CN105368936B - 一种利用囊胚培养液检测胚胎染色体异常的方法 - Google Patents

一种利用囊胚培养液检测胚胎染色体异常的方法 Download PDF

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CN105368936B
CN105368936B CN201510746098.XA CN201510746098A CN105368936B CN 105368936 B CN105368936 B CN 105368936B CN 201510746098 A CN201510746098 A CN 201510746098A CN 105368936 B CN105368936 B CN 105368936B
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CN105368936A (zh
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陆思嘉
蔡立义
姚兵
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Medical technology (Suzhou) Co., Ltd.
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Xukang Medical Technology Suzhou Co ltd
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Abstract

本发明涉及一种利用囊胚培养液检测胚胎染色体异常的方法，通过从胚胎早期体外培养液即囊胚培养液中检测胚胎来源的游离DNA，对微量DNA进行均匀的全基因组扩增，再利用二代测序等方法对扩增后的DNA产物进行分析，从而判断胚胎的染色体情况，即是否出现染色体整体或局部的非整倍体。本发明选择囊胚培养液这种体外受精操作过程中胚胎体外培养阶段的废弃物作为检测样本，这种非侵入性无创的方法既避免了常规卵裂球活检或囊胚滋养层细胞活检取样时对胚胎造成的细胞损伤，又不给临床增添额外的麻烦，同时简化了样本获取时的操作，安全可靠性及简便性更高。

Description

一种利用囊胚培养液检测胚胎染色体异常的方法
技术领域
本发明涉及生物医学和分子细胞生物学领域，具体涉及一种利用囊胚培养液对胚胎染色体的状态进行检测和分析的方法。
背景技术
试管婴儿技术是一项对抗不孕不育的强大技术手段，其技术过程为，首先从母亲获得多个卵子(通常8-15个)，再以父亲的精子对卵子进行体外受精，受精卵在体外培养液中生长5天时，胚胎是由约80～100个细胞组成的囊状结构即囊胚，将2-3个囊胚放置入母亲子宫后，理想情况下，置入子宫的囊胚可有1个至3个按正常孕期成功发育，直至出生。但由于各种原因，从囊胚置入子宫到胎儿出生的成功率不高，通常只有40％左右，除母亲自身的健康原因以外，受精卵质量是导致囊胚发育失败的重要原因之一。
受精卵的染色体来自于母源的卵子和父源的精子，任何一方的染色体异常均导致受精卵的染色体异常。正常人的受精卵、每个胚胎细胞及胎儿、婴幼儿直至成人的每个细胞内均有44条即22对常染色体(称为二倍体)和两条性染色体(男性为XY，女性为XX)。在异常情况下，任何染色体的全部或局部均可能出现多于或少于二倍体的情况，称为非整倍体异常，非整倍体异常是最常见的导致胚胎发育失败的染色体异常形式。在常规试管婴儿技术中，仅依靠显微镜下的形态观察，从多个(通常8-15个)胚胎中挑选2-3个相对“正常”者置入母亲子宫。而显微镜下的形态正常并无法反映染色体是否正常，错误地挑选形态正常而染色体异常的胚胎置入母亲子宫导致了很多试管婴儿受孕失败。
近年以来，人们已建立了一些技术，统称为胚胎植入前遗传学筛查(Preimplantation Genetic Screen，PGS)对体外培养胚胎的染色体状态进行检测，以筛选染色体正常的胚胎置入母亲子宫,从而提高受孕成功率。有研究表明，经过PGS再置入子宫的试管婴儿操作可将成功率提高到60％以上，各种PGS方法包括免疫荧光检测(FISH)，芯片检测，二代测序检测等。上述各种检测所需的生物样本都是从体外培养胚胎中采集的一个至数个细胞，通过对这少量细胞的检测反映整个胚胎的染色体是否正常。
