CN117511854B - 一种未成熟卵母细胞培养液及其制备方法 - Google Patents
一种未成熟卵母细胞培养液及其制备方法 Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
本发明涉及一种未成熟卵母细胞培养液及其制备方法。所述未成熟卵母细胞培养液包括质量分数如下的组分:无机盐1‑3%、氨基酸0.1‑0.2%、能量物质0.05‑0.25%、维生素≤0.01%、生长因子≤0.01%、褪黑素0.01‑0.2%、改性细胞外基质0.1‑5%以及余量的水。其中,所述改性细胞外基质由细胞外基质经谷胱甘肽改性所得,谷胱甘肽有助于保持线粒体膜完整,保证细胞能量代谢,其与生长因子、褪黑素协同,使得未成熟卵细胞在体内外生长的条件高度一致。本发明的未成熟卵母细胞培养液有利于未成熟卵母细胞在体外的发育,促进未成熟卵母细胞的成熟及囊胚的形成,可以获得较高的囊胚率。
Description
技术领域
本发明属于辅助生殖技术领域,具体涉及一种未成熟卵母细胞培养液及其制备方法。
背景技术
在辅助生殖的过程中,人们广泛采用激素等外界刺激,对女性患者做超数排卵的操作,然后通过卵母细胞的体外受精形成受精卵,从而实现孕育后代的目标。然而,在取得的卵子中,有一部分卵子是未成熟的细胞,这些未成熟的卵子不可能直接用于受精,因此,需要对该类卵子进行体外培养液中的培养后,再进一步进行受精过程。然而,现在对于卵母细胞培养液的研究还未能完全成熟产业化,从而这部分未成熟的卵母细胞不能加以利用。
如现有技术CN111518749A公开了一种未成熟卵母细胞的体外培养液,包括丁内酯Ⅰ 0.01-1 mg/L、雌二醇0.001-2 mg/L、促卵泡素0.001-1 IU/L、促黄体生成素0.001-1 IU/L、表皮生长因子0.001-1 IU/L、类胰岛素生长因子0.001-1 mg/L和人绒毛膜促性腺激素0.001-1 IU/L。然而,诸如此类的技术往往较难在临床上获得成功,主要原因在于:未成熟的卵母细胞往往线粒体能力不足,而研究表明足够多的线粒体对于蛋白质的化学修饰和纺锤体的形成至关重要,线粒体能力受到限制时,体外培养的卵母细胞成熟率明显下降;而且体外环境与体内差异较大,未成熟卵母细胞不适应体外环境,因而使得成熟过程中受到损伤。
基于此,亟需找到一种技术方案,来解决上述技术难题。
发明内容
为解决现有技术的不足,本发明提供了一种未成熟卵母细胞培养液及其制备方法。在本发明中,在培养液中新增了经谷胱甘肽改性的细胞外基质,其与生长因子、褪黑素协同,可以充分模拟卵细胞在卵巢中的生长环境,并且提供与之相匹配的营养物质,从而使得未成熟的卵细胞在体外的生长与在体内生长的条件高度一致,有利于未成熟的卵母细胞在体外的发育。
本发明的一个目的在于,提供一种未成熟卵母细胞培养液,所述未成熟卵母细胞培养液包括质量分数如下的组分:
无机盐 1-3%
氨基酸 0.1-0.2%
能量物质 0.05-0.25%
维生素 ≤0.01%
生长因子 ≤0.01%
褪黑素 0.01-0.2%
改性细胞外基质 0.1-5%
水 余量;
其中,
所述改性细胞外基质为谷胱甘肽改性后的细胞外基质。
进一步地,所述无机盐选自氯化钙、氯化钠、氯化钾、硫酸镁、碳酸氢钠、磷酸二氢钠、柠檬酸钠的一种或多种。
进一步地,所述氨基酸包括必须氨基酸,和非必须氨基酸。
进一步地,所述能量物质选自丙酮酸钠、葡萄糖、乳酸钠的一种或多种。
进一步地,所述维生素选自D-泛酸钙、氯化胆碱、叶酸、肌醇、烟酰胺、抗坏血酸的一种或多种。
