技术领域Technical field
本发明属于胚胎培养技术领域，具体涉及一种透明质酸钠双相凝胶及其应用。The invention belongs to the technical field of embryo culture, and specifically relates to a sodium hyaluronate biphasic gel and its application.
背景技术Background technique
卵细胞的发育在滤泡中进行，当第一层滤泡细胞层完全包被住卵细胞后，在卵细胞的外方开始形成非细胞的膜，称为透明带。受精卵在不断的卵裂过程中，其发育潜能的维持需要确保卵裂球之间保持相互的联系以及一定的空间结构。而透明带在维持卵裂间相互联系和空间结构中起着非常关键的作用。在辅助生殖技术实施过程中，会存在透明带缺失或异常的现象；另外在体外操作过程中，透明带结构也存在脱落的现象。缺乏透明带的受精卵利用常规的胚胎培养方法，会导致细胞分裂后卵裂球平铺生长，从而影响胚胎的后期发育潜能。The development of the egg cell proceeds in the follicle. When the first follicular cell layer completely surrounds the egg cell, a non-cellular membrane, called the zona pellucida, begins to form outside the egg cell. During the continuous cleavage process of the fertilized egg, maintaining its developmental potential requires ensuring that the blastomeres maintain mutual connections and a certain spatial structure. The zona pellucida plays a very critical role in maintaining the interconnection and spatial structure between cleavages. During the implementation of assisted reproductive technology, the zona pellucida may be missing or abnormal; in addition, during in vitro operations, the zona pellucida structure may also fall off. Conventional embryo culture methods for fertilized eggs lacking zona pellucida will cause the blastomeres to grow flat after cell division, thus affecting the embryo's later developmental potential.
针对无透明带胚胎，目前也有文献报道使用悬滴法和琼脂糖凝胶等方法进行培养获得成功的案例。但在使用中均存在一定的缺陷：悬滴法操作比较复杂，而且培养液的量非常少，很难保证胚胎培养环境的稳定，故其最终所获得的可用胚胎效率仍然较低；使用琼脂糖或其他凝胶状物质虽然可以在一定程度上起到维持胚胎发育空间结构的作用，但由于其本身不透气性，并且强度很难控制，导致胚胎发育效果的稳定性较差，而且琼脂糖等凝胶成分对胚胎发育以及后期着床方面的负面影响也很难评估。For embryos without zona pellucida, there are also reported cases of successful culture using methods such as hanging drop method and agarose gel. However, there are certain flaws in its use: the hanging drop method is complicated to operate, and the amount of culture medium is very small, making it difficult to ensure the stability of the embryo culture environment, so the efficiency of the usable embryos finally obtained is still low; using agarose Although it can maintain the spatial structure of embryonic development to a certain extent, it is not breathable and its strength is difficult to control, resulting in poor stability of embryonic development effects, and agarose, etc. The negative effects of gel composition on embryonic development and later implantation are also difficult to assess.
因此，急需开发一种能有效维持无透明带受精卵或胚胎发育潜能和稳定性的产品和培养方法。Therefore, there is an urgent need to develop a product and culture method that can effectively maintain the developmental potential and stability of zona-free fertilized eggs or embryos.
发明内容Contents of the invention
基于此，本发明的目的在于提供一种透明质酸钠双相凝胶，其能有效维持无透明带受精卵或胚胎发育潜能和稳定性，使无透明带受精卵或胚胎在培养过程中能正常发育。Based on this, the object of the present invention is to provide a sodium hyaluronate biphasic gel that can effectively maintain the developmental potential and stability of zona-free fertilized eggs or embryos, so that the zona-free fertilized eggs or embryos can be cultured during the culture process. normal development.
具体技术方案如下。The specific technical solutions are as follows.
一种透明质酸钠双相凝胶，所述透明质酸钠双相凝胶由交联透明质酸钠和游离透明质酸钠构成。A sodium hyaluronate biphasic gel, which is composed of cross-linked sodium hyaluronate and free sodium hyaluronate.
在一些实施例中，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比为1：(1.2～6)。In some embodiments, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel is 1: (1.2-6).
在一些实施例中，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比为1：(1.2～4)。In some embodiments, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel is 1: (1.2-4).
在优选的实施例中，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比为1：(1.5～3.5)。In a preferred embodiment, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel is 1: (1.5-3.5).
在更优选的实施例中，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比为1：(2～3.5)。In a more preferred embodiment, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel is 1: (2-3.5).
