WO2019061560A1 - 未成熟卵母细胞的体外成熟培养液及其应用 - Google Patents
未成熟卵母细胞的体外成熟培养液及其应用 Download PDFInfo
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Definitions
- the invention relates to an in vitro maturation culture liquid, a related composition and a culture method for immature oocytes, and belongs to the field of medical biology.
- gonadotropin In human clinical assisted reproduction, gonadotropin is widely used to perform superovulation operation in female patients, and then the obtained mature oocytes are subjected to in vitro fertilization or single sperm injection to achieve the goal of breeding offspring.
- gonadotropin due to environmental, mental stress and individual responsiveness to gonadotropin, some patients often have a large part of the resulting oocytes that are not mature after the implementation of superovulation, which greatly reduces the number of oocytes.
- the success rate of assisted reproductive assisted pregnancy in this patient reduces the utilization efficiency of the collected oocytes.
- the oocyte maturation culture method and the culture medium used in clinical practice cannot solve the above problems to some extent.
- the embryo engineering technology system is used to transplant the embryos obtained by in vitro fertilization into a technology platform and direction to promote the development of animal husbandry.
- a commonly used method is to obtain immature oocytes from hormone-free ovaries, and culture them in mature culture systems for embryo production and animal reproduction. But unfortunately, the number of embryos produced in vitro is significantly lower than that of the body, which reflects that the in vitro culture system of oocytes is still ignored. miss you.
- the quality of in vitro fertilized embryos also affects the pregnancy rate after embryo transfer, and the survival rate of the offspring after transplantation. Therefore, optimizing and improving the in vitro maturation culture system of oocytes helps to increase the number of oocytes that can be used for in vitro fertilization to produce embryos, and to promote the improvement of embryo quality. Therefore, the improvement of the culture medium and culture method of immature oocytes in vitro in the livestock embryo engineering system can promote the increase of the in vitro maturation rate of the oocytes, which can improve the efficiency of embryo engineering and promote the reproduction of livestock.
- IVM in vitro maturation
- the basic approach is to simulate the in vivo environment to develop two-stage in vitro culture methods for oocytes, ie, to temporarily reversibly block oocyte meiosis recovery by using meiotic inhibitors in vitro, while promoting oocyte growth and cytoplasmic maturation.
- the oocyte is then removed from the meiotic inhibitory environment for in vitro maturation.
- the purpose of this method is to prolong the time between granulosa cells and oocytes for material and information exchange through gap junctions, and to promote the accumulation of mRNA and protein in oocytes.
- the patent CN102140435A discloses a buffalo oocyte in vitro maturation medium which improves the maturation rate of buffalo oocytes by adding insulin, transferrin and selenium.
- CN101591637A discloses a bovine oocyte in vitro maturation culture liquid, which comprises adding amnion epithelial cells and a supernatant of the culture liquid thereof through the IVM medium, and utilizing the characteristics of the epithelial cells to utilize the secretory and synthetic bovine oocyte in vitro maturation. Bioactive substances, thereby increasing the rate of bovine oocyte maturation.
- CN100432219C discloses a bovine oocyte in vitro maturation culture liquid, which mainly uses tea polyphenol as an antioxidant, and utilizes the characteristics of anti-free radical and activated intracellular antioxidant defense system, thereby obtaining a bovine egg.
- the nutrient base of maternal cells in vitro discloses a bovine oocyte in vitro maturation medium by adding CNP to TCM199 medium, and CNP can promote nuclear maturation and cytoplasmic maturation synchronization, thereby improving in vitro development ability.
- the main object of the present invention is to provide a promotion failure. Mature maturation of mature oocytes and in vitro maturation of developmental potential.
- the present invention provides a medium which promotes in vitro maturation of immature oocytes, specifically comprising follicular granulosa cells and a culture medium for culturing the follicular granulosa cells, the culture medium for culturing the follicular granulosa cells Contains CNP or a variant thereof or an analog thereof and an HDAC (histone deacetylase) inhibitor.
