CN111956657A - 一种改善卵巢功能、提高卵子和胚胎质量的组合物及其应用 - Google Patents
一种改善卵巢功能、提高卵子和胚胎质量的组合物及其应用 Download PDFInfo
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- CN111956657A CN111956657A CN202010774124.0A CN202010774124A CN111956657A CN 111956657 A CN111956657 A CN 111956657A CN 202010774124 A CN202010774124 A CN 202010774124A CN 111956657 A CN111956657 A CN 111956657A
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Abstract
本发明公开了一种改善卵巢功能、提高卵子和胚胎质量的组合物及其应用。本发明提供了一种含有烟酰胺单核苷酸、褪黑素和辅酶Q10的组合物,能够显著改善老龄动物的排卵率,提高卵子的成熟率、受精率和早期胚胎的发育率,对于女性不孕不育的预防和治疗以及动物胚胎的体外生产具有重大意义。该组合物中的三种活性成分均为生物机体的内源性物质,从已公开发表的文献来看不会产生副作用,因此作为药物、保健品或生物制剂的安全性很高。
Description
技术领域
本发明属于生物医药领域,涉及一种改善卵巢功能、提高卵子和胚胎质量的组合物及其应 用。
背景技术
卵母细胞的正常发育是确保卵子成熟,受精以及胚胎发育的前提条件,是生命启始的物质 基础。卵母细胞的质量不仅影响人类的生殖健康,同时对动物胚胎的体外生产具有重大意义。
体外培养过程中,由于卵母细胞本身的质量下降,以及老化等因素的影响,卵母细胞的成 熟受到很大的影响,不仅表现在卵母细胞成熟率低,同时卵子质量的下降也导致受精能力及之 后的胚胎发育潜能受到很大的影响。
高龄妇女是指年龄在35周岁以上的妇女,其生育能力降低的主要原因为卵巢的卵泡数量 减少以及卵子质量下降。处于育龄期女性的卵巢储备功能会随着年龄的增长而逐渐降低,原始 卵泡会以1000个/月的速度减少,并且当妇女年龄达到35周岁以后,卵巢卵泡减少的速度会 逐渐加快,直到妇女年龄到达更年期时,卵巢内卵泡的剩余数量已经在1000个以下。相关流 行病学分析结果显示,在年龄为35周岁的妇女中,不孕发生概率<20%,而在40、45周岁的 女性中,其不孕的发生概率为40%、50%。此外,随着女性年龄的增长,其卵巢内卵母细胞发 生减数分裂异常的情况增加,导致非整倍体胚胎的概率也增加,进而降低高龄妇女的卵子质量 并增加流产率。
生育年龄推迟已经是全球趋势,我国准备生育二胎的群体快速增加,其中大部分为高龄妇 女。高龄女性的卵子和胚胎质量难以逆转,卵巢储备不足、生育功能低下成为生殖医学的难题。 女性年龄增加导致卵子质量下降通常是由于卵子减数分裂染色体不分离,导致早期胚胎的非整 倍体率增加,自然流产率也随之升高。研究发现,黏连蛋白作为调节染色体分离的关键蛋白在 高龄女性卵子中的含量降低,以至于不能维持姐妹染色单体交叉点的稳定。着丝粒的丢失与哺 乳动物发生年龄相关的减数分裂错误有关。尽管目前有很多研究试图改善高龄妇女卵巢功能及 卵子质量,但是效果都不太理想。因此,找到合适有效的治疗方法是我们亟待解决的问题。
发明内容
本发明要解决的技术问题就是针对现有的由于老龄化、疾病以及环境污染等引起的卵母细 胞质量下降、女性不孕不育以及动物胚胎的体外生产能力低下,提供一种烟酰胺单核苷酸的应 用以及一种含有烟酰胺单核苷酸、褪黑素和辅酶Q10的新药物组合物。我们经过大量实验研 究发现,烟酰胺单核苷酸及药物组合物中其它组分能够显著改善老龄动物的排卵率,提高卵子 的成熟率、受精率和早期胚胎的发育率,对于预防和治疗女性不孕不育以及动物胚胎的体外生 产具有重大意义,经过进一步的研究和实验,完成本发明。
烟酰胺单核苷酸在制备改善卵巢功能、提高卵子和胚胎质量的药物或保健品中的应用。
烟酰胺单核苷酸在制备改善卵子和胚胎质量的生物试剂中的应用。
一种改善卵巢功能、提高卵子和胚胎质量的组合物,包含烟酰胺单核苷酸、褪黑素和辅酶 Q10。
所述的组合物优选由烟酰胺单核苷酸、褪黑素和辅酶Q10组成。