在一些实施例中，所述交联透明质酸钠由选自丁二醇缩水甘油醚、二乙烯基砜、双磺基琥珀酰亚胺辛二酸酯、3,3′-二硫双磺基琥珀酰亚胺丙酸酯、4,4′二硫双磺基琥珀酰亚胺丁酸酯中的至少一种的交联剂制备而成。In some embodiments, the cross-linked sodium hyaluronate is selected from the group consisting of butanediol glycidyl ether, divinyl sulfone, disulfosuccinimide suberate, 3,3′-dithiodisulfonate It is prepared from at least one cross-linking agent selected from succinimide propionate and 4,4′ disulfide disulfosuccinimide butyrate.
在优选的实施例中，所述交联剂选自丁二醇缩水甘油醚。In a preferred embodiment, the cross-linking agent is selected from butanediol glycidyl ether.
本发明还提供了上述透明质酸钠双相凝胶在无透明带受精卵或胚胎的培养中的应用。The present invention also provides the application of the above sodium hyaluronate biphasic gel in the culture of zona pellucida-free fertilized eggs or embryos.
本发明还提供了一种哺乳动物无透明带受精卵或胚胎培养方法，包括以下步骤：(1)向培养皿中加入上述透明质酸钠双相凝胶，然后依次在所述透明质酸钠双相凝胶上面覆盖培养液以及矿物油；(2)将所述培养皿置于培养箱中，使所述透明质酸钠双相凝胶的pH值与所述培养液的pH值相同；(3)取出所述培养皿，用拉细的胚胎转移管将哺乳动物无透明带受精卵或胚胎嵌入所述透明质酸钠双相凝胶中，然后将培养皿放入培养箱中继续培养。The invention also provides a method for cultivating mammalian zona pellucida fertilized eggs or embryos, which includes the following steps: (1) adding the above-mentioned sodium hyaluronate biphasic gel to the culture dish, and then sequentially adding the sodium hyaluronate The biphasic gel is covered with culture fluid and mineral oil; (2) Place the culture dish in an incubator so that the pH value of the sodium hyaluronate biphasic gel is the same as the pH value of the culture fluid; (3) Take out the culture dish, use a thinned embryo transfer tube to embed mammalian zona-free fertilized eggs or embryos into the sodium hyaluronate biphasic gel, and then put the culture dish into an incubator to continue culturing. .
在一些实施例中，所述培养液选自胚胎培养液、受精液培养液、卵裂期培养液、囊胚培养液、体外操作培养液中的至少一种。In some embodiments, the culture medium is selected from at least one of embryo culture medium, sperm culture medium, cleavage stage culture medium, blastocyst culture medium, and in vitro operation culture medium.
在优选的实施例中，所述培养液选自胚胎培养液。In a preferred embodiment, the culture medium is selected from embryo culture medium.
在一些实施例中，所述哺乳动物为小鼠、大鼠、兔、猪、猴、牛、羊、狗或马。In some embodiments, the mammal is a mouse, rat, rabbit, pig, monkey, cow, sheep, dog, or horse.
本发明提供了一种可以有效维持无透明带受精卵或胚胎发育潜能和稳定性的透明质酸钠双相凝胶，所述透明质酸钠双相凝胶可作为无透明带受精卵或胚胎发育的“支架”，替代透明带对受精卵或胚胎形成约束作用，使其分裂过程能以正常的细胞排列方式进行，有效解决了无透明带受精卵或胚胎在发育过程中由于无法维持空间结构而导致胚胎发育潜能差的难题，从而使无透明带受精卵或胚胎能正常发育。进一步地，发明人还发现，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的比例会对培养效果产生明显的影响，并优化获得了两者合适的比例，使得所述透明质酸钠双相凝胶既能满足对无透明带受精卵或胚胎的束缚作用，又能满足营养物质交换的需要，从而取得好的培养效果。The invention provides a sodium hyaluronate biphasic gel that can effectively maintain the development potential and stability of zona-free fertilized eggs or embryos. The sodium hyaluronate bi-phase gel can be used as a zona-free fertilized egg or embryo The "scaffold" of development replaces the zona pellucida to constrain the fertilized egg or embryo, allowing the division process to proceed in a normal cell arrangement, effectively solving the problem of the inability to maintain the spatial structure of a fertilized egg or embryo without a zona pellucida during development. This leads to the problem of poor embryo development potential, so that fertilized eggs or embryos without zona pellucida can develop normally. Furthermore, the inventor also found that the ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel will have a significant impact on the culture effect, and optimized the ratio of both. The ratio is such that the sodium hyaluronate biphasic gel can not only satisfy the binding effect on fertilized eggs or embryos without zona pellucida, but also meet the needs of nutrient exchange, thereby achieving good culture results.
本发明还提供了利用所述透明质酸钠双相凝胶对哺乳动物无透明带受精卵或胚胎进行培养的方法，所述方法具有操作上更加简单、有效和便利的优点。The present invention also provides a method for culturing mammalian zona pellucida-free fertilized eggs or embryos using the sodium hyaluronate biphasic gel. The method has the advantages of being simpler, more effective and convenient in operation.
