CN114836373A - 一种卵细胞激活培养基及其培育方法和应用 - Google Patents
一种卵细胞激活培养基及其培育方法和应用 Download PDFInfo
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- CN114836373A CN114836373A CN202210602983.0A CN202210602983A CN114836373A CN 114836373 A CN114836373 A CN 114836373A CN 202210602983 A CN202210602983 A CN 202210602983A CN 114836373 A CN114836373 A CN 114836373A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本申请涉及辅助生殖技术领域,更具体地说,它涉及一种卵细胞激活培养基及其培育方法和应用。所述卵细胞激活培养基包括基础培养液和激活组分:所述激活组分包括钙离子载体2‑6μmol/L、IGF‑2 1‑100 nmol/L、人血清蛋白0.005‑5 g/L、β‑烟酰胺单核苷酸0.02‑0.05 mg/L、辅酶Q10 0.1‑2.0 mg/L。本申请中通过激活组分与基础培养基的复配,可有效提高卵细胞的激活率,并达到减少因精卵结合时因卵子激活缺陷,导致诱导减数无法正常进行分裂的现象,可将受精失败率降至10‑30%。
Description
技术领域
本申请涉及辅助生殖技术领域,更具体地说,它涉及一种卵细胞激活培养基及其培育方法和应用。
背景技术
辅助生殖技术是人类辅助生殖技术的简称,是指将患者双方的卵子与精子取出后,在体外进行受精,然后再将受精卵或者胚胎移植回母体子宫内发育成胎儿的技术,其中卵母细胞的激活状态则是决定着最终的受精率的关键之一,因此也成为了现阶段所首要攻克的技术问题。
相关技术中的卵母细胞培育激活即先通过成熟促进因子(MPF)和细胞生长抑制因子(CSF)的调节作用,促使卵母细胞发育停滞在MII期(第二次减数分裂中期),再在受精前通过增加Ca2+浓度形成钙波及震荡,以破坏CSF和MPF活性,继而使得卵母细胞内的各种代谢活动重新活跃的技术。
经上述激活步骤处理后的卵母细胞可自MII期完成减数分裂,形成原核(PN),释放第二极体(2Pb),完成受精过程,但受限于卵母细胞的激活率,其受精失败率普遍高达40%或以上,因此迫切需要提供一种能有效保障卵子激活率,诱导减数分裂正常进行的激活培养基及培育技术。
发明内容
为保障卵细胞的激活率,以减少因精卵结合时因卵子激活缺陷,导致诱导减数无法正常进行分裂的现象,本申请提供一种卵细胞激活培养基及其培育方法。
第一方面,本申请提供一种卵细胞激活培养基,采用如下的技术方案:
一种卵细胞激活培养基,所述卵细胞激活培养基包括基础培养液和激活组分:
所述激活组分包括钙离子载体2-6μmol/L、IGF-2 1-100nmol/L、人血清蛋白0.005-5g/L、β-烟酰胺单核苷酸0.02-0.05mg/L、辅酶Q10 0.1-2.0mg/L。
通过采用上述技术方案,该配比及组成的激活组分其与基础培养基的复配后,可通过多种组分的协同效应有效提高卵细胞的激活率,并显著减少因精卵结合时因卵子激活缺陷,导致诱导减数无法正常进行分裂的现象,可将受精失败率降至10-30%;
推测其原因可能是由于该组分下的激活组分除能在激活阶段有效模拟体内环境降低并抑制CSF和MPF的活性外,还可促使钙波的形成,以及增强对CSF和MPF震荡破坏效果,继而使得卵母细胞内的各种代谢活动被重启且有利于后续精子的穿透结合。