本发明所述的组合物在制备改善卵巢功能、提高卵子和胚胎质量的药物或保健品中的应 用。
本发明所述的组合物在制备改善卵子和胚胎质量的生物试剂中的应用。
卵母细胞体外成熟培养液,包含所述的组合物,且所述体外成熟培养液中烟酰胺单核苷酸 的浓度为100~500μM,褪黑素浓度为5~50μM,辅酶Q10浓度为20~200μM;优选烟酰胺单核 苷酸的浓度为200μM,褪黑素浓度为10μM,辅酶Q10浓度为50μM。
所述卵母细胞体外成熟培养液选取M16(sigma,货号M7292)。
一种培养卵母细胞的方法,包括如下的步骤:
(1)将本发明所述的组合物按照对应浓度配比后加入培养液中,放入培养箱中平衡预热 2-3小时;
(2)将卵母细胞取出后放入含有本发明所述的组合物的体外成熟培养液中洗三遍,移入 本发明所述的体外成熟培养液中继续培养;
(3)成熟后的卵母细胞可以继续完成体外受精及体外胚胎发育。
本发明的体外实验显示烟酰胺单核苷酸、褪黑素和辅酶Q10均具有抗氧化并防止卵母细 胞凋亡的作用。在老龄鼠的体内实验中也发现烟酰胺单核苷酸、褪黑素和辅酶Q10具有抑制 活性氧ROS产生,从而提高卵母细胞质量,增强卵母细胞受精能力及后续胚胎发育潜能的作 用。
本发明中的烟酰胺单核苷酸、褪黑素和辅酶Q10三种活性成分均为生物机体的内源性物 质,从已公开发表的文献来看不会产生副作用,因此作为药物、保健品或生物制剂的安全性很 高。当用于口服时,可将其制备成常规的固体制剂如片剂、粉剂或者胶囊剂等,用于注射时, 可将其制备成注射液。在各种制剂中,活性成分的重量含量为1%-99.9%,优选的重量含量为 0.5%-90%。
本发明的烟酰胺单核苷酸的制备以及含有烟酰胺单核苷酸、褪黑素和辅酶Q10的新药物 组合物可用于制备高效卵母细胞成熟培养液、制备女性不孕不育的预防和治疗的药物或保健品 以及制备提高动物胚胎体外生产的药物或是饲料添加剂。可以按剂型通过培养液添加、口服、 腹腔注射、皮下注射、静脉注射、肌肉注射、食品添加、饲料添加等途径用于需要治疗的卵母 细胞或者生物个体。个体可以是人或者动物。剂量根据个体差异调整。
本发明所用的原料或者试剂均市售可得。
有益效果
(1)一种烟酰胺单核苷酸的制备以及一种含有烟酰胺单核苷酸、褪黑素和辅酶Q10的新 药物组合物的体外成熟培养液可以显著改善卵巢功能、提高卵子的成熟率、受精率和早期胚胎 的发育率,特别是老龄动物卵母细胞的成熟率及卵子的质量。
(2)本发明还提供了一种烟酰胺单核苷酸的制备以及一种含有烟酰胺单核苷酸、褪黑素 和辅酶Q10的新药物组合物,能够显著改善老龄动物的排卵率,提高卵子质量,对于女性不 孕不育的预防和治疗以及动物胚胎的体外生产具有重大意义。该组合物中的三种活性成分均为 生物机体的内源性物质,从已公开发表的文献来看不会产生副作用,因此作为药物、保健品或 生物制剂的安全性很高。
附图说明
图1为老龄组和给药组小鼠卵母细胞排极体的典型图片;
图2为老龄组和给药组小鼠卵母细胞体外受精后发育至二细胞的典型图片;
图3为老龄组和给药组小鼠胚胎体外培养后囊胚发育的典型图片;
具体实施方式
为使本发明更加容易理解,下面将进一步阐述本发明的具体实施例。下列实施例举例了发 明人的标准实验室实践,用于示例本发明的模式,而不应将本发明理解为限定于这些实施例的 范围。本实施例根据本发明公开和本领域技术人员的普遍水平,技术人员将理解下列仅用于示 例,可以在不超过本发明的范围内进行各种变动、修饰和改造。其中所涉及的技术,除非特别 说明,均是本领域技术人员熟知的常规实验技术;若未特别指明,以下实施例中使用的试剂和 材料均可从商业途径获得。
实施例
一、老龄鼠卵母细胞体外成熟液的配置及组合物的添加
体外成熟液为M16(sigma,货号M7292),组合物由烟酰胺单核苷酸、褪黑素和辅酶Q10 组成,且所述体外成熟培养液中烟酰胺单核苷酸的浓度为200μM,褪黑素浓度为10μM,辅酶 Q10浓度为50μM。
二、老龄鼠卵母细胞的获取及体外培养和成熟率的统计
老龄鼠注射10IU/mL孕马血清促性腺激素(宁波市第三激素制品有限公司,中国)48个小 时后,利用脱颈法处死小鼠。取出卵巢剁碎后用口吸管将GV期卵母细胞取出,将卵母细胞取 出后放入体外成熟培养液或者含有组合物的体外成熟培养液中洗三遍,移入含有组合物的体外 成熟培养液中继续培养。培养14小时后统计卵母细胞成熟率,通过卵母细胞排出第一极体判 断卵母细胞是否达到第二次减数分裂中期。