附图说明Description of the drawings
图1为对比例4培养的正常小鼠受精卵的发育状态。Figure 1 shows the development status of normal mouse fertilized eggs cultured in Comparative Example 4.
图2为本发明实施例1所述透明质酸钠双相凝胶培养的小鼠无透明带受精卵的发育状态。Figure 2 shows the development status of mouse zona-free fertilized eggs cultured in sodium hyaluronate biphasic gel according to Example 1 of the present invention.
图3为对比例3中常规方法培养的小鼠无透明带受精卵的发育状态。Figure 3 shows the development status of mouse zona-free fertilized eggs cultured by conventional methods in Comparative Example 3.
图4为本发明实施例4所述透明质酸钠双相凝胶培养的小鼠无透明带受精卵的发育状态。Figure 4 shows the development status of mouse zona-free fertilized eggs cultured in sodium hyaluronate biphasic gel according to Example 4 of the present invention.
图5为本发明实施例5所述透明质酸钠双相凝胶培养的小鼠无透明带受精卵的发育状态。Figure 5 shows the development status of mouse zona-free fertilized eggs cultured in sodium hyaluronate biphasic gel according to Example 5 of the present invention.
图6为对比例1中培养的小鼠无透明带受精卵的发育状态。Figure 6 shows the development status of the mouse zona-free fertilized eggs cultured in Comparative Example 1.
图7为对比例2中培养的小鼠无透明带受精卵的发育状态。Figure 7 shows the development status of the mouse zona-free fertilized eggs cultured in Comparative Example 2.
具体实施方式Detailed ways
本发明下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。Experimental methods without specifying specific conditions in the following examples of the present invention usually follow conventional conditions or conditions recommended by the manufacturer. Various commonly used chemical reagents used in the examples are all commercially available products.
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings commonly understood by those skilled in the technical field belonging to the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention.
本发明的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤的过程、方法、装置、产品或设备没有限定于已列出的步骤或模块，而是可选地还包括没有列出的步骤，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤。The terms "including" and "having" and any variations thereof herein are intended to cover a non-exclusive inclusion. For example, a process, method, device, product or equipment that includes a series of steps is not limited to the listed steps or modules, but optionally also includes unlisted steps, or optionally also includes steps for these processes, Other steps inherent to the method, product, or device.
本发明提供了一种透明质酸钠双相凝胶，所述透明质酸钠双相凝胶由交联透明质酸钠和游离透明质酸钠构成。The invention provides a sodium hyaluronate biphasic gel, which is composed of cross-linked sodium hyaluronate and free sodium hyaluronate.
透明质酸钠状态包括包括液相和固相。未交联的透明质酸钠是液态的，用交联剂将游离的透明质酸钠连接起来形成网状结构，使透明质酸钠的物理性质发生变化，变成果冻状的固态，即为交联透明质酸钠。既含有交联透明质酸钠又含有游离状态透明质酸钠的凝胶称为透明质酸钠双相凝胶。交联透明质酸钠具有一定的硬度和结构，并且透明质酸钠本身是细胞外基质的主要成分组成，因此其对胚胎发育本身是没有负面作用的。制备交联透明质酸钠使用的交联剂可选自但不限于以下交联剂：丁二醇缩水甘油醚(BDDE)、二乙烯基砜(DVS)、双磺基琥珀酰亚胺辛二酸酯(BS3)、3,3′-二硫双磺基琥珀酰亚胺丙酸酯(DTSSP)、4,4′二硫双磺基琥珀酰亚胺丁酸酯(DTSSB)。所述交联透明质酸钠可直接从市面上购买成品，也可根据本领域常规技术手段制备获得。The state of sodium hyaluronate includes liquid phase and solid phase. Uncross-linked sodium hyaluronate is liquid. Cross-linking agents are used to connect free sodium hyaluronate to form a network structure, which changes the physical properties of sodium hyaluronate and turns it into a jelly-like solid state, which is Cross-linked sodium hyaluronate. Gels containing both cross-linked sodium hyaluronate and free sodium hyaluronate are called sodium hyaluronate biphasic gels. Cross-linked sodium hyaluronate has a certain hardness and structure, and sodium hyaluronate itself is the main component of the extracellular matrix, so it has no negative effect on embryonic development itself. The cross-linking agent used to prepare cross-linked sodium hyaluronate can be selected from, but is not limited to, the following cross-linking agents: butanediol glycidyl ether (BDDE), divinyl sulfone (DVS), bis-sulfosuccinimide octadiene Acid ester (BS3), 3,3′-disulfisulfosuccinimide propionate (DTSSP), 4,4′-disulfisulfosuccinimidebutyrate (DTSSB). The cross-linked sodium hyaluronate can be purchased directly from the market as a finished product, or can be prepared according to conventional technical means in this field.