具体而言，当体外培养5天的受精卵发育至囊胚期时可提取胚胎滋养层细胞(trophoblast)。一般操作为在显微镜下，用毛细玻璃管吸取一个至数个滋养层细胞将细胞裂解，释放其中的微量DNA，将微量DNA进行全基因组扩增后，可采用核酸芯片或二代测序等方法检测细胞的染色体状态(参见发明专利申请CN104711362A，公开日为2015年6月17日)。理论上讲，吸取出来的几个细胞中的染色体状态与胚胎中其它细胞一致，检测这几个细胞即可了解该胚胎的染色体状态是否正常。一般认为，此时吸取几个滋养层细胞不会对胚胎发育造成不良影响，从已出生婴儿的健康状况来看，这一操作也确实没有健康影响。但是，该技术出现的时间尚短(仅数年)，其对人的终生健康是否存在远期影响仍然有待观察。
此外，人们也已研发出利用囊胚腔液进行胚胎质量检测的方法，首先在囊胚腔液中获取游离的DNA，即在显微操作仪下使用微穿刺技术，利用无菌的针头吸取得到囊胚腔液游离DNA(参见发明专利申请CN104450923A，公开日为2015年3月25日；以及期刊文献LucaGianaroli,M.Cristina Magli,Alessandra Pomante,et al.Blastocentesis:a sourceof DNA for preimplantation genetic testing.Results from a pilotstudy.Fertility and Sterility,2014,102(6):1692-1698.)。然而，囊胚腔液是囊胚腔中的液体，欲获取囊胚腔液仍需要在囊胚上打洞或刺穿，其介入性仍会对胚胎造成不可避免的损伤。
综上所述，现有技术所存在的主要缺点为：
1.细胞取样时对胚胎操作技术要求较高，如果操作失误、粗暴操作会导致胚胎严重损伤，损伤过于严重可造成胚胎发育终止。
2.即使良好操作，细胞取样时不可避免地对胚胎造成细胞损失和轻微损伤。尽管现在尚无证据表明细胞损失和轻微损伤会对胚胎发育和出生后健康发生不良影响，但该技术出现的时间尚短(仅数年)，其对人的终生健康是否存在远期影响仍然有待观察。
3.在少数情况下，存在取样获得的几个细胞的染色体状态与胚胎中其它细胞的染色体状态不同的情况，导致检测结果失误。
因此，一种不损伤胚胎本身，而又可以对胚胎染色体状况进行检查的非侵入性的技术手段，是消除健康影响隐患、确保胚胎检测安全性的现实需求。
发明内容
本发明的目的就是针对上述现有技术中的不足，提供一种利用囊胚培养液检测胚胎染色体异常的方法，其不会对胚胎带来任何损伤，操作简单，安全性和可靠性更高。
为了实现上述目的，本发明提供了一种利用囊胚培养液检测胚胎染色体异常的方法，其包括如下步骤：
(1)囊胚培养液的获得：通过单精子注射方法获得受精卵，将其培养至第3天卵裂球期之后转移至新制备的囊胚培养微滴中进行囊胚培养，此时在第3天进行换液是必须的，目的是去除脱落的颗粒细胞及未受精精子的污染；
将形成囊胚的胚胎吸出，转移至新的囊胚培养液中或者进入玻璃化冻存流程，剩余的原囊胚培养液约1微升至500微升，优选10微升至200微升，即为进行胚胎植入前遗传学筛查(PGS)需要收集的样本；
(2)囊胚培养液的采集：将步骤(1)中获取的原囊胚培养液转移至裂解液中，离心后，样本进入下一步的全基因组扩增步骤；
(3)囊胚培养液中微量DNA的全基因组扩增：向步骤(2)中获得的囊胚培养液与裂解液的混合物中加入裂解酶，混匀后孵育，而后使裂解酶失活，取出裂解产物加入PCR反应管中进行PCR反应；
(4)将全基因组扩增后的DNA产物进行分析，以鉴定胚胎的染色体状态是否正常：分析时采用二代测序、核酸芯片或者免疫荧光检测。
其中，步骤(2)中裂解液的成分是PH为7.0～8.0的Tris-Cl 25-45mM，EDTA0.5-3mM，KCl 10-25mM以及浓度为0.05％-5％的去污剂，所述去污剂为Triton X-100、TritonX-114、吐温20、NP40和SDS中的一种或多种。优选地，裂解液的成分为PH为7.2的Tris-Cl40mM，EDTA 1mM，KCl 15mM以及3％的Triton X-100。
步骤(3)中的裂解酶选自蛋白酶K、Qiagen Protease、胃蛋白酶、木瓜蛋白酶、胰蛋白酶和溶菌酶中的一种或多种，所述裂解酶的浓度为1-25μg/ml，优选为20μg/ml；步骤(3)中孵育温度为30-60℃，孵育时间为1min至12h，失活温度为75-95℃，失活时间为1-15min；优选地，孵育温度为40℃，孵育时间为3h，失活温度为90℃，失活时间为5min。
