasing the level of T helper lymphocytes Disorders related to tolerance disorders.
  • exosomes of the present invention can effectively increase their development rate by multiples, regardless of whether they are fertilized in vivo or in vitro fertilized fertilized eggs, and at the same time, it also improves the implantation rate and birth rate of in vitro fertilization rate transplantation. , It plays an active auxiliary role in the treatment of infertility.
  • the exosomes in the present invention not only have a certain therapeutic or alleviating effect on related diseases that cause infertility, but also can actively assist them by increasing the normal development rate of fertilized eggs, which is infertility.
  • the treatment of infertility provides a new way.
  • the statistical calculations in this embodiment and the following embodiments select the different statistical modes of software SPSS 22.0 version to calculate the p value according to the comparison between two groups, multiple groups, and the comparison requirements of rates. P value less than 0.05 is considered statistically significant.
  • NK cell isolation and flow cytometric sorting can refer to the literature [ Fu B, et al. Immunity, 2017, 47(6): 1100-1113.e6.].
  • An example is as follows: digested with 1 mg/mL collagenase IV (Sigma-Aldrich) and 0.01 mg/mL DNase I (Shanghai Sangon) for 1 h, then obtained lymphocytes by Percoll (GE Healthcare) density gradient centrifugation, and cultured at 37°C for 2 h in a petri dish Remove stromal cells and macrophages, and then isolate NK cells using flow cytometry.
  • First screening uses CD56 antibody, CD3 antibody, CD14 antibody to preliminarily sort NK cells, and then use CD16 antibody, CD49a antibody to further screen decidual NK cells, that is, decidual NK cells with a phenotype of CD56 bright CD16 - CD49a + (CD56 bright CD16 - CD49a + CD3 - CD14 - ).
  • the NK cells obtained in the normal group and the abortion group were lysed. After the protein concentration was determined by the Bradford method, the two groups of lysates were analyzed for protein expression using the iTRAQ-nano-HPLC-MS/MS method to confirm that the two groups of NK cells contained different membrane surface markers. .
  • Example 1 Use healthy non-medical reasons to terminate the early pregnancy decidual tissue to prepare decidual NK cells, and implement the NK cell isolation method described in Example 1, briefly as follows: After 1 mg/mL collagenase IV (Sigma-Aldrich) and 0.01 mg/mL DNaseI(Shanghai Sangon) was digested for 1h, then Percoll(GE Healthcare) density gradient centrifugation was used to obtain lymphocytes. Incubate at 37°C for 2h in a petri dish to remove stromal cells and macrophages, and then isolate NK cells using flow cytometry. The decidual NK cells with the phenotype of CD56 bright CD16 - CD49a + are obtained.
  • the freshly isolated decidual NK cells, decidual NK cell subsets, and control NK cells described in Example 2 were cultured in serum-free 1640 medium for 24 hours.
  • the cells were removed by centrifugation, the medium supernatant was filtered with a 0.45 ⁇ m filter, and the supernatant was collected by centrifugation at 4°C, 1000g for 10 minutes; the collected supernatant was centrifuged at 4°C and 2000g for 20 minutes, and the supernatant was collected; the collected supernatant was 4°C Centrifuge at 10,000g for 30min to collect the supernatant; centrifuge the collected supernatant at 110,000g for 100min, discard the supernatant, and resuspend the pellet with phosphate buffer; centrifuge again at 110,000g for 100min, discard the supernatant, and resuspend the pellet with a small amount of phosphate buffer.
  • exosomes were lyophilized and stored at -80°C.
  • CD56 bright CD16 - CD49a + decidual NK cell-derived exosomes CD56 bright CD16 - CD49a + CD39 + decidual NK cell-derived exosomes
  • CD56 bright CD16 - CD49a + CD27 + Decidual NK cell-derived exosomes CD56 bright CD16 - CD49a + CD160 + decidual NK cell-derived exosomes
  • CD56 bright CD16 - CD49a + CD39 + TIGIT + decidual NK cells are derived from exosomes.
  • the exosomes derived from peripheral NK cells described in Example 2 were used as control exosomes.
  • Example 4 Exosomes enhance endometrial cell viability, reduce endometrial cell damage, and increase VEGF expression
  • the amount of exosomal protein is 0.02% (that is, the relative mass-volume ratio) of the medium volume matrix .
  • the control group used the control exosomes described in Example 3, and the blank group did not contain exosomes.
  • stromal cells and media were sampled for analysis.
  • the cell viability of the stromal cells was detected by the PrestoBlue method (Thermo Fisher Scientific), and the measurement was performed 48 hours after the treatment, and the value was expressed as the mean% of the normalized control (Table 1).
  • the decidual NK cells and exosomes derived from cell subgroups of the present invention have the ability to increase the viability of uterine stromal cells, and can be used as a product for enhancing endometrial proliferation.
  • Lactate dehydrogenase detection method is used to measure cell damage by colorimetric method, so that cell damage can be quantified based on the measurement of lactate dehydrogenase activity in damaged cells in the culture medium. Increased cell membrane damage and cell lysis lead to an increase in lactate dehydrogenase activity, which is proportional to the number of lysed cells. After 48 hours of exosomal treatment, lactate dehydrogenase activity was measured in the culture medium, and the value was expressed as a normalized mean% of the control (Table 2).
  • decidual NK cells and exosomes derived from cell subgroups of the present invention have the ability to reduce membrane damage and can be used as products for enhancing the activity of stromal cells.
  • the decidual NK cells and exosomes derived from cell subgroups of the present invention can promote the expression of VEGF, which has the effect of enhancing endometrial angiogenesis.
  • Example 5 Exosomes promote the expression of endometrial stromal cell markers
  • Example 3 After culturing the non-pathological endometrial stromal cells (stroma cells) for 24 hours, adding the various exosomes described in Example 3, the amount of exosomal protein is 0.02% (that is, the relative mass-to-volume ratio) . As the control group, the control exosomes described in Example 3 were used. After culturing in an incubator at 37°C and 5% CO 2 for 24 hours, flow cytometry was used to detect the ALDH positive rate and Ki67 positive rate of stromal cells. The results are shown in Table 4 and Table 5.
  • the patient's endometrial thickness is less than 8mm due to induced abortion, curettage, infection and other factors, and the clinical diagnosis is that the endometrium is thin.
  • the treatment of anti-infection and other treatments to patients is ineffective.
  • the active ingredients were administered with various exosomes prepared in Example 3, and the dosage was 10 mg/kg of exosomes derived from decidual NK cells and decidual NK cell subgroups, such as intravenous infusion or intrauterine perfusion.