优选的,所述基础培养液为包括如下组分的水溶液:无机盐缓冲组分30-80g/L、氨基酸组分1-22g/L、葡萄糖0.5-1.2g/L、乳酸钙0.1-2.0g/L、乳酸钠1.0-5.0g/L、丙酮酸钠0.1-0.8g/L、四元羧酸乙二胺四乙酸0.001-000.5g/L、丙氨酰-谷氨酰胺0.1-1.5g/L。
优选的,所述无机盐缓冲组分由氯化钠、氯化钾、磷酸二氢钠、硫酸镁、碳酸氢钠、氯化钙和水按摩尔比(5-10):(0.3-0.8):(0.05-0.1):(0.2-1.6):(1.0-3.5):(0.05-0.3):(900-1000)组成。
优选的,所述氨基酸组分由L-苯丙氨酸、L-羟脯氨酸、丝氨酸、甘氨酸、L-缬氨酸、L-天门冬氨酸、天冬酰胺、谷氨酸按摩尔比(0.01-0.1):(0.01-0.1):(0.01-0.1):(0.01-0.1):
(0.01-0.1):(0.01-0.16):(0.06-0.24):(0.06-0.24)组成。
优选的,还包括生物碱组分0.1-0.5g/L,所述生物碱组分由腺嘌呤、硫酸腺嘌呤、鸟嘌呤、盐酸鸟嘌呤、尿嘧啶和腺苷三磷酸二钠按摩尔比(0.1-0.2):(0.05-0.08):(0.1-0.2):(0.2-0.4):(0.2-0.4):(0.06-0.12)组成。
通过采用上述技术方案,上述组分及比例生物碱的掺入可以与激活组分复配,有效模拟体内环境降低并进一步抑制CSF和MPF的活性,从而保障了卵细胞的激活效果。
优选的,所述激活组分还包括褪黑素0.1-5nmol/L、辅酶NADH 0.05-0.5mg/L,所述钙离子载体为A23187。
通过采用上述技术方案,褪黑素与辅酶NADH的掺入,除能辅助抑制CSF和MPF的活性外,还可增强钙波及其震荡的破坏效果,继而使得卵母细胞内的各种代谢活动被完全重启。
第二方面,本申请提供一种卵细胞激活培养基的制备方法,采用如下的技术方案:一种卵细胞激活培养基的使用方法,具体步骤如下:
S1、先将激活培养基加入培养器皿中;
S2、再将S1中的培养器皿置于培养箱中,以37±0.2℃、6±0.1%CO2、湿度95±1%的条件平衡4-6h;
S3、待卵细胞激活培养基平衡完全后,将卵细胞放入激活培养液中,37±0.2℃、6±0.1%CO2、湿度95±1%的条件培养箱中培育10-30min,即可完成激活。
第三方面,本申请提供一种卵细胞激活培养基的应用,采用如下的技术方案:
一种卵细胞激活培养基的应用,所述的卵细胞激活培养基用于卵母细胞的激活、ICSI后的卵子或者受精卵培育,所用卵细胞选自人或哺乳动物。
综上所述,本申请具有以下有益效果:
1、本申请中通过激活组分与基础培养基的复配协同作用,有效提高了卵细胞的激活率,并显著减少因精卵结合时因卵子激活缺陷,导致诱导减数无法正常进行分裂的现象,可将受精失败率降至10-30%;
2、本申请通过生物碱的掺入,并将其与激活组分复配,有效模拟了体内环境的同时,进一步抑制了CSF和MPF的活性,从而保障了卵细胞的激活效果,保障了受精的成功率;3、本申请通过褪黑素与辅酶NADH的掺入,除辅助抑制CSF和MPF的活性外,还进一步增强了钙波及其震荡的破坏效果,继而使得卵母细胞内的各种代谢活动被完全重启;4、本申请中的卵细胞激活培养基其应用方法简易,各项指标易于控制和达到,且具有较高的应用价值,能够用于卵母细胞的激活、ICSI后的卵子或者受精卵培育,所用卵细胞选自人或哺乳动物。
具体实施方式
以下结合应用例对本申请作进一步详细说明。
本申请的各应用例中所用的原料和试剂,均为市售的常规试剂。
实施例
实施例1-6
一种卵细胞激活培养基,所述卵细胞激活培养基包括基础培养液和激活组分,其各组分及含量如表1所示:
