CN113862219B - 一种用于猪胎盘滋养层类器官培养的分子培养基 - Google Patents
一种用于猪胎盘滋养层类器官培养的分子培养基 Download PDFInfo
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Abstract
本发明公开了一种用于猪胎盘滋养层类器官培养的分子培养基，它是在F12高糖培养基中添加谷氨酰胺、B27血清替代物、N‑乙酰基半胱氨酸、EGF表皮生长因子、成纤维细胞生长因子‑2、HGF肝细胞生长因子、R‑spondin1、A83‑01、SB‑431542、尼克酰胺、乙醇胺、糖原合酶激酶‑3抑制剂、ROCK激酶抑制剂配制而成；所述谷氨酰胺、B27血清替代物、N‑乙酰基半胱氨酸、EGF表皮生长因子、成纤维细胞生长因子‑2、HGF肝细胞生长因子、R‑spondin1、A83‑01、SB‑431542、尼克酰胺、乙醇胺、糖原合酶激酶‑3抑制剂、ROCK激酶抑制剂在F12高糖培养基中的浓度或体积百分比浓度分别为：1.5~2mM、1~3%、1~2mM、50~150ng/ml、100~300ng/ml、100~300ng/ml、60~120ng/ml、1~2μM、400~600ng/ml、5~10mM、25~50μM、0.6~1.5mM、10~15μM。
Description
技术领域
本发明属于细胞培养技术领域，具体涉及一种用于猪胎盘滋养层类器官培养的分子培养基。
背景技术
对于母猪营养来说，胎盘的发育与功能对于仔猪健康至关重要。胎盘是支持胎儿宫内生命的胚外器官。胎盘功能障碍会导致严重的妊娠紊乱，对母猪和仔猪造成直接和终生的后果，但由于缺乏功能性实验模型，我们对母猪胎盘的了解是有限的。胚胎植入后，胚泡的滋养外胚层迅速增殖并生成滋养层细胞，滋养层细胞是胎盘独特的细胞类型。在体内，增殖的绒毛细胞滋养层细胞分化为两个主要亚群：合体滋养层细胞和绒毛外滋养层细胞，合体滋养层细胞负责营养交换和激素产生，绒毛外滋养层细胞负责将胎盘固定在母体蜕膜上，并转化母体螺旋动脉。体滋养层细胞代表母体和胎儿循环之间的边缘界限，将营养物质和氧气输送到发育中的胚胎，并产生维持妊娠的激素。绒毛外滋养层重塑促进低压血液流向发育中的胎盘，从而适应胎儿生长所需的氧气和营养供应。在不同的母猪妊娠障碍中已经检测到EVT侵袭和重构的异常变化，例如严重形式的仔猪宫内生长受限(IUGR)和流产。
绒毛外滋养层前体形成和分化的缺陷可能是一个潜在的原因。然而，能够充分模拟这些早期发育过程的母猪滋养层细胞模型还没有建立起来。在人体医学领域，从妊娠早期胎盘中分离出的原代合体滋养层细胞很难被操控，因为它们在培养中迅速停止增殖。可供选择的模型，如滋养层细胞系、绒毛膜癌细胞和BMP4处理的人胚胎干细胞(HESCs)已经建立。然而，BMP4处理的hESCs和滋养层特异性标记的全球基因表达谱，以及细胞系的人类白细胞抗原(HLA)状态，与原始组织有很大的不同。因此，妊娠期间人类滋养层干细胞和祖细胞的定位和特征在很大程度上仍然难以捉摸，对于母猪来说更是如此。种猪资源一直非常宝贵，很少有通过大规模屠宰母猪进行繁殖性能相关的研究，也缺乏猪源的胎盘细胞系列供体外实验。尽管前期我们通过小鼠和人类的技术体系建立了猪胎盘类器官3D培养方法取得了一些进展，但是对于体外培养胎盘类器官来说，需要一个成分明确且无血清干扰的环境才能真正模拟出营养素的胎盘转运吸收。
发明内容
本发明为了能够建立标准化可重复的猪胎盘类器官，针对猪胎盘发育的特点与营养需要特征，提出了一种用于猪胎盘滋养层类器官培养的分子培养基，旨在促进胎盘营养和功能研究的标准化和工程化，为母猪繁殖性能研究提供新的工具和载体，促进类器官技术在动物营养领域的应用与推广普及。
为了达到上述目的，本发明提供的技术方案为：
