CN111272517A - 一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法 - Google Patents
一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法 Download PDFInfo
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Abstract
本发明提供了一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法,将小鼠胚胎体外培养至形成囊胚,通过小鼠胚胎囊胚染色操作观察囊胚细胞数量,其染色操作包括:固定:小鼠囊胚细胞在染色前先用特定pH值缓冲液冲洗,再用浓度为3.0~5.0%多聚甲醛固定5~10分钟后,用特定pH值缓冲液冲洗;染色:小鼠囊胚固定后用苏木素染色2~5分钟,再用纯化水冲洗;分色、蓝化:小鼠囊胚染色后用0.5~1.5%的盐酸酒精分色5~30秒,再用纯化水冲洗;冲洗后用1.0~3.0%的氨水蓝化5~15分钟,再用纯化水冲洗;固定、封片:小鼠囊胚蓝化后,滴加无水甘油,再盖上盖玻片、加指甲油封片等操作;染色结果判断:囊胚细胞分散均匀、染色清晰。
Description
技术领域
本发明涉及人类辅助生殖类耗材产品的质量控制技术,具体涉及小鼠胚胎囊胚染色方法。
背景技术
人类体外辅助生殖技术(Assisted Reproductive technology,ART)是指将卵子与精子取出后,置于体外使其受精(IVF,in vitro fertilization)或通过胞浆精子穿刺(intracytoplasmic sperm injection,ICSI),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发育而诞生婴儿,它是解决不育不孕症乃至实现优生优育的重要技术手段。在ART的过程中需要一系列的医疗器械实现配子的获得、受精、发育等过程,以便能够安全的应用于ART。要求用囊胚计数法判断质控用鼠胚的发育情况,用于评价产品的安全性。
囊胚计数法判断质控用鼠胚的发育情况,实质是将体外培养至囊胚期的小鼠胚胎染色并计数。染色是生物标本制作中最重要的环节之一,需要把生物组织浸入染色剂内,使组织细胞的某一部分染上与其他部分不同的颜色或深度不同的颜色,产生不同的折射率,以便显微镜观察。
目前采用的方法为:免疫荧光染色,通过对囊胚期胚胎细胞表达胚胎细胞转录因子1(UTF1)或NANOG(Nanog homeobox,NANOG)的内细胞团细胞进行染色并用显微镜观察计数。免疫荧光染色的主要原理是利用抗原抗体之间的特异性结合来显示目的蛋白,主要包括蛋白和一抗结合,其次是带有荧光基团的二抗识别并结合一抗,荧光显微镜下即可观察到荧光。该方法使用了双苯酰亚胺(Hoechst33342)试剂、DABCO(1,4-Diazabicyclo[2.2.2]octane solution,DABCO)抗淬灭剂、anti-UTF1抗体、与anti-UTF1抗体相对应的二抗、DNA染色液等高级试剂,而且同时需要体式显微镜、荧光显微镜搭配使用;该方法不仅技术程序比较复杂,而且还存在质控成本高等缺点。
通过相关文献资料查明,鼠胚囊胚期胚胎细胞包括内细胞团(ICM)细胞和滋养层细胞(TE),内细胞团细胞表达未分化的胚胎细胞转录因子1(UTF1)或NANOG(Nanoghomeobox,NANOG)。其主要成分是DNA,在DNA双螺旋结构中,两条核苷酸链上的磷酸基向外,使DNA双螺旋的外侧带负电荷,呈酸性,很容易与带正电荷的苏木素碱性染料以离子键或氢键结合而被染色。而苏木精-伊红(H&E)染色技术是组织样本病理学可视化的最常用的组织学技术。一般的H&E染色系统由包括铝基苏木精(aluminum based hematoxylin)、伊红、分化液以及上蓝剂的溶液构成。
发明内容
本发明的目的在于提供一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法。
本发明采用的技术方案为,一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法,其染色操作包括:
⑴固定:小鼠囊胚细胞在染色前先用特定pH值缓冲液冲洗1~3分钟,再用浓度为3.0~6.0%多聚甲醛固定5~10分钟后,用特定pH值缓冲液冲洗1~3分钟;
⑵染色:小鼠囊胚固定后用苏木素染色2~5分钟,再用纯化水冲洗1~3分钟;
⑶分色、蓝化:小鼠囊胚染色后用0.5~1.5%的盐酸酒精分色5~30秒,再用纯化水冲洗1~3分钟;冲洗后用1.0~3.0%的氨水蓝化5~15分钟,再用纯化水冲洗1~3分钟;
⑷固定、封片:小鼠囊胚蓝化后,滴加无水甘油,再盖上盖玻片、加指甲油封片等操作;
⑸染色结果判断:囊胚细胞分散均匀、染色清晰。
优选的,在染色操作“⑴固定”可重复操作2~3次。
优选的,“用纯化水冲洗1~3分钟”可重复操作2~3次。
优选的,用特定pH值缓冲液为pH值7.0~7.5的磷酸盐缓冲液。
优选的,滴加的无水甘油为5~30ul,滴加的指甲油为50~100ul。
本发明是在常规苏木精-伊红(H&E)染色技术基础上进行改进,实现体外培养至囊胚期的鼠胚染色并计数,不仅能简化操作步骤、缩短操作时间,而且能解决免疫荧光染色中存在质控成本高和技术程序复杂的问题。为用于人类辅助生殖类耗材产品的质量控制实现快速评价产品的安全性提供了有利条件。
附图说明
图1为实施例1的染色操作照片;
图2为实施例2的染色操作照片;
具体实施方式
