CN112980779A - 一种从孕妇宫颈脱落细胞中分离胎盘滋养层细胞的方法 - Google Patents
一种从孕妇宫颈脱落细胞中分离胎盘滋养层细胞的方法 Download PDFInfo
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Abstract
本发明公开了一种从孕妇宫颈脱落细胞中分离胎盘滋养层细胞的方法。该方法基于研究确定的滋养层细胞表面或胞内表达的特异性抗原及组合,并利用设计的微流控分选芯片或流式细胞仪,对胎盘滋养层样本的细胞悬浮液进行细胞分选,获得分离纯化的胎盘滋养层细胞。本发明与传统方法相比,具有无创获取标本和特异性好的优势,而且该方法取材时间较早,导致感染及流产的风险低,能实现同时对多个抗原进行同步标记和特征荧光信号的识别和分选,准确性得到较大提升,检测结果又具有更高的可信度和更广的覆盖范围。
Description
技术领域
本发明属于细胞分离技术领域。更具体地,涉及一种从孕妇宫颈脱落细胞中分离筛选滋养层细胞的方法。
背景技术
孕前、产前和新生儿三级防控体系是我国降低出生缺陷、提高人口素质的重要手段之一,其中产前筛查和诊断是最为复杂且难度最高的环节。
目前临床上用于产前诊断的羊膜腔穿刺术或者脐静脉穿刺术,以及胎儿游离核酸测序等无创产前筛查技术都存在各种不足;羊膜腔穿刺术或者脐静脉穿刺术取材方法操作感染和流产的风险高、分析时间长,检测项目和范围有限,而且诊断时间限制在孕中晚期,孕妇的接受度低且不利于临床处理。胎儿游离核酸测序技术检验范围极其有限,且存在仅可检测3-5条指定染色体倍性,假阳性、假阴性情况难以避免,对常见变异检出能力不足,母血胎儿游离核酸的含量也存在个体差异性等问题。
申请人的专利申请CN111304153A披露了一种分离滋养层细胞的方法,该方法通过确定滋养层细胞表面表达的特异性抗原,利用带有对应特异性抗体的免疫磁珠技术,从胎盘滋养层样本细胞悬浮液中分离纯化胎盘滋养层细胞。该技术与传统的羊膜腔穿刺和绒毛穿刺相比,具有无创的优势,且取材时间较早,感染及流产的风险低,检测结果又具有相似的可信度。另一方面看,该技术仍有改进的空间,所披露适用的抗体组合有限,准确性和特异性扔有待更好的提升。申请人团队对该项目一直在进行持续的研究开发。
发明内容
本发明的目的是提供一种基于流式细胞分离或微流控技术从孕妇宫颈脱落细胞中分离筛选滋养层细胞的方法,不仅克服传统方法的问题和缺陷,还可实现同时对更多多个抗原进行同步标记和特征荧光信号的识别和分选,准确性得到较大提升,同时特异性也优于之前的免疫磁珠法分离方案。
本发明上述目的通过以下技术方案实现:
一种分离滋养层细胞的方法,包括如下步骤:
(1)将宫颈脱落细胞液样本制备成样本细胞悬浮液;
(2)向样本细胞悬浮液中加入特异性抗体进行孵育;
所述特异性抗体是对应滋养层细胞表面或胞内表达的特异性抗原的抗体组合;优选地特异性抗体组合为HLA-G+CK7、HLA-G+CK18、HLA-G+β-HCG、CD31+HPL、MMP9+CD31、HLA-G+HPL、HLA-G+MMP9、HLA-G+CD31、HLA-G+P、CD31+P、HLA-G+CDH5、CD31+CDH5、CD31+CK7+HLA-G、HLA-G+CK18+CD31、HLA-G+β-HCG+CD31、CD31+HPL+HLA-G、MMP9+CD31+HLA-G、CD31+P+HLA-G或HLA-G+CDH5+CD31的组合;
(3)利用流式细胞仪将步骤(2)孵育完成的细胞重悬液进行分选,即可获得分离纯化的胎盘滋养层细胞;
或利用微流控分选芯片,将步骤(2)孵育完成的细胞重悬液进行荧光标记微流控细胞分选,即可获得分离纯化的胎盘滋养层细胞。
优选地,步骤(3)所用微流控分选芯片的结构为:包括基片和与之贴合的盖片;选用包括但不限于亚克力作为基本材质通过注塑成型技术制成;
所述基片的一面上设有主流道、侧流道A和侧流道B,两个侧流道分别对应靠近主流道的左、右两端部;
所述基片的另一面上设有C入口、S入口、N出口和T出口;四个口均贯穿到基片另一面与流道连通;且C入口的位置对应于主流道的左端部,S入口的位置对应于侧流道A端部,N出口的位置对应于主流道的右端部,T出口的位置对应于侧流道B端部;
