CN115011551B - 一种改良的全程胚胎培养液及其制备方法 - Google Patents
一种改良的全程胚胎培养液及其制备方法 Download PDFInfo
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- CN115011551B CN115011551B CN202210767924.9A CN202210767924A CN115011551B CN 115011551 B CN115011551 B CN 115011551B CN 202210767924 A CN202210767924 A CN 202210767924A CN 115011551 B CN115011551 B CN 115011551B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种改良的全程胚胎培养液及其制备方法，包括基础物料及用于溶解的水，改良的全程胚胎培养液还包括关键物料，该关键物料包括磷脂酰丝氨酸或/和连翘酯苷A。本发明改良的全程胚胎培养液提高了胚胎体外培养96小时后的囊胚形成率。
Description
技术领域
本发明涉及属于辅助生殖领域，具体涉及一种全程胚胎培养液及其制备方法。
背景技术
体外受精-胚胎移植InVitro Fertilization-Embryo Transplantation简称IVF-ET，是指分别将卵子与精子取出后，体外受精并培养至囊胚期，再将囊胚移植回母体子宫内发育成胎儿。试管婴儿是用人工方法让卵子和精子在体外受精并进行早期胚胎发育，然后移植到母体子宫内继续发育而诞生的婴儿。
自从世界上第一个“试管”婴孩于1978年7月25日在英国出生后，到2012年7月1日已有500万个“试管”婴孩诞生。IVF为全球超过占5000万不孕症夫妇带来了福音。然而，三十多年的临床实践并没有显著提高IVF的成功率。2011年，美国妊娠协会统计35岁病人IVF成功率(受孕率)在25-30％左右，而1997年也在30％左右，而婴儿出生率更低。其中一个重要的原因是IVF胚胎培养液的质量，这是IVF胚胎质量最重要的决定性因素之一。
IVF培养通常分为两种流程即两种培养液系列：全程式培养液系列和分程式培养液系列。利用双培养液系列流程对胚胎早期和后期阶段提供不同的培养液成份，但也丢失了由胚胎分泌入培养液中的重要的有效成份，如胚胎营养因子。全程培养液系列流程保留了这些重要的有效成份，但也保留了培养液中有害的分解产物。
组织或胚胎在体外培养或者经过液氮超低温玻璃化冷冻和玻璃化解冻的过程中以及胚胎体外培养过程中，组织或胚胎细胞表面常会产生大量的自由基，对组织细胞膜表面、胚胎细胞膜和细胞器膜等造成不可逆的毒性损伤，最终导致组织细胞坏死或者胚胎体外培养终止发育和凋亡。
发明内容
基于上述问题，本发明提供一种改良的全程胚胎培养液，该改良的全程胚胎培养液提高了胚胎体外培养96小时后的囊胚形成率。
一种改良的全程胚胎培养液，包括基础物料及用于溶解的水，改良的全程胚胎培养液还包括关键物料，该关键物料包括磷脂酰丝氨酸或/和连翘酯苷A。
作为优选，所述基础物料：关键物料重量比为10～50：0.01～1。
作为优选，所述基础物料：关键物料重量比为15～25：0.04～0.6。
作为优选，所述磷脂酰丝氨酸：连翘酯苷A重量比为5～95：0.5～10。
作为优选，所述磷脂酰丝氨酸：连翘酯苷A重量比为30～70：1～5。
作为优选，所述基础物料包括氯化钠、甘氨酸、氯化钾、L-天冬氨酸、硫酸镁、L-谷氨酸、氯化钙、L-胱氨酸、九水硝酸铁、L-丝氨酸、磷酸二氢钾、L-组氨酸盐酸盐、磷酸氢二钠、L-精氨酸盐酸盐、丙酮酸钠、L-苏氨酸、乳酸钠、L-脯氨酸、葡萄糖、L-酪氨酸、碳酸氢钠、L-缬氨酸、乙酸钠、L-甲硫氨酸、牛磺酸、L-异亮氨酸、氯化胆碱、L-亮氨酸、D-泛酸钙、L-苯基丙氨酸、叶酸、L-赖氨酸单盐酸盐、烟酰胺、L-色氨酸、对氨基苯甲酸、L-谷氨酰胺、盐酸吡哆醛、L-丙氨酸、盐酸吡哆醇、L-半胱氨酸盐酸盐、烟酸、人血白蛋白、核黄素、白藜芦醇、盐酸硫胺素、还原型谷胱甘肽、生育酚、三磷酸腺苷、柠檬酸钠、ATP钠盐、辅酶Q10、胆固醇、D-脱氧核糖、生物素、次黄嘌呤、抗坏血酸、胸腺嘧啶、丙氨酰谷氨酰胺、β-雌二醇、肌醇、黄体生成素LH和促卵泡激素FSH。
作为优选，所述氯化钠：甘氨酸：氯化钾：L-天冬氨酸：硫酸镁：L-谷氨酸：氯化钙：L-胱氨酸：九水硝酸铁：L-丝氨酸：磷酸二氢钾：L-组氨酸盐酸盐：磷酸氢二钠：L-精氨酸盐酸盐：丙酮酸钠：L-苏氨酸：乳酸钠：L-脯氨酸：葡萄糖：L-酪氨酸：碳酸氢钠：L-缬氨酸：乙酸钠：L-甲硫氨酸：牛磺酸：L-异亮氨酸：氯化胆碱：L-亮氨酸：D-泛酸钙：L-苯基丙氨酸：叶酸：L-赖氨酸单盐酸盐：烟酰胺：L-色氨酸：对氨基苯甲酸：L-谷氨酰胺：盐酸吡哆醛：L-丙氨酸：盐酸吡哆醇：L-半胱氨酸盐酸盐：烟酸：人血白蛋白：核黄素：白藜芦醇：盐酸硫胺素：还原型谷胱甘肽：生育酚：三磷酸腺苷：柠檬酸钠：ATP钠盐：辅酶Q10：胆固醇：D-脱氧核糖：生物素：次黄嘌呤：抗坏血酸：胸腺嘧啶：丙氨酰谷氨酰胺：β-雌二醇：肌醇：黄体生成素LH和促卵泡激素FSH重量比为：52～78：0.26～0.41：3.44～4.36：0.17～0.29：0.71～0.99：0.44～0.62：1.73～2.25：0.13～0.23：0.01～0.02：0.14～0.21：1.14～1.31：0.11～0.19：3.67～4.15：0.32～0.41：0.42～0.48：0.18～0.22：6.11～6.56：0.20～0.29：10.28～11.88：0.37～0.44：25.75～26.86：0.13～0.20：0.81～0.95：0.08～0.13：0.11～0.15：0.21～0.24：0.007～0.013：0.41～0.48：0.0003～0.0009：0.13～0.19：0.0004～0.0009：0.42～0.49：0.0008～0.0012：0.054～0.063：0.0008～0.0012：0.51～0.55：0.0006～0.0010：0.16～0.22：0.0006～0.0010：0.002～0.006：0.0008～0.0012：41～58：0.0003～0.0007：0.17～0.22：0.0003～0.0005：0.008～0.012：0.0001～0.0005：0.01～0.03：0.84～0.96：0.11～0.15：0.04～0.07：0.01～0.03：0.004～0.008：0.0003～0.0007：0.008～0.012：0.001～0.005：0.011～0.017：0.54～0.61：0.012～0.016：3.53～4.16：0.0001～0.0004：0.00004～0.00007。
另一方面，本发明还提供一种上述改良的全程胚胎培养液的制备方法。
一种制备上述的改良的全程胚胎培养液的方法，包括以下步骤：
S1：称取配方各物料，将除HSA外的加入纯化，搅拌得到混合溶液；
S2：用过滤上杯，将S1混合溶液过滤，得到过滤溶液；
S3：调过滤溶液溶液的pH至7.2-7.4，然后添加HSA，并将溶液定容，得到初溶液；
S4：用过滤上杯，将初溶液进行无菌过滤，最终得到全程胚胎培养液溶液。
作为优选，所述S1中搅拌条件为37℃、300-500转/分钟。
作为优选，所述S2中，过滤上杯为0.45μm规格的PES材质的过滤上杯；S4中，过滤上杯为0.22μm规格的PES材质的过滤上杯。
发明原理及有益效果
磷脂酰丝氨酸由于其强亲脂性，在细胞膜表面形成一道脂质性保护膜，通过牺牲自身来抵抗自由基的氧化作用，减少自由基对胚胎细胞膜和细胞器膜造成的毒害。连翘酯苷A则利用自身的还原能力直接与自由基发生相互作用，降低培养液中和胚胎细胞内外的自由基浓度，促进体外培养胚胎健康发育，提高胚胎体外培养的囊胚形成率和体内移植的妊娠成功率。
磷脂酰丝氨酸与连翘酯苷A联合用于制备一种改良的全程胚胎培养液，对胚胎体外培养表现出协同促进效应。尤其对于经过液氮超低温玻璃化冷冻的一细胞、二细胞胚胎，解冻复苏后利用这种改良的全程胚胎培养液继续培养，可显著改善囊胚形成率和孵化囊胚率。
连翘酯苷A(Forsythoside A)，分子式为C29H36O15，是本犀科连翘属植物连翘中的一种活性药物成分。
羟自由基·OH，是活性氧中的一种。羟自由基能杀死红细胞，降解DNA、细胞膜和多糖化合物，继而又发现许多由它所致有害效应当加入羟自由基的清除剂后会明显降低。
超氧阴离子自由基O2 —·：是生物体内寿命最长的自由基，通常作为自由基链式反应的促进剂，产生活性更强的O2 —·，由此对机体造成更严重的毒害。
连翘酯苷A作为连翘的一种有效成分，长期以来被用于消炎、抗内毒素以及作为食品、饲料添加剂。连翘酯苷A对脂质过氧化的抑制作用：在生物体系中，细胞膜和细胞器膜中含有大量的不饱和脂肪酸，在自由基的作用下，很容易引起脂质过氧化，如卵磷脂C-2位上含有极低密度脂蛋白和低密度脂蛋白的过不饱和脂肪酸在亚铁离子的催化下，经振荡能诱发脂质过氧化产生烷氧基LO·和烷基过氧基LOO·，由此导致多种相关疾病和衰老的发生。
磷脂酰丝氨酸又称复合神经酸，英文名Phosphatidylserine，简称PS，通常从天然大豆榨油剩余物中提取得到，是细胞膜的活性物质，尤其存在于大脑细胞中。磷脂酰丝氨酸作为真核生物细胞膜的必须组分，是真核细胞中最丰富的阴离子磷脂，在细胞质膜中的含量最高，其含量可达到细胞脂质总量的10-15％，在内质网和线粒体中的含量很低，分别为4％和1％左右。其功能主要是改善神经细胞功能，调节神经脉冲的传导，增进大脑记忆功能，由于其具有很强的亲脂性，吸收后能够迅速通过血脑屏障进入大脑，起到舒缓血管平滑肌细胞，增加脑部供血的作用。作为细胞中重要的膜磷脂，还能够调控细胞膜关键蛋白功能状态，控制细胞信号转导。因此长期以来，磷脂酰丝氨酸被用于阿尔兹海默病和帕金森等疾病的治疗，用以增强记忆力，提高脑功能。目前，其最为熟知的是外翻磷脂酰丝氨酸在细胞凋亡和凝血中起作用，并且在几种胞内信号途径中起着至关重要的作用，如滋养层细胞在母体与胎儿的交界面融合形成大的胎盘合胞体滋养层，精子/卵子融合形成受精卵等。
本发明中，磷脂酰丝氨酸或连翘酯苷A单独使用于一种改良的全程胚胎培养液中时均表现出胚胎体外培养的良好促进作用；磷脂酰丝氨酸和连翘酯苷A联合应用大大提高了胚胎体外培养96小时后的囊胚形成率。
解释及术语说明
％，本发明中，如无特别说明，％指质量体积百分比。
HSA，Human Serum Albumin，人血清白蛋白。
PES，Polyethersulfone，聚醚砜树脂。
本发明中，如无特别说明，采用的物品均为市售渠道所购。
附图说明
图1为Day1-1细胞胚胎；
图2为Day2-2细胞胚胎；
图3为Day5-囊胚期胚胎(囊胚Ⅳ、Ⅴ、Ⅵ期)。
具体实施方式
下面将结合附图对本发明作进一步说明。
本发明提供的实施例仅为进一步解释本发明，不应解释为对本发明的任何限制。
本领域技术人员应当清楚，在下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。
实施例1-11
S1：按照表1各实施例的配方称取除HSA外的所有物料，其中，基础物料见表2，加入800mL纯化水，恒温磁力搅拌器搅拌1小时，得到溶液1，搅拌条件为37℃、300-500转/分钟。
S2：用0.45μm规格的PES过滤上杯，将溶液1过滤，得到溶液2。
S3：用1M的盐酸溶液(或1M的氢氧化钠溶液)调节溶液2的pH至7.2-7.4，然后添加HSA，并将溶液定容至1L，得到溶液3。
S4：用0.22μm规格的过滤上杯，将溶液3进行无菌过滤，最终得到全程胚胎培养液溶液。
表1
表2基础物料表
对比实施例1
S1：按照表3的配方称取除HSA外的所有物料，加入800mL纯化水，恒温磁力搅拌器搅拌1小时，得到溶液1，搅拌条件为37℃、300-500转/分钟。
S2：用0.45μm规格的PES过滤上杯，将溶液1过滤，得到溶液2。
S3：用1M的盐酸溶液(或1M的氢氧化钠溶液)调节溶液2的pH至7.2-7.4，然后添加HSA，并将溶液定容至1L，得到溶液3。
S4：用0.22μm规格的PES过滤上杯，将溶液3进行无菌过滤，最终得到全程胚胎培养液溶液。
表3
实施例12体外鼠胚试验
1)超数排卵
选择6～8周龄B6D2F1雌鼠，经腹腔注射PMSG 10IU/只；48h后经腹腔注射hCG10IU/只，注射hCG当日雌鼠与同品系雄鼠合笼过夜。
2)准备培养皿
细胞培养皿中盛有实施例1-11、对比实施例1及品牌为Global营养液(Global为国外的一个培养液品牌厂商，本实施例采用的为该品牌批号为220211-004436的加蛋白一步培养液)任一制备的全程胚胎培养液溶液(30～50μL，微滴形态)，再在培养液的表面覆盖培养用油，在37℃、6％CO2、5％O2，饱和湿度的培养箱内预平衡4～18小时。
3)鼠胚采集
合笼第二天早上检查交配情况，选择见栓小鼠备用。
1-细胞胚胎收集：注射hCG 18～22小时后处死见栓雌鼠，在输卵管壶腹部收集1-细胞胚胎。将收集到的含透明带和颗粒细胞的受精卵团放置到37℃提前预热好的透明质酸酶中，当胚胎周围的卵丘和颗粒细胞被消化分离后立即转移、清洗后，挑选出正常形态的受精1细胞胚胎(Day1)，转移到2)的全程胚胎培养液微滴中，连续培养至96小时(Day5)，每组鼠胚数不少于50个；或者将新鲜采集的1-细胞胚胎按照标准程序进行液氮超低温玻璃化冷冻和玻璃化解冻复苏后按照相同程序继续进行体外培养。
4)体外培养
采用微滴法培养，将1-细胞胚胎置于已平衡的培养液中，于37℃、6％CO2、5％O2、饱和湿度的培养箱中培养，每组鼠胚数不少于50个。
5)试验结果
1-细胞胚胎体外培养96小时后观察并记录囊胚数量。观察指标：囊胚形态观察。结果见表4-5及图1-3，其中表4为正常体内受精后，体外1细胞胚胎培养96h至囊胚期，表5为正常体内受精后，体外1细胞胚胎经过玻璃化冷冻和玻璃化解冻后培养96h至囊胚期。图1-3为1-细胞胚胎体外培养实验图片，其中图1为Day1-1细胞胚胎，图2为Day2-2细胞胚胎，图3为Day5-囊胚期胚胎(囊胚Ⅳ、Ⅴ、Ⅵ期)。
表4
表5
从表4-5可知，磷脂酰丝氨酸或连翘酯苷A单独使用于一种改良的全程胚胎培养液中时均表现出胚胎体外培养的良好促进作用。经过实施例1-10的实验结果可知，500mg/L的磷脂酰丝氨酸和50mg/L的连翘酯苷A分别为各自的最适浓度。通过实施例11的实验结果可知，磷脂酰丝氨酸和连翘酯苷A联合应用于一种改良的全程胚胎培养液中，两种物料表现出了协同促进作用，大大提高了胚胎体外培养96小时后的囊胚形成率，而且，从表4-5可知，在同样的实验条件下，本申请磷脂酰丝氨酸和连翘酯苷A联合应用的囊胚形成率不低于Global，且还提高了2％。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种改良的全程胚胎培养液，其特征在于，由基础物料、关键物料及用于溶解的水组成，关键物料为连翘酯苷A或连翘酯苷A和磷脂酰丝氨酸；
基础物料：关键物料重量比为10~50：0.01~1；
基础物料由氯化钠、甘氨酸、氯化钾、L-天冬氨酸、硫酸镁、L-谷氨酸、氯化钙、L-胱氨酸、九水硝酸铁、L-丝氨酸、磷酸二氢钾、L-组氨酸盐酸盐、磷酸氢二钠、L-精氨酸盐酸盐、丙酮酸钠、L-苏氨酸、乳酸钠、L-脯氨酸、葡萄糖、L-酪氨酸、碳酸氢钠、L-缬氨酸、乙酸钠、L-甲硫氨酸、牛磺酸、L-异亮氨酸、氯化胆碱、L-亮氨酸、D-泛酸钙、L-苯基丙氨酸、叶酸、L-赖氨酸单盐酸盐、烟酰胺、L-色氨酸、对氨基苯甲酸、L-谷氨酰胺、盐酸吡哆醛、L-丙氨酸、盐酸吡哆醇、L-半胱氨酸盐酸盐、烟酸、人血清白蛋白、核黄素、白藜芦醇、盐酸硫胺素、还原型谷胱甘肽、生育酚、三磷酸腺苷、柠檬酸钠、ATP钠盐、辅酶Q10、胆固醇、D-脱氧核糖、生物素、次黄嘌呤、抗坏血酸、胸腺嘧啶、丙氨酰谷氨酰胺、β-雌二醇、肌醇、黄体生成素LH和促卵泡激素FSH组成。
2.根据权利要求1所述的改良的全程胚胎培养液，其特征在于，所述基础物料：关键物料重量比为15~25：0.04~0.6。
3.根据权利要求1-2任一所述的改良的全程胚胎培养液，其特征在于，所述磷脂酰丝氨酸：连翘酯苷A重量比为5~95：0.5~10。
4.根据权利要求3所述的改良的全程胚胎培养液，其特征在于，所述磷脂酰丝氨酸：连翘酯苷A重量比为30~70：1~5。
5. 根据权利要求1所述的改良的全程胚胎培养液，其特征在于，所述氯化钠：甘氨酸：氯化钾：L-天冬氨酸：硫酸镁：L-谷氨酸：氯化钙：L-胱氨酸：九水硝酸铁：L-丝氨酸：磷酸二氢钾：L-组氨酸盐酸盐：磷酸氢二钠：L-精氨酸盐酸盐：丙酮酸钠：L-苏氨酸：乳酸钠：L-脯氨酸：葡萄糖：L-酪氨酸：碳酸氢钠：L-缬氨酸：乙酸钠：L-甲硫氨酸：牛磺酸：L-异亮氨酸：氯化胆碱：L-亮氨酸：D-泛酸钙：L-苯基丙氨酸：叶酸：L-赖氨酸单盐酸盐：烟酰胺：L-色氨酸：对氨基苯甲酸：L-谷氨酰胺：盐酸吡哆醛：L-丙氨酸：盐酸吡哆醇：L-半胱氨酸盐酸盐：烟酸：人血清白蛋白：核黄素：白藜芦醇：盐酸硫胺素：还原型谷胱甘肽：生育酚：三磷酸腺苷：柠檬酸钠：ATP钠盐：辅酶Q10：胆固醇：D-脱氧核糖：生物素：次黄嘌呤：抗坏血酸：胸腺嘧啶：丙氨酰谷氨酰胺：β-雌二醇：肌醇：黄体生成素LH：促卵泡激素FSH重量比为：52~78：0.26~0.41：3.44~4.36：0.17~0.29：0.71~0.99：0.44~0.62：1.73~2.25：0.13~0.23：0.01~0.02：0.14~0.21：1.14~1.31：0.11~0.19：3.67~4.15：0.32~0.41：0.42~0.48：0.18~0.22：6.11~6.56：0.20~0.29：10.28~11.88：0.37~0.44：25.75~26.86：0.13~0.20：0.81~0.95：0.08~0.13：0.11~0.15：0.21~0.24：0.007~0.013：0.41~0.48：0.0003~0.0009：0.13~0.19：0.0004~0.0009：0.42~0.49：0.0008~0.0012：0.054~0.063：0.0008~0.0012：0.51~0.55：0.0006~0.0010：0.16~0.22：0.0006~0.0010：0.002~0.006：0.0008~0.0012：41~58：0.0003~0.0007：0.17~0.22：0.0003~0.0005：0.008~0.012：0.0001~0.0005：0.01~0.03：0.84~0.96：0.11~0.15：0.04~0.07：0.01~0.03：0.004~0.008：0.0003~0.0007：0.008~0.012：0.001~0.005：0.011~0.017：0.54~0.61：0.012~0.016：3.53~4.16：0.0001~0.0004：0.00004~0.00007。
6.根据权利要求1、2、4、5任一所述的改良的全程胚胎培养液，其特征在于，所述改良的全程胚胎培养液由以下步骤制成：
S1：称取配方各物料，将除人血清白蛋白外的物料加入纯化水中，搅拌得到混合溶液；
S2：用过滤上杯，将S1混合溶液过滤，得到过滤溶液；
S3：调过滤溶液的pH至7.2-7.4，然后添加人血清白蛋白，并将溶液定容，得到初溶液；
S4：用过滤上杯，将初溶液进行无菌过滤，最终得到全程胚胎培养液。
7.根据权利要求3所述的改良的全程胚胎培养液，其特征在于，所述改良的全程胚胎培养液由以下步骤制成：
S1：称取配方各物料，将除人血清白蛋白外的物料加入纯化水中，搅拌得到混合溶液；
S2：用过滤上杯，将S1混合溶液过滤，得到过滤溶液；
S3：调过滤溶液的pH至7.2-7.4，然后添加人血清白蛋白，并将溶液定容，得到初溶液；
S4：用过滤上杯，将初溶液进行无菌过滤，最终得到全程胚胎培养液。
8.一种制备权利要求1-5任一所述的改良的全程胚胎培养液的方法，包括以下步骤：
S1：称取配方各物料，将除人血清白蛋白外的物料加入纯化水中，搅拌得到混合溶液；
S2：用过滤上杯，将S1混合溶液过滤，得到过滤溶液；
S3：调过滤溶液的pH至7.2-7.4，然后添加人血清白蛋白，并将溶液定容，得到初溶液；
S4：用过滤上杯，将初溶液进行无菌过滤，最终得到全程胚胎培养液。
9.根据权利要求8所述的制备改良的全程胚胎培养液的方法，其特征在于，所述S1中搅拌条件为37℃、300-500转/分钟。
10.根据权利要求8-9任一所述的制备改良的全程胚胎培养液的方法，其特征在于，所述S2中，过滤上杯为0.45μm规格的PES材质的过滤上杯；S4中，过滤上杯为0.22μm规格的PES材质的过滤上杯。
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