在优选的是实施例中，所述交联剂为丁二醇缩水甘油醚(BDDE)。In a preferred embodiment, the cross-linking agent is butanediol glycidyl ether (BDDE).
在一些实施例中，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比为1：(1.2～6)。In some embodiments, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel is 1: (1.2-6).
在一些实施例中，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比为1：(1.2～4)。In some embodiments, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel is 1: (1.2-4).
进一步地，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比为1：(1.5～3.5)。Further, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel is 1: (1.5-3.5).
在优选的实施例中，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比为1：(2～3.5)。In a preferred embodiment, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel is 1: (2-3.5).
在更优选的实施例中，所述透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比为1：3。In a more preferred embodiment, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel is 1:3.
本发明所述透明质酸钠双相凝胶制备方法如下：将所述交联透明质酸钠打碎后与所述游离透明质酸钠按比例混合，即得所述透明质酸钠双相凝胶。The preparation method of the sodium hyaluronate biphasic gel of the present invention is as follows: crush the cross-linked sodium hyaluronate and mix it with the free sodium hyaluronate in proportion to obtain the sodium hyaluronate biphasic gel. gel.
本发明所述透明质酸钠双相凝胶可用于培养无透明带受精卵或胚胎。其可作为无透明带受精卵或胚胎发育的“支架”，替代透明带对受精卵或胚胎形成约束作用。The sodium hyaluronate biphasic gel of the present invention can be used to culture fertilized eggs or embryos without zona pellucida. It can be used as a "scaffold" for the development of fertilized eggs or embryos without zona pellucida, replacing the zona pellucida to constrain fertilized eggs or embryos.
利用本发明所述透明质酸钠双相凝胶对哺乳动物无透明带受精卵或胚胎进行培养，既能为无透明带受精卵或胚胎的发育提供“支架”，又能满足营养物质的交换需求，从而有效维持无透明带受精卵或胚胎的发育潜能和稳定性。Utilizing the sodium hyaluronate biphasic gel of the present invention to culture mammalian zona-free fertilized eggs or embryos can not only provide a "scaffold" for the development of zona-free fertilized eggs or embryos, but also satisfy the exchange of nutrients. needs, thereby effectively maintaining the developmental potential and stability of zona-free fertilized eggs or embryos.
在一些实施例中，所述哺乳动物无透明带受精卵或胚胎的培养方法，包括以下步骤：(1)向培养皿中加入上述透明质酸钠双相凝胶，然后依次在所述透明质酸钠双相凝胶上面覆盖培养液以及矿物油；(2)将所述培养皿置于培养箱中，使所述透明质酸钠双相凝胶的pH值与所述培养液的pH值相同；(3)取出所述培养皿，用拉细的胚胎转移管将哺乳动物无透明带受精卵或胚胎嵌入所述透明质酸钠双相凝胶中，然后将培养皿放入培养箱中继续培养。In some embodiments, the method for culturing mammalian zona pellucida fertilized eggs or embryos includes the following steps: (1) adding the above-mentioned sodium hyaluronate biphasic gel to the culture dish, and then sequentially adding the above-mentioned sodium hyaluronate biphasic gel to the culture dish; The sodium hyaluronate biphasic gel is covered with culture fluid and mineral oil; (2) Place the culture dish in an incubator so that the pH value of the sodium hyaluronate biphasic gel is consistent with the pH value of the culture fluid. Same; (3) Take out the culture dish, use a thinned embryo transfer tube to embed the mammalian zona pellucida fertilized eggs or embryos into the sodium hyaluronate biphasic gel, and then put the culture dish into the incubator Continue to cultivate.
在一些实施例中，所述步骤(2)中将所述培养皿置于培养箱中至少6h。In some embodiments, in step (2), the culture dish is placed in an incubator for at least 6 hours.
在一些实施例中，所述培养液选自胚胎培养液、受精液培养液、卵裂期培养液、囊胚培养液、体外操作培养液中的至少一种。In some embodiments, the culture medium is selected from at least one of embryo culture medium, sperm culture medium, cleavage stage culture medium, blastocyst culture medium, and in vitro operation culture medium.
在优选的实施例中，所述培养液选自胚胎培养液。In a preferred embodiment, the culture medium is selected from embryo culture medium.
在一些实施例中，所述哺乳动物为卵生哺乳动物，包括但不限于小鼠、大鼠、兔、猪、猴、牛、羊、狗或马。In some embodiments, the mammal is an oviparous mammal, including but not limited to mouse, rat, rabbit, pig, monkey, cow, sheep, dog, or horse.
在一些实施例中，所述培养皿可以为普通细胞培养皿、96孔板、48孔板、24孔板、12孔板、4孔皿，以及其他含有固定孔槽的培养皿。In some embodiments, the culture dish can be an ordinary cell culture dish, a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, a 4-well dish, and other culture dishes containing fixed wells.