- a medium which promotes in vitro maturation of immature oocytes specifically comprising follicular granulosa cells and a culture medium for culturing the follicular granulosa cells
- the culture medium for culturing the follicular granulosa cells Contains CNP or a variant thereof or an analog thereof and an HDAC (histone deacetylase) inhibitor.
- the medium is obtained by culturing the follicular granulosa cells in a culture medium of the follicular granulosa cells for 4-6 h.
- the HDAC inhibitor is selected from the group consisting of an HDAC1 inhibitor, a HDAC2 inhibitor, and a HDAC3 inhibitor.
- the HDAC inhibitor is an HDAC3 inhibitor; the HDAC3 inhibitor is selected from one or more of HDACi 4b, Entinostat (MS-275), BG45, RG2833 (RGFP109), RGFP966.
- the CNP is a C-type natriuretic peptide.
- the culture medium of the follicular granulosa cells is cultured containing 30-120 ng/mL of CNP or a variant thereof or analog thereof and 1-10 ⁇ M of HDAC inhibitor.
- the culture fluid of the follicular granulosa cells is cultured containing 40-80 ng/mL of CNP or a variant thereof or analog thereof and a 3-7 ⁇ M HDAC inhibitor.
- the culture medium for culturing the follicular granule cells further comprises: 5-15% FBS, 0.01-0.5 mg/mL glutamine, 10-200 IU/mL penicillin, 10-200 IU/mL streptavidin Prime.
- the culture medium for culturing the follicular granule cells is 60 ng/mL CNP or a variant thereof or an analog thereof, 5 ⁇ M HDAC inhibitor, 10% FBS, 0.1 mg/ mL glutamine, 100 IU/mL penicillin and 100 IU/mL streptomycin in TCM199 broth.
- the present invention also provides an in vitro maturation culture solution which can be used for in vitro maturation culture of immature oocytes containing CNP or a variant thereof or an analog thereof and an HDAC inhibitor. It forms a medium for in vitro maturation of immature oocytes by contact with follicular granulosa cells.
- the HDAC inhibitor is selected from the group consisting of an HDAC1 inhibitor, an HDAC2 inhibitor, and a HDAC3 inhibitor.
- the HDAC inhibitor is an HDAC3 inhibitor; the HDAC3 inhibitor is selected from one or more of HDACi 4b, Entinostat (MS-275), BG45, RG2833 (RGFP109), RGFP966.
- the CNP is a C-type natriuretic peptide.
- the culture fluid contains 30-120 ng/mL CNP or a variant thereof or analog thereof and 1-10 ⁇ M HDAC inhibitor.
- the culture fluid contains 40-80 ng/mL of CNP or a variant thereof or analog thereof and a 3-7 ⁇ M HDAC inhibitor.
- the culture solution further comprises: 5-15% FBS, 0.01-0.5 mg/mL glutamine, 10-200 IU/mL penicillin, 10-200 IU/mL streptomycin or More species.
- the culture solution further comprises: 5-10% FBS, 0.05-0.2 mg/mL glutamine, 50-150 IU/mL penicillin, 50-150 IU/mL streptomycin. Or more.
- the culture solution further comprises: one or more of 10% FBS, 0.1 mg/mL glutamine, 100 IU/mL penicillin, and 100 IU/mL streptomycin.
- the culture broth is a 60 ng/mL CNP or variant thereof or an analog thereof, 5 ⁇ M HDAC inhibitor, 10% FBS, 0.1 mg/mL glutamine, 100 IU /mL penicillin and 100 IU/mL streptomycin in TCM199 broth.
- the present invention also provides a composition for in vitro maturation culture of immature oocytes consisting of CNP or a variant thereof or an analog thereof and an HDAC inhibitor.
- the composition can be used for in vitro maturation of immature oocytes by addition to a medium conventional in the art.
- the composition when used for in vitro maturation of immature oocytes, is in the medium at a concentration of 30-120 ng/mL of CNP or a variant thereof or analog thereof and 1- 10 ⁇ M HDAC inhibitor.
