CN103275930B - 一种卵母细胞体外成熟培养液及其制备方法 - Google Patents
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Abstract
本发明属于辅助生殖技术领域，具体涉及一种卵母细胞体外成熟培养液及其制备方法。本发明所述卵母细胞体外成熟培养液由商品化的组织培养液TCM199为基础，精确添加明确的已经商品化的成分包括0.075-0.10IU/ml FSH和0.5-0.8IU/ml HCG，0.1-0.2μg/ml的17β-雌二醇，和10%-20%体积百分比的人商品化替代血清或者人白蛋白。本发明所述卵母细胞体外成熟培养液成分稳定，质量稳定，可以产业化生产。通过添加商品化的人替代血清，血清白蛋白以及提高培养液中雌二醇，FSH，HCG的浓度，模拟人卵母细胞体内成熟的环境获得了很好的临床效果，卵母细胞体外成熟率达到了72.08%，临床妊娠率达到了23.71%。
Description
技术领域
本发明属于辅助生殖技术领域，具体涉及一种卵母细胞体外成熟培养液及其制备方法。
背景技术
现有的用于培养女性生殖细胞在体外快速成熟的培养液中大多添加胎牛血清或者人成熟卵泡液，容易导致异种病原物的传播以及同种间不明原因疾病的散播，易导致新生儿的缺陷，不利于优生优育。另外各种添加物的细化成分不明确，导致临床结果不稳定以及无法产业化。
发明内容
本发明的目的是提供一种成分稳定，质量稳定，可以产业化生产的卵母细胞体外成熟培养液及其制备方法，以解决现有技术中存在的由于各种添加物的细化成分不明确，导致临床结果不稳定以及无法产业化问题。
本发明所采用的技术方案是：
本发明所述卵母细胞体外成熟培养液是以商品化的组织培养液TCM199（Sigma,St.Louis.USA）为基础，精确添加明确的已经商品化的成分包括0.075-0.10IU/ml FSH和0.5-0.8IU/ml HCG（Serono公司，瑞士），0.1-0.2μg/ml的17β-雌二醇（Sigma,St.Louis,USA），和10%-20%体积百分比的人商品化替代血清或者人白蛋白。
本发明所述卵母细胞体外成熟培养液的制备方法是将基本细胞培养液TCM199添加0.075-0.10IU/ml FSH，0.5-0.8IU/ml HCG，用0.22μm的过滤器过滤，4℃低温保存，3周内使用。使用前再依次添加0.1-0.2μg/ml的17β-雌二醇，10%-20%的人商品化替代血清或者人白蛋白，充分混匀。培养卵母细胞前培养液于37℃培养箱，5%二氧化碳浓度条件下平衡12小时以上。
TCM199是一种经典的细胞体外培养系统，用其作为母液安全、可靠。
FSH，HCG以及17β-雌二醇为人体内卵母细胞成熟的必须激素，其含量是依据其体内成熟环境以及多年的临床试验数据设定。
人商品化替代血清或人白蛋白与体内组织液环境类似，商品组分稳定安全。
FSH，HCG以及17β-雌二醇主要是促进未成熟卵母细胞周围颗粒细胞松散，促进未成熟卵母细胞核成熟排出极体。与人商品化替代血清或人白蛋白合用能进一步促进细胞质的成熟。为人卵母细胞成熟后的受精及形成胚胎后的体外发育以及临床妊娠打下坚实的基础。
本发明的有益效果是：
本发明所述卵母细胞体外成熟培养液，培养女性生殖细胞可以在体外快速成熟，该培养液成分稳定，质量稳定，可以产业化生产。通过添加商品化的人替代血清，血清白蛋白以及提高培养液中雌二醇，FSH，HCG的浓度，模拟人卵母细胞体内成熟的环境获得了很好的临床效果，卵母细胞体外成熟率达到了72.08%，临床妊娠率达到了23.71%。
具体实施方式
1.卵母细胞体外成熟培养液的制备
本发明所述卵母细胞体外成熟培养液的制备方法：
将基本细胞培养液TCM199添加0.075-0.10IU/ml FSH，0.5-0.8IU/mlHCG，用0.22μm的过滤器过滤，4℃低温保存，3周内使用。使用前再依次添加0.1-0.2μg/ml的17β-雌二醇，10%-20%的人商品化替代血清或者人白蛋白，充分混匀。培养卵母细胞前培养液于37℃培养箱，5%二氧化碳浓度条件下平衡12小时以上。