  • the composition It is administered to the patient one or more times to promote the increase in the thickness of the endometrium.
  • CBA/J female mice and DBA/2J male mice were used to establish a stress abortion model.
  • This abortion model is a classic research model of maternal-fetal immune tolerance.
  • the establishment methods, experimental methods and observation time points are equivalent to the literature (Blois S M ,et al.. Nature Medicine, 2007,13(12):1450-1457.).
  • CBA/J female mice were divided into negative control group, stress pressure group, control group, and treatment group before being caged.
  • the treatment group was intravenously administered the decidual NK cells and decidual NK cell subgroups (150 ⁇ g/mouse) of the present invention, once every 3 days, for a total of 3 administrations.
  • the control group was given control exosomes with the same dosage and administration method.
  • the cages were closed 3 days after the first application.
  • Example 9 The effect of decidual NK cells and decidual NK cell subsets on T helper cells
  • mice in the control group, the stress pressure group, the control exosome group and the NK cell exosome treatment groups described in Example 8 were separated, and the level of Foxp3-positive T helper lymphocytes was detected.
  • the isolation and detection methods are the same as those in the literature (Kim B J, et al.. Proceedings of the National Academy of Sciences, 2015, 112(5): 1559-1564.
  • the results show that decidual NK cells and decidual NK cell subgroups are derived from Exosomal treatment can effectively increase the level of Foxp3-positive T helper lymphocytes (Table 9).
  • mice To construct an animal endometrial injury model (C57 mice), divide 8-week-old female mice into groups, each with 10 mice, and use the double (infection + mechanical) injury method to construct the endometrial injury model, namely After the mouse is anesthetized, a longitudinal incision of about 2 cm in the middle of the abdomen is taken into the abdomen, and a 0.5 cm longitudinal incision is made in the middle and lower 1/3 of the uterus. An endometrial spatula is used to scrape the middle and upper uterine cavity.
  • a blank control group (sham operation group) is set up, with only saline injection (model) group; model + control exosomes and NK cell exosomes treatment group.
  • the treatment group was intravenously administered the decidual NK cells of the present invention and exosomes derived from the decidual NK cell subsets (150 ⁇ g/head), once every 3 days, for a total of 3 administrations.
  • mice were mated with male mice after 3 cycles of estrus. One month later, the samples were taken for HE staining and Masson staining to evaluate the function of the endometrium. Three months later, the mouse pregnancy results were evaluated. Results: The histological function evaluation at 1 month after operation showed that compared with the control groups, the degree of fibrosis of decidual NK cells and decidual NK cell subgroups was significantly reduced; compared with the control groups, the number of secretory gland All are higher than the control groups. The evaluation of pregnancy results showed that the pregnancy rates of the exosomes derived from decidual NK cells and decidual NK cell subgroups were higher than those of the control exosomes group. The results are shown in Table 10.
  • C57BL/6J female mice (21 weeks old to 27 weeks old, body weight 20.0-24.5 g) were used as female mice for collecting fertilized eggs.
  • C57BL/6J male mice (32-38 weeks old, body weight 31.0-35.5 g) were used as male mice for mating.
  • 5 U (unit) of horse chorionic gonadotropin was administered intraperitoneally to each female mouse, and after 45-48 hours, 5 U (unit) of human chorionic gonadotropin was administered intraperitoneally to each female mouse.
  • Female mice Immediately after the administration of human chorionic gonadotropin, each female mouse was mated with each of the above-mentioned male mice.
  • the fertilized egg with separated cumulus cells is recovered, washed with M16 medium without hyaluronidase, and removed. Hyaluronidase.
  • the resulting fertilized eggs from which the cumulus cells have been removed are placed in a 37°C, CO 2 incubator.
  • a 100 ⁇ L drop of M16 medium was made in a 35mm petri dish, and the drop was layered with mineral oil (manufactured by Sigma-Aldrich) and added with decidual NK cells and decidual NK cell subgroups derived from Example 3
  • the final concentration of exosomes and control exosomes is 0.02% by mass volume. Place it in a 37°C, CO 2 incubator. Thirty fertilized eggs from which the cumulus cells were removed were transferred to the spot, and cultured in vitro at 37°C in a CO 2 incubator.
  • the number of fertilized eggs (0 hour) is 100%, and the ratio of the number of embryos that develop normally after 24 hours, 48 hours, 72 hours, and 96 hours is calculated as the normal development rate.
  • the number of fertilized eggs recovered (0 hours) includes a certain degree of unfertilized eggs, even if it is a colony of fertilized eggs (0 hours) recovered in the same experiment, it is allocated to each condition group In the colony of fertilized eggs (0 hours), the content of unfertilized eggs may also change occasionally.
  • the development rate calculated by taking the number of embryos at the 2-cell stage 24 hours after fertilization as 100% is calculated to exclude the influence of the unfertilized egg content rate that accidentally changes among the condition groups.
  • the number of eggs at the 2-cell stage after 24 hours was set to 100%.
  • the development rate in this case is shown in Table 13.
  • the statistical analysis of each result was performed by chi-square test, and there was a statistically significant difference when p ⁇ 0.05(*) and p ⁇ 0.01(**). Since the control exosomes relatively reduced embryo development, the medium group without any exosomes was statistically compared.
  • the control exosomes may have certain cytotoxicity because they are derived from peripheral blood NK cells.
  • Sperm was recovered from the epididymal tail of C57BL/6J male mice, and cultured in mHTF medium at 37°C and 5% CO 2 for 40 minutes to 1 hour to capacitate the sperm. Into the medium in which the collected eggs are added, 2 ⁇ l to 4 ⁇ l of mHTF medium containing sperm are added.
  • Insemination was carried out and cultured at 37°C and 5% CO 2. 4 hours to 6 hours after fertilization, wash the fertilized eggs with K SOM medium to remove cumulus cells and sperm. Temporarily use KSOM or mWM to culture in an incubator at 37°C and 5% CO2 until all fertilized eggs are recovered.
  • Example 13 The effect of exosomes on in vitro fertilized egg transplantation
  • Embryos that reached blastocyst stage embryos in each group in Example 12 were subjected to embryo transfer.
  • Recipient mice were C57BL/6J female mice aged 6 to 10 weeks.
  • the female mice of C57BL/6J who had undergone vas deferens ligation were bred on the day before the egg collection day.