- the present invention provides a culture method for promoting in vitro maturation of an oocyte, which comprises the step of culturing an immature oocyte isolated from a subject with the aforementioned medium.
- the culturing method is specifically: collecting granulosa cells in the follicle of the subject, performing cell monolayer adherent culture with the above-mentioned in vitro maturation broth for use as a feeder layer cell, and then collecting in a raw foaming period.
- Oocytes cultured directly in mature medium containing feeder cells 20h-25h.
- the subject comprises humans, cows, pigs, sheep, rodents, and the like.
- the mature oocyte can be used for parthenogenetic activation, in vitro fertilization or single sperm injection, followed by embryo culture followed by embryo transfer for progestation or livestock production.
- the method specifically includes the following steps:
- the oocytes which are still in the foaming stage are cultured in the mature culture medium containing the feeder cells after the step 2).
- the present invention also provides the use of the aforementioned in vitro maturation medium for promoting in vitro maturation of immature oocytes.
- the present invention provides a composition comprising CNP or a variant thereof or an analogue thereof and an HDAC inhibitor, which is useful for promoting recovery of meiosis and developmental potential of immature oocytes after promotion of clinical female superovulation application.
- the present invention adopts the maturation of the oocytes which are still immature after treatment with gonadotropin for the first time, and improves the maturation rate and late development potential of the oocyte. Not only make full use of the oocyte resources after super-discharge, it improves the early embryo development ability after fertilization, and also increases the chance of success in assisting reproductive patients. Similar effects were also observed for the culture of immature oocytes in animals (in the case of rodent mice).
- the invention adopts granulosa cells as a feeding layer to induce oocyte maturation, and uses CNP or its variant or its analog and HDAC inhibitor to treat ovarian follicle-derived granulosa cells, and promotes oocyte development in granulosa cells.
- the expression and secretion of factors enhance the in vitro development of oocytes.
- the method can thus be a novel oocyte-promoting drug, and is popularized and applied in human assisted reproduction.
- the CNP involved in the present invention is an active peptide substance in the follicle, which has little toxic effect on oocytes, and the HDAC inhibitor has also proved to be very weak in toxicity to biological individuals and oocytes.
- Figure 1 shows that the HDACi inhibitor HDACi 4b promotes human oocyte maturation and developmental effects.
- FIG 1A shows that HDACi 4b promotes maturation of human GV stage oocytes to the second metaphase of meiosis (MII).
- 1B shows that the number of fertile eggs of the double pronucleus (2PN) after MSI stage oocyte fertilization by ICSI showed that the fertilization rates of the control and treatment groups were substantially the same.
- Figure 1C shows a significant increase in cleavage ability of HDACi 4b treated oocytes after fertilization.
- Figure 1D shows that the control group and HDACi 4b induced mature oocytes to be cultured in vitro to obtain blastocysts after fertilization.
- Figure 1E shows that blastocyst counting results show that HDACi 4b induces a significant increase in embryo developmental ability after maturation of mature oocytes.
- Figure 1F shows that inhibition of human ovarian granulosa cells can significantly promote AREG expression.
- Figure 1G shows that the cytoplasmic cell-specific protein OCT4 and the trophoblastic-specific protein CDX2 immunofluorescence results show that HDACi 4b induces a significant increase in the quality of mature oocytes to blastocysts.
- FIG. 2 shows that HDACi inhibitor HDACi 4b can significantly promote mouse oocyte maturation and developmental ability.
- Figure 2A shows that HDACi 4b can significantly promote oocyte meiotic recovery in mouse follicles under in vitro follicular model culture.
- Figure 2B shows that the oocyte recovery meiosis ability after HDACi 4b treatment was similar to that of the control group in in vitro maturation medium.
- FIG. 2C shows that HDACi 4b significantly promotes oocyte crossing the first meiosis and arrests in the second meiotic metaphase (MII).
- Figure 2D shows that the HDACi 4b-induced mature oocyte group has substantially the same cleavage ability as the control oocyte after fertilization.