所述用于猪胎盘滋养层类器官培养的分子培养基是在F12高糖培养基中添加谷氨酰胺、B27血清替代物、N-乙酰基半胱氨酸、EGF表皮生长因子、成纤维细胞生长因子-2、HGF肝细胞生长因子、R-spondin1、A83-01、SB-431542、尼克酰胺、乙醇胺、糖原合酶激酶-3抑制剂、ROCK激酶抑制剂配制而成；所述谷氨酰胺、B27血清替代物、N-乙酰基半胱氨酸、EGF表皮生长因子、成纤维细胞生长因子-2、HGF肝细胞生长因子、R-spondin1、A83-01、SB-431542、尼克酰胺、乙醇胺、糖原合酶激酶-3抑制剂、ROCK激酶抑制剂在F12高糖培养基中的浓度或体积百分比浓度分别为：1.5～2mM、1～3％、1～2mM、50～150ng/ml、100～300ng/ml、100～300ng/ml、60～120ng/ml、1～2μM、400～600ng/ml、5～10mM、25～50μM、0.6～1.5mM、10～15μM。
优选地，所述谷氨酰胺、B27血清替代物、N-乙酰基半胱氨酸、EGF表皮生长因子、成纤维细胞生长因子-2、HGF肝细胞生长因子、R-spondin1、A83-01、SB-431542、尼克酰胺、乙醇胺、糖原合酶激酶-3抑制剂、ROCK激酶抑制剂在F12高糖培养基中的浓度或体积百分比浓度分别为：2mM、1％、1mM、50ng/ml、150ng/ml、100ng/ml、80ng/ml、2μM、500ng/ml、10mM、50μM、1.5mM、10μM。
本发明提出了一种用于猪胎盘滋养层类器官培养的分子培养基和应用，为胎盘滋养层类器官的培养提供了标准化的培养体系，具有以下两个特点：
(1)根据猪胎盘结构，提出了滋养层类器官的培养，而不再是笼统的胎盘组织全培养。本发明的培养基专门针对猪胎盘滋养层尤其是绒毛外滋养层进行优化，更符合猪胎盘发育特征与生理。
(2)分子培养基配方是完全不含血清的，能够很好的用于微量元素转运等动物营养研究，从最大限度上减少了血清因素的干扰。
附图说明
图1为本发明所述分子培养基对小鼠滋养层类器官进行培养第3天的形态图；
图2为本发明所述分子培养基(Serum Free TOM)与有血清培养基(L-WRN TOM)效果对比结果图。
具体实施方式
实施例1
A.滋养层干细胞分离方法
实验开始前，对已经蒸汽灭菌后干燥的剪刀、镊子等耗材用100％酒精消毒，将24孔细胞培养板放到37℃培养箱预热至少30分钟，冰上解冻基质胶。
1.冲洗液的配制:洗涤培养基为Advanced/F12+1×青霉素/链霉素(100单位青霉素+0.1mg/mL链霉素)。
2.将胎盘样本转移到室温(RT，21-24℃)的洗涤介质中，并在1小时内处理。
3.用搅拌器在洗涤介质中清洗胎盘组织，轻轻搅拌>10min，以尽可能清除污染的血液和其他母体子宫细胞。彻底但轻柔地清洗胎盘组织，以避免绒毛受损。从手术切除的胎盘样本开始时，确保不存在蜕膜组织，因为这会加重类器官污染。
4.将胎盘样本倒入洗涤介质中的培养皿中，并用钳子将组织转移到干净的培养皿中。
5.用钳子清除任何可见的血块，注意不要损伤绒毛。
6.用手术刀按住胎盘的一端，从绒毛膜上刮下绒毛。丢弃剩余的胎膜。
7.将刮下的绒毛放入75毫升室温GCDR中，充分搅拌。盖上盖子，放在37℃摇床上，轻轻搅拌消化15分钟。
8.将分解的细胞悬浮液通过无菌滤网(100μm)过滤，并立即用消化停止液(洗涤介质中添加20％胎牛血清)冲洗，以阻止GCDR过度消化。
9.在0.4RCF的4℃温度下离心5分钟，将细胞颗粒过滤并重新悬浮在洗涤介质中。
10.去除上清部分，加入10ml带有0.1％BSA的DMEM/F12,按0.3RCF继续离心3分钟。去除上清部分，即获得了含有胎盘滋养层干细胞的微组织。
B.所述用于猪胎盘滋养层类器官培养的分子培养基配方及其制备
所述用于猪胎盘滋养层类器官培养的分子培养基组成见表1：
表1
本发明提出的用于猪胎盘滋养层类器官培养的分子培养基需将上述条件培养基按照表中配方比例添加，并按照表中所列组成成分调整其他分子浓度后，充分混匀，现配现用，或放置于-20℃一星期内用完。
C.三维(3D)胎盘滋养层类器官培养
(1)用10mL冷(2 8℃)F-12高糖培养基重悬滋养层沉淀并计数。室温(15-25℃)加100μL本发明胎盘滋养层类器官分子培养基到每个管中,不要用冷的培养基，对于分装的培养基，使用前必须加入P/S双抗并充分混匀。加入150μL未稀释的(已预先冰上解冻)基质胶到每个管中，使用相同的移液枪头，小心地上下吸打十次以重悬沉淀。主意避免产生气泡。