在一些实施例中，所述培养箱为二氧化碳培养箱或三气培养箱。In some embodiments, the incubator is a carbon dioxide incubator or a three-gas incubator.
下面结合具体实施例对本发明进行进一步说明。The present invention will be further described below with reference to specific embodiments.
以下实施例使用的交联透明质酸钠购于中国华熙生物；胚胎培养液包括卵裂期培养液和囊胚期培养液，购于瑞典vitrolife公司，卵裂期培养液货号为10128，囊胚期培养液货号为10132。The cross-linked sodium hyaluronate used in the following examples was purchased from China Huaxi Biotech; the embryo culture medium includes cleavage stage culture medium and blastocyst stage culture medium, purchased from the Swedish company Vitrolife, the cleavage stage culture medium product number is 10128, and the blastocyst stage culture medium is 10128. The item number for embryonic stage culture medium is 10132.
实施例1Example 1
本实施例提供一种透明质酸钠双相凝胶，所述透明质酸钠双相凝胶由丁二醇缩水甘油醚交联的透明质酸钠(交联透明质酸钠)和游离透明质酸钠构成，且交联透明质酸钠和游离透明质酸钠的重量比为1：3。This embodiment provides a sodium hyaluronate biphasic gel, which is composed of butylene glycol glycidyl ether cross-linked sodium hyaluronate (cross-linked sodium hyaluronate) and free transparent It is composed of sodium hyaluronate, and the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate is 1:3.
实施例2Example 2
本实施例提供一种透明质酸钠双相凝胶，所述透明质酸钠双相凝胶由丁二醇缩水甘油醚交联的透明质酸钠(交联透明质酸钠)和游离透明质酸钠构成，且交联透明质酸钠和游离透明质酸钠的重量比为1：1.5。This embodiment provides a sodium hyaluronate biphasic gel, which is composed of butylene glycol glycidyl ether cross-linked sodium hyaluronate (cross-linked sodium hyaluronate) and free transparent It is composed of sodium hyaluronate, and the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate is 1:1.5.
实施例3Example 3
本实施例提供一种透明质酸钠双相凝胶，所述透明质酸钠双相凝胶由丁二醇缩水甘油醚交联的透明质酸钠(交联透明质酸钠)和游离透明质酸钠构成，且交联透明质酸钠和游离透明质酸钠的重量比为1：5。This embodiment provides a sodium hyaluronate biphasic gel, which is composed of butylene glycol glycidyl ether cross-linked sodium hyaluronate (cross-linked sodium hyaluronate) and free transparent It is composed of sodium hyaluronate, and the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate is 1:5.
实施例4Example 4
本实施例提供一种透明质酸钠双相凝胶，所述透明质酸钠双相凝胶由丁二醇缩水甘油醚交联的透明质酸钠(交联透明质酸钠)和游离透明质酸钠构成，且交联透明质酸钠和游离透明质酸钠的重量比为1：1。This embodiment provides a sodium hyaluronate biphasic gel, which is composed of butylene glycol glycidyl ether cross-linked sodium hyaluronate (cross-linked sodium hyaluronate) and free transparent It is composed of sodium hyaluronate, and the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate is 1:1.
实施例5Example 5
本实施例提供一种透明质酸钠双相凝胶，所述透明质酸钠双相凝胶由丁二醇缩水甘油醚交联的透明质酸钠(交联透明质酸钠)和游离透明质酸钠构成，且交联透明质酸钠和游离透明质酸钠的重量比为1：6.5。This embodiment provides a sodium hyaluronate biphasic gel, which is composed of butylene glycol glycidyl ether cross-linked sodium hyaluronate (cross-linked sodium hyaluronate) and free transparent It is composed of sodium hyaluronate, and the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate is 1:6.5.
利用上述实施例1～5所述的透明质酸钠双相凝胶对小鼠无透明带受精卵进行培养，测试培养效果。The sodium hyaluronate biphasic gel described in the above Examples 1 to 5 was used to culture mouse zona pellucida fertilized eggs, and the culture effect was tested.
同时，实验过程中设置以下对比例：At the same time, the following comparison ratios were set during the experiment:
对比例1：使用丁二醇缩水甘油醚交联的透明质酸钠(交联透明质酸钠)对小鼠无透明带受精卵进行培养；Comparative Example 1: Using butylene glycol glycidyl ether cross-linked sodium hyaluronate (cross-linked sodium hyaluronate) to culture mice without zona pellucida fertilized eggs;
对比例2：使用游离透明质酸钠对小鼠无透明带受精卵进行培养；Comparative Example 2: Use free sodium hyaluronate to culture mouse zygotes without zona pellucida;
对比例3：使用常规培养方法对小鼠无透明带受精卵进行对照培养；Comparative Example 3: Use conventional culture methods to conduct control culture on mouse fertilized eggs without zona pellucida;
对比例4：使用常规培养方法对正常小鼠受精卵进行培养。Comparative Example 4: Normal mouse fertilized eggs were cultured using conventional culture methods.