- Figure 2E shows that the ability of mouse GV stage oocytes to develop to embryos after induction by HDACi 4b is significantly enhanced.
- Figure 2F shows that the ability of HDACi 4b-treated oocytes to develop into blastocysts after fertilization is significantly high.
- Figure 2G shows the results of blastocyst culture in the control and HDACi 4b treated groups.
- Figure 2H shows that blastocyst count results show a significant increase in the ability of GV stage oocytes to fertilize to blastocysts after treatment with HDACi 4b.
- Figure 2I shows that the inner cell mass-specific protein OCT4 immunofluorescence results show a significant increase in blastocyst quality of HDACi 4b-treated oocytes.
- the term "medium” is understood to mean a liquid environment that maintains and/or proliferates an oocyte or embryo.
- isolated is understood to mean that the oocyte is sometimes removed or purified (at least in part) from its natural environment.
- An example of an isolated oocyte is an oocyte or denuded oocyte isolated from a subject as part of a follicle, cumulus oocyte complex.
- variant is understood to mean an amino acid sequence having one or more amino acid changes. Variants may have a "conservative" change in which the substituted amino acid has similar structural or chemical properties of the substituted amino acid (eg, replacing the leucine with isoleucine). Variants may also have "non-conservative" changes in one or more amino acids (eg, replacing tryptophan with tryptophan) or deletions and/or insertions.
- analog is understood to mean a molecule having structural, regulatory or biochemical functions similar to a reference molecule, including biologically active fragments of the reference molecule.
- subject is understood to mean a woman, a female mammal, including primates, livestock (eg, horses, cows, sheep, pigs, goats), companion animals (eg, dogs, cats), laboratory test animals (eg, , mouse, rat, guinea pig) or veterinary meaningful animal.
- livestock eg, horses, cows, sheep, pigs, goats
- companion animals eg, dogs, cats
- laboratory test animals eg, mouse, rat, guinea pig
- the term "assisted reproduction” is understood to mean any technique involving the production of an implantable embryo, including techniques using in vitro cultured oocytes or embryos (eg, in vitro maturation of oocytes), in vitro fertilization (IVF; aspiration) Oocytes, fertilization in the laboratory and transfer of embryos into recipients), intrauterine gamete transplantation (GIFT; placement of oocytes and sperm in the fallopian tubes), transfer of fertilized oviducts (ZIFT; fertilized oocytes) Cells are placed in the fallopian tubes), tubal embryo transfer (TET; placing the split embryos in the fallopian tubes), oocytes and sperm intraperitoneal transplantation (POST; placing oocytes and sperm in the pelvic cavity) ), sperm intracytoplasmic injection (ICSI), testicular sperm extraction (TESE), microsurgical epididymal sperm aspiration (MESA), nuclear transfer, pluripotent stem cell expansion, and parthenogenic activ
- LH is critical for oocyte cytoplasmic maturation induced by somatic cells (Chen J, Torcia S, Xie F, Lin CJ, Cakmak H, Franciosi F, Horner K, Onodera C, Song JS, Cedars MI, Ramalho-Santos M, Conti M. Nat Cell Biol. 2013 Dec; 15(12): 1415-23. doi: 10.1038/ncb2873. Epub 2013 Nov 24).
- our experimental method is: in the in vitro culture environment, simulate the effect of LH in vivo on granulosa cells to promote oocyte maturation, in order to achieve the purpose of promoting oocyte maturation while improving its developmental potential.
- the results showed that in the presence of CNP, the oocyte nuclear maturation was inhibited for a short time, and by adding HDAC3 inhibitor to the culture medium with the granulated cell feeder layer, an LH-like effect was obtained, which promoted the secretion of EGF by the granulosa cells.
- Growth factors such as AREG, EREG, etc.
- the media, cultures and compositions in the various combinations of the invention may be packaged individually, in a multi-purpose or unit form, in a suitable container (usually sterile), such as in ampoules, vials or vials. These containers can be sealed after filling. Other additives may be included for the stability and/or application of the protein. Methods of packaging various components are known in the art.