  • General anesthesia is given with somnopentyl anesthetics, and the uterus is exposed by incising the back. Fix the uterus with forceps, and use a 30G injection to open a hole at the oviduct junction, insert the glass capillary that attracts the blastocyst, and transfer the embryo into the uterus.
  • the uterus was carefully put back into the body, and the posterior peritoneum and skin were sutured.
  • blastocysts obtained from each group of mice were transplanted to recipient mice individually.
  • the second day of egg collection was set as the first day, and a caesarean section was performed on the nineteenth day.
  • the recipient mice were euthanized and opened, the uterus was removed and the fetuses were taken out.
  • the number of implantation marks/the number of embryo transfers was used to calculate the implantation rate (Table 16), and the number of litters/the number of embryo transfers was used to calculate the birth rate (Table 17).
  • the results showed that the implantation rate and farrowing rate of exosomes derived from decidual NK cells in each group were increased compared with the control product without addition of exosomes.
  • the control exosomes may have certain cytotoxicity because they are derived from peripheral blood NK cells.

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Abstract

本发明提供了蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途。通过实验证实，该外泌体通过促进子宫内膜厚度增加、增强子宫内膜细胞活力、降低内膜细胞损伤、促进VEGF表达、维持子宫内膜基质细胞干性和刺激增殖的方式治疗子宫内膜生长障碍性疾病，使得子宫内膜损伤模型小鼠的受孕成功率由20％提高至50～70％；通过发挥免疫耐受性作用、降低自发性流产率以及提高T辅助淋巴细胞水平治疗母胎免疫耐受障碍相关性疾病。此外，本发明所述外泌体无论对体内受精的受精卵，还是体外受精的受精卵，均能够以倍数方式有效提高其发育率，同时还提高了体外受精率移植的着床率和产仔率，对于不孕不育的治疗起到积极的辅助作用。

Description

蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途 技术领域
本发明属于生物医药领域，涉及蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途、该外泌体的制备方法以及含有该外泌体的药物组合物。
背景技术
子宫内膜损伤主要为内膜基底层的损伤，其主要原因与妊娠期刮宫有关，与正常子宫内膜相比，妊娠期子宫内膜基底层疏松，更容易受到损伤。基于我国国情以及人工流产率逐渐增加的现状，子宫内膜受损的发生情况不容忽视。子宫内膜基底层受损，可能导致子宫内膜干细胞受损或缺失；与此同时，损伤内膜局部的感染和无菌性炎症会破坏干细胞的壁龛微环境，造成上皮和间质细胞再生修复发生障碍，血管形成受阻，致密纤维组织形成，最终导致宫腔仅覆盖少量内膜，甚至无内膜，腺体萎缩，宫腔失去正常形态及功能。进一步的，宫腔内的免疫情况紊乱进一步介导子宫内膜免疫性流产的风险。
NK细胞是存在子宫内膜一种固有免疫细胞。在妊娠早期，NK细胞占蜕膜中淋巴细胞总数的40-70％，是正常蜕膜中含量较高的一类的淋巴细胞。现代生殖医学研究认为，人类蜕膜NK(dNK)细胞具有独特的CD56 brightCD16 -KIR +CD9 +CD49a +表型，与外周NK细胞不同，几乎没有细胞毒性。蜕膜NK细胞在蜕膜组织局部的参与促进新生血管生成、组织重塑、免疫调节和胎盘形成。
近年来的前沿热点研究显示，活细胞能分泌大量的外泌体(exosome)，外泌体是胞内体逆出芽形成多囊泡胞内体，多囊泡胞内体与细胞膜融合，向细胞外释放形成外泌体。几乎所有的细胞都可以分泌外泌体。这些外泌体的特点是：直径在30-150nm之间；密度在1.13-1.19g/mL之间；表达特异性的蛋白，携带了亲本细胞的重要信号分子，包括蛋白质、脂质和RNA等；保持着与亲代细胞相似的生活学活性。
根据现有探究，并未公开或提示利用蜕膜NK细胞来源的外泌体具有何功能，更无公开或提示蜕膜NK细胞来源的外泌体制备产品以用于疾病的治疗的技术方案，这些都是未知的和需要证实的。另一方面，蜕膜NK细胞群本身是具有极强的异质性的，即具 有CD56 brightCD16 -KIR +CD9 +CD49a +表型的蜕膜组织内NK细胞还可以根据其表面标志物的表达不同分为多群，且功能各异。现有技术没有公开或提示表达特异性标志物的蜕膜NK细胞亚群来源的外泌体制备产品以用于疾病治疗的技术方案。
发明内容
本发明是为解决上述技术问题进行的，目的在于提供一种蜕膜NK细胞来源外泌体的用途、制备方法以及含有该外泌体药物组合物。
本发明的第一方面在于提供蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途。