(2)从含有300-500个胎盘滋养层微组织结构的悬液中吸取150μL，并加入到提前预热的6孔板小室中。将点好的6孔培养板于37℃培养箱孵育10分钟直到基质胶凝固，将平板转放入培养箱时，应注意不要破坏凝固后的液滴。
(3)使用移液枪沿着孔侧壁向每个孔中轻轻地加入4mL室温(15-25℃)本发明胎盘滋养层类器官分子培养基。将培养板的盖子盖好，并在37℃和5％CO2下进行培养。
(4)每周进行两次完全换液，换液时将移液枪头放在孔底边缘小心地将原有液体培养基吸出，并加入为4mL新鲜的室温(15-25℃)本发明分子培养基。观察滋养层类器官的生长情况与状态，并记录拍照。
实施例2
用本发明所述的分子培养基分离培养小鼠胎盘滋养层类器官，步骤如下：
(1)将小鼠胎盘样本转移到室温(RT，21-24℃)的洗涤介质中，用搅拌器在洗涤介质中清洗胎盘组织，轻轻搅拌>10min，以尽可能清除污染的血液和其他母体子宫细胞。
(2)将胎盘样本倒入洗涤介质中的培养皿中，并用钳子将组织转移到干净的培养皿中。
(3)用钳子清除任何可见的血块，注意不要损伤绒毛。用手术刀按住胎盘的一端，从绒毛膜上刮下绒毛。丢弃剩余的胎膜。
(4)将刮下的绒毛放入75毫升室温GCDR中，充分搅拌。盖上盖子，放在37℃摇床上，轻轻搅拌消化10分钟。
(5)将分解的细胞悬浮液通过无菌滤网(70μm)过滤，并立即用消化停止液(洗涤介质中添加20％胎牛血清)冲洗，以阻止GCDR过度消化。
(6)在0.4RCF的4℃温度下离心5分钟，将细胞颗粒过滤并重新悬浮在洗涤介质中。
(7)去除上清部分，加入10ml带有0.1％BSA的DMEM/F12,按0.3RCF继续离心3分钟。
(8)将所得的胎盘组织沉淀物与基质胶按1：1.5混合，以体积为125微升接种到6孔板。
(9)每个孔加入37℃预热的本发明的分子培养基4mL，然后置入37℃，5％CO2培养箱培养。
实施例3
本发明无血清分子培养基(Serum Free TOM)与有血清培养基(L-WRN TOM)效果对比。这里我们比较的是本团队之前发布的胎盘3D类器官培养方法所用的有血清培养基。有血清培养基具体配方如表2。
表2
按照实施例2的步骤，用两种培养基对胎盘滋养层类器官进行传代对比培养3天，类器官形成率显著高于有血清体系。
Claims (2)
1.一种用于猪胎盘滋养层类器官培养的分子培养基，其特征在于，所述分子培养基是在F12高糖培养基中添加谷氨酰胺、B27血清替代物、N-乙酰基半胱氨酸、EGF表皮生长因子、成纤维细胞生长因子-2、HGF肝细胞生长因子、R-spondin1、A83-01、SB-431542、尼克酰胺、乙醇胺、糖原合酶激酶-3抑制剂、ROCK激酶抑制剂配制而成；所述谷氨酰胺、B27血清替代物、N-乙酰基半胱氨酸、EGF表皮生长因子、成纤维细胞生长因子-2、HGF肝细胞生长因子、R-spondin1、A83-01、SB-431542、尼克酰胺、乙醇胺、糖原合酶激酶-3抑制剂、ROCK激酶抑制剂在F12高糖培养基中的浓度或体积百分比浓度分别为：1.5~2mM、1~3%、1~2mM、50~150ng/ml、100~300ng/ml、100~300ng/ml、60~120ng/ml、1~2μM、400~600ng/ml、5~10mM、25~50μM、0.6~1.5mM、10~15μM。
2.如权利要求1所述用于猪胎盘滋养层类器官培养的分子培养基，其特征在于，所述谷氨酰胺、B27血清替代物、N-乙酰基半胱氨酸、EGF表皮生长因子、成纤维细胞生长因子-2、HGF肝细胞生长因子、R-spondin1、A83-01、SB-431542、尼克酰胺、乙醇胺、糖原合酶激酶-3抑制剂、ROCK激酶抑制剂在F12高糖培养基中的浓度分别为： 2mM、1%、1mM、50ng/ml、150ng/ml、100ng/ml、80ng/ml、2μM、500ng/ml、10mM、50μM、1.5mM、10μM。
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