表1实施例1-6卵细胞激活培养基各组分及含量
其中钙离子载体为A23187;
无机盐缓冲组分由氯化钠、氯化钾、磷酸二氢钠、硫酸镁、碳酸氢钠、氯化钙和水按摩尔比3:0.1:0.01:0.1:0.5:0.01:800组成;
氨基酸组分由L-苯丙氨酸、L-羟脯氨酸、丝氨酸、甘氨酸、L-缬氨酸、L-天门冬氨酸、天冬酰胺、谷氨酸按摩尔比0.01:0.01:0.01:0.01:0.01:0.01:0.06:0.06组成。
实施例7
一种卵细胞激活培养基,与实施例1的不同之处在于,无机盐缓冲组分由氯化钠、氯化钾、磷酸二氢钠、硫酸镁、碳酸氢钠、氯化钙和水按摩尔比5:0.3:0.05:0.2:1.0:0.05:900组成。
实施例8
一种卵细胞激活培养基,与实施例1的不同之处在于,无机盐缓冲组分由氯化钠、氯化钾、磷酸二氢钠、硫酸镁、碳酸氢钠、氯化钙和水按摩尔比8:0.5:0.08:1.2:2.0:0.15:900组成。
实施例9
一种卵细胞激活培养基,与实施例1的不同之处在于,无机盐缓冲组分由氯化钠、氯化钾、磷酸二氢钠、硫酸镁、碳酸氢钠、氯化钙和水按摩尔比10:0.8:0.1:1.6:3.5:0.3:1000组成。
实施例10
一种卵细胞激活培养基,与实施例1的不同之处在于,无机盐缓冲组分由氯化钠、氯化钾、磷酸二氢钠、硫酸镁、碳酸氢钠、氯化钙和水按摩尔比12:1:0.2:2.0:4:0.4:1000组成。
实施例11
一种卵细胞激活培养基,与实施例1的不同之处在于,氨基酸组分由L-苯丙氨酸、L-羟脯氨酸、丝氨酸、甘氨酸、L-缬氨酸、L-天门冬氨酸、天冬酰胺、谷氨酸按摩尔比0.01:0.01:0.01:0.01:0.01:0.01:0.06:0.06组成。
实施例12
一种卵细胞激活培养基,与实施例1的不同之处在于,氨基酸组分由L-苯丙氨酸、L-羟脯氨酸、丝氨酸、甘氨酸、L-缬氨酸、L-天门冬氨酸、天冬酰胺、谷氨酸按摩尔比0.05:0.05:0.05:0.05:0.05:0.08:0.15:0.15组成。
实施例13
一种卵细胞激活培养基,与实施例1的不同之处在于,氨基酸组分由L-苯丙氨酸、L-羟脯氨酸、丝氨酸、甘氨酸、L-缬氨酸、L-天门冬氨酸、天冬酰胺、谷氨酸按摩尔比0.1:0.1:0.1:0.1:0.1:0.16:0.24:0.24组成。
实施例14
一种卵细胞激活培养基,与实施例1的不同之处在于,氨基酸组分由L-苯丙氨酸、L-羟脯氨酸、丝氨酸、甘氨酸、L-缬氨酸、L-天门冬氨酸、天冬酰胺、谷氨酸按摩尔比0.2:0.2:0.2:0.2:0.2:0.16:0.24:0.24组成。
实施例15-19
一种卵细胞激活培养基,与实施例1的不同之处在于,还包括生物碱组分,其各组分及含量如表2所示:
表2实施例15-19卵细胞激活培养基各组分及含量
其中生物碱组分由腺嘌呤、硫酸腺嘌呤、鸟嘌呤、盐酸鸟嘌呤、尿嘧啶和腺苷三磷酸二钠按摩尔比0.05:0.03:0.05:0.1:0.1:0.03组成。
实施例20
一种卵细胞激活培养基,与实施例17的不同之处在于,生物碱组分由腺嘌呤、硫酸腺嘌呤、鸟嘌呤、盐酸鸟嘌呤、尿嘧啶和腺苷三磷酸二钠按摩尔比0.1:0.05:0.1:0.2:0.2:0.06组成。
实施例21
一种卵细胞激活培养基,与实施例17的不同之处在于,生物碱组分由腺嘌呤、硫酸腺嘌呤、鸟嘌呤、盐酸鸟嘌呤、尿嘧啶和腺苷三磷酸二钠按摩尔比0.15:0.06:0.15:0.3:0.3:0.08组成。
实施例22
一种卵细胞激活培养基,与实施例17的不同之处在于,生物碱组分由腺嘌呤、硫酸腺嘌呤、鸟嘌呤、盐酸鸟嘌呤、尿嘧啶和腺苷三磷酸二钠按摩尔比0.2:0.08:0.2:0.4:0.4:0.12组成。
实施例23