添加和配置要求在GMP车间环境下进行，要求无菌，无毒，无尘。
2.卵母细胞体外成熟培养液的急毒和长毒试验
采用人精子存活实验，小鼠胚胎培养实验进行急毒和长毒实验。
人精子存活实验：
使用正常水平的精液标本，利用上游法分离活动精子。分两管，分别加入待检测的培养液（T）和对照培养液（C），混匀并调整精子密度至5×106／ml。取混匀后的精子悬液用Markler精子计数板做精液常规检查，测定两组的精子活率。将精子悬液置于室温或CO2培养箱中孵育，分别于24小时、48小时、72小时和96小时后测定两组精子活率。计算生存指数（活率指数）＝T组精子活率/C组精子活率，生存指数＞75%为合格。
鼠胚实验：
选择4～7周龄雌性小鼠，每只腹腔注射10IU PMSG，48小时后注射10IUhCG。当晚按雌雄比例3:1合笼。第二日观察阴道栓，阳性者记为第一天。第三天颈椎脱臼法处死雌鼠，撕开腹部皮肤，打开腹腔。更换剪刀和镊子，剪下输卵管置于培养液中。在解剖显微镜下撕开输卵管壶腹部，取出受精卵，充分洗涤后移入待检测的培养液（T）和对照培养液（C），CO2培养箱中孵育。72小时后观察囊胚形成率，2细胞胚胎形成囊胚的比例超过70%视为合格。
3.卵母细胞体外成熟培养液的临床观察
应用自行研配的成熟培养液系统，进行未成熟卵母细胞在体外成熟培养的研究和临床运用。收集5例行小卵泡穿刺的PCOS患者的未成熟卵母细胞作为试验对象，进行体外成熟、胚胎发育的研究。
表1.培养液对小卵泡穿刺术卵子成熟及胚胎发育的影响
表2.培养液对卵子成熟及胚胎发育的结果
表3.培养液培养的卵子的胚胎种植及临床妊娠的能力
Claims (2)
1.一种卵母细胞体外成熟培养液，其特征是由基本细胞培养液TCM199添加0.075-0.10IU/ml FSH，0.5-0.8IU/ml HCG，0.1-0.2μg/ml的17β-雌二醇，10%-20%体积百分比的人商品化替代血清或者人白蛋白组成。
2.根据权利要求1所述卵母细胞体外成熟培养液的制备方法，其特征是将基本细胞培养液TCM199添加0.075-0.10IU/ml FSH，0.5-0.8IU/ml HCG，用0.22μm的过滤器过滤，4℃低温保存，3周内使用；使用前再依次添加0.1-0.2μg/ml的17β-雌二醇，10%-20%体积百分比的人商品化替代血清或者人白蛋白，充分混匀；培养卵母细胞前培养液于37℃培养箱，5%二氧化碳浓度条件下平衡12小时以上。
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| CN104140952B (zh) * | 2014-08-08 | 2018-03-16 | 山东威高新生医疗器械有限公司 | 透明质酸酶及其制备方法 |
| CN105518124A (zh) * | 2015-06-26 | 2016-04-20 | 深圳市第二人民医院 | 一种猪卵母细胞体外成熟培养液及其制备方法和培养方法 |
| CN107058215A (zh) * | 2017-01-20 | 2017-08-18 | 江苏省中医院 | 一种添加中药单体的猪卵母细胞体外受精液及培养方法 |
| CN108823152A (zh) * | 2018-07-25 | 2018-11-16 | 河南牧业经济学院 | 一种小鼠卵母细胞的体外成熟培养液及体外成熟培养方法 |
| CN108949670A (zh) * | 2018-08-16 | 2018-12-07 | 中国人民解放军第四军医大学 | 一种卵母细胞体外成熟培养液的制备方法 |
| CN109628386A (zh) * | 2019-01-18 | 2019-04-16 | 周桦 | 一种人卵母细胞的体外成熟培养液制备方法和培养方法 |
| CN115029303B (zh) * | 2022-06-22 | 2023-08-15 | 安徽第肆纪医学科技有限公司 | 一种用于coh周期中人未成熟卵母细胞ivm能量培养液 |
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