- Oocytes and the medium are expected to not only improve the outcome of implant and assisted reproductive technologies, but also reduce implant failure, miscarriage, including spontaneous abortion, pre-eclampsia, intrauterine growth restriction, preterm birth, and Placental abruption.
- implant failure miscarriage, including spontaneous abortion, pre-eclampsia, intrauterine growth restriction, preterm birth, and Placental abruption.
- implantation may be facilitated by improving placental development and/or reducing the risk and/or likelihood of pregnancy complications to improve post-partum outcomes.
- the various forms of oocytes of the invention are human oocytes or mammalian oocytes.
- suitable mammals include primates, livestock (eg, horses, cows, sheep, pigs, goats), companion animals (eg, dogs, cats), laboratory test animals (eg, mice, rats, guinea pigs) .
- the oocyte is a human oocyte.
- the oocyte can be, for example, an oocyte that is part of a follicle, a part of a mound oocyte complex (COC), or can be a naked oocyte.
- COC mound oocyte complex
- the medium of the present invention is not only suitable for use in humans, but also for culturing oocytes and embryos of animals. Therefore, the present invention can be applied not only to human assisted reproductive technology, but also to assisted reproductive technologies of non-human animals, as well as other techniques for producing embryos in non-human animals, such as the use of parthenogenetic activation, nuclear transfer, and utilization of omnipotence. stem cell.
- a variant of CNP is a CNP molecular form having one or more natural amino acid changes in the amino acid sequence.
- the variant has greater than 75% homology to the native CNP at the amino acid level.
- the variant has greater than 90% homology to native CNP.
- the variant has greater than 95% homology to native CNP.
- Analogs of CNP are structures (i.e., structural analogs), regulatory functions (i.e., regulatory analogs) or biochemical functions (i.e., functional analogs) that are similar to CNP, including biologically active fragments of CNP.
- the CNP or a variant thereof or an analog thereof is in the culture medium, the culture solution or the group In a concentration of 30-120 ng/mL, in a specific embodiment, the concentration of the CNP or variant or analog thereof in the medium, culture solution or composition is 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 ng/ml.
- the HDAC inhibitor is in the medium, culture or composition at a concentration of 1-10 ⁇ M, and in particular embodiments, the HDAC inhibitor is in the medium, culture or composition
- concentration is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ⁇ M.
- FBS, HDACi 4b, CNP, glutamine, penicillin and streptomycin were dissolved in TCM-199 to prepare 10% FBS, 5 ⁇ M HDACi 4b, 60 ng/mL CNP, 0.1 mg/mL glutamine, 100 IU/
- the TCM-199 culture solution of mL penicillin and 100 IU/mL streptomycin is an in vitro maturation medium.
- FBS, HDACi 4b, CNP, glutamine, penicillin and streptomycin were dissolved in TCM-199 to prepare 5% FBS, 3 ⁇ M HDACi 4b, 80 ng/mL CNP, 0.05 mg/mL glutamine, 100 IU/
- the TCM-199 culture solution of mL penicillin and 100 IU/mL streptomycin is an in vitro maturation medium.
- FBS, HDACi 4b, CNP, glutamine, penicillin and streptomycin were dissolved in TCM-199 to prepare 15% FBS, 7 ⁇ M HDACi 4b, 40 ng/mL CNP, 0.2 mg/mL glutamine, 100 IU/
- the TCM-199 culture solution of mL penicillin and 100 IU/mL streptomycin is an in vitro maturation medium.
- the HDACi 4b and CNP were dissolved in TCM-199 to prepare a TCM-199 culture solution containing 5 ⁇ M HDACi 4b, 60 ng/mL CNP, which is an in vitro maturation medium.
- the HDACi 4b and CNP were dissolved in TCM-199 to prepare a TCM-199 culture solution containing 1 ⁇ M HDACi 4b, 100 ng/mL CNP, which is an in vitro mature culture solution.