一种卵细胞激活培养基,与实施例17的不同之处在于,生物碱组分由腺嘌呤、硫酸腺嘌呤、鸟嘌呤、盐酸鸟嘌呤、尿嘧啶和腺苷三磷酸二钠按摩尔比0.3:0.1:0.3:0.5:0.5:0.15组成。
实施例24-28
一种卵细胞激活培养基,与实施例1的不同之处在于,还包括生物碱组分,其各组分及含量如表3所示:
表2实施例24-28卵细胞激活培养基各组分及含量
性能检测试验先选取ICSI(卵胞浆内单精子显微注射)受精失败的卵子和精子,再分别对卵子和精子进行预处理:脱去卵子外周颗粒细胞,经密度梯度离心处理选取形态正常的优质精子,然后采用单精子胞浆注射的技术方法进行人为干预受精,待优质精子注射入卵子内后;
采用下列应用例中的方式对同源的卵子(考虑到卵子活力、取样难度等因素数量小幅度波动)进行激活,培育第二天观察卵子是否受精(观察原核形成及是否有卵裂),并计算受精率%=受精卵数/卵子总数。
应用例
应用例1
一种卵细胞激活培养基的应用,其具体使用步骤如下:
S1、先将实施例1中所得的激活培养基加入培养器皿中;
S2、再将S1中的培养器皿置于培养箱中,以37±0.2℃、6±0.1%CO2、湿度95±1%的条件平衡6h;
S3、待卵细胞激活培养基平衡完全后,将卵细胞放入激活培养液中,37±0.2℃、6±0.1%CO2、湿度95±1%的条件培养箱中培育30min,即可完成激活。
应用例2-6
一种卵细胞激活培养基的应用,与应用例1的不同之处在于,所用卵细胞激活培养基的使用情况不同,具体对应关系下表所示。
表:应用例2-6中卵细胞激活培养基使用情况对照表
组别 | 卵细胞激活培养基 |
应用例2 | 由实施例2制得 |
应用例3 | 由实施例3制得 |
应用例4 | 由实施例4制得 |
应用例5 | 由实施例5制得 |
应用例6 | 由实施例6制得 |
抽取经上述应用例1-6中激活方法所得的卵细胞,按上述测量步骤和测量标准测试其受精率%,测试结果记入下表。
表:应用例1-6中卵细胞激活培养基性能测试结果
从上表中可以看出,经上述应用例1-6中激活方法所得的卵细胞,均能被有效激活,其受精率较高,为75.0-84.6%;
可见该配比及组成的激活组分其与基础培养基的复配后,可通过多种组分的协同效应有效提高卵细胞的激活率,并显著减少因精卵结合时因卵子激活缺陷,导致诱导减数无法正常进行分裂的现象。
特别是,应用例2中制得的卵细胞激活培养基,其激活效果最优,其受精率高达84.6%;
可见该组分及配比为优选配备,其复配效果最优,分析其原因可能是由于由于该组分下的激活组分除能在激活阶段有效模拟体内环境降低并抑制CSF和MPF的活性外,还可促使钙波的形成,以及增强对CSF和MPF震荡破坏效果,继而使得卵母细胞内的各种代谢活动被重启且有利于后续精子的穿透结合。
应用例7-10
一种卵细胞激活培养基的应用,与应用例1的不同之处在于,所用卵细胞激活培养基的使用情况不同,具体对应关系下表所示。
表:应用例7-10中卵细胞激活培养基使用情况对照表
组别 | 卵细胞激活培养基 |
应用例7 | 由实施例7制得 |
应用例8 | 由实施例8制得 |
应用例9 | 由实施例9制得 |
应用例10 | 由实施例10制得 |
抽取经上述应用例7-10中激活方法所得的卵细胞,按上述测量步骤和测量标准测试其受精率%,测试结果记入下表。
表:应用例7-10中卵细胞激活培养基性能测试结果
从上表中可以看出,经上述应用例7-10中激活方法所得的卵细胞,均能被有效激活,其受精率较高,为75.0-76.9%;