三、老龄鼠卵母细胞的体外受精及受精率的统计
成熟的卵母细胞通过与体外获能的精子共孵育,从而完成体外受精。具体操作是将公鼠附 睾尾取出,放入HTF获能滴中,用尖头镊子撕开后放入培养箱,待精子获能1小时左右,将 成熟卵母细胞用受精液洗三遍后移入受精滴中,吸取50ul获能完成的精子加入放入卵母细胞 的受精滴中,受精6-8小时后,将受精的卵母细胞用胚胎培养液KSOM洗三遍,移入胚胎培 养滴中继续培养。待培养24小时后,即可观察记录二细胞所占比例,从而得出卵母细胞的受 精率。
四、老龄鼠胚胎的体外培养及囊胚率的统计
将发育至二细胞的胚胎留下,其余移出胚胎培养滴,待培养至受精后第五天的时候观察记 录囊胚所占比例,进而得出胚胎的囊胚率。
五、结果
1、卵母细胞成熟率的统计
表1:老化组与给药组3次重复实验得到卵母细胞成熟率的统计结果
表1结果表明小鼠体外成熟培养液中添加组合物可以显著提升老龄鼠卵母细胞的体外成 熟率,由老化组平均56.1±0.13%n=59升至给药组81.2±0.56%,n=70;P<0.05。
2、卵母细胞体外受精率的统计
表2:老化组与给药组3次重复实验得到卵母细胞外受精率的统计结果
表2结果表明小鼠体外成熟培养液中添加组合物可以显著提升老龄鼠卵母细胞的体外受 精率,由老化组平均39.5±0.35%n=33升至老化挽救组64.6±0.57%,n=56;P<0.05。
3、胚胎体外发育囊胚率的统计
表3:老化组与给药组3次重复实验得到胚胎体外发育囊胚率的统计结果
表3结果表明小鼠体外成熟培养液中添加组合物可以显著提升老龄鼠胚胎的囊胚率,老化 组由于卵母细胞质量低下,难以发育至囊胚期。给药组胚胎约有25.3%可以发育至囊胚。
本技术领域中的普通技术人员应当认识到,以上的实施例仅是用来说明本发明,而并非用 作为对本发明的限定,只要在本发明的实质精神范围内,对以上所述实施例的变化、变型都将 落在本发明的权利要求范围内。
Claims (9)
1.烟酰胺单核苷酸在制备改善卵巢功能、提高卵子和胚胎质量的药物或保健品中的应用。
2.烟酰胺单核苷酸在制备改善卵子和胚胎质量的生物试剂中的应用。
3.一种改善卵巢功能、提高卵子和胚胎质量的组合物,其特征在于包含烟酰胺单核苷酸、褪黑素和辅酶Q10。
4.根据权利要求3所述的组合物,特征在于所述的组合物由烟酰胺单核苷酸、褪黑素和辅酶Q10组成。
5.权利要求3或4所述的组合物在制备改善卵巢功能、提高卵子和胚胎质量的药物或保健品中的应用。
6.权利要求3或4所述的组合物在制备改善卵子和胚胎质量的生物试剂中的应用。
7.卵母细胞体外成熟培养液,其特征在于包含权利要求3或4所述的组合物,且所述体外成熟培养液中烟酰胺单核苷酸的浓度为100~500μM,褪黑素浓度为5~50μM,辅酶Q10浓度为20~200μM。
8.根据权利要求7所述的卵母细胞体外成熟培养液,其特征在于所述卵母细胞体外成熟培养还包括TCM199基础培养液、葡萄糖、丙酮酸钠、半胱氨酸、表皮生长因子、黄体生成素、促卵泡生成素以及10%血清替代物。
9.一种培养卵母细胞的方法,其特征在于包括如下的步骤:
(1)将权利要求3或4所述的组合物按照对应浓度配比后加入培养液中,放入培养箱中平衡预热2-3小时;
(2)将卵母细胞取出后放入含有权利要求5或6所述的组合物的体外成熟培养液中洗三遍,移入权利要求7或8所述的体外成熟培养液中继续培养;
(3)成熟后的卵母细胞可以继续完成体外受精及体外胚胎发育。
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CN115247146A (zh) * | 2021-12-31 | 2022-10-28 | 温州医科大学 | 一种人类胚胎体外培养基及提高体外培养人类胚胎发育潜能的方法 |
CN114836373A (zh) * | 2022-05-30 | 2022-08-02 | 上海洲康医疗器械有限公司 | 一种卵细胞激活培养基及其培育方法和应用 |
CN114836373B (zh) * | 2022-05-30 | 2023-11-10 | 上海洲康医疗器械有限公司 | 一种卵细胞激活培养基及其培育方法和应用 |
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