CN110669719A - 一种体外卵泡培养方法 - Google Patents
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Abstract
本发明涉及一种哺乳动物的体外卵泡培养方法，它包括以下连续操作：从哺乳动物的卵巢中选择合适直径范围内的卵泡，所述卵泡至少包括外层的卵泡膜细胞，中层的卵巢颗粒细胞，以及内层的***；将符合以上要求的卵泡在96孔板中培养；培养的第一阶段，即前三天，使用第一培养基，在培养的第二阶段，即第四天，使用第二培养基。培养最终可以得到成熟的卵泡。
Description
技术领域
本发明涉及一种哺乳动物的体外卵泡培养方法，为期4天，培养周期后，体外培养的卵泡可成功排出卵丘-***复合物(COCs)。
背景技术
卵泡数量随着年龄的增长而逐渐减少。新生儿两侧卵巢皮质中共有70万-200万个原始卵泡，7-9岁减少到约30万个，***开始有4万个左右，至更年期仅剩几百个。从***到更年期的整个生育期内(30-40年)，卵巢在脑垂体周期分泌的***的影响下，一般每隔28天左右有15-20个卵泡生长发育，但通常只有1个卵泡发育成熟并且***。人的一生中，卵巢总共可以***400-500个，其余卵泡均在发育的不同阶段退化。为了得到更多发育成熟的卵泡，我们建立了一种卵泡培养模型。
发明内容
本发明克服了传统方法卵泡培养周期较长的缺点，建立了短期体外卵泡培养模型。
为了实现以上发明目的，本发明方法通过机械法分离符合要求的哺乳动物卵泡，总共体外培养4天，在培养的第3天更换培养基诱导***。
本发明方法按照如下步骤操作(均在无菌超净台中操作)：第一步，腹部开口，取出肾脏下包含有卵巢、输卵管的组织部分，放于灭菌的PBS缓冲液中；第二步，在显微镜下通过显微镊剥除除卵巢外的其他部分，将卵巢组织转移到另一装有αMEM培养基的培养皿中；第三步，在显微镜下使用机械法分离卵巢中合适的卵泡，转移到事先装有培养基的96孔板中，每孔中培养基的量为200μl。每个卵巢大约能分离20-30枚卵泡用于后续培养；第四步，在培养的第三天更换培养基，诱导***，观察COCs的排出情况。
附图说明
图1为本发明中所包括的从哺乳动物中分离卵泡到发育成熟并***的操作过程示意图。
图2为从哺乳动物卵巢中初始分离的卵泡。
图3为培养过程中产生的有腔卵泡。有单个大液泡腔卵泡，且卵泡中有明显的卵丘结构。
图4为诱导***后的卵泡，可见排出的卵丘-***复合物(COCs)。
具体实施方式
实施例
一种体外哺乳动物卵泡培养方法，其特征在于培养方法的具体步骤包括：
1)卵泡的分离，从35日龄的ICR小鼠中分离卵巢组织，并把其放于装有PBS(phosphate buffer saline)的中皿中，这种培养基中含有0.1％青霉素-链霉素混合物。用胰岛素针分离卵巢中的卵泡，一般每个卵巢可以分离得到20-30枚合格的卵泡。
2)将合适的小鼠卵泡从卵巢中分离出来，放入事先装有培养基的96孔板中培养四天。培养的前三天使用第一培养基，第三天换液，使用第二培养基。培养基成分见表1。
优选地，步骤1)中分离的卵泡尺寸范围为200-400μm。
优选地，步骤2)的具体方法是：配制第一培养基，在基本培养基αMEM中加入5％的胎牛血清FBS，并加入浓度为5IU/ml的***FSH以及1×ITS[1.0mg/ml recombinanthuman insulin、0.55mg/ml human transferrin(substantially iron-free)、0.5μg/mlsodium selenite]；将获取的卵泡在灭菌后的PBS中清洗后移入加入第一培养基的96孔板中，在37℃、5％CO2、饱和湿度的二氧化碳培养箱中进行培养；配制第二培养基，在第一培养基的基础上加入1.5IU/mL HCG和5ng/mL EGF；在培养的第三天，将96孔板中的第一培养基换成第二培养基；培养的第四天，观察COCs排出情况。
优选地，第一培养基中加入FSH，且浓度为5IU/ml。
优选地，第二培养基中加入EGF，且浓度为5ng/ml。
特定培养基功能和组成见下表：
FBS＝胎牛血清，ITS＝胰岛素-转铁蛋白-***钠，FSH＝促卵泡激素，HCG＝人绒毛膜***，EGF＝表皮生长因子，αMEM可以用任何适宜的基础细胞培养基代替，例如Waymouth培养基。
本发明已经试验实施了多次，试验的结果是成功的，实现了发明的目的。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (3)
1.一种体外哺乳动物卵泡培养方法，其特征在于培养方法的具体步骤包括：
1)卵泡的分离，从35日龄的ICR小鼠中分离卵巢组织，并把其放于装有PBS(phosphatebuffer saline)的中皿中，这种培养基中含有0.1％青霉素-链霉素混合物。用胰岛素针分离卵巢中的卵泡，一般每个卵巢可以分离得到20-30枚合格的卵泡。
2)将合适的小鼠卵泡从卵巢中分离出来，放入事先装有培养基的96孔板中培养四天。培养的前三天使用第一培养基，第三天换液，使用第二培养基。培养基成分见表1。
2.根据权利要求1所述的培养方法，其特征在于步骤1)分离卵泡的直径范围为200-400μm。
3.根据权利要求1或2所述的培养方法，其特征在于步骤2)中的第一培养基中加入的FSH浓度为5IU/ml，第二培养基中加入的EGF浓度为5ng/ml。
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| 陈原等: "小鼠窦前卵泡与卵泡膜***的3 种共培养方法比较", 《中国妇幼保健》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980773A (zh) * | 2021-03-15 | 2021-06-18 | 中山大学附属第六医院 | 一种体外培养小鼠次级卵泡的方法 |
| CN112980773B (zh) * | 2021-03-15 | 2023-11-17 | 中山大学附属第六医院 | 一种体外培养小鼠次级卵泡的方法 |
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