本发明培养方法如下：(1)向培养皿中分别加入实施例1～5所述透明质酸钠双相凝胶作为“支架”，然后依次在所述透明质酸钠双相凝胶上面覆盖适量的卵裂期培养液以及矿物油；(2)将所述培养皿放入6％CO2细胞培养箱中平衡8h，使所述透明质酸钠双相凝胶和卵裂期培养液达到相同pH值；(3)取出所述培养皿，用拉细的胚胎转移管吸取哺乳动物无透明带受精卵并嵌入步骤(2)获得的培养皿的透明质酸钠双相凝胶支架，使无透明受精卵将被固定在凝胶支架中；然后将培养皿放入细胞培养箱中继续培养。继续培养过程同常规胚胎培养一致，等胚胎发育到融合胚期或桑葚胚期再更换囊胚期培养液。The culture method of the present invention is as follows: (1) Add the sodium hyaluronate biphasic gel described in Examples 1 to 5 as a "scaffold" to the culture dish, and then cover the sodium hyaluronate biphasic gel in sequence Appropriate amount of cleavage stage culture medium and mineral oil; (2) Place the culture dish into a 6% CO2 cell culture incubator for 8 hours to balance the sodium hyaluronate biphasic gel and cleavage stage culture medium. The same pH value; (3) Take out the culture dish, use a thinned embryo transfer tube to absorb the mammalian zona pellucida fertilized eggs and embed them in the sodium hyaluronate biphasic gel scaffold of the culture dish obtained in step (2), so that Non-transparent fertilized eggs will be fixed in the gel holder; the dish is then placed in a cell culture incubator to continue culturing. Continue the culture process in the same manner as conventional embryo culture, and replace the blastocyst stage culture medium when the embryos develop to the fusion embryo stage or morula stage.
对比例1～2的培养方法：除了步骤(1)中向培养皿中加入的分别是交联透明质酸钠和游离透明质酸钠外，其他均与本发明培养方法一致。Culture methods of Comparative Examples 1 to 2: Except for the cross-linked sodium hyaluronate and free sodium hyaluronate added to the culture dish in step (1), the others are consistent with the culture method of the present invention.
对比例3～4中的常规培养方法如下：(1)向培养皿中加入适量的卵裂期培养液，培养液上方覆盖矿物油；(2)将所述培养皿放入6％CO2细胞培养箱中平衡8h；(3)用拉细的胚胎转移管吸取小鼠无透明带受精卵放入培养液中；然后将培养皿放入培养箱中继续培养，等胚胎发育到融合胚期或桑葚胚期再更换囊胚期培养液。The conventional culture methods in Comparative Examples 3 to 4 are as follows: (1) Add an appropriate amount of cleavage stage culture medium to the culture dish, and cover the top of the culture medium with mineral oil; (2) Place the culture dish into 6% CO2 cells Equilibrate in the incubator for 8 hours; (3) Use a thinned embryo transfer tube to absorb the mouse zona-free fertilized eggs and put them into the culture medium; then put the culture dish into the incubator to continue culturing until the embryos develop to the fusion embryo stage or At the morula stage, replace the blastocyst stage culture medium.
本发明实施例1～3所述透明质酸钠双相凝胶培养的无透明带受精卵发育状态良好，可正常扩张成囊胚。其中，以实施例1培养效果最好，可获得优质囊胚(图2)。常规方法培养的正常小鼠受精卵(对比例4)发育状态如图1所示，可培养出优质囊胚。常规方法培养的无透明带小鼠受精卵发育状态(对比例3)如图3所示。由此可知，采用本实施例1所培养的无透明带受精卵培养效果与正常受精卵差异不大，均可获得优质囊胚，而常规方法培养的则无法培养出优质囊胚。The zona pellucida-free fertilized eggs cultured in the sodium hyaluronate biphasic gel described in Examples 1 to 3 of the present invention are in good development status and can normally expand into blastocysts. Among them, Example 1 has the best culture effect and can obtain high-quality blastocysts (Figure 2). The development status of normal mouse fertilized eggs (Comparative Example 4) cultured by conventional methods is shown in Figure 1, and high-quality blastocysts can be cultured. The development status of fertilized eggs of mice without zona pellucida cultured by conventional methods (Comparative Example 3) is shown in Figure 3. It can be seen from this that the culture effect of the zona-free fertilized eggs cultured in Example 1 is not much different from that of normal fertilized eggs, and high-quality blastocysts can be obtained, while high-quality blastocysts cannot be cultured by conventional methods.
