CN115247146A - 一种人类胚胎体外培养基及提高体外培养人类胚胎发育潜能的方法 - Google Patents
一种人类胚胎体外培养基及提高体外培养人类胚胎发育潜能的方法 Download PDFInfo
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Abstract
本发明属于人类辅助生殖领域,具体涉及一种人类胚胎体外培养基及提高体外培养人类胚胎发育潜能的方法。所述人类胚胎体外培养基中添加有NMN(β‑烟酰胺单核苷酸)。本发明发现在G1培养基中添加NMN,可以有效提升着床前胚胎中NAD+水平,有效的提升了人类胚胎的发育潜能。
Description
技术领域
本发明属于人类辅助生殖领域,具体涉及一种人类胚胎体外培养基及提高体外培养人类胚胎发育潜能的方法。
背景技术
“试管婴儿技术”即“体外受精和胚胎移植”(IVF-ET)技术”。体外受精是将卵子和精子置于体外,让它们在体外人工控制的环境中完成受精过程,然后发育成早期胚胎,再移植到女性的子宫内。这种体外培养的人类胚胎,一般不超过7天,也称之为着床前胚胎,即处于卵裂期或囊胚期:一般精卵受精结合后前3天为卵裂期胚胎,受精3天后胚胎继续分裂、增殖,就形成了由内细胞团和滋养外胚层构成的囊胚。目前人类胚胎体外培养主要通过在八细胞期前使用G1培养基,G2用于培养八细胞后人类胚胎,而当前培养基主要通过提供人类胚胎着床前发育所必需的成分进行配比,缺少增强发育潜能的重要辅因子。
发明内容
本发明的目的是为了克服现有技术存在的缺点和不足,而提供一种人类胚胎体外培养基及提高体外培养人类胚胎发育潜能的方法。
本发明所采取的技术方案如下:一种人类胚胎体外培养基,所述人类胚胎体外培养基中添加有NMN(β-烟酰胺单核苷酸)。
所述人类胚胎体外培养基为G1培养基。
所述NMN浓度为0.5µM-5uM。
一种提高体外培养人类胚胎发育潜能的方法,包括以下步骤:在八细胞期前使用添加有NMN的G1培养基进行培养。
NMN在G1培养基中的浓度为0.5µM-5uM。
本发明的有益效果如下:本发明发现在G1培养基中添加NMN,可以有效提升着床前胚胎中NAD+水平,有效的提升了人类胚胎的发育潜能。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,根据这些附图获得其他的附图仍属于本发明的范畴。
图1.小鼠着床前胚胎发育中NAD+/NADH比例;
图2. NAD+对小鼠着床前胚胎发育的影响,其中,A为NAD+应急补偿通路模式图,B为NAD+应急补偿通路抑制和补偿对小鼠早期胚胎发育的影响,C为NAD+应急补偿通路抑制和补偿对小鼠早期胚胎体外发育率的影响,D为NAD+应急补偿通路抑制和补偿对小鼠囊胚细胞数的影响;
图3. 人类成熟卵母细胞单精子胞浆内注射流程;
图4. NMN有效提升人类胚胎着床前胚胎发育潜能,其中,A为NMN添加对人早期胚胎体外发育率的影响,B为NMN添加获得人早期各阶段胚胎图片。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步地详细描述。
下文所述的G1培养基为直接购买得到的G1细胞专用培养基
一、实验过程:
1. 小鼠受精卵获取与体外培养
于第一天下午16:00 腹腔注射孕马血清促性腺激素(PMSG),于第三天下午 15:00腹腔注射人绒毛膜促性腺激素(hCG第四天早7:00通过检查雌鼠阴道口确定是否已经交配。通过外科手术获取小鼠输卵管,在显微镜下划开输卵管壶腹部,将卵丘卵母细胞复合体置于透明质酸酶中消化1分钟挑取 高质量受精卵在KSOM培养基中进行体外培养。
2. 人类卵母细胞与ICSI胚胎获取
本实验所收集人类卵母细胞均为患者正常周期取卵,取卵后MII期卵母细胞用于正常治疗方案,GV期卵母细胞放入G1培养基中培养,当患者已具备正常可移植胚胎后,体外成熟的GV期卵母细胞才获准用于本实验。体外成熟的MII卵母细胞在MOPS PLUS操作液中进行ICSI,受精胚胎置于G1培养基中进行培养,培养基在八细胞期更换为G2,随后在囊胚期检查发育率。
3. 小鼠胚胎NAD+/NADH比例检测
NAD+/NADH比例使用Glo cycling assay(G9071; Promega, Madison, WI, USA)试剂盒进行检测。待检测胚胎在100µl 0.2M NaOH中室温裂解10分钟,随后分为两管(50µl/管)分别检测NAD+和NADH。在NAD+检测中,管中继续添加25 µL 0.4N HCl后在60℃孵育15分钟,随后在室温孵育10分钟后加入25µl 0.5 M Trizma base。在NADH检测中,样本在60℃孵育15分钟后,继续在室温孵育10分钟,随后加入25 µL 0.5M Trizma base 和 25 µL 0.4NHCl。完成上述反应后每管样品加入100µl检测液,室温孵育3h,利用酶标仪进行化学发光检测。
4. FK866和NMN处理胚胎细胞
为了对早期胚胎进行处理,利用DMSO稀释FK866制备10mM浓储液,按0.01µM终浓度添加至KSOM培养基中,将含有抑制剂的培养液做成30µl的微滴,上方覆盖矿物油进行过夜平衡后,第二天将小鼠体内胚胎取出进行体外培养。在人类胚胎体外培养中,NMN用水稀释制备10mM浓储液,按0.5µM-5uM终浓度添加至G1培养基中,添加矿物油后放入培养箱过夜平衡。第二天ICSI胚胎放入G1培养基中培养,八细胞更换的G2培养基中不包含NMN。
5. 利用免疫荧光对胚胎细胞进行计数
为了对胚胎细胞进行计数,将胚胎置于4%多聚甲醛中固定30分钟,固定后胚胎在Hoechst溶液中染色5分钟,随后在PBS中清洗3次,在荧光显微镜下对细胞进行计数。
二、实验结果:
图1结果表明,在小鼠早期胚胎发育过程中NAD+/NADH比例分别在合子期和桑椹胚期呈现峰值表达,NAD+/NADH在发育过程中的高度动态表达暗示着NAD+在早期胚胎发育具有重要功能。
图2A显示NAD+体内应急补偿通路,已有文献表明哺乳动物体内约有90%左右NAD+产生于应急通路。因此挑选应急通路中核心催化酶-Nampt作为靶标,挑选其特异性抑制剂-FK866进行研究。如图2B-C显示,在小鼠胚胎培养基中添加0.01µM FK866后,胚胎发生严重阻滞。如图2D显示,小鼠胚胎大多阻滞在四细胞到八细胞时期。最重要的是上述发育阻滞可以通过添加NMN进行补偿,图2B-C显示NMN可以基本完全补偿有FK866导致的发育阻滞。上述结果表明NAD+对小鼠胚胎早期发育具有至关重要的功能。
图3显示人类成熟卵母细胞进行单精子胞浆内注射的流程。
图4A-B显示在G1培养基中添加1.0 uM NMN对体外培养人类胚胎的囊胚率的影响,可以说明1.0 uM NMN显著提升体外培养人类胚胎的囊胚率。另外,在0.5µM-5uM终浓度范围内的不同浓度NMN处理胚胎细胞的结果均发现处理后的体外培养人类胚胎的囊胚率相比空白组有显著提升。
以上所揭露的仅为本发明较佳实施例而已,当然不能以此来限定本发明之权利范围,因此依本发明权利要求所作的等同变化,仍属本发明所涵盖的范围。
Claims (5)
1.一种人类胚胎体外培养基,其特征在于:所述人类胚胎体外培养基中添加有NMN。
2.根据权利要求1所述的人类胚胎体外培养基,其特征在于:所述人类胚胎体外培养基为G1培养基。
3.根据权利要求2所述的人类胚胎体外培养基,其特征在于:所述NMN浓度为0.5µM-5uM。
4.一种提高体外培养人类胚胎发育潜能的方法,其特征在于包括以下步骤:在八细胞期前使用添加有NMN的G1培养基进行培养。
5.根据权利要求4所述的提高体外培养人类胚胎发育潜能的方法,其特征在于:NMN在G1培养基中的浓度为0.5µM-5uM。
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US20130059384A1 (en) * | 2011-06-29 | 2013-03-07 | President And Fellows Of Harvard College | Compositions and methods for enhancing bioenergetic status in female germ cells |
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