步骤(3)中进行PCR反应时的PCR反应管中含有扩增混合液、0.5％-20％的PCR抑制物对抗剂、5-20mM dNTP、5-100μM NG和NT引物、50-200μM扩增引物、0.5-10单位核酸聚合酶，所述PCR抑制物对抗剂选自DMSO、甜菜碱、甲酰胺、甘油和白蛋白中的一种或多种，所述核酸聚合酶选自Phi29DNA聚合酶、Bst DNA聚合酶、Vent聚合酶、Deep Vent聚合酶、KlenowFragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、
Figure BDA0000839652430000041
超保真DNA聚合酶、Taq聚合酶、E.coli DNA聚合酶、LongAmp Taq DNA聚合酶和OneTaq DNA聚合酶中的一种或多种。
所述扩增混合液的成分为10-25mM Tris-HCl，5-25mM(NH4)2SO4，5-30mM KCl，0.5-5mM MgSO4，0.1％-20％DMSO和0.05-5％Triton X-100。优选地，所述扩增混合液的成分为15mM Tris-HCl，15mM(NH4)2SO4，20mM KCl，1mM MgSO4，5％DMSO和2％Triton X-100。
所述NG和NT引物从5’端到3’端包含通用序列和可变序列，其中所述通用序列由G、A、C和T四种碱基中的三种或者两种组成，条件是所述通用序列不同时包括G和C；所述扩增引物包含所述通用序列且不包含所述可变序列。所述可变序列选自下组：(N)nGGG、(N)nTTT，(N)mTNTNG，(N)xGTGG(N)y，其中N为任意的可与天然核酸进行碱基配对的核苷酸，n是选自3-17的正整数，m是选自3-15的正整数，x和y分别是选自3-13的正整数。
优选地，所述NG和NT引物包括SEQ ID NO:1[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNNN]、SEQ ID NO:2[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG]、SEQ ID NO:3[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT]、SEQ ID NO:4[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNTNTNG]或SEQ ID NO:5[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNGTGGNN]的序列，其中N为任意的可与天然核酸进行碱基配对的核苷酸；所述扩增引物从5’到3’具有SEQ ID NO:6[GTGAGTGATGGTTGAGGTAGTGTGGAG]的序列。
步骤(3)中全基因组扩增的热循环程序如下所述：
(1)在介于90-98℃之间的第一变性温度反应5-20s；
(2)在介于5-15℃之间的第一退火温度反应5-60s，在介于15-25℃之间的第二退火温度反应5-60s，在介于25-35℃之间的第三退火温度反应30-80s，在介于35-45℃之间的第四退火温度反应5-60s，在介于45-55℃之间的第五退火温度反应5-60s；
(3)在介于55-80℃之间的第一延伸温度反应10-150min；
(4)在介于90-98℃之间的第二变性温度反应5-30s；
(5)在介于45-70℃之间的第六退火温度反应10-30s；
(6)在介于60-80℃之间的第二延伸温度反应1-10min；
(7)重复步骤(4)到(6)5至50个循环；
(8)在介于60-80℃之间的温度下继续延伸反应1-10min；
(9)将扩增后的产物在0-5℃下冷藏保存。
优选地，步骤(3)中全基因组扩增的热循环程序如下所述：
(1)在第一变性温度95℃下反应10s；
(2)在第一退火温度10℃下反应45s，在第二退火温度20℃下反应45s，在第三退火温度30℃下反应60s，在第四退火温度40℃下反应45s，在第五退火温度50℃下反应45s；
(3)在第一延伸温度62℃下反应90min；
(4)在第二变性温度95℃下反应20s；
(5)在第六退火温度59℃下反应20s；
(6)在第二延伸温度72℃下反应3min；
(7)重复步骤(4)到(6)10至30个循环；
(8)在72℃下继续延伸反应5min；
(9)将扩增后的产物在4℃下冷藏保存。
本发明从胚胎早期体外培养液(囊胚培养液)中检测胚胎来源的游离DNA，从而判断该胚胎的染色体情况(是否出现染色体整体或局部的非整倍体)。由于胚胎在早期体外培养发育过程中会向囊胚培养液中释放极少量(约几十皮克)的DNA，要想利用如此微量的DNA进行染色体非整倍体检测，必须先对DNA进行大幅度的均一扩增。而囊胚培养液的体积约为30微升，因此培养液中的胚胎来源DNA是高度稀释的，同时胚胎培养液成份复杂，其中的某些成份会对DNA的扩增产生抑制作用。本发明中的技术方案克服了上述各种技术难题，成功地建立了从囊胚培养液中检测胚胎染色体非整倍体的技术方法。
因此，与现有技术相比，本发明避免了常规PGS检测取样方法对胚胎造成的细胞损失和损伤，并且简化了PGS样本获取时的操作；此外，由于囊胚培养液本来是进行试管婴儿的操作过程中胚胎体外培养阶段的废弃物，本发明技术对这一废弃物进行检测，几乎不给临床增添额外麻烦而实现了对相应胚胎染色体状态进行评估。
附图说明
下面结合附图和具体实施方式对本发明作进一步详细的说明。
图1为本发明的实施例1中分别利用囊胚培养液与囊胚细胞对A样本进行染色体检测的结果分析；
图2为本发明的实施例1中分别利用囊胚培养液与囊胚细胞对B样本进行染色体检测的结果分析。
具体实施方式
下面将结合本发明的附图和实施例，对本发明的具体实施方式作进一步详细的描述。
实施例1
选择A、B两个体外受精的胚胎样本，分别采用囊胚细胞检测的方法和囊胚培养液检测的方法评估其染色体状态，具体步骤如下所述：
1.囊胚培养液的获得
1)通过单精子注射方法获得的受精卵培养至第3天卵裂球期后，将胚胎转移至新制备的囊胚培养微滴中进行囊胚培养。
2)将形成囊胚的胚胎吸出，转移至新的囊胚培养液中/或者进入玻璃化冻存流程，剩余的原囊胚培养液(约30ul)即为进行PGS需要收集的样本A及样本B。
2.囊胚培养液的采集
1)将装有10微升裂解液(PH为7.2的Tris-Cl 40mM，EDTA 1mM，KCl 15mM以及3％的Triton X-100)的采集管室温放置2min，待裂解液解冻后，将样本采集管置于迷你离心机中，离心30s，保证裂解液全部聚集在管底。
2)将步骤一1.2)中的原囊胚培养液用口吸管全部转移至裂解液中。
3)用记号笔在采集管上标记样本名称，微型离心机离心30s，样本可立即进入下一步全基因组扩增步骤或放入-20℃或-80℃冷冻保存。
3.囊胚培养液中微量DNA的全基因组扩增
1)将囊胚培养液与裂解液的混合物于室温下融解。
2)向管中加入蛋白酶，上下吹打混匀。
3)将管子置于40℃孵育3h。
4)将管子置于90℃5min以失活裂解酶。
5)从管中取出裂解产物加入一个PCR反应管中。
6)向PCR反应管中加入扩增混合液(15mM Tris-HCl，15mM(NH4)2SO4，20mM KCl，1mMMgSO4，5％DMSO和2％Triton X-100)，5％DMSO，10mM dNTP，50μM NG(5′-GT GAG TGA TGGTTG AGG TAG TGT GGA GNNNNNGGG-3′)和NT(5′-GT GAG TGA TGG TTG AGG TAG TGT GGAGNNNNNTTT-3′)引物，100μM扩增引物(5′-GT GAG TGA TGG TTG AGG TAG TGT GGA G-3′)，1单位BstDNA聚合酶，1单位DeepVentR。
7)将PCR反应管置于PCR仪中进行全基因组扩增，热循环程序为：
Figure BDA0000839652430000071
4.将扩增后的DNA产物按常规方法进行二代测序以鉴定胚胎的染色体状态是否正常。
二代测序数据结果表明，在A样本中，囊胚培养液检测方法(图A1)与囊胚细胞检测方法(图A2)均能检出多条染色体异常；而在B样本中，囊胚培养液检测方法(图B1)与囊胚细胞检测方法(图B2)均判定为染色体正常。上述结果表明，采用囊胚培养液检测与囊胚细胞检测这两种方法对鉴定胚胎染色体状态取得相同的结果，进一步证实了该非侵入性的检测方法准确、可靠。
本发明所公开的实施例的上述说明，使本领域技术人员能够实现或使用本发明，同时，以上仅为本发明的较佳实施例而已，并不用以限制本发明实施例，凡在本发明实施例的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明实施例的保护范围之内。
Figure IDA0000839652520000011
Figure IDA0000839652520000021
Figure IDA0000839652520000031

Claims (10)

1.一种非治疗性和非诊断性地利用囊胚培养液来判断胚胎染色体非整倍体异常的方法，其特征在于，由如下步骤组成：
(1)通过单精子注射方法获得受精卵，将其培养至第3天卵裂球期之后转移至新制备的囊胚培养微滴中以换液并进行囊胚培养，将形成囊胚的胚胎吸出，转移至新的囊胚培养液中或者进入玻璃化冻存流程，剩余的原囊胚培养液即检测时所需要收集的样本，剩余的原囊胚培养液为10微升至200微升，包含来自胚胎的几十皮克的DNA；