本发明所述蜕膜NK细的表面标志物为CD56 brightCD16 -CD49a +，所述细胞亚群是表面标志物为CD39 +，CD27 +，CD160 +及TIGIT +中的任意一种或多种的组合的蜕膜NK细胞。
该蜕膜NK细胞来源的外泌体具有如下特征：直径约为30-150nm，具有脂质膜，内包裹蛋白质和mRNA、microRNA等遗传物质，由分离得到的蜕膜NK细胞，和表达特异性标志物的蜕膜NK细胞亚群体外培养基中进一步富集而来。
不孕不育相关疾病包括子宫内膜生长障碍性疾病以及母胎免疫耐受障碍相关性疾病两类。子宫内膜生长障碍相关疾病包括子宫内膜损伤、卵巢早衰、性激素紊乱、多囊卵巢综合症、盆腔炎性疾病、子宫内膜容受性下降、子宫内膜炎、内膜息肉、宫腔粘连、子宫内膜腺体减少、内膜性纤维化、闭经、异常性子宫出血、子宫腺肌症和子宫内膜异位症、生殖系统感染、子宫肌瘤等；母胎免疫耐受障碍相关性疾病包括反复性自发流产、先兆流产或辅助生殖技术治疗失败等。
因此，该治疗不孕不育相关疾病药物主要包括治疗子宫内膜生长障碍性疾病的药物或治疗母胎免疫耐受障碍相关性疾病的药物。
具体的，治疗子宫内膜生长障碍性疾病的药物为促进子宫内膜厚度增加的药物、增强子宫内膜细胞活力的药物、降低内膜细胞损伤的药物、促进VEGF表达的药物、维持子宫内膜基质细胞干性和刺激增殖药物中的任意一种或多种组合。治疗母胎免疫耐受障碍相关性疾病的药物为发挥免疫耐受性作用的药物、治疗自发性流产的药物、以及提高T辅助淋巴细胞水平药物的任意一种或多种组合。
不孕不育相关疾病辅助治疗剂为提高受精卵正常发育率的试剂组合物，该组合物可以为促进受精卵、胚泡或囊胚发育成熟的试剂，也可以是含有该外泌体的受精卵培养基 或体外受精用培养基。
促进受精卵、胚泡或囊胚发育成熟的试剂优选将试剂用于受精卵的培养基中以提高受精卵发育率。将试剂经口、舌下、皮下、静脉内、肌肉内、鼻、卵泡、阴道施用于作为卵子来源的人或非人动物母本。与未使用本发明所述的用于提高发育率组合物的情况相比，在使用了本发明所述组合物的情况下，受精卵的正常发育率得到有意义的提高，进而受孕率也得到提高。还可以将组合物添加到用于进行取卵卵子的体外成熟的培养基中、卵子冷冻保存剂中等。
对于含有该外泌体的动物受精卵培养基或体外受精用培养基，没有特别限定，只要是能培养哺乳动物受精卵的培养基即可，例如可以是HTF培养基、m-HTF培养基、Ham培养基、Ham F-10培养基、MEM培养基、199培养基、BME培养基、CMRL1066培养基、McCoy-5A培养基、Weymouth培养基、TrowellT-8培养基、Leibovitz L-15培养基、NCTC培养基、William-E培养基、Kane and Foote培养基、Brinster培养基、m-Tyrode培养基、BWW培养基、WK Whitten培养基、TYH培养基、Hoppes&Pitts培养基、m-KRB培养基、BO培养基、T6培养基、GPM培养基、KSOM、HECM培养基以及这些培养基的改良培养基。还可以使用CARD MEDIUM小鼠体外受精用培养基、用于猪胚胎发育培养的培养基(PZM-5)等市售的专用体外受精用培养基。
本发明的第二方面在于，提供了该蜕膜NK细胞来源的外泌体的制备方法，由包括如下步骤的方法制备得到：
A、分离蜕膜NK细胞和蜕膜NK细胞亚群
(i)蜕膜组织细胞分离：蜕膜组织经胶原酶Ⅳ(Sigma-Aldrich)以及DNaseⅠ(Shanghai Sangon)消化获取细胞悬液
(ii)淋巴细胞获取：Percoll(GE Healthcare)密度梯度离心得到。
(iii)以流式细胞仪分离蜕膜NK细胞，首先利用通用技术分离一些杂质细胞，然后利用anti-CD56抗体、anti-CD16抗体和anti-CD49a抗体，实施流式细胞仪法或磁珠法分离蜕膜NK细胞，蜕膜NK细胞的标志物为CD56 brightCD16 -CD49a +。
(iv)根据需要进一步利用anti-CD39抗体，anti-CD27抗体，anti-CD160抗体，和anti-TIGIT抗体，实施流式细胞仪法或磁珠法分离蜕膜NK细胞亚群。蜕膜NK细胞亚群标志物为CD39 +，CD27 +，CD160 +，和TIGIT +中的任一种和/或至少两种的组合。
B、NK细胞体外培养
将NK细胞或细胞亚群转移至不添加血清的CTS AIM-V培养基，1640培养基或 DMEM培养基中，37℃、5％CO 2环境中培养时间为24-96小时，培养基一般不添加血清。
C、外泌体分离
将步骤B中的液体培养基0.45μm滤膜过滤后，依次经4℃，1000g离心10min；4℃，2000g离心20min；4℃，10000g离心30min；110000g离心90min后，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。
本发明的第三方面在于，提供了一种不孕不育相关疾病的药物组合物，由蜕膜NK细胞来源外泌体以及药学上可接受的辅料组成。该外泌体是其主要甚至唯一活性成分。辅料有助于外泌体更稳定地发挥疗效，这些制剂可以保证本发明公开的外泌体的构像完整性，同时还要保护的外泌体的活性官能团，防止其降解。
通常情况下，液体制剂可以在2℃-8℃条件下保存至少稳定三个月，冻干制剂在-30℃至少六个月保持稳定。
在药物形式上，该药物组合物包括制药领域常用的片剂、丸剂、散剂、注射剂、酊剂、溶液剂、浸膏剂、软膏剂等；还包括子宫黏膜给药制剂，例如膜剂、栓剂、片剂、泡腾片剂、凝胶剂、及支架等子宫内药物释放系统等；还可包括黏膜吸收促进剂，例如表面活性剂、螯合剂、脂肪酸、脂醇、脂肪酸酯、环糊精衍生物、蛋白酶抑制剂等。
与外泌体的用途类似，本发明还提供了该药物组合物在制备治疗和预防不孕不育相关疾病产品中的用途。给具有不孕不育相关疾病的受试者(人或动物)施用有效量的外泌体和/或组合物，也可以给具有不孕不育风险的健康受试者预防性施用有效量的外泌体和/或组合物。
本发明的第四方面，提供了一种不孕不育相关疾病的辅助治疗剂，由蜕膜NK细胞来源外泌体以及药学上可接受的辅料组成，或者为含有该外泌体的动物受精卵培养基或体外受精用培养基。
优选的，在该辅助治疗剂中，所述外泌体的质量体积比为0.02％(外泌体质量指外泌体所含有蛋白质量)，也可以是其他数值，只要能取得受精卵正常发育率提高的效果即可，对此没有特别限定。
本发明的第五方面，提供了为提高受精卵正常发育率的用于体外发育或体内发育的试剂盒，只要包含所述外泌体作为活性成分即可，对其没有特别限定，可以使用还包含一种或多种哺乳动物受精卵的体外培养基的试剂盒。
相比现有技术，本发明的技术效果如下：
本发明提供了蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途。通过实验证实，该外泌体通过促进子宫内膜厚度增加、促进子宫内膜细胞活力、降低内膜细胞损伤、促进VEGF表达、维持子宫内膜基质细胞干性和刺激增殖的方式治疗子宫内膜生长障碍性疾病，使得子宫内膜损伤模型小鼠的受孕成功率由20％提高至50～70％；通过发挥免疫耐受性作用、降低自发性流产率以及提高T辅助淋巴细胞水平治疗母胎免疫耐受障碍相关性疾病。