本发明的另一个目的在于,提供上述未成熟卵母细胞培养液的制备方法,其制备方法包括如下步骤:
S1、将动物卵巢组织进行脱细胞处理,然后冻干,得到细胞外基质;
S2、将所述细胞外基质浸入EDC溶液中,然后加入NHS溶液、谷胱甘肽得到混合物;
S3、用缓冲剂将所述混合物的pH调节至5.5-6.0,并低温浸泡;
S4、将所述混合物取出,洗涤干燥,得到改性细胞外基质,与其余组分混合,得到所述未成熟卵母细胞培养液。
进一步地,所述EDC和NHS的摩尔比为2:1-4:1。
进一步地,所述细胞外基质和谷胱甘肽的质量比为1:0.05-1:1。
进一步地,所述低温为0-4℃。
本发明具有以下有益效果:
本发明提供的未成熟卵母细胞培养液同时含有改性细胞外基质、生长因子和褪黑素。其中,细胞外基质经过谷胱甘肽改性,谷胱甘肽是细胞内重要的调节代谢物质,可参与机体多种重要的生化反应,有助于保持线粒体膜完整,保护体内重要酶蛋白巯基不被氧化、灭活,保证细胞能量代谢。经过谷胱甘肽改性后的细胞外基质可以更好地诱导细胞增殖、迁移和分化,充分模拟卵细胞在卵巢中的生长环境,并且提供与之相匹配的营养物质,从而使得未成熟的卵细胞在体内外生长的条件高度一致,有利于未成熟卵母细胞在体外的发育。所述改性细胞外基质与生长因子、褪黑素起到协同作用,共同促进未成熟卵母细胞的生长发育。
附图说明
图1为未成熟老鼠卵母细胞外观图;
图2为实施例1培养后的成熟老鼠卵母细胞外观图;
图3为实施例1培养后的鼠胚培养至囊胚阶段外观图。
具体实施方式
下面结合实施例对本发明进行具体描述,以便于所属技术领域的人员对本发明的理解。有必要在此特别指出的是,实施例只是用于对本发明做进一步说明,不能理解为对本发明保护范围的限制,所属领域技术熟练人员,根据上述发明内容对本发明作出的非本质性的改进和调整,应仍属于本发明的保护范围。同时下述所提及的原料未详细说明的,均为市售产品;未详细提及的工艺步骤或制备方法为均为本领域技术人员所知晓的工艺步骤或制备方法。
本实施例中的环境温度,如不特别指明,均指室温。
本实施例中,选用以下原料:
无机盐,选用氯化钠、氯化钾、二水氯化钙、七水硫酸镁、碳酸氢钠按110:5:2:0.8:28的质量比复配而成,
其中,
氯化钠,采购自Sigma,牌号为S3014;
氯化钾,采购自Sigma,牌号为P9541;
二水氯化钙,采购自Sigma,牌号为C7902;
七水硫酸镁,采购自Sigma,牌号为M1880;
碳酸氢钠,采购自Sigma,牌号为S5761;
氨基酸,选用非必须氨基酸、必须氨基酸按1.5:1的质量比复配而成,
其中,
非必须氨基酸,采购自Gibco,牌号为11140050;
必须氨基酸,采购自Gibco,牌号为12492021;
能量物质,选用葡萄糖、乳酸钠、丙酮酸钠按16:2:1的质量比复配而成,
其中,
葡萄糖,采购自Sigma,牌号为1181302;
乳酸钠,采购自Sigma,牌号为1614308;
丙酮酸钠,采购自Sigma,牌号为P2256;
维生素,选用抗坏血酸、泛酸钙、氯化胆碱、叶酸、烟酰胺按1.5:0.2:18:0.1:1的质量比复配而成,
其中,
抗坏血酸,采购自Sigma,牌号为1043003;
泛酸钙,采购自Sigma,牌号为1087009;
氯化胆碱,采购自Sigma,牌号为C7527;
叶酸,采购自Sigma,牌号为F8758;
烟酰胺,采购自Sigma,牌号为N0636;