可见该配比及组成的无机盐组分仅会轻微影响到多种组分的协同效应,主要用于维持卵细胞的正常生理状态和渗透压,从上表还可以看出其中应用例7-9为优选例,无机盐缓冲组分的优选配比为:氯化钠、氯化钾、磷酸二氢钠、硫酸镁、碳酸氢钠、氯化钙和水按摩尔比计为(5-10):(0.3-0.8):(0.05-0.1):(0.2-1.6):(1.0-3.5):(0.05-0.3):(900-1000)。
应用例11-14
一种卵细胞激活培养基的应用,与应用例1的不同之处在于,所用卵细胞激活培养基的使用情况不同,具体对应关系下表所示。
表:应用例11-14中卵细胞激活培养基使用情况对照表
组别 | 卵细胞激活培养基 |
应用例11 | 由实施例11制得 |
应用例12 | 由实施例12制得 |
应用例13 | 由实施例13制得 |
应用例14 | 由实施例14制得 |
抽取经上述应用例11-14中激活方法所得的卵细胞,按上述测量步骤和测量标准测试其受精率%,测试结果记入下表。
表:应用例11-14中卵细胞激活培养基性能测试结果
从上表中可以看出,经上述应用例11-14中激活方法所得的卵细胞,均能被有效激活,其受精率较高,为75.0-83.3%;
可见该配比及组成的氨基酸组分仅会轻微影响到多种组分的协同效应,主要用于维持卵细胞的正常发育供给,从上表还可以看出其中应用例11-13为优选例,氨基酸组分的优选配比为:L-苯丙氨酸、L-羟脯氨酸、丝氨酸、甘氨酸、L-缬氨酸、L-天门冬氨酸、天冬酰胺、谷氨酸按摩尔比计为(0.01-0.1):(0.01-0.1):(0.01-0.1):(0.01-0.1):(0.01-0.1):(0.01-0.16):(0.06-0.24):(0.06-0.24)。
应用例15-23
一种卵细胞激活培养基的应用,与应用例1的不同之处在于,所用卵细胞激活培养基的使用情况不同,具体对应关系下表所示。
表:应用例15-23中卵细胞激活培养基使用情况对照表
组别 | 卵细胞激活培养基 |
应用例15 | 由实施例15制得 |
应用例16 | 由实施例16制得 |
应用例17 | 由实施例17制得 |
应用例18 | 由实施例18制得 |
应用例19 | 由实施例19制得 |
应用例20 | 由实施例20制得 |
应用例21 | 由实施例21制得 |
应用例22 | 由实施例22制得 |
应用例23 | 由实施例23制得 |
抽取经上述应用例15-23中激活方法所得的卵细胞,按上述测量步骤和测量标准测试其受精率%,测试结果记入下表。
表:应用例15-23中卵细胞激活培养基性能测试结果
从上表中可以看出,经上述应用例15-23中激活方法所得的卵细胞,均能被有效激活,其受精率较高,为75.0-84.6%;
可见上述组分及比例生物碱的掺入可以与激活组分复配,有效模拟体内环境降低并进一步抑制CSF和MPF的活性,从而保障了卵细胞的激活效果,其中应用例17为优选例,受精率高达84.6%。
从上表还可以看出其中应用例20-22为生物碱组分复配的优选例,其受精率高达83.3%,可见生物碱组分的优选配比为:腺嘌呤、硫酸腺嘌呤、鸟嘌呤、盐酸鸟嘌呤、尿嘧啶和腺苷三磷酸二钠按摩尔比计为(0.1-0.2):(0.05-0.08):(0.1-0.2):(0.2-0.4):(0.2-0.4):(0.06-0.12)。
应用例24-28
一种卵细胞激活培养基的应用,与应用例1的不同之处在于,所用卵细胞激活培养基的使用情况不同,具体对应关系下表所示。
表:应用例24-28中卵细胞激活培养基使用情况对照表
组别 | 卵细胞激活培养基 |
应用例24 | 由实施例24制得 |
应用例25 | 由实施例25制得 |
应用例26 | 由实施例26制得 |
应用例27 | 由实施例27制得 |
应用例28 | 由实施例28制得 |
抽取经上述应用例24-28中激活方法所得的卵细胞,按上述测量步骤和测量标准测试其受精率%,测试结果记入下表。