此外，本发明的外泌体无论对体内受精的受精卵，还是体外受精的受精卵，均能够以倍数方式有效提高其发育率，同时还提高了体外受精率移植的着床率和产仔率，对于不孕不育的治疗起到积极的辅助作用。
因此，本发明中的外泌体不仅对引起不孕不育的相关疾病具有一定的治疗或缓解作用，同时能够通过提高受精卵正常发育率的途径对其起到积极地辅助作用，为不孕不育的治疗提供了一种新的途径。
具体实施方式
以下实施例、实验例对本发明进行进一步的说明，不应理解为对本发明的限制，同时实施例不包括对传统方法的详细描述。
实施例1子宫蜕膜NK细胞候选标志物筛选
本实施例及后述实施例中的统计学计算根据两组比较、多组比较、率的比较需求选择软件SPSS 22.0版本的不同统计模式计算p值。P值小于0.05认为有统计学显著性。
首先利用10例健康非医学原因终止早期妊娠蜕膜组织(正常组)和5例自然流产早期妊娠蜕膜组织(流产组)分离NK细胞，NK细胞的分离和流式细胞分选可以参考文献[Fu B,et al.Immunity,2017,47(6):1100-1113.e6.]。举例如下：经1mg/mL胶原酶Ⅳ(Sigma-Aldrich)和0.01mg/mL DNaseⅠ(Shanghai Sangon)消化1h，再经Percoll(GE Healthcare)密度梯度离心获取淋巴细胞，利用培养皿37℃培养2h以去除基质细胞和巨噬细胞，然后利用流式细胞仪分离NK细胞。首先筛选利用CD56抗体，CD3抗体，CD14抗体初步分选NK细胞，然后利用CD16抗体，CD49a抗体进一步筛选蜕膜NK细胞，即得到表型为CD56 brightCD16 -CD49a +的蜕膜NK细胞 (CD56 brightCD16 -CD49a +CD3 -CD14 -)。裂解正常组和流产组获得的NK细胞，Bradford法测定蛋白质浓度后两组裂解物以iTRAQ-nano-HPLC-MS/MS方法进行蛋白质表达分析，确认两组NK细胞内含量不同的膜表面标志物。方法参考文献[Jiang,Hong-Lin,et al.Cancer research 76.4(2016):952-964.]。经聚类统计分析膜表面标志物，正常组蜕膜NK细胞中CD39(UniProtKB:P49961)、CD27(UniProtKB:P26842)、CD160(UniProtKB:O95971)、TIGIT(UniProtKB:Q495A1)表达均显著高于流产组蜕膜细胞。
实施例2子宫蜕膜NK细胞及细胞亚群制备
利用健康非医学原因终止早期妊娠蜕膜组织制备蜕膜NK细胞，按照实施例1所述NK细胞分离方法实施，简述如下：经1mg/mL胶原酶Ⅳ(Sigma-Aldrich)和0.01mg/mL DNaseⅠ(Shanghai Sangon)消化1h,再经Percoll(GE Healthcare)密度梯度离心获取淋巴细胞。利用培养皿37℃培养2h以去除基质细胞和巨噬细胞，然后利用流式细胞仪分离NK细胞。即得到表型为CD56 brightCD16 -CD49a +的蜕膜NK细胞。进一步利用抗体磁珠分选CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群、CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群、CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群、CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群、和CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群。获得的细胞可以直接进行实验、使用或冻存。采集同一名志愿者的外周血，按照通用方法采集分离NK细胞作为对照细胞。
实施例3子宫蜕膜NK细胞及细胞亚群来源的外泌体制备
将实施例2所述的新鲜分离的蜕膜NK细胞和蜕膜NK细胞亚群、对照NK细胞在无血清1640培养基中培养24小时。离心去除细胞，培养基上清用0.45μm滤膜过滤，在4℃，1000g离心10min，收集上清；收集的上清液4℃，2000g离心20min，收集上清；收集的上清液4℃，10000g离心30min，收集上清；收集的上清液，110000g离心100min，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心100min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。用Bradford法检测外泌体总蛋白量(Bio-Rad Protein Assay Reagent)。外泌体冻干后储存于-80℃。即获得以下六种外泌体：CD56 brightCD16 -CD49a +的蜕膜NK细胞来源外泌体、CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞来源外泌体、CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞来源外泌体、CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞来源外泌体、CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞来源外泌体、和 CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞来源外泌体。实施例2所述外周NK细胞来源的外泌体作为对照外泌体。
实施例4外泌体增强子宫内膜细胞活力、降低内膜细胞损伤、增加VEGF表达
将非病理性子宫内膜基质细胞(stroma cell)培养24小时后，加入实施例3所述各种外泌体，外泌体蛋白量比培养基体积基质为0.02％(即相对质量体积比)。对照组采用实施例3所述对照外泌体，空白组不含有外泌体。处理48小时后，对基质细胞和培养基采样用于分析。
通过PrestoBlue方法(Thermo Fisher Scientific)检测基质细胞的细胞存活力，处理48小时后进行测定，值表示为对照进行标准化后的平均值％(表1)。
表1基质细胞相对细胞活力
组别(外泌体组处理，0.02％) 平均值 SD p值
空白(仅培养基) 100.00 14.63  
对照外泌体 101.66 4.55  
CD56 brightCD16 -CD49a +的蜕膜NK细胞来源外泌体 158.37 24.99 p＜0.05
CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群来源外泌体 140.49 8.32 p＜0.05
CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群来源外泌体 172.69 24.81 p＜0.05
CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群来源外泌体 159.90 11.93 p＜0.05
CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群来源外泌体 161.14 18.66 p＜0.05
CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群来源外泌体 179.38 23.61 p＜0.05
结论：本发明所述的蜕膜NK细胞及细胞亚群来源的外泌体具有增加子宫基质细胞存活力的能力，能作为增强子宫内膜增殖的产品使用。
进一步评估基质细胞的细胞损伤水平：利用乳酸脱氢酶检测法，通过比色法测量细胞损伤，使得能够基于培养基中受损细胞中乳酸脱氢酶活性的测量来定量细胞损伤。细胞膜损伤的增加和细胞溶解导致乳酸脱氢酶活性增加，其与裂解细胞的数量成比例。外泌体处理48小时后，在培养基中进行了乳酸脱氢酶活性测量，值表示为对照进行标准化后的平均值％(表2)。
表2乳酸脱氢酶活性
组别(外泌体组处理，0.02％) 平均值 SD p值
空白(仅培养基) 100.00 15.75  
对照外泌体 103.35 7.65  
CD56 brightCD16 -CD49a +的蜕膜NK细胞来源外泌体 67.77 7.11 p＜0.05
CD56 brightCD16 -CD49a +CD39 +蜕膜NK细胞群来源外泌体 63.54 7.64 p＜0.05
CD56 brightCD16 -CD49a +CD27 +蜕膜NK细胞群来源外泌体 49.01 7.10 p＜0.05
CD56 brightCD16 -CD49a +CD160 +蜕膜NK细胞群来源外泌体 36.12 5.86 p＜0.05
CD56 brightCD16 -CD49a +TIGIT +蜕膜NK细胞群来源外泌体 49.50 7.33 p＜0.05
CD56 brightCD16 -CD49a +CD39 +TIGIT +蜕膜NK细胞群来源外泌体 32.71 5.08 p＜0.05
结论：本发明所述的蜕膜NK细胞及细胞亚群来源的外泌体具有减轻膜损伤的能力，能作为增强基质细胞活性的产品使用。
进一步研究了外泌体对子宫内膜基质细胞VEGF的潜在增强影响。结果呈现于表3中。外泌体处理48小时后，在培养基中进行了VEGF浓度的ELISA检测。
表3：VEGF表达
Figure PCTCN2020119327-appb-000001
结论：本发明所述的蜕膜NK细胞及细胞亚群来源的外泌体具有促进VEGF表达，其具有增强子宫内膜血管新生的作用。
实施例5外泌体促进子宫内膜基质细胞标志物表达
将非病理性子宫内膜基质细胞(stroma cell)培养24小时后，加入实施例3所述各类外泌体，外泌体蛋白量比培养基体积基质为0.02％(即相对质量体积比)。对照组采用 实施例3所述对照外泌体。在37℃、5％CO 2条件的培养箱中培养24小时后，利用流式细胞术检测基质细胞的ALDH阳性率和Ki67阳性率。其结果如表4和表5。
表4.ALDH阳性率
Figure PCTCN2020119327-appb-000002
表5.Ki67阳性率
Figure PCTCN2020119327-appb-000003
结果显示本发明所述的蜕膜NK细胞及细胞亚群来源的外泌体具有非常强的维持基质细胞干性和刺激增殖的作用。
实施例6外泌体治疗子宫内膜损伤
患者因人工流产刮宫、感染等因素子宫内膜厚度低于8mm，临床诊断为子宫内膜薄。给与患者抗感染等治疗无效。给与活性成分为用实施例3所制备的各种外泌体，给药量为蜕膜NK细胞和蜕膜NK细胞亚群来源的外泌体10mg/kg，如静脉输入或宫腔灌注所述组合物。一次或多次施用于患者，以促进子宫内膜厚度增加。
实施例7.外泌体对蜕膜DC细胞的作用
从人非医学原因终止妊娠的蜕膜组织分离树突状细胞DC(CD1c阳性)，分离及筛选方法等同文献(Guo P F,et al.Blood,2010,116(12):2061-2069.)对DC细胞分为阴性对照组(采用实施例3所述对照外泌体，对质量体积比为0.02％)，处理组(采用实施例3所述蜕膜NK细胞和蜕膜NK细胞亚群来源的外泌体，对质量体积比为0.02％)，LPS处理组(100纳克/毫升)，空白组不含有外泌体。48小时后检测培养体系中白介素10(IL-10)和肿瘤坏死因子α(TNFα)水平，检测方法同文献(Guo P F,et al.Blood,2010,116(12):2061-2069.)结果显示外泌体可以显著增加IL-10分泌水平而不增加TNFα水平(表6-7)。这些结果进一步证实蜕膜NK细胞和蜕膜NK细胞亚群来源的外泌体可以通过DC发挥免疫耐受作用。
表6.IL-10含量
Figure PCTCN2020119327-appb-000004
表7.TNFα含量
Figure PCTCN2020119327-appb-000005
Figure PCTCN2020119327-appb-000006
实施例8.外泌体对自发性流产模型的治疗作用
利用CBA/J雌性小鼠和DBA/2J雄性小鼠建立应激流产模型，该流产模型为经典的母胎免疫耐受障碍的研究模型，建立方法、实验方法和观察时间点等同文献(Blois S M,et al..Nature Medicine,2007,13(12):1450-1457.)。CBA/J雌性小鼠合笼前分为阴性对照组，应激压力组，对照组，处理组。处理组分别经静脉施用本发明所述蜕膜NK细胞和蜕膜NK细胞亚群(150μg/只)，3天一次，共施用3次。对照组给与对照外泌体，剂量和给药方式一致。第一次施用后3天后合笼。确定阴栓怀孕后立即将小鼠分笼(有效n＝10)。
实验结果显示(表8)，治疗组的流产率显著低于应激压力流产组，说明采用蜕膜NK细胞和蜕膜NK细胞亚群来源的外泌体具有良好的治疗效果。
表8各组小鼠胚胎吸收率(流产)分析
Figure PCTCN2020119327-appb-000007
Figure PCTCN2020119327-appb-000008
实施例9.蜕膜NK细胞和蜕膜NK细胞亚群对T辅助细胞影响
将实施例8所述对照组、应激压力组、对照外泌体组和NK细胞外泌体处理各组的小鼠主动脉旁淋巴结分离，检测其中的Foxp3阳性T辅助淋巴细胞水平。分离和检测的方法同文献(Kim B J,et al..Proceedings of the National Academy of Sciences,2015,112(5):1559-1564。结果显示蜕膜NK细胞和蜕膜NK细胞亚群来源的外泌体处理可以有效增加Foxp3阳性T辅助淋巴细胞水平(表9)。