利用实施例4～5所述透明质酸钠双相凝胶对哺乳动物无透明带受精卵的培养效果如图4～5所示，囊胚能生成，但囊胚发育后无法依靠自身的力量扩张，难以形成优质胚胎。由实施例1～5所述透明质酸钠双相凝胶对无透明带受精卵的培养结果可知，透明质酸钠双相凝胶中交联透明质酸钠和游离透明质酸钠的重量比会影响培养效果，当两者重量比为1：(1.2～6)时，既能满足对无透明带受精卵的束缚作用，又能满足营养物质交换的需要；尤其是两者重量比为1：3(实施例1)时，培养效果更好(图2)。当两者重量比过高时(例如实施例4为1：1)，此时束缚力过强，又会影响营养物质的交换，难以形成优质胚胎(图4)。而当两者重量比过低时(例如实施例5为1：6.5时)，无法有效满足对无透明带受精卵的束缚作用，各卵裂球之间的细胞连接较为松散，也无法形成优质的囊胚(图5)。The culture effect of the sodium hyaluronate biphasic gel described in Examples 4-5 on mammalian zona pellucida fertilized eggs is shown in Figures 4-5. Blastocysts can be generated, but blastocysts cannot rely on their own strength after development. Expansion makes it difficult to form high-quality embryos. From the culture results of sodium hyaluronate biphasic gel on zona-free fertilized eggs described in Examples 1 to 5, it can be seen that the weight of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate biphasic gel The ratio will affect the culture effect. When the weight ratio of the two is 1: (1.2~6), it can not only satisfy the binding effect of fertilized eggs without zona pellucida, but also meet the needs of nutrient exchange; especially the weight ratio of the two is When 1:3 (Example 1), the culture effect is better (Figure 2). When the weight ratio between the two is too high (for example, 1:1 in Example 4), the binding force is too strong and the exchange of nutrients will be affected, making it difficult to form high-quality embryos (Figure 4). When the weight ratio between the two is too low (for example, 1:6.5 in Example 5), the binding effect on fertilized eggs without a zona pellucida cannot be effectively satisfied, the cell connections between the blastomeres are relatively loose, and high-quality blastomeres cannot be formed. of blastocyst (Figure 5).
当仅使用交联透明质酸钠进行培养时(对比例1)，高浓度的交联透明质酸无法有效的交换培养液，导致卵裂球在发育过程中逐渐凋亡，无法发育到囊胚阶段(图6)；当仅使用游离透明质酸钠进行培养时(对比例2)，游离透明质酸与培养液混合后几乎无束缚效果，导致卵裂球之间无法达成紧密的细胞连接，分布比较分散，最终也无法发育到囊胚阶段(图7)。When only cross-linked sodium hyaluronate is used for culture (Comparative Example 1), the high concentration of cross-linked hyaluronic acid cannot effectively exchange the culture medium, causing the blastomeres to gradually undergo apoptosis during development and fail to develop into blastocysts. stage (Figure 6); when only free sodium hyaluronate is used for culture (Comparative Example 2), free hyaluronic acid has almost no binding effect after being mixed with the culture medium, resulting in the inability to achieve tight cell connections between blastomeres. The distribution is relatively scattered, and ultimately they cannot develop to the blastocyst stage (Figure 7).
综上所述，本发明所述透明质酸钠双相凝胶和培养方法能有效维持无透明带受精卵或胚胎发育潜能和稳定性，使无透明带受精卵或胚胎能正常发育。In summary, the sodium hyaluronate biphasic gel and culture method of the present invention can effectively maintain the development potential and stability of zona-free fertilized eggs or embryos, so that the zona-free fertilized eggs or embryos can develop normally.
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对以上实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。The technical features of the above embodiments can be combined in any way. To simplify the description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, All should be considered to be within the scope of this manual.