表:应用例24-28中卵细胞激活培养基性能测试结果
从上表中可以看出,经上述应用例24-28中激活方法所得的卵细胞,均能被有效激活,其受精率较高,为75.0-91.6%;
可见褪黑素与辅酶NADH的掺入,除能辅助抑制CSF和MPF的活性外,还可增强钙波及其震荡的破坏效果,继而使得卵母细胞内的各种代谢活动被完全重启,其中应用例27为优选例,该用量下的效果最优,其受精率高大91.6%,具有极高的商业价值和经济效益。
本具体应用例仅仅是对本申请的解释,其并不是对本申请的限制,本领域技术人员在阅读完本说明书后可以根据需要对本应用例做出没有创造性贡献的修改,但只要在本申请的权利要求范围内都受到专利法的保护。
Claims (8)
1.一种卵细胞激活培养基,其特征在于,所述卵细胞激活培养基包括基础培养液和激活组分:
所述激活组分包括钙离子载体 2-6 μmol/L、IGF-2 1-100 nmol/L、人血清蛋白0.005-5 g/L、β-烟酰胺单核苷酸0.02-0.05 mg/L、辅酶Q10 0.1-2.0 mg/L。
2.根据权利要求1所述的卵细胞激活培养基,其特征在于,所述基础培养液为包括如下组分的水溶液:无机盐缓冲组分30-80g/L、氨基酸组分1-22g/L、葡萄糖0.5-1.2 g/L、乳酸钙0.1-2.0 g/L、乳酸钠1.0-5.0 g/L、丙酮酸钠0.1-0.8 g/L、四元羧酸乙二胺四乙酸0.001-000.5 g/L、丙氨酰-谷氨酰胺0.1-1.5 g/L。
3.根据权利要求2所述的卵细胞激活培养基,其特征在于,所述无机盐缓冲组分由氯化钠、氯化钾、磷酸二氢钠、硫酸镁、碳酸氢钠、氯化钙和水按摩尔比(5-10):(0.3-0.8):(0.05-0.1):(0.2-1.6):(1.0-3.5):(0.05-0.3):(900-1000)组成。
4.根据权利要求2所述的卵细胞激活培养基,其特征在于,所述氨基酸组分由L-苯丙氨酸、L-羟脯氨酸、丝氨酸、甘氨酸、L-缬氨酸、L-天门冬氨酸、天冬酰胺、谷氨酸按摩尔比(0.01-0.1):(0.01-0.1):(0.01-0.1):(0.01-0 .1):(0.01-0.1):(0.01-0.16):(0.06-0.24):(0.06-0.24)组成。
5.根据权利要求2所述的卵细胞激活培养基,其特征在于,还包括生物碱组分0.1-0.5g/L,所述生物碱组分由腺嘌呤、硫酸腺嘌呤、鸟嘌呤、盐酸鸟嘌呤、尿嘧啶和腺苷三磷酸二钠按摩尔比(0.1-0.2):(0.05-0.08):(0.1-0.2):(0.2-0.4):(0.2-0.4):(0.06-0.12)组成。
6.根据权利要求1所述的卵细胞激活培养基,其特征在于,所述激活组分还包括褪黑素0.1-5nmol/L、辅酶NADH 0.05-0.5mg/L,所述钙离子载体为A23187。
7.一种权利要求1-6任一项所述的卵细胞激活培养基的使用方法,其特征在于,具体步骤如下:
S1、先将权利要求1-6任一项所述的激活培养基加入培养器皿中;
S2、再将S1中的培养器皿置于培养箱中,以37±0.2℃、6±0.1%CO2、湿度95±1%的条件平衡4-6h;
S3、待激活培养基平衡完全后,将卵细胞放入激活培养液中,37±0.2℃、6±0.1%CO2、湿度95±1%的条件培养箱中培育10-30min,即可完成激活。
8.一种权利要求1-6任一项所述的卵细胞激活培养基的应用,其特征在于,所述的卵细胞激活培养基用于卵母细胞的激活、ICSI后的卵子或者受精卵培育,所用卵细胞选自人或哺乳动物。
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