- HDACi 4b and CNP were dissolved in TCM-199 to prepare 10 ⁇ M HDACi 4b.
- 30 ng/mL CNP TCM-199 broth is an in vitro mature broth.
- Granular cells and oocytes from different patients were taken at the hospital assisted reproductive center.
- the donor semen comes from the sperm center.
- Microdroplet culture of granulosa cells granulosa cells obtained by digesting the cumulus oocyte complex obtained by superovulation from a patient with hyaluronidase are centrifuged, and then in vitro mature broth of Examples 1-6 are separately placed for microfiltration. Drip adherent culture. The droplets are covered with paraffin oil. After the granulosa cells are attached, they can be used as an in vitro maturation medium 1-6 for immature oocytes.
- the oocyte obtained after superovulation of the patient is identified as immature, and is placed in an in vitro maturation medium 1 containing the feeder layer cells (that is, the medium obtained by using the in vitro maturation medium prepared in Example 1).
- an in vitro maturation medium 1 containing the feeder layer cells that is, the medium obtained by using the in vitro maturation medium prepared in Example 1).
- Microdroplet culture Each droplet was placed in 1-2 oocytes for a culture time of 20h-25h.
- the culture conditions were 37 ° C, air containing 5% CO 2 and 6% O 2 , humidity 100%; the developmental effect of immature oocytes after 1 microdrop culture in vitro maturation medium is shown in FIG. 1 .
- ICSI cytoplasmic sperm injection
- G-1PLUS Plantasset Culture medium
- G-1PLUS Plantasset Culture medium
- This culture is suitable for culturing embryos from fertilization (Day 0) to 8-cell.
- G-2PLUS (Vitrolife, Sweden) embryo culture broth was prepared in a 35 mm culture dish to make several 50 ⁇ L droplets. Cover the surface of the droplet with mineral oil. The culture is suitable for culturing embryos that have developed to 8 cells until the blastocyst stage.
- the maturity rate and early embryo development rate of human immature oocytes obtained by the method were compared with the conventional methods of the control group, as shown in Table 1.
- the experimental results showed that HDACi4b+CNP was cultured in vitro for 20h, and the cleavage rate and blastocyst rate of ICSI were significantly higher than that of the control group (P ⁇ 0.01).
- cleavage rate number of cleavage / number of oocytes
- blastocyst rate number of blastocysts / number of cleavage.
- the conventional mature medium is M199 medium.
- the preparation of the in vitro maturation medium 2-6 (that is, the medium obtained by using the in vitro maturation medium prepared in the examples 2-6) of the present invention also achieves the technical effect similar to that in the embodiment 8, that is,
- the addition of HDACi 4b+CNP to the conventional culture medium can significantly promote the maturation rate of immature oocytes, and also increase the cleavage rate and blastocyst development rate of the oocyte after fertilization to the control group. Times -3 times. And the effect is still valid after replacing the HDAC3 inhibitor type.
- Example 8 For specific culture methods, see Example 8, except that patient-derived granulosa cells are replaced with granulosa cells of C57/BL6J strain mice, and patient-derived oocytes are replaced with cumulus oocyte complexes derived from the strain-derived mouse. See Figure 2 for the in vitro development of mouse oocytes.