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the patent scope of the present invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210726398.1ACN114874976B (en) | 2022-06-23 | 2022-06-23 | A kind of sodium hyaluronate biphasic gel and its application |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210726398.1ACN114874976B (en) | 2022-06-23 | 2022-06-23 | A kind of sodium hyaluronate biphasic gel and its application |
| Publication Number | Publication Date |
|---|---|
| CN114874976A CN114874976A (en) | 2022-08-09 |
| CN114874976Btrue CN114874976B (en) | 2024-02-02 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210726398.1AActiveCN114874976B (en) | 2022-06-23 | 2022-06-23 | A kind of sodium hyaluronate biphasic gel and its application |
| Country | Link |
|---|---|
| CN (1) | CN114874976B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999021415A1 (en)* | 1997-10-28 | 1999-05-06 | Stem Cell Sciences Pty. Ltd. | Nuclear transfer for production of transgenic animal embryo |
| CN107522881A (en)* | 2017-08-16 | 2017-12-29 | 杭州协合医疗用品有限公司 | The method for preparing single-phase modification hyaluronic acid sodium gel |
| CN108250457A (en)* | 2017-05-08 | 2018-07-06 | 上海利康瑞生物工程有限公司 | Controllable two-phase cross-linking sodium hyaluronate gel of a kind of shear viscosity and preparation method thereof and preparation |
| CN111269877A (en)* | 2020-02-21 | 2020-06-12 | 南京市江宁医院 | A kind of method of preimplantation without zona pellucida embryo aggregation and in vitro culture |
| CH717854A2 (en)* | 2020-09-10 | 2022-03-15 | Hyamed Biotechnology Zhuhai Co Ltd | Process for the preparation of sodium hyaluronate gel. |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3241895A1 (en)* | 2016-05-04 | 2017-11-08 | ETH Zürich, ETH Transfer | Transglutaminase mediated high molecular weight hyaluronan hydrogels |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999021415A1 (en)* | 1997-10-28 | 1999-05-06 | Stem Cell Sciences Pty. Ltd. | Nuclear transfer for production of transgenic animal embryo |
| CN108250457A (en)* | 2017-05-08 | 2018-07-06 | 上海利康瑞生物工程有限公司 | Controllable two-phase cross-linking sodium hyaluronate gel of a kind of shear viscosity and preparation method thereof and preparation |
| CN107522881A (en)* | 2017-08-16 | 2017-12-29 | 杭州协合医疗用品有限公司 | The method for preparing single-phase modification hyaluronic acid sodium gel |
| CN111269877A (en)* | 2020-02-21 | 2020-06-12 | 南京市江宁医院 | A kind of method of preimplantation without zona pellucida embryo aggregation and in vitro culture |
| CH717854A2 (en)* | 2020-09-10 | 2022-03-15 | Hyamed Biotechnology Zhuhai Co Ltd | Process for the preparation of sodium hyaluronate gel. |
| Publication number | Publication date |
|---|---|
| CN114874976A (en) | 2022-08-09 |
| Publication | Publication Date | Title |
|---|---|---|
| Yu et al. | Effects of combined epidermal growth factor, brain-derived neurotrophic factor and insulin-like growth factor-1 on human oocyte maturation and early fertilized and cloned embryo development | |
| Ye et al. | High quality clinical grade human embryonic stem cell lines derived from fresh discarded embryos | |
| Zhang et al. | Generation of artificial gamete and embryo from stem cells in reproductive medicine | |
| JP2008017840A (en) | Growth of embryonic stem cell | |
| Reuter et al. | Fact or fiction: In vitro spermatogenesis | |
| CN104004708A (en) | Method for separation and purification, long-term passage cultivation, cryopreservation and recovery of sheep spermatogonia stem cells | |
| WO2014043835A1 (en) | Method for maturation of oocytes in vitro | |
| ES2703751T3 (en) | Formulation and method of passage and collection of pluripotent stem cells | |
| Zehorai et al. | From fertilised oocyte to cultivated meat–harnessing bovine embryonic stem cells in the cultivated meat industry | |
| CN114874976B (en) | A kind of sodium hyaluronate biphasic gel and its application | |
| CN115161265B (en) | A method of obtaining mammalian embryos of the same genetic background | |
| CN104946581B (en) | A special medium and method for cultivating porcine trophoblast stem cells | |
| Stenberg et al. | Sustained embryoid body formation and culture in a non-laborious three dimensional culture system for human embryonic stem cells | |
| Hovatta | Derivation of human embryonic stem cell lines, towards clinical quality | |
| Shan et al. | Aggregation of pre‐implantation embryos improves establishment of parthenogenetic stem cells and expression of imprinted genes | |
| Vichera et al. | Sperm genome cloning used in biparental bovine embryo reconstruction | |
| CN101423817A (en) | Method for constructing cell line by using insect egg | |
| Morovic et al. | Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes | |
| Chang et al. | Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos | |
| AU2022387152A1 (en) | Derivation of naïve bovine embryonic stem cells | |
| Xu et al. | Bioengineering and the ovarian follicle | |
| Liu et al. | Effect of bFGF on fibroblasts derived from the golden snub-nosed monkey | |
| Nandi et al. | Isolation and Culture of Ovine and Bubaline Small and Large Pre‐antral Follicles: Effect of Cyclicity and Presence of a Dominant Follicle | |
| Nakami et al. | Culture of spermatogonial stem cells and use of surrogate sires as a breeding technology to propagate superior genetics in livestock production: a systematic review | |
| Haraguchi et al. | Electroporation‐mediated genome editing in vitrified/warmed porcine zygotes obtained in vitro |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |