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CN118389410B - Composition and method for regulating in vitro embryo DNA methylation modification and application of composition and method in improving embryo development efficiency and quality - Google Patents

️Tue Oct 22 2024

具体实施方式DETAILED DESCRIPTION

现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为具体公开了该范围的上限和下限以及它们之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

除非另有说明，否则本文使用的所有技术和科学术语具有本发明所属领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

本文中，术语“胚胎”是由受精卵经发育而成的初期幼体。由于发育是一个连续的过程，因此，本文的胚胎包括任何发育过程或阶段生成的不同形态的幼体，一般是指附植前胚胎或着床前胚胎，例如2胞胚胎、4胞胚胎、8胞胚胎、桑椹胚、囊胚等。Herein, the term "embryo" refers to an early stage larvae developed from a fertilized egg. Since development is a continuous process, the embryo herein includes larvae of different forms generated in any developmental process or stage, generally referring to pre-implantation embryos or pre-implantation embryos, such as 2-cell embryos, 4-cell embryos, 8-cell embryos, morulae, blastocysts, etc.

本文中，术语“体外”是指在人工环境，例如在试管或反应容器中、在细胞培养物中、在培养皿中等发生，而不是在生物体(例如动物、植物或微生物)中发生的事件。As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (eg, an animal, plant, or microorganism).

本文中，术语“DNA甲基化水平”是指从胚胎获得的样品的DNA如基因组或其区域或基因的甲基化程度，即甲基基团添加到特定碱基上的程度。DNA甲基化或去甲基化是基因表达调控的重要机制，能够在不改变DNA序列的前提下改变遗传表现或发育进展。Herein, the term "DNA methylation level" refers to the degree of methylation of DNA, such as a genome or a region or gene thereof, of a sample obtained from an embryo, i.e., the degree to which methyl groups are added to specific bases. DNA methylation or demethylation is an important mechanism for regulating gene expression, which can change genetic expression or developmental progression without changing the DNA sequence.

本文中，术语“调控”，有时也可称作“调节”，是指任何改变甲基化水平行为，包括上调特定碱基的甲基化水平，或下调特定碱基的甲基化水平，还包括优化甲基化水平使其接近或达到某一标准。这些标准可以是人为设定的标准或者为天然状态的标准，如体内发育时胚胎的甲基化水平。Herein, the term "regulation", sometimes also referred to as "regulation", refers to any behavior that changes the methylation level, including increasing the methylation level of a specific base, or decreasing the methylation level of a specific base, and also includes optimizing the methylation level to make it close to or reach a certain standard. These standards can be artificially set standards or standards of natural states, such as the methylation level of an embryo during in vivo development.

本文中，术语“维生素C”或称作“抗坏血酸”，缩写“VitC”，其与多种疾病相关，并被世界卫生组织确定为基本药物清单中的基本药物。Herein, the term "vitamin C" or "ascorbic acid", abbreviated as "VitC", is associated with a variety of diseases and is identified as an essential medicine in the list of essential medicines by the World Health Organization.

本文中，术语“前体”是指任何物质、分子或实体，其在经化学或物理改变后能够充当或产生维生素C。前体可以以某种方式共价结合或螯合，并且在施用给受试者或靶细胞或组织之前、同时或之后释放或转化成活性成分，特别是维生素C。前体可以通过修饰化合物中存在的官能团来制备，修饰方式使得修饰物可以在常规操作中或在体内裂解为母体化合物。前体包括这样的化合物，其中羟基、氨基、巯基或羧基与当施用于哺乳动物受试者时遭受裂解以分别形成游离羟基、氨基、巯基或羧基的任何基团结合。Herein, the term "precursor" refers to any substance, molecule or entity that can act as or produce vitamin C after chemical or physical changes. The precursor can be covalently bound or chelated in some manner and released or converted into an active ingredient, particularly vitamin C, before, simultaneously or after administration to a subject or target cell or tissue. Precursors can be prepared by modifying the functional groups present in the compound in a manner that allows the modification to be cleaved into the parent compound in routine manipulation or in vivo. Precursors include compounds in which hydroxyl, amino, sulfhydryl or carboxyl groups are bound to any group that is cleaved to form free hydroxyl, amino, sulfhydryl or carboxyl groups, respectively, when administered to a mammalian subject.

本文中，术语“RELN”，表示络丝蛋白或其活成片段、核酸。其中，络丝蛋白是指全长形式的络丝蛋白或具有或含有完整序列的络丝蛋白，还包括与人络丝蛋白具有同源和/或直系同源的蛋白或同工型。活性片段是指全长形式络丝蛋白的一部分，即具有活性的肽、多肽等。此处，络丝蛋白可以天然分离得到，也可通过人工合成得到，包括基因工程或生物技术、化学合成方法得到。在分离的情况下，络丝蛋白的来源不限定，可以是与受试者或其组织、细胞相同来源，也可以是不同来源，对此不限定。As used herein, the term "RELN" refers to reelin or its active fragments or nucleic acids. Reelin refers to a full-length reelin or a reelin having or containing a complete sequence, and also includes proteins or isoforms that are homologous and/or orthologous to human reelin. An active fragment refers to a portion of the full-length reelin, i.e., an active peptide or polypeptide. Here, reelin can be isolated from nature or obtained by artificial synthesis, including genetic engineering or biotechnology, or chemical synthesis methods. In the case of separation, the source of reelin is not limited, and may be the same source as the subject or its tissues or cells, or may be a different source, and there is no limitation on this.

本文中，术语“FGF2”或“bFGF”，表示成纤维细胞生长因子II(有时也称作“碱性成纤维细胞生长因子”，表示成纤维细胞生长因子II或其活成片段、核酸。其中，纤维细胞生长因子II是指全长形式的纤维细胞生长因子II或具有或含有完整序列的纤维细胞生长因子II，还包括与人纤维细胞生长因子II具有同源和/或直系同源的蛋白或同工型。此处的纤维细胞生长因子II可以天然分离得到，也可通过人工合成得到，包括基因工程或生物技术、化学合成方法得到。在分离的情况下，纤维细胞生长因子II的来源不限定，可以是与受试者或其组织、细胞相同来源，也可以是不同来源，对此不限定。As used herein, the term "FGF2" or "bFGF" refers to fibroblast growth factor II (sometimes also referred to as "basic fibroblast growth factor"), and refers to fibroblast growth factor II or its active fragments or nucleic acids. Fibroblast growth factor II refers to the full-length form of fibroblast growth factor II or fibroblast growth factor II having or containing the complete sequence, and also includes proteins or isoforms that are homologous and/or orthologous to human fibroblast growth factor II. The fibroblast growth factor II herein can be isolated from nature or obtained by artificial synthesis, including genetic engineering or biotechnology, or chemical synthesis methods. In the case of isolation, the source of fibroblast growth factor II is not limited, and can be the same source as the subject or its tissues or cells, or a different source, and there is no limitation on this.

本文中，术语“核酸”包括DNA和RNA或其修饰形式，包括多核苷酸、寡核苷酸。优选能够产生活性蛋白如RELN和FGF2的mRNA。此处，修饰形式是指核酸的组分，即糖、碱基和磷酸酯结构的一或多个成份，与天然的成份不一样，优选为不同于产生自人体的天然成份。核苷替代物是这样一些分子，其中核糖磷酸酯主链被非核糖磷酸酯构造所替代，该构造使碱基处于正确的空间关系，以便杂交与所见到的与核糖磷酸酯主链的杂交基本相似，例如，非带电的核糖磷酸酯主链的类似物。Herein, the term "nucleic acid" includes DNA and RNA or modified forms thereof, including polynucleotides, oligonucleotides. Preferably, the mRNA capable of producing active proteins such as RELN and FGF2 is used. Here, the modified form refers to the components of nucleic acid, i.e. one or more components of sugar, base and phosphate structure, which are different from natural components, preferably different from natural components produced from human body. Nucleoside substitutes are molecules in which the ribose phosphate backbone is replaced by a non-ribose phosphate structure, which places the base in the correct spatial relationship, so that hybridization is substantially similar to that seen with the ribose phosphate backbone, for example, an analog of a non-charged ribose phosphate backbone.

本文中，术语“工作浓度”也称作“使用浓度”，是指使调节剂与胚胎接触并处理有效处理时的初始浓度。浓度单位不限定，可以是例如ng/ml、μg/ml、mg/ml等。Herein, the term "working concentration" is also referred to as "use concentration", which refers to the initial concentration when the regulator is brought into contact with the embryo and effectively treated. The concentration unit is not limited, and can be, for example, ng/ml, μg/ml, mg/ml, etc.

[组合物][Composition]

本发明的第一方面，提供一种体外调控胚胎DNA甲基化水平的组合物，本文有时简称为“本发明的组合物”，其包括不同调节剂组合，其中调节剂选自维生素C或其前体、RELN和FGF2中的两种以上。本发明的调节剂组合中各调节剂相互作用，产生协同效应，与单独一种调节剂相比，组合后大大提升调控效果。本发明的组合物形式不限定，可以是固体如干燥粉末，也可以是液体如溶液形式。The first aspect of the present invention provides a composition for regulating embryonic DNA methylation levels in vitro, sometimes referred to herein as the "composition of the present invention", which includes a combination of different regulators, wherein the regulators are selected from two or more of vitamin C or its precursor, RELN and FGF2. The regulators in the regulator combination of the present invention interact with each other to produce a synergistic effect, and the combination greatly improves the regulatory effect compared to a single regulator. The form of the composition of the present invention is not limited, and can be a solid such as a dry powder, or a liquid such as a solution.

在某些实施方案中，本发明的组合物可以是上述调节剂的组合，即除调节剂外不包含除避不可免的杂质外的任何成分。本发明的组合物中各调节剂可以混合形式存在，也可以是两种以上的调节剂单独存在的形态存在。在使用时单独存在的调节剂可以预先混合再使用，也可以单独同时或分别依次使用各调节剂。In certain embodiments, the composition of the present invention may be a combination of the above-mentioned regulators, i.e., it does not contain any component other than the regulators except for the inevitable impurities. The regulators in the composition of the present invention may exist in a mixed form, or two or more regulators may exist in a form of separate existence. When used, the separate regulators may be pre-mixed and then used, or each regulator may be used separately, simultaneously or separately in sequence.

在某些实施方案中，本发明的组合物包含上述调节剂组合，且除此之外进一步包含其他成分。其他成分的组成或类型不限定，可根据需要自由选择。此类其他成分可以选择已知的任何成分，特别是胚胎培养相关的试剂、组合物，如培养基、培养液或其添加剂等。In certain embodiments, the composition of the present invention comprises the above-mentioned combination of regulators, and further comprises other ingredients in addition. The composition or type of other ingredients is not limited and can be freely selected as needed. Such other ingredients can select any known ingredients, particularly reagents and compositions related to embryo culture, such as culture medium, culture fluid or its additives, etc.

在某些实施方案中，本发明的调节剂组合为维生素C或其前体和RELN的组合。优选地，维生素C与RELN的重量比为800-1500:1，例如850:1、900:1、950:1、1000:1、1050:1、1100:1、1150:1、1200:1、1250:1、1300:1、1350:1、1400:1、1450:1等。在上述范围内能够实现维生素C与RELN两者的有效协同，大大提升调控作用。In certain embodiments, the regulator combination of the present invention is a combination of vitamin C or its precursor and RELN. Preferably, the weight ratio of vitamin C to RELN is 800-1500:1, such as 850:1, 900:1, 950:1, 1000:1, 1050:1, 1100:1, 1150:1, 1200:1, 1250:1, 1300:1, 1350:1, 1400:1, 1450:1, etc. Within the above range, effective synergy between vitamin C and RELN can be achieved, greatly improving the regulatory effect.

在某些实施方案中，本发明的调节剂组合为维生素C或其前体和FGF2的组合。优选地，维生素C与FGF2的重量比为50-500:1，例如55:1、60:1、65:1、70:1、75:1、80:1、85:1、90:1、95:1、100:1、110:1、120:1、130:1、140:1、150:1、160:1、170:1、180:1、190:1、200:1、210:1、220:1、230:1、240:1、250:1、260:1、270:1、280:1、290:1、300:1、320:1、340:1、360:1、380:1、400:1、420:1、440:1、460:1、480:1等。在上述范围内能够实现维生素C与FGF2两者的有效协同，大大提升调控作用。In certain embodiments, the modulator combination of the present invention is a combination of vitamin C or its precursor and FGF2. Preferably, the weight ratio of vitamin C to FGF2 is 50-500:1, for example, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1 , 190:1, 200:1, 210:1, 220:1, 230:1, 240:1, 250:1, 260:1, 270:1, 280:1, 290:1, 300:1, 320:1, 340:1, 360:1, 380:1, 400:1, 420:1, 440:1, 460:1, 480:1, etc. Within the above range, effective synergy between vitamin C and FGF2 can be achieved, greatly enhancing the regulatory effect.

在某些实施方案中，本发明的调节剂组合为RELN和FGF2的组合。优选地，RELN与FGF2的重量比为1:1-10，例如1:1.1、1:1.2、1:1.5、1:1.8、1:2.0、1:2.5、1:3、1:3.5、1:4、1:4.5、1:5、1:5.5、1:6、1:6.5、1:7、1:7.5、1:8、1:8.5、1:9等。在上述范围内能够实现RELN与FGF2两者的有效协同，大大提升调控作用。In certain embodiments, the modulator combination of the present invention is a combination of RELN and FGF2. Preferably, the weight ratio of RELN to FGF2 is 1:1-10, such as 1:1.1, 1:1.2, 1:1.5, 1:1.8, 1:2.0, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, etc. Within the above range, effective synergy between RELN and FGF2 can be achieved, greatly enhancing the regulatory effect.

在某些实施方案中，本发明的维生素C或其前体的量能够使维生素C在使用时的工作浓度达到0.5-5000μg/ml。只要能够使维生素C在使用时达到上述范围，则对于组合物中的浓度或量不限定。在组合物为溶液且作为添加成分的情况下，维生素C或其前体在组合物中的浓度可以工作浓度，从而当将组合物添加到胚胎培养液中时，能够稀释达到上述工作浓度。在组合物为溶液且直接用作培养液的情况下，维生素C或其前体在组合物中的浓度通常基本与工作浓度相等。维生素C的工作浓度优选1-4000μg/ml，如1.5μg/ml、2μg/ml、2.5μg/ml、3μg/ml、3.5μg/ml、4μg/ml、4.5μg/ml、5μg/ml、5.5μg/ml、6μg/ml、6.5μg/ml、7μg/ml、7.5μg/ml、8μg/ml、8.5μg/ml、9μg/ml、9.5μg/ml、10μg/ml、11μg/ml、12μg/ml、13μg/ml、14μg/ml、15μg/ml、16μg/ml、17μg/ml、18μg/ml、19μg/ml、20μg/ml、21μg/ml、22μg/ml、25μg/ml、30μg/ml、35μg/ml、40μg/ml、50μg/ml、60μg/ml、70μg/ml、80μg/ml、90μg/ml、100μg/ml、120μg/ml、140μg/ml、160μg/ml、180μg/ml、200μg/ml、220μg/ml、240μg/ml、260μg/ml、280μg/ml、300μg/ml、350μg/ml、400μg/ml、450μg/ml、500μg/ml、550μg/ml、600μg/ml、650μg/ml、700μg/ml、750μg/ml、800μg/ml、900μg/ml、1000μg/ml、1500μg/ml、2000μg/ml、2500μg/ml、3000μg/ml、3500μg/ml、4000μg/ml、4500μg/ml。In certain embodiments, the amount of vitamin C or its precursor of the present invention can make the working concentration of vitamin C reach 0.5-5000 μg/ml when used. As long as vitamin C can reach the above range when used, the concentration or amount in the composition is not limited. In the case where the composition is a solution and is used as an added component, the concentration of vitamin C or its precursor in the composition can be a working concentration, so that when the composition is added to the embryo culture medium, it can be diluted to reach the above working concentration. In the case where the composition is a solution and is directly used as a culture medium, the concentration of vitamin C or its precursor in the composition is usually substantially equal to the working concentration. The working concentration of vitamin C is preferably 1-4000 μg/ml, such as 1.5 μg/ml, 2 μg/ml, 2.5 μg/ml, 3 μg/ml, 3.5 μg/ml, 4 μg/ml, 4.5 μg/ml, 5 μg/ml, 5.5 μg/ml, 6 μg/ml, 6.5 μg/ml, 7 μg/ml, 7.5 μg/ml, 8 μg/ml, 8.5 μg/ml, 9 μg/ml, 9 .5μg/ml, 10μg/ml, 11μg/ml, 12μg/ml, 13μg/ml, 14μg/ml, 15μg/ml, 16μg/ml, 17μg/ml, 18μg/ml, 19μg/ml, 20μg/ml, 21μg/ml, 22μg/ml, 25μg/ml, 30μg/ml, 35μg/ml, 40 μg/ml, 50μg/ml, 60 μg/ml, 70μg/ml, 80μg/ml, 90μg/ml, 100μg/ml, 120μg/ml, 140μg/ml, 160μg/ml, 180μg/ml, 200μg/ml, 220μg/ml, 240μg/ml, 260μg/ml, 280μg/ml, 300μg/ml, 350μg/ml, 400μg/ml, 450μg/m l, 500μg/ml, 550μg/ml, 600μg/ml, 650μg/ml, 700μg/ml, 750μg/ml, 800μg/ml, 900μg/ml, 1000μg/ml, 1500μg/ml, 2000μg/ml, 2500μg/ml, 3000μg/ml, 3500μg/ml, 40 00μg/ml, 4500μg/ml.

在某些实施方案中，本发明的FGF2的量能够使FGF2的工作浓度达到0.001-5μg/ml。只要能够使FGF2在使用时达到上述范围，则对于组合物中FGF2的浓度或量不限定。在组合物为溶液且作为添加成分的情况下，FGF2在组合物中的浓度可以工作浓度，从而当将组合物添加到胚胎培养液中时，能够稀释达到上述工作浓度。在组合物为溶液且直接用作培养液的情况下，FGF2在组合物中的浓度通常基本与工作浓度相等。FGF2的工作浓度优选0.05-5μg/ml，如0.06μg/ml、0.07μg/ml、0.08μg/ml、0.09μg/ml、0.1μg/ml、0.15μg/ml、0.20μg/ml、0.25μg/ml、0.3μg/ml、0.35μg/ml、0.4μg/ml、0.45μg/ml、0.5μg/ml、0.55μg/ml、0.6μg/ml、0.65μg/ml、0.7μg/ml、0.75μg/ml、0.8μg/ml、0.85μg/ml、0.9μg/ml、0.95μg/ml、1μg/ml、1.5μg/ml、2μg/ml、2.5μg/ml、3μg/ml、3.5μg/ml、4μg/ml、4.5μg/ml、5μg/ml。In certain embodiments, the amount of FGF2 of the present invention can make the working concentration of FGF2 reach 0.001-5 μg/ml. As long as FGF2 can reach the above range when used, the concentration or amount of FGF2 in the composition is not limited. When the composition is a solution and is used as an added component, the concentration of FGF2 in the composition can be a working concentration, so that when the composition is added to the embryo culture medium, it can be diluted to reach the above working concentration. When the composition is a solution and is directly used as a culture medium, the concentration of FGF2 in the composition is usually substantially equal to the working concentration. The working concentration of FGF2 is preferably 0.05-5 μg/ml, such as 0.06 μg/ml, 0.07 μg/ml, 0.08 μg/ml, 0.09 μg/ml, 0.1 μg/ml, 0.15 μg/ml, 0.20 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.5 5μg/ml, 0.6μg/ml, 0.65μg/ml, 0.7μg/ml, 0.75μg/ml, 0.8μg/ml, 0.85μg/ml, 0.9μg/ml, 0.95μg/ml, 1μg/ml, 1.5μg/ml, 2μg/ml, 2.5μg/ml, 3μg/ml, 3.5μg/ml, 4μg/ml, 4.5μg/ml, 5μg/ml.

在某些实施方案中，本发明的RELN的量能够使RELN的工作浓度达到0.001-5μg/ml。RELN的工作浓度优选0.05-5μg/ml，如0.06μg/ml、0.07μg/ml、0.08μg/ml、0.09μg/ml、0.1μg/ml、0.15μg/ml、0.20μg/ml、0.25μg/ml、0.3μg/ml、0.35μg/ml、0.4μg/ml、0.45μg/ml、0.5μg/ml、0.55μg/ml、0.6μg/ml、0.65μg/ml、0.7μg/ml、0.75μg/ml、0.8μg/ml、0.85μg/ml、0.9μg/ml、0.95μg/ml、1μg/ml、1.5μg/ml、2μg/ml、2.5μg/ml、3μg/ml、3.5μg/ml、4μg/ml、4.5μg/ml、5μg/ml。In certain embodiments, the amount of RELN of the present invention can make the working concentration of RELN reach 0.001-5 μg/ml. The working concentration of RELN is preferably 0.05-5 μg/ml, such as 0.06 μg/ml, 0.07 μg/ml, 0.08 μg/ml, 0.09 μg/ml, 0.1 μg/ml, 0.15 μg/ml, 0.20 μg/ml, 0.25 μg/ml, 0.3 μg/ml, 0.35 μg/ml, 0.4 μg/ml, 0.45 μg/ml, 0.5 μg/ml, 0.5 5μg/ml, 0.6μg/ml, 0.65μg/ml, 0.7μg/ml, 0.75μg/ml, 0.8μg/ml, 0.85μg/ml, 0.9μg/ml, 0.95μg/ml, 1μg/ml, 1.5μg/ml, 2μg/ml, 2.5μg/ml, 3μg/ml, 3.5μg/ml, 4μg/ml, 4.5μg/ml, 5μg/ml.

[体外调控胚胎DNA甲基化水平的方法][Methods for regulating embryonic DNA methylation levels in vitro]

本发明的第二方面，提供体外调控胚胎DNA甲基化水平的方法，其包括在体外使胚胎与第一方面所述的组合物接触的步骤。The second aspect of the present invention provides a method for regulating embryonic DNA methylation levels in vitro, comprising the step of contacting the embryo with the composition of the first aspect in vitro.

在某些实施方案中，本发明的接触在液体中进行，即使胚胎在含有调节剂组合的培养液中进行处理。In certain embodiments, the contacting of the present invention is performed in liquid, ie, the embryos are treated in a culture medium containing a combination of modulators.

在某些实施方案中，本发明的调控包括提高胚胎DNA中5hmC的量。其中，5hmC是指5-羟甲基胞嘧啶，提高胚胎DNA中5hmC的量是指DNA碱基中5-羟甲基胞嘧啶的相对量(特别是相对于未处理组或单一调节剂的处理组)或绝对量得到提升。In certain embodiments, the regulation of the present invention includes increasing the amount of 5hmC in embryonic DNA. Wherein, 5hmC refers to 5-hydroxymethylcytosine, and increasing the amount of 5hmC in embryonic DNA means that the relative amount (especially relative to an untreated group or a group treated with a single regulator) or the absolute amount of 5-hydroxymethylcytosine in DNA bases is increased.

在某些实施方案中，本发明的调控包括降低5mC的量。其中，5mC是指5-甲基胞嘧啶，降低5mC的量是指DNA碱基中5-甲基胞嘧啶的相对量(特别是相对于未处理组或单一调节剂的处理组)或绝对量得到提升。In certain embodiments, the regulation of the present invention includes reducing the amount of 5mC, wherein 5mC refers to 5-methylcytosine, and reducing the amount of 5mC means that the relative amount (especially relative to the untreated group or the group treated with a single regulator) or the absolute amount of 5-methylcytosine in the DNA base is increased.

在某些实施方案中，本发明的调控是指提高5hmC/5mC的比值。其中，5hmC相对于5mC增加或提高，包括在5hmC量不变的情况下，5mC量变少或降低；包括在5mC量不变的情况下，5hmC量变多或提高；还包括5hmC量变多或提高，同时5mC量变少或降低。In certain embodiments, the regulation of the present invention refers to increasing the ratio of 5hmC/5mC. The increase or increase of 5hmC relative to 5mC includes the decrease or reduction of 5mC when the amount of 5hmC remains unchanged; the increase or increase of 5hmC when the amount of 5mC remains unchanged; and the increase or increase of 5hmC while the amount of 5mC decreases or decreases.