下面将结合本发明实施方式中的附图,对本发明实施方式中的技术方案进行清楚、完整地描述,显然,所描述的实施方式仅仅是本发明的一部分实施方式,而不是全部的实施方式。
实施例1:一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法,染色操作如下:
⑴固定:吸取3-5个囊胚,置于载玻片中央,形成直径3-5mm(20-30ul)液滴,用pH值7.0~7.5的磷酸盐缓冲液清洗1分钟,再换成4.0%多聚甲醛(20-30ul)固定5分钟,吸去4.0%多聚甲醛,再滴加4.0%多聚甲醛(20-30ul)固定5分钟。用pH值5.0~7.0的纯化水(20-30ul)清洗1分钟,吸去多余液体,反复水洗三次,进入下一步。
⑵染色:加入20ul苏木素染色液,显微镜下观察,根据囊胚细胞核染色情况,控制染色时间为3分钟。用pH值5.0~7.0的纯化水洗1分钟,吸去多余液体,反复水洗三次,第4次水洗时留置在水滴中,镜下观察直至囊胚细胞核变成深蓝色。吸取多余液体。
⑶分色、蓝化:加0.5%盐酸酒精(20-30ul)分色30秒。注意严格控制时间,避免分色过度。分色后用pH值5.0~7.0的纯化水洗1分钟,吸去多余液体,反复水洗三次,然后在水滴中观察囊胚返蓝程度。如果分色后囊胚细胞返蓝不足,蓝色过浅,需要蓝化。用1.0%氨水(20-30ul)蓝化15分钟。
⑷固定、封片:在液滴位置加入10ul无水甘油,液滴周边等距4个角点上直径1-2mm的指甲油,指甲油滴之间的距离为15-18mm(正方形盖玻片边长20mm)。用平头镊夹住盖玻片一边,将盖玻片一边先盖上邻边两个指甲油滴,再缓慢放下另一边,直至盖住4个油滴。用两个食指分别压住盖玻片的两个侧边,显微镜下找到并观察囊胚,同时增加压迫力度,过程中可以发现囊胚被压扁,囊胚细胞散开。用指甲油涂抹盖玻片四周,隔离囊胚和外环境。用记号笔圈出囊胚所在位置。
⑸染色结果判断:囊胚细胞分散均匀、染色清晰。
实施例2:一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法,染色操作如下:
⑴固定:吸取3-5个囊胚,置于载玻片中央,形成直径3-5mm(20-30ul)液滴,用pH值7.0~7.5的磷酸盐缓冲液清洗3分钟,再换成5.0%多聚甲醛(20-30ul)固定10分钟,吸去5.0%多聚甲醛。用pH值5.0~7.0的纯化水(20-30ul)清洗3分钟,吸去多余液体,反复水洗二次,进入下一步。
⑵染色:加入20ul苏木素染色液,显微镜下观察,根据囊胚细胞核染色情况,控制染色时间为5分钟。用pH值5.0~7.0的纯化水洗3分钟,吸去多余液体,反复水洗二次,第三次水洗时留置在水滴中,镜下观察直至囊胚细胞核变成深蓝色。吸取多余液体。
⑶分色、蓝化:加1.5%盐酸酒精(20-30ul)分色5秒。注意严格控制时间,避免分色过度。分色后用pH值5.0~7.0的纯化水洗3分钟,吸去多余液体,反复水洗二次,然后在水滴中观察囊胚返蓝程度。如果分色后囊胚细胞返蓝不足,蓝色过浅,需要蓝化。用3.0%氨水(20-30ul)蓝化5分钟。
⑷固定、封片:在液滴位置加入10ul无水甘油,液滴周边等距4个角点上直径1-2mm的指甲油,指甲油滴之间的距离为15-18mm(正方形盖玻片边长20mm)。用平头镊夹住盖玻片一边,将盖玻片一边先盖上邻边两个指甲油滴,再缓慢放下另一边,直至盖住4个油滴。用两个食指分别压住盖玻片的两个侧边,显微镜下找到并观察囊胚,同时增加压迫力度,过程中可以发现囊胚被压扁,囊胚细胞散开。用指甲油涂抹盖玻片四周,隔离囊胚和外环境。用记号笔圈出囊胚所在位置。
⑸染色结果判断:囊胚细胞分散均匀、染色清晰。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (5)
1.一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法,其特征在于,小鼠胚胎体外培养至形成囊胚,通过小鼠胚胎囊胚染色操作观察囊胚细胞数量,其染色操作包括:
⑴固定:小鼠囊胚细胞在染色前先用特定pH值缓冲液冲洗1~3分钟,再用浓度为3.0~5.0%多聚甲醛固定5~10分钟后,用特定pH值缓冲液冲洗1~3分钟;
⑵染色:小鼠囊胚固定后用苏木素染色2~5分钟,再用纯化水冲洗1~3分钟;
⑶分色、蓝化:小鼠囊胚染色后用0.5~1.5%的盐酸酒精分色5~30秒,再用纯化水冲洗1~3分钟;冲洗后用1.0~3.0%的氨水蓝化5~15分钟,再用纯化水冲洗1~3分钟;
⑷固定、封片:小鼠囊胚蓝化后,滴加无水甘油,再盖上盖玻片、加指甲油封片等操作;
⑸染色结果判断:囊胚细胞分散均匀、染色清晰。
2.根据权利要求1所述的一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法,其特征在于,在染色操作“⑴固定”可重复操作2~3次。
3.根据权利要求1所述的一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法,其特征在于,“用纯化水冲洗1~3分钟”可重复操作2~3次。
4.根据权利要求1所述的一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法,其特征在于,用特定pH值缓冲液为pH值7.0~7.5的磷酸盐缓冲液。
5.根据权利要求1所述的一种用于人类辅助生殖类耗材产品的质量控制的小鼠胚胎囊胚染色法,其特征在于,滴加的无水甘油为5~30ul,滴加的指甲油为50~100ul。
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