表9各组小鼠Foxp3％表达分析
Figure PCTCN2020119327-appb-000009
实施例10蜕膜NK细胞和蜕膜NK细胞亚群治疗小鼠子宫内膜损伤模型
构建动物子宫内膜损伤模型(C57小鼠)，取8周龄大的雌性小鼠分为组，每组10只小鼠，采用双重(感染+机械)损伤法构建子宫内膜损伤模型，即小鼠麻醉后，取下腹部正中长约2cm纵切口进腹，于子宫中下1/3处作0.5cm纵行切口，采用子宫内膜刮勺搔刮中上段子宫腔，当刮勺进出子宫凹凸感消失，四壁感觉粗糙时，停止刮宫，刮宫后宫腔留置脂多糖棉线，缝合腹部切口，48h后取出脂多糖棉线。建模完成后，设置空白对照组(假手术组)，仅注射生理盐水(模型)组；模型+对照外泌体和NK细胞外泌体处理组。处理组分别经静脉施用本发明所述蜕膜NK细胞和蜕膜NK细胞亚群来源的外泌体(150μg/只)，3天一次，共施用3次。在小鼠动情3个周期后将小鼠与雄性小鼠进行交配。1个月后取材行HE染色和Masson染色进行子宫内膜组织功能学评估，3个月后行小鼠妊娠结果评估。结果：术后1个月组织功能学评估显示，与各对照组相比，蜕膜NK细胞和蜕膜NK细胞亚群组的纤维化程度明显减少；与各对照组相比，分泌腺体数量均高于各对照组。妊娠结果评估显示，蜕膜NK细胞和蜕膜NK细胞亚群来源外泌体各组的妊娠率高于对照外泌体组，结果如表10所示。
表10各组小鼠妊娠结果分析
Figure PCTCN2020119327-appb-000010
实施例11:外泌体对受精卵作用
使用C57BL/6J系的雌性小鼠(21周龄-27周龄，体重20.0-24.5g)作为采集受精卵的雌性小鼠。使用C57BL/6J系的雄性小鼠(32-38周龄，体重31.0-35.5g)作为交配用的雄性小鼠。根据诱导过量排卵的常规方法，将5U(单位)的马绒毛膜促性腺激素腹腔内给予各雌性小鼠，45-48小时后将5U(单位)的人绒毛膜促性腺激素经腹腔内给予各雌性小鼠。给予人绒毛膜促性腺激素后立即使各雌性小鼠与上述各雄性小鼠交配。
次日，确定交配后的雌性小鼠是否有乳白色树脂样的阴道栓，从确认有阴道栓的雌性小鼠采集输卵管。将采集到的输卵管在生理盐水(0.9％(w/v)NaCl)中静置15分钟左右，然后将输卵管移至添加了约300μg/mL透明质酸酶(西格玛奥德里奇公司生产)的M16培养基中，以处理除去卵丘细胞。切开输卵管，取出受精卵，在CO 2保温箱内于37℃静置5-10分钟，之后回收已分离卵丘细胞的受精卵，用未添加透明质酸酶的M16培养基洗净，除去透明质酸酶。将所得除去了卵丘细胞的受精卵静置于37℃、CO 2保温箱内。
另外，在35mm培养皿中制作100μL的M16培养基的点滴，该点滴层叠了矿物油(西格玛奥德里奇公司生产)并添加施例3所述蜕膜NK细胞和蜕膜NK细胞亚群来源的外泌体、对照外泌体，最终浓度为质量体积比为0.02％。将其静置于37℃、CO 2保温箱内。向所述点滴移送30个上述除去了卵丘细胞的受精卵，在CO 2保温箱内于37℃进行体外培养。
从点滴内的体外培养开始时(0小时)起，经过24小时、48小时、72小时、96小时后，通过立体显微镜观察，确定各胚胎的发育阶段，算出正常进行发育的胚胎的数、发育率的变化。具体就是，作为在各个阶段正常进行发育的胚胎，计算培养开始24小时后2细胞期的卵数，48小时后3细胞期、4细胞期和8细胞期的卵数，72小时后桑椹胚和胚泡的数，96小时后胚泡的数。各阶段的胚胎数记录在表11中，以受精卵(0小时)的数为100％时的发育率显示在表12中。对于发育率的评价，是以受精卵(0小时)的卵数为100％，算出24小时后、48小时后、72小时后、96小时后正常进行发育的胚胎数的比例，作为正常发育率。另外，由于是自然交配，回收到的受精卵(0小时)的数目包含一定程度的未受精卵，即使是同一实验中回收到的受精卵(0小时)的集落，在分配到各条件群间的受精卵(0小时)集落中，未受精卵的含有率也可能偶然变化。以受精24小时后的2细胞期的胚胎数为100％所算出的发育率，是为了排除在各条件群间偶然变化的未受精卵含有率的影响而进行的计算。也就是说，为了以确实开始了胚胎发育的卵数为基础计算发育率，将24小时后的2细胞期的卵数设定为100％，这种情况下的发育率显示在表13中。各结果的统计分析通过卡方检验进行，p<0.05(*)、p<0.01(**)时具有统计学 上的显著差异。由于对照外泌体相对减少了胚胎发育，故统计学对比不添加任何外泌体的培养基组。
表11各组发育胚胎数
Figure PCTCN2020119327-appb-000011
表12以受精卵0小时为100％计数各组发育率(％)
Figure PCTCN2020119327-appb-000012
Figure PCTCN2020119327-appb-000013
表13以受精卵24小时为100％计数各组发育率(％)
Figure PCTCN2020119327-appb-000014
(vs仅培养基卡方检验**:p<0.01,*:p<0.05)
结果显示，各组外泌体当以受精卵(0小时)的数为100％时，与未添加外泌体的对照品相比，分别添加了各组蜕膜NK细胞及亚群来源的外泌体的的情况下的发育率提高了。对照外泌体因为来自外周血NK细胞，可能具有一定的细胞毒性。
实施例12:外泌体对体外受精卵作用
将5U(单位)的马绒毛膜促性腺激素腹腔内给予C57BL/6J的雌性小鼠(3.9周龄-4.0周龄)，45-48小时后腹腔内给予5U(单位)的人绒毛膜促性腺激素，ASKA制药株式会社生产)，诱导其过量排卵。给予人绒毛膜促性腺激素15小时后，开腹回收输卵管。在矿物油中，用解剖针切开输卵管膨大部，将卵子回收到mHTF培养基的点滴中。从C57BL/6J雄性小鼠的附睾尾部回收精子，在mHTF培养基中于37℃、5％CO 2条件下培养40分钟到1小时，使精子获能。向加入了所回收的卵子的培养基点滴中，加入2μl～4μl含有精子的mHTF培养基。
进行授精，在37℃、5％CO  2条件下进行培养。受精之后4小时到6小时后，用K SOM培养基涤受精卵去除卵丘细胞和精子。暂且用KSOM或mWM培养在37℃、5％CO2的保温箱内，直至回收完全部受精卵。
向未添加外泌体或添加了实施例3所述蜕膜NK细胞和蜕膜NK细胞亚群来源的外泌体、对照外泌体，最终浓度为质量体积比为0.02％的100μl培养基形成的点滴(层叠有矿物油(西格玛奥德里奇公司生产))每个点滴中移入25个受精卵，各处理组共计200个受精卵。此后，进行培养。计测卵子回收24小时后的2细胞期胚胎个数、96小时后的胚泡个数。并且，由于从体外受精获得的受精卵的发育会稍迟，对卵子回收120小时后的胚泡个数也进行计测。以受精24小时后的2细胞期的胚胎数为100％计，以此为基础计算各培养时间的胚胎发育率。结果显示在表14中。