在某些实施方案中，本发明的调控是指使5hmC/5mC的比值接近体内发育胚胎的水平。与体内发育的胚胎相比，在体外培养的胚胎中DNA甲基化异常，特别是5hmC/5mC比值降低。本发明的组合物能够使5hmC/5mC比值接近体内发育胚胎的对应水平，或基本上与体内发育胚胎的对应水平一致。此处“接近”是指与体内发育胚胎中DNA甲基化水平相差经10%以内，优选6%以内，更优选5%以内，如4%以内、3%以内、2%以内、1%以内、0.5%以内、0.1%以内、甚至0.05%以内。此处“基本上”是指与体内发育胚胎的对应水平达到90%以上一致，优选92%以上一致，更优选95%以上一致，如96%以上、97%以上、98%以上、99%以上、99.5%以上99.8%以上、99.9%以上。In certain embodiments, the regulation of the present invention refers to making the ratio of 5hmC/5mC close to the level of the embryo developed in vivo. Compared with the embryo developed in vivo, the DNA methylation in the embryo cultured in vitro is abnormal, especially the 5hmC/5mC ratio is reduced. The composition of the present invention can make the 5hmC/5mC ratio close to the corresponding level of the embryo developed in vivo, or substantially consistent with the corresponding level of the embryo developed in vivo. Here, "close" means that the difference with the DNA methylation level in the embryo developed in vivo is within 10%, preferably within 6%, more preferably within 5%, such as within 4%, within 3%, within 2%, within 1%, within 0.5%, within 0.1%, and even within 0.05%. Here, "substantially" means that the corresponding level of the embryo developed in vivo is consistent with more than 90%, preferably more than 92%, more preferably more than 95%, such as more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, more than 99.8%, and more than 99.9%.

[用于改善体外胚胎发育的方法][Methods for improving in vitro embryo development]

本发明的第三方面，提供用于改善体外胚胎发育的方法，其包括在体外使胚胎与第一方面所述的组合物接触的步骤。The third aspect of the present invention provides a method for improving in vitro embryo development, comprising the step of contacting the embryo with the composition of the first aspect in vitro.

本发明的改善包括提高胚胎发育效率和/或质量，包括提高体外的卵裂率、得到的囊胚数量或囊胚率以及移植后的附植率和生产的胎儿数。The improvements of the present invention include improving the efficiency and/or quality of embryo development, including improving the in vitro cleavage rate, the number of blastocysts obtained or the blastocyst rate, the implantation rate after transplantation, and the number of fetuses produced.

[用途][use]

本发明的第四方面，提供第一方面所述组合物在动物遗传改良、良种扩繁、体外胚胎生产中的用途。组合物及其调节剂已在第一方面进行了详细说明，在此不再赘述。The fourth aspect of the present invention provides the use of the composition of the first aspect in animal genetic improvement, breeding and in vitro embryo production. The composition and its regulator have been described in detail in the first aspect and will not be repeated here.

在某些实施方案中，本发明的动物是指非人类动物。在某些实施方案中，非人类动物是哺乳动物(例如，啮齿动物、小鼠、大鼠、兔、猴、犬、猫、羊、牛、灵长类动物或猪)。在一些实施方案中，动物包括但不限于哺乳动物、禽、爬行动物、两栖动物、鱼和蠕虫。在一些实施方案中，动物是转基因动物、基因工程化动物或克隆。在某些实施方案中，本发明的动物为家畜，包括猪、牛、羊。In certain embodiments, the animal of the present invention refers to a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, or a pig). In some embodiments, animals include but are not limited to mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, animals are transgenic animals, genetically engineered animals, or clones. In certain embodiments, animals of the present invention are livestock, including pigs, cows, and sheep.

在某些实施方案中，本发明的遗传改良和良种扩繁包括依赖于体外胚胎生产技术进行的遗传改良或扩繁。In certain embodiments, the genetic improvement and enrichment of the present invention include genetic improvement or enrichment relying on in vitro embryo production technology.

实施例1Example 1

本实施例为VitC、RELN、FGF2的联合施用在改善小鼠体外受精胚胎DNA甲基化修饰以及提高小鼠体外受精胚胎发育效率和质量中的应用。This example is the application of the combined administration of VitC, RELN and FGF2 in improving DNA methylation modification of mouse in vitro fertilization embryos and improving the development efficiency and quality of mouse in vitro fertilization embryos.

1、实验动物及试剂：1. Experimental animals and reagents:

若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。实施例中培养液KSOM+AA购自美国Millipore公司，VitC、RELN以及FGF2均购自美国Sigma-Aldrich公司，ICR小鼠购自斯贝福(北京)生物技术有限公司，M2培养基购自中科迈晨(北京)科技有限公司。If not otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The culture medium KSOM+AA in the examples was purchased from Millipore, USA, VitC, RELN and FGF2 were purchased from Sigma-Aldrich, USA, ICR mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., and M2 culture medium was purchased from Zhongke Maichen (Beijing) Technology Co., Ltd.

2、实验方法2. Experimental methods

(1)实验分组(1) Experimental groups

体内组：体内受精组；In vivo group: in vivo fertilization group;

对照组：正常体外受精组；Control group: normal in vitro fertilization group;

处理组1：VitC单独施用组；Treatment group 1: VitC alone administration group;

处理组2：FGF2单独施用组；Treatment group 2: FGF2 alone administration group;

处理组3：RELN单独施用组；Treatment group 3: RELN alone administration group;

处理组4：VitC和FGF2联合施用组；Treatment group 4: VitC and FGF2 combined administration group;

处理组5：VitC和RELN联合施用组Treatment group 5: VitC and RELN combined administration group

处理组6：RELN和FGF2联合施用组；Treatment group 6: RELN and FGF2 combined administration group;

处理组7：VitC、RELN、FGF2联合施用组。Treatment group 7: VitC, RELN, and FGF2 combined administration group.