生长因子,选用促卵泡素、表皮生长因子、人绒毛膜促性腺激素、类胰岛素生长因子、促黄体生成素按2:1.5:2:1:2的质量比复配而成,
其中,
促卵泡素,采购自Aladdin,牌号为rp174041;
表皮生长因子,采购自Sigma,牌号为E4127;
人绒毛膜促性腺激素,采购自Sigma,牌号为230734;
类胰岛素生长因子,采购自Aladdin,牌号为R283957;
促黄体生成素,采购自Aladdin,牌号为L464334;
褪黑素,采购自Sigma,牌号为M5250;
细胞外基质,取自猪卵巢组织,经脱细胞处理制备而成;
EDC,1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐溶液,采购自Sigma,牌号为800907;
NHS,N-羟基丁二酰亚胺溶液,采购自Sigma,牌号为804518;
HTF溶液,人类输卵管液,用于体外受精的细胞培养,采购自Sigma,牌号为MR-070;
M16溶液,常用的哺乳动物细胞培养基,采购自Sigma,牌号为MR-016;
谷胱甘肽,采购自Sigma,牌号为1294820;
缓冲剂,采购自Sigma,牌号为P3563;
本实施例中的水,均为去离子水。
实施例1
一种未成熟卵母培养液,所述未成熟卵母细胞培养液包括质量分数如下的组分:
无机盐 2%
氨基酸 0.15%
能量物质 0.2%
维生素 0.01%
生长因子 0.01%
褪黑素 0.1%
改性细胞外基质 4%
水 余量;
所述未成熟卵母培养液的制备方法包括如下步骤:
S1、取20 g猪卵巢组织进行脱细胞处理,然后在-65℃冻干,得到细胞外基质;
S2、取5 g所述细胞外基质浸入50 ml EDC溶液(0.1 mol/L)中,然后加入50 mlNHS溶液(0.05 mol/L)、2.5 g谷胱甘肽并搅拌,得到混合物;
S3、将所述混合物用缓冲剂调节pH至5.5,在4℃低温浸泡;
S4、将所述混合物取出,过滤并洗涤干燥,得到所述改性细胞外基质,按上述质量分数与其余组分混合,得到所述未成熟卵母细胞培养液。
实施例2
一种未成熟卵母细胞培养液,所述未成熟卵母细胞培养液包括质量分数如下的组分:
无机盐 2.5%
氨基酸 0.2%
能量物质 0.25%
维生素 0.01%
生长因子 0.01%
褪黑素 0.2%
改性细胞外基质 5%
水 余量;
所述未成熟卵母细胞培养液制备方法、步骤同实施例1,仅按上述组合物改变原料质量分数。
实施例3
一种未成熟卵母细胞培养液,所述未成熟卵母细胞培养液包括质量分数如下的组分:
无机盐 1.5%
氨基酸 0.1%
能量物质 0.2%
维生素 0.01%
生长因子 0.01%
褪黑素 0.15%
改性细胞外基质 2%
水 余量;
所述未成熟卵母细胞培养液制备方法、步骤同实施例1,仅按上述组合物改变原料质量分数。
对比例1
一种未成熟卵母细胞培养液,所述未成熟卵母细胞培养液制备方法、步骤同实施例1,仅删去改性细胞外基质,其他成分及制备方法与实施例1相同。
对比例2
一种未成熟卵母细胞培养液,所述未成熟卵母细胞培养液制备方法、步骤同实施例1,仅删去生长因子,其他成分及制备方法与实施例1相同。
对比例3
一种未成熟卵母细胞培养液,所述未成熟卵母细胞培养液制备方法、步骤同实施例1,仅删去褪黑素,其他成分及制备方法与实施例1相同。
对比例4
一种未成熟卵母细胞培养液,所述未成熟卵母细胞培养液制备方法、步骤同实施例1,仅将改性细胞外基质替换为等质量的细胞外基质,其他成分及制备方法与实施例1相同。
测试例