(2)将步骤(1)中获取的原囊胚培养液转移至裂解液中，离心；
(3)向步骤(2)中获得的囊胚培养液与裂解液的混合物中加入裂解酶，混匀后孵育，而后使裂解酶失活，取出裂解产物加入包含PCR抑制物对抗剂的PCR反应管中进行全基因组扩增反应；
(4)将全基因组扩增后的DNA产物进行分析，并判断胚胎是否是具有染色体非整倍体异常的胚胎，其中分析时采用二代测序、核酸芯片或者免疫荧光检测；
其中，步骤(3)中进行PCR反应时的PCR反应管中含有扩增混合液、0.5％-20％的PCR抑制物对抗剂、5-20mM dNTP、5-100μM NG和NT引物、50-200μM扩增引物、0.5-10单位核酸聚合酶，所述PCR抑制物对抗剂选自DMSO、甜菜碱、甲酰胺、甘油和白蛋白中的一种或多种，所述核酸聚合酶选自Phi29DNA聚合酶、Bst DNA聚合酶、Vent聚合酶、Deep Vent聚合酶、KlenowFragment DNA聚合酶I、MMLV反转录酶、AMV反转录酶、HIV反转录酶、超保真DNA聚合酶、Taq聚合酶、E.col i DNA聚合酶、LongAmp Taq DNA聚合酶和OneTaq DNA聚合酶中的一种或多种，
其中所述NG引物从5’到3’为SEQ ID NO：2[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG]的序列，所述NT引物从5’到3’为SEQ ID NO：3[GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT]的序列，其中N为任意的可与天然核酸进行碱基配对的核苷酸；所述扩增引物从5’到3’为SEQID NO：6[GTGAGTGATGGTTGAGGTAGTGTGGAG]的序列，
其中，步骤(3)中全基因组扩增的热循环程序如下所述：
(1)在第一变性温度95℃下反应10s；
(2)在第一退火温度10℃下反应45s，在第二退火温度20℃下反应45s，在第三退火温度30℃下反应60s，在第四退火温度40℃下反应45s，在第五退火温度50℃下反应45s；
(3)在第一延伸温度62℃下反应90min；
(4)在第二变性温度95℃下反应20s；
(5)在第六退火温度59℃下反应20s；
(6)在第二延伸温度72℃下反应3min；
(7)重复步骤(4)到(6)10至30个循环；
(8)在72℃下继续延伸反应5min；
(9)将扩增后的产物在4℃下冷藏保存。
2.如权利要求1所述的方法，其特征在于，所述剩余的原囊胚培养液为10微升至30微升。
3.根据权利要求1所述的方法，其特征在于，步骤(2)中裂解液的成分是pH为7.0～8.0的Tris-Cl 25-45mM，EDTA 0.5-3mM，KCl 10-25mM以及浓度为0.05％-5％的去污剂，所述去污剂为Triton X-100、Triton X-114、吐温20、NP40和SDS中的一种或多种。
4.根据权利要求3所述的方法，其特征在于，裂解液的成分为pH为7.2的Tris-Cl 40mM，EDTA 1mM，KCl 15mM以及3％的Triton X-100。
5.根据权利要求1所述的方法，其特征在于，步骤(3)中的裂解酶选自蛋白酶K、QiagenProtease、胃蛋白酶、木瓜蛋白酶、胰蛋白酶和溶菌酶中的一种或多种，所述裂解酶的浓度为1-25μg/ml。
6.根据权利要求5所述的方法，其特征在于，所述裂解酶的浓度为20μg/ml。
7.根据权利要求1所述的方法，其特征在于，步骤(3)中孵育温度为30-60℃，孵育时间为1min至12h，失活温度为75-95℃，失活时间为1-15min。
8.根据权利要求7所述的方法，其特征在于，步骤(3)中，孵育温度为40℃，孵育时间为3h，失活温度为90℃，失活时间为5min。
9.根据权利要求1所述的方法，其特征在于，所述扩增混合液的成分为10-25mM Tris-HCl，5-25mM(NH4)2SO4，5-30mM KCl，0.5-5mM MgSO4，0.1％-20％DMSO和0.05-5％Triton X-100。
10.根据权利要求9所述的方法，其特征在于，所述扩增混合液的成分为15mM Tris-HCl，15mM(NH4)2SO4，20mM KCl，1mM MgSO4，5％DMSO和2％Triton X-100。
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