(2)小鼠体内受精胚胎收集(2) Collection of fertilized embryos in mice

选取健康状况良好的8周龄雌性小鼠，腹腔注射5 IU的孕马血清促性腺激素(PMSG)进行同期发情处理，48 h后腹腔注射5 IU人绒毛膜促性腺激素(hCG)进行超数排卵处理。然后将雌鼠与10周龄雄鼠合笼，第二天早上将带有阴道栓的雌鼠单独放在鼠笼中，默认受精时间为夜间24时，此时胚胎记为E0.5。收集囊胚时，在注射hCG96 h后，外科手术法打开雌性小鼠腹腔，剪取子宫置于M2培养液内，使用冲卵针冲取囊胚。Eight-week-old female mice in good health were selected and injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin (PMSG) for estrus synchronization. 48 hours later, 5 IU of human chorionic gonadotropin (hCG) was injected intraperitoneally for superovulation. The female mice were then caged with 10-week-old male mice. The female mice with vaginal plugs were placed alone in the mouse cage the next morning. The default fertilization time was 24:00 at night, and the embryos at this time were recorded as E0.5. When collecting blastocysts, 96 hours after the injection of hCG, the abdominal cavity of the female mice was opened surgically, the uterus was cut and placed in M2 culture medium, and the blastocysts were flushed out using an egg flushing needle.

(3)小鼠体外受精胚胎收集(3) Mouse in vitro fertilization embryo collection

以8周龄青年母鼠为实验材料，每只母鼠腹腔注射5 IU孕马血清促性腺激素(PMSG)，48 h后每只母鼠腹腔注射5 IU人绒毛膜促性腺激素(hCG)，12 h后断颈处死母鼠，取出输卵管，置于预热的37℃ M2中，用1 ml注射器将输卵管壶腹部的COCs(卵丘-卵母细胞复合物)划入受精滴中，放入CO₂培养箱平衡30分钟，然后将8周龄的青年公鼠断颈处死，剪取附睾，用镊子将精子从附睾轻轻挤入获能滴中，放入CO₂培养箱获能1 h。精子获能后，用移液枪吸取适量的精液加入到含COCs的受精滴中，使精子与COCs孵育4 h，之后根据极体排出情况挑选受精卵用于体外培养，在体外培养后96 h收取囊胚。Eight-week-old young female mice were used as experimental materials. Each female mouse was intraperitoneally injected with 5 IU pregnant mare serum gonadotropin (PMSG). After 48 hours, each female mouse was intraperitoneally injected with 5 IU human chorionic gonadotropin (hCG). After 12 hours, the female mice were killed by cervical dislocation, and the oviducts were removed and placed in preheated 37℃ M2. The COCs (cumulus oocyte complex) in the ampulla of the oviduct were drawn into the fertilization drop with a 1 ml syringe and placed in a CO ₂ incubator for equilibration for 30 minutes. Then, the 8-week-old young male mice were killed by cervical dislocation, the epididymis was cut, and the sperm was gently squeezed from the epididymis into the capacitation drop with forceps, and placed in a CO ₂ incubator for capacitation for 1 hour. After sperm capacitation, an appropriate amount of semen was pipetted with a pipette and added to the fertilization drop containing COCs. The sperm and COCs were incubated for 4 hours. After that, the fertilized eggs were selected for in vitro culture according to the discharge of polar bodies, and the blastocysts were collected 96 hours after in vitro culture.

(4)胚胎5-羟甲基胞嘧啶(5-hmC)和5-甲基胞嘧啶(5-mC)免疫荧光染色(4) Immunofluorescence staining of embryonic 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC)

将步骤(2)获得的胚胎，用0.1% PBS-PVA清洗三次，用酸性Tyrode溶液(T1788，美国sigma公司)去除透明带，然后将胚胎转移到4%多聚甲醛固定液中室温固定1 h，之后用含0.5%Triton X-100的DPBS(杜尔贝科磷酸盐缓冲液)室温下通透1 h，之后用0.1%PBS-PVA清洗一次，之后用4M HCl中处理20分钟，之后用100 mM Tris-HCl中处理10分钟，之后用含1%BSA的DPBS溶液4℃过夜封闭胚胎。第二天用一级抗体(5-羟甲基胞嘧啶和5-甲基胞嘧啶抗体)室温孵育1 h，之后用含0.5%Triton X-100的DPBS室温下清洗三次，之后用二级抗体室温孵育1 h。最后，用DAPI(4',6-二脒基-2-苯基吲哚)孵育15分钟以浸染细胞核，在BX51显微镜下观察荧光信号、拍照，使用Image J软件统计5-羟甲基胞嘧啶和5-甲基胞嘧啶的荧光强度，计算比值。The embryos obtained in step (2) were washed three times with 0.1% PBS-PVA, and the zona pellucida was removed with acidic Tyrode solution (T1788, Sigma, USA). The embryos were then transferred to 4% paraformaldehyde fixative and fixed at room temperature for 1 h. They were then permeabilized with DPBS (Dulbecco's phosphate buffered saline) containing 0.5% Triton X-100 at room temperature for 1 h. They were then washed once with 0.1% PBS-PVA, treated with 4M HCl for 20 minutes, treated with 100 mM Tris-HCl for 10 minutes, and blocked with DPBS solution containing 1% BSA at 4°C overnight. The next day, the embryos were incubated with primary antibodies (5-hydroxymethylcytosine and 5-methylcytosine antibodies) at room temperature for 1 h, washed three times with DPBS containing 0.5% Triton X-100 at room temperature, and incubated with secondary antibodies at room temperature for 1 h. Finally, the cells were incubated with DAPI (4',6-diamidino-2-phenylindole) for 15 min to stain the cell nucleus, and the fluorescence signals were observed and photographed under a BX51 microscope. The fluorescence intensities of 5-hydroxymethylcytosine and 5-methylcytosine were counted using Image J software, and the ratio was calculated.

其中，所用的一级抗体如下：5-羟甲基胞嘧啶抗体(1：500稀释，36769，ActiviteMotif，美国)，5-甲基胞嘧啶抗体(1：250稀释度，36649，Activite Motif，美国)，使用的二级抗体如下：Alexafluor 594羊抗兔抗体(1：1000稀释，美国Invitrogen，A-11034)，Alexafluor 488羊抗鼠抗体(1：1000稀释，美国Invitrogen，A-11030)。Among them, the primary antibodies used were as follows: 5-hydroxymethylcytosine antibody (1:500 dilution, 36769, Activite Motif, USA), 5-methylcytosine antibody (1:250 dilution, 36649, Activite Motif, USA), and the secondary antibodies used were as follows: Alexafluor 594 goat anti-rabbit antibody (1:1000 dilution, Invitrogen, USA, A-11034), Alexafluor 488 goat anti-mouse antibody (1:1000 dilution, Invitrogen, USA, A-11030).

(5)小鼠囊胚子宫移植(5) Mouse blastocyst uterine transplantation

在体外受精的当天，选择自然发情的青年雌鼠与输精管切除的结扎青年雄鼠交配。第二天早上，有阴道栓的雌鼠视为假孕受体，视为第0.5天。将步骤(2)获得的第4.5天囊胚进行胚胎移植，选取6个发育良好的囊胚移植到假孕受体的两侧子宫角位置。在胚胎第19.5天(胚胎移植的14天)，获取胎儿以评估每个胚胎的发育能力。On the day of in vitro fertilization, young female mice in natural estrus were selected to mate with young male mice with vasectomy and ligation. The next morning, female mice with vaginal plugs were regarded as pseudopregnant recipients and regarded as day 0.5. The 4.5-day blastocysts obtained in step (2) were used for embryo transfer, and 6 well-developed blastocysts were selected and transplanted into the uterine horns on both sides of the pseudopregnant recipients. On embryonic day 19.5 (14 days after embryo transfer), fetuses were obtained to evaluate the developmental capacity of each embryo.

(6)VitC、RELN、FGF2的联合施用调节体外胚胎DNA甲基化提高体外受精胚胎发育质量(6) Combined administration of VitC, RELN, and FGF2 regulates DNA methylation of in vitro embryos to improve the quality of in vitro fertilized embryo development

具体实验分组：Specific experimental groups:

体内组：体内受精组；In vivo group: in vivo fertilization group;

对照组：正常体外受精组；Control group: normal in vitro fertilization group;

处理组1：VitC(100 μg/ml)单独施用组；Treatment group 1: VitC (100 μg/ml) alone administration group;

处理组2：FGF2(500 ng/ml)单独施用组；Treatment group 2: FGF2 (500 ng/ml) alone administration group;

处理组3：RELN(100 ng/ml)单独施用组；Treatment group 3: RELN (100 ng/ml) alone administration group;

处理组4：VitC(50 μg/ml)和FGF2(250 ng/ml)联合施用组；Treatment group 4: VitC (50 μg/ml) and FGF2 (250 ng/ml) combined administration group;

处理组5：VitC(50 μg/ml)和RELN(50 ng/ml)联合施用组；Treatment group 5: VitC (50 μg/ml) and RELN (50 ng/ml) combined administration group;

处理组6：RELN(50 ng/ml)和FGF2(250 ng/ml)联合施用组；Treatment group 6: RELN (50 ng/ml) and FGF2 (250 ng/ml) combined administration group;

处理组7：VitC(33.3 μg/ml)、RELN(33.3 ng/ml)和FGF2(166.7 ng/ml)联合施用组。Treatment group 7: VitC (33.3 μg/ml), RELN (33.3 ng/ml), and FGF2 (166.7 ng/ml) combined administration group.

在体外培养96h后，统计并计算各组的囊胚率。同时分别比较体内组、对照组与处理组1/2/3/4/5/6/7囊胚的5-hmC/5-mC免疫荧光强度比值。After 96 hours of in vitro culture, the blastocyst rate of each group was counted and calculated. At the same time, the 5-hmC/5-mC immunofluorescence intensity ratios of blastocysts 1/2/3/4/5/6/7 in the in vivo group, control group, and treatment group were compared.

将体内组囊胚和在体外培养96h后各组的囊胚进行胚胎移植。在胚胎第19.5天(胚胎移植的14天)，处死受体母鼠后，统计附植位点数，之后将胎儿和胎盘分别取出，记录活胎儿数。The blastocysts of the in vivo group and the blastocysts of each group after 96 hours of in vitro culture were used for embryo transfer. On embryonic day 19.5 (14 days of embryo transfer), the recipient mothers were killed, the number of implantation sites was counted, and then the fetuses and placentas were taken out respectively, and the number of live fetuses was recorded.

3、实验结果3. Experimental results

表1 VitC、RELN、FGF2的联合施用对小鼠体外受精囊胚率和5hmC/5mC比值的影响Table 1 Effects of combined administration of VitC, RELN and FGF2 on the blastocyst rate and 5hmC/5mC ratio of mice in vitro fertilization

受精卵(N)Fertilized egg (N) 囊胚(%)Blastocyst(%) 5hmC/5mC比值5hmC/5mC ratio 体内组In vivo group -- -- 2.44±0.08c2.44±0.08c 对照组Control group 350350 114(32.5±3.3)a114(32.5±3.3)a 1.89±0.07a1.89±0.07a 处理组1Treatment Group 1 350350 144(41.1±3.0)b144(41.1±3.0)b 2.99±0.14b2.99±0.14b 处理组2Treatment Group 2 350350 143(40.9±2.2)b143(40.9±2.2)b 1.82±0.16a1.82±0.16a 处理组3Treatment Group 3 350350 112(31.7±3.4)a112(31.7±3.4)a 2.02±0.06a2.02±0.06a 处理组4Treatment Group 4 350350 176(50.7±3.2)c176(50.7±3.2)c 2.62±0.12c2.62±0.12c 处理组5Treatment Group 5 350350 168(48.0±2.8)c168(48.0±2.8)c 2.64±0.16c2.64±0.16c 处理组6Treatment Group 6 350350 172(49.1±2.2)c172(49.1±2.2)c 2.26±0.07c2.26±0.07c 处理组7Treatment Group 7 350350 186(53.1±2.0)c186(53.1±2.0)c 2.58±0.13c2.58±0.13c

注：a、b、c上标表示不同显著差异，显著性使用单因素方差分析来检验。囊胚率=囊胚数/受精卵数。Note: The superscripts a, b, and c indicate significant differences, and the significance was tested using one-way analysis of variance. Blastocyst rate = number of blastocysts/number of fertilized eggs.

由表1可见，与对照组(体外受精组)(32.5±3.3%)相比，处理组1(41.1±3.0%)、处理组2(40.9±2.2%)、处理组4(50.7±3.2%)、处理组6(49.1±2.2%)或处理组7(53.1±2.0%)均可显著提升体外囊胚发育效率，此外，VitC+FGF2、VitC+RELN、RELN+FGF2、VitC+FGF2 +RELN组合处理产生协同效应，均显著高于VitC、FGF2和RELN单独添加的囊胚发育效率；且VitC+FGF2 +RELN三者组合效果与体内组最为接近。可见VitC、FGF2和RELN联合使用有助于提高小鼠体外受精胚胎的发育效率。As shown in Table 1, compared with the control group (IVF group) (32.5±3.3%), treatment group 1 (41.1±3.0%), treatment group 2 (40.9±2.2%), treatment group 4 (50.7±3.2%), treatment group 6 (49.1±2.2%) or treatment group 7 (53.1±2.0%) can significantly improve the efficiency of in vitro blastocyst development. In addition, the combination of VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2+RELN produced a synergistic effect, which was significantly higher than the blastocyst development efficiency of VitC, FGF2, and RELN added alone; and the combination of VitC+FGF2+RELN was the closest to the in vivo group. It can be seen that the combined use of VitC, FGF2, and RELN helps to improve the development efficiency of mouse IVF embryos.

与对照组(体外受精组)(1.89±0.07)相比，处理组1/4/5/6/7均可显著提升体外囊胚中5hmC/5mC的比值。并且，VitC+FGF2、VitC+RELN、RELN+FGF2、VitC+FGF2 +RELN组合处理后5hmC/5mC的比值与体内组相比无显著性差异，这说明VitC、RELN、FGF2的两者联合或者三者联合施用对体外受精胚胎的DNA甲基化修饰起到了有效的矫正作用。Compared with the control group (IVF group) (1.89±0.07), treatment groups 1/4/5/6/7 can significantly increase the ratio of 5hmC/5mC in in vitro blastocysts. Moreover, the ratio of 5hmC/5mC after treatment with VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2 +RELN combination was not significantly different from that in the in vivo group, indicating that the combination of VitC, RELN, and FGF2 or the combination of the three has an effective correction effect on DNA methylation modification in IVF embryos.

表2 VitC、RELN、FGF2的联合施用对小鼠体外受精胚胎移植后的附植率和出生后代数目的影响Table 2 Effects of combined administration of VitC, RELN, and FGF2 on the implantation rate and number of offspring born after in vitro fertilization embryo transfer in mice

附植率(%)Implantation rate (%) 平均胎儿数(个)Average number of fetuses 体内组In vivo group 79.1±5.3c79.1±5.3c 8.9±0.76c8.9±0.76c 对照组Control group 50.0±4.3a50.0±4.3a 4.2±1.40a4.2±1.40a 处理组1Treatment Group 1 60.8±3.4b60.8±3.4b 5.8±0.30b5.8±0.30b 处理组2Treatment Group 2 62.3±1.2b62.3±1.2b 6.0±0.34b6.0±0.34b 处理组3Treatment Group 3 57.0±8.0a57.0±8.0a 5.4±0.55b5.4±0.55b 处理组4Treatment Group 4 71.8±4.0c71.8±4.0c 7.1±0.44c7.1±0.44c 处理组5Treatment Group 5 69.8±4.0c69.8±4.0c 7.4±0.34c7.4±0.34c 处理组6Treatment Group 6 70.8±1.2c70.8±1.2c 7.2±0.46c7.2±0.46c 处理组7Treatment Group 7 78.5±2.5c78.5±2.5c 8.0±0.24c8.0±0.24c

注：a、b、c上标表示不同显著差异，显著性使用单因素方差分析来检验。附植率=附植位点数/移植胚胎数，平均胎儿数=总胎儿数/妊娠受体数。Note: The superscripts a, b, and c indicate significant differences, and the significance was tested using one-way analysis of variance. Implantation rate = number of implantation sites/number of transferred embryos, average number of fetuses = total number of fetuses/number of pregnant recipients.

由表2可见，对于附植率，处理组1/2/4/5/6/7均显著高于对照组(体外受精组)(50.0±4.3%)，其中，VitC+FGF2、VitC+RELN、RELN+FGF2、VitC+FGF2 +RELN组合处理产生协同效应，均显著高于VitC、FGF2和RELN单独添加的胚胎附植率；且VitC+FGF2 +RELN三者组合效果与体内组最为接近。As can be seen from Table 2, for the implantation rate, the treatment groups 1/2/4/5/6/7 were significantly higher than the control group (in vitro fertilization group) (50.0±4.3%), among which, the combination treatments of VitC+FGF2, VitC+RELN, RELN+FGF2, and VitC+FGF2 +RELN produced a synergistic effect, which were significantly higher than the embryo implantation rates of VitC, FGF2, and RELN added alone; and the combination effect of VitC+FGF2 +RELN was closest to the in vivo group.

以上结果表明，VitC、RELN、FGF2的两者联合或者三者联合施用可以显著提高小鼠体外受精胚胎移植后的胚胎附植率和出生后代数目。The above results indicate that the combined administration of two or three of VitC, RELN and FGF2 can significantly increase the embryo implantation rate and the number of offspring born after in vitro fertilization embryo transfer in mice.

实施例2Example 2

本实施例为VitC、RELN、FGF2的联合施用在提高绵羊体外受精胚胎发育效率和质量中的应用。This example is the application of combined administration of VitC, RELN and FGF2 in improving the development efficiency and quality of sheep in vitro fertilization embryos.

1、实验动物及试剂：1. Experimental animals and reagents:

若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。实施例中VitC、RELN、FGF2及基础培养液中成分均购自美国Sigma-Aldrich公司，绵羊品种为黑头萨福克，取自内蒙古赛诺种羊科技有限公司。Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. VitC, RELN, FGF2 and the components in the basal culture medium in the examples were purchased from Sigma-Aldrich, USA, and the sheep breed was Black-headed Suffolk, which was obtained from Inner Mongolia Sino Sheep Breeding Technology Co., Ltd.