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Description
Claims (21)
- 一种培养基,其包含卵泡颗粒细胞和培养该卵泡颗粒细胞的培养液,其中培养该卵泡颗粒细胞的培养液中含有CNP或其变体或其类似物和HDAC抑制剂。
- 根据权利要求1所述的培养基,其中所述培养基通过培养该卵泡颗粒细胞的培养液培养卵泡颗粒细胞4-6h而获得。
- 根据权利要求1所述的培养基,其中所述HDAC抑制剂选自HDAC1抑制剂、HDAC2抑制剂和HDAC3抑制剂中的一种或更多种。
- 根据权利要求3所述的培养基,其中所述HDAC抑制剂为HDAC3抑制剂;所述HDAC3抑制剂选自HDACi 4b、Entinostat(MS-275)、BG45、RG2833(RGFP109)、RGFP966中的一种或更多种。
- 根据权利要求1所述的培养基,其中培养该卵泡颗粒细胞的培养液含有30-120ng/mL的CNP或其变体或其类似物和1-10μM的HDAC抑制剂。
- 根据权利要求5所述的培养基,其中培养该卵泡颗粒细胞的培养液含有40-80ng/mL的CNP或其变体或其类似物和3-7μM的HDAC抑制剂。
- 根据权利要求5或6所述的培养基,其中培养该卵泡颗粒细胞的培养液还含有:5-15%FBS,0.01-0.5mg/mL谷氨酰胺,10-200IU/mL青霉素,10-200IU/mL链霉素。
- 一种体外成熟培养液,其可用于未成熟卵母细胞的体外成熟培养,所述培养液中含有CNP或其变体或其类似物和HDAC抑制剂,其通过与卵泡颗粒细胞接触进而构成一种用于未成熟卵母细胞的体外成熟培养的培养基。
- 根据权利要求8所述的体外成熟培养液,其中所述HDAC抑制剂选自HDAC1抑制剂、HDAC2抑制剂和HDAC3抑制剂中的一种或更多种。
- 根据权利要求9所述的体外成熟培养液,其中所述HDAC抑制剂为HDAC3抑制剂;所述HDAC3抑制剂选自HDACi 4b、Entinostat(MS-275)、BG45、RG2833(RGFP109)、RGFP966中的一种或更多种。
- 根据权利要求8所述的体外成熟培养液,其中所述培养液含有30-120ng/mL的CNP或其变体或其类似物和1-10μM的HDAC抑制剂。
- 根据权利要求11所述的体外成熟培养液,其中所述培养液含有 40-80ng/mL的CNP或其变体或其类似物和3-7μM的HDAC抑制剂。
- 根据权利要求11或12所述的体外成熟培养液,其中所述培养液还含有:5-15%FBS,0.01-0.5mg/mL谷氨酰胺,10-200IU/mL青霉素,10-200IU/mL链霉素中的一种或更多种。
- 一种组合物,其用于未成熟卵母细胞的体外成熟培养,所述组合物由CNP或其变体或其类似物和HDAC抑制剂组成;所述组合物通过添加到本领域常规的培养基中来用于对未成熟卵母细胞的体外成熟培养。
- 根据权利要求14所述的组合物,其中所述组合物在培养基中的浓度为30-120ng/mL的CNP或其变体或其类似物和1-10μM的HDAC抑制剂。
- 一种促进卵母细胞体外成熟的培养方法,其包括采用权利要求1-7任一所述的培养基对从对象分离的未成熟卵母细胞培养的步骤。
- 根据权利要求16所述的促进卵母细胞体外成熟的培养方法,所述培养方法具体为:采集对象的卵泡中的颗粒细胞,用前述体外成熟培养液进行细胞单层贴壁培养以用做饲养层细胞,然后收取处于生发泡期的卵母细胞,直接在含饲养层细胞的成熟液中培养20h-25h。
- 根据权利要求16所述的促进卵母细胞体外成熟的培养方法,所述对象包括人、牛、猪、羊和啮齿类动物等。
- 根据权利要求17所述的促进卵母细胞体外成熟的培养方法,当所述对象为人时,所述方法具体包括如下步骤:1)对透明质酸酶消化后的颗粒细胞和卵母细胞分类采集;2)将颗粒细胞放在所述体外成熟培养液中进行贴壁培养;3)将消化后体外培养4-6h后,仍处于生发泡期的卵母细胞,放入步骤2)处理后的含饲养层细胞的成熟培养液中进行培养。
- 权利要求8-13任一所述体外成熟培养液在促进未成熟卵母细胞体外成熟的应用。
- 权利要求14或15任一所述组合物在制备用于促进临床女性超排后未成熟卵母细胞恢复减数分裂和提高发育潜能中的应用。
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