表14各组发育胚胎数
Figure PCTCN2020119327-appb-000015
Figure PCTCN2020119327-appb-000016
表15以受精卵24小时为100％计数各组发育率(％)
Figure PCTCN2020119327-appb-000017
(vs仅培养基卡方检验**:p<0.01,*:p<0.05)
结果显示，各组外泌体当以受精卵(24小时)的数为100％时，与未添加外泌体的对照品相比，分别添加了各组蜕膜NK细胞来源的外泌体的的情况下的发育率提高了。对照外泌体因为来自外周血NK细胞，可能具有一定的细胞毒性。
实施例13:外泌体对体外受精卵移植的作用
将实施例12中各组达到胚泡期胚胎的胚胎进行了胚胎移植。受体小鼠为6～10周龄 的C57BL/6J雌性小鼠，使用了在采卵日的前一天与被进行了输精管结扎的C57BL/6J的雌性小鼠雄性小鼠交配，并在第二天能确认阴道栓的个体。用戊巴比妥(somnopentyl)麻醉药实施全身麻醉，通过切开后背部而使子宫露出。用镊子固定子宫，并用30G的注射针对输卵管连接部进行开孔，插入吸引了胚泡的玻璃毛细管，将胚胎移植至子宫内部。在移植后，将子宫小心地放回体内，缝合了后腹膜以及皮肤。在移植时，将从各组小鼠得到的胚泡按照个体分别移植至受体小鼠。将采卵的第二天设为第一天，在第十九天实施了剖腹产。对受体小鼠实施安乐死并进行开腹，摘除子宫并取出胎仔。以着床痕迹数/胚胎移植数来计算出着床率(表16)，以产仔数/胚胎移植数来计算出产仔获得率(表17)。
表16各组着床率
Figure PCTCN2020119327-appb-000018
(vs仅培养基卡方检验**:p<0.01,*:p<0.05)
表17各组产仔率
Figure PCTCN2020119327-appb-000019
Figure PCTCN2020119327-appb-000020
(vs仅培养基卡方检验**:p<0.01,*:p<0.05)
结果显示，各组外泌体与未添加外泌体的对照品相比，分别添加了各组蜕膜NK细胞来源的外泌体的的情况下的着床率和产仔率提高了。对照外泌体因为来自外周血NK细胞，可能具有一定的细胞毒性。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。

Claims (10)

  1. 蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途。
  2. 根据权利要求1所述的蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途，其特征在于：
    其中，所述蜕膜NK细胞的表面标志物为CD56 brightCD16 -CD49a +，所述细胞亚群是表面标志物为CD39 +，CD27 +，CD160 +及TIGIT +中的任意一种或多种的组合的蜕膜NK细胞。
  3. 根据权利要求1所述的蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途，其特征在于：
    其中，所述不孕不育相关疾病治疗药物为治疗子宫内膜生长障碍性疾病的药物或治疗母胎免疫耐受障碍相关性疾病的药物；
    所述辅助治疗剂为提高受精卵正常发育率的试剂组合物。
  4. 根据权利要求3所述的蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途，其特征在于：
    其中，所述治疗子宫内膜生长障碍性疾病的药物为促进子宫内膜厚度增加的药物、增强子宫内膜细胞活力的药物、降低内膜细胞损伤的药物、促进VEGF表达的药物、维持子宫内膜基质细胞干性和刺激增殖药物中的任意一种或多种组合；
    所述治疗母胎免疫耐受障碍相关性疾病的药物为维持或增强免疫耐受性作用的药物、治疗自发性流产的药物、以及提高T辅助淋巴细胞水平的药物的任意一种或多种组合；
    所述提高受精卵正常发育率的试剂组合物为促进受精卵、胚泡或囊胚发育成熟的试剂，或含有该外泌体的受精卵培养基或体外受精用培养基。
  5. 根据权利要求1～4任一项所述的蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途，其特征在于，该外泌体的制备方法如下：
    A、分离蜕膜NK细胞和蜕膜NK细胞亚群
    将蜕膜组织酶解消化后获取细胞悬液，而后通过密度梯度离心法得到淋巴细胞，然后利用anti-CD56抗体、anti-CD16抗体和anti-CD49a抗体，从中分离标志物为CD56 brightCD16 -CD49a +的蜕膜NK细胞；再根据需要利用anti-CD39抗体，anti-CD27抗体，anti-CD160抗体和anti-TIGIT抗体，分离蜕膜NK细胞亚群；
    B、NK细胞体外培养
    将NK细胞或细胞亚群转移至不添加血清的CTS AIM-V培养基，1640培养基或DMEM培养基中，37℃、5％CO 2环境中培养时间为24-96小时；
    C、外泌体分离
    将步骤B中的液体培养基滤膜过滤后，依次经4℃，1000g离心10min；4℃，2000g离心20min；4℃，10000g离心30min；110000g离心90min后，弃上清，使用磷酸盐缓冲液重悬沉淀；再次110000g离心90min，弃上清，少量磷酸盐缓冲液重悬沉淀，0.45μm滤膜过滤，得到外泌体。
  6. 根据权利要求1所述的蜕膜NK细胞及其细胞亚群来源外泌体在制备不孕不育相关疾病药物及辅助治疗剂中的用途，其特征在于：
    其中，所述药物是以外泌体作为唯一活性成分或者是包含该外泌体的药物组合物，
    所述辅助治疗剂是含有该外泌体的试剂组合物。
  7. 根据权利要求6所述的蜕膜NK细胞来源外泌体在制备治疗不孕不育相关疾病药物及辅助治疗剂中的用途，其特征在于：
    其中，所述药物组合物为片剂、丸剂、散剂、注射剂、酊剂、溶液剂、浸膏剂、软膏剂、膜剂、栓剂、片剂、泡腾片剂、凝胶剂、黏膜吸收促进剂或子宫内药物释放系统。
  8. 一种治疗不孕不育相关疾病的药物组合物，其特征在于，由蜕膜NK细胞来源外泌体以及药学上可接受的辅料组成。
  9. 一种不孕不育相关疾病的辅助治疗剂，其特征在于，由蜕膜NK细胞来源外泌体以及药学上可接受的辅料组成，或者为含有该外泌体的动物受精卵培养基或体外受精用培养基。
  10. 根据权利要求9所述的不孕不育相关疾病的辅助治疗剂，其特征在于：
    其中，在所述辅助治疗剂中，所述外泌体的质量体积比为0.02％。
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