测试方法:
对实施例和测试例进行卵母细胞活性测试,测试方法参考YY/T 1434-2016的标准。测试方法如下:
S1、处死雌鼠,选择未成熟的卵母细胞分成7组,分别用上述培养液培养20 h,观察成熟卵母细胞个数,计算卵母细胞成熟率;
S2、将卵母细胞放到HTF溶液中做体外受精,计算受精率;
S3、将卵母细胞放到M16溶液中培养72 h至囊胚,计算囊胚率。
所得结果如表1所示。
表 1 细胞活性测试结果
图1为未成熟老鼠卵母细胞外观图;
图2为实施例1培养后的成熟老鼠卵母细胞外观图;
图3为实施例1培养后的鼠胚培养至囊胚阶段外观图。
从表1的测试结果可以得出,与对比例相比,实施例的卵母细胞均具有更优良的生物活性。对比例说明,谷胱甘肽改性后的细胞外基质能够促进未成熟卵母细胞的成熟及囊胚的形成,生长因子、褪黑素等成分的复配能够进一步促进上述过程。
综上所述,改性细胞外基质、生长因子更多地影响未成熟卵母细胞的成熟,而褪黑素主要影响囊胚的形成。其中,改性细胞外基质、生长因子、褪黑素三者互相协同,使得所述未成熟卵母细胞培养液能够获得最佳的细胞培养效果,获得较高的囊胚率。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (2)
1. 一种未成熟卵母细胞培养液,其特征在于,所述未成熟卵母细胞培养液包括质量分数如下的组分:
无机盐 1.5-2.5%
氨基酸 0.1-0.2%
能量物质 0.2-0.25%
维生素 0.01%
生长因子 0.01%
褪黑素 0.1-0.2%
改性细胞外基质 2-5%
水 余量;
其中,
所述无机盐选用氯化钠、氯化钾、二水氯化钙、七水硫酸镁、碳酸氢钠按110:5:2:0.8:28的质量比复配而成;
所述氨基酸选用非必须氨基酸、必须氨基酸按1.5:1的质量比复配而成;
所述能量物质选用葡萄糖、乳酸钠、丙酮酸钠按16:2:1的质量比复配而成;
所述维生素选用抗坏血酸、泛酸钙、氯化胆碱、叶酸、烟酰胺按1.5:0.2:18:0.1:1的质量比复配而成;
所述生长因子选用促卵泡素、表皮生长因子、人绒毛膜促性腺激素、类胰岛素生长因子、促黄体生成素按2:1.5:2:1:2的质量比复配而成;
所述改性细胞外基质为谷胱甘肽改性后的细胞外基质,其制备包括如下步骤:
S1、取20 g猪卵巢组织进行脱细胞处理,然后在-65℃冻干,得到细胞外基质;
S2、取5 g所述细胞外基质浸入50 ml 0.1 mol/L EDC溶液中,然后加入50 ml 0.05mol/L NHS溶液、2.5 g谷胱甘肽并搅拌,得到混合物;
S3、将所述混合物用缓冲剂调节pH至5.5,在4℃低温浸泡;
S4、将所述混合物取出,过滤并洗涤干燥,得到所述改性细胞外基质。
2.如权利要求1所述未成熟卵母细胞培养液的制备方法,其特征在于,所述未成熟卵母细胞培养液的制备方法包括如下步骤:
S1、取20 g猪卵巢组织进行脱细胞处理,然后在-65℃冻干,得到细胞外基质;
S2、取5 g所述细胞外基质浸入50 ml 0.1 mol/L EDC溶液中,然后加入50 ml 0.05mol/L NHS溶液、2.5 g谷胱甘肽并搅拌,得到混合物;
S3、将所述混合物用缓冲剂调节pH至5.5,在4℃低温浸泡;
S4、将所述混合物取出,过滤并洗涤干燥,得到所述改性细胞外基质,按上述质量分数与其余组分混合,得到所述未成熟卵母细胞培养液。
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