所述主流道内、在N出口与T出口汇合处,还设有偏转电极装置。
优选地,所有主流道、侧流道A和侧流道B的流道宽度均不超过1000μm,深度均不超过500μm。
更优选地,所有主流道、侧流道A和侧流道B的流道宽度均为500-1000μm。
更优选地,所有主流道、侧流道A和侧流道B的流道宽度均为1000μm。
微流控分选芯片中,C入口可通入待分选混合细胞样品、S入口可通入缓冲液、T出口为目标细胞收集,N出口为非目标细胞收集。
分选过程中,含有目标细胞的混合样品由C入口处流入芯片主流道,缓冲液于S入口进入芯片侧流道,二者在流道交汇处混合后继续沿主流道向同一方向流动。混合细胞流经偏转电极装置时,以电极开闭控制进入的管道,目标细胞被分选而到达T出口,而非目标细胞则沿主流道继续到达N出口,分选完成。
另外,优选地,步骤(2)中一抗孵育条件为:4℃反应30-90min(优选4℃,60min),二抗-荧光标记复合物孵育条件为2℃-8℃反应20min。
优选地,步骤(2)的具体方法是:通过孵育将一抗、二抗-荧光标记复合物先后分步与目标抗原进行特异性连接,中间使用洗涤和离心分离技术避免交叉污染。
优选地,步骤(3)的具体方法是:孵育完成的细胞重悬液通入微流控分选芯片的C入口,并在S入口通入缓冲液;然后将微流控分选芯片放置于细胞分选仪并运行分选程序,程序结束后,搜集T出口的标本,获得分选的滋养层细胞。
优选地,步骤(3)中细胞分选液相体系为0.2%-0.4%Triton-X-100(优选0.3%Triton-X-100)。更优选地,利用PBS配置。
优选地,步骤(3)所述利用流式细胞仪进行分选的条件:调整上样速度为1000-2000 events/秒,收集速率为5.0。
优选地,步骤(1)中细胞悬浮液的最佳体系为含0.2%-0.4% FBS 的1xPBS(优选含0.3% FBS 的1xPBS)。
另外所述方法在构建人类STR鉴定、人类染色体倍性检测、地中海贫血基因检测、耳聋基因检测、全外显子组基因测序、染色体微缺失/重复检测(高通量测序法)、或染色体结构变异检测(高密度芯片法)的产品方面的应用,也应当在本发明的保护范围之内。
本发明具有以下有益效果:
本发明提供的基于流式细胞分离或微流控技术分离胎盘滋养层细胞的方法,与传统的羊膜腔穿刺和绒毛穿刺相比,具有无创获取标本的优势,且取材时间较早,感染及流产的风险低,检测结果又具有更高的可信度和更广的覆盖范围。通过该种类的标本可获取胎儿完整的基因组核酸样本,使检测分析所有遗传性疾病成为可能。对技术人员和实验室设备的要求不高,可在更多的医疗机构开展,能够得到大范围的推广。
本发明提供的方法,与申请人另一技术(专利申请CN111304153A披露的方法)相比,本方法能实现同时对更多多个抗原进行同步标记和特征荧光信号的识别和分选,准确性得到较大提升,同时特异性也更好。
附图说明
图1为微流控分选芯片的基片结构示意图;(a)图和(b)图分别为基片的两个面。
图2为流式细胞分选中选择的单荧光标记阳性细胞群。
图3为流式细胞分选中选择的双荧光标记阳性细胞群。
图4为FAM标记的通道1,“822-”为分选后剩余标本,“822+”为分选出的标本,“822”为分选前标本。
图5为HEX标记的通道2。
图6为TAMRA标记的通道3。
图7为ROX标记的通道4。
图8为对分选后合体滋养层细胞中含有Y染色体的标本进行Y-STR检测的结果示意图。
图9为分选标本进行耳聋基因检测的结果。
图10为分选标本进行地中海基因检测的结果。
图11为检测家系致病突变携带家系图。
图12为全染色体示意图。
图13为异常染色体示意图。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1 微流控分选芯片设计
用于从孕妇宫颈脱落细胞中分离筛选滋养层细胞的微流控分选芯片的示意图如图1所示。
微流控分选芯片的结构设计描述如下:芯片选用包括但不限于亚克力作为基本材质进行制备,通过注塑成型技术在一面基材上形成图1之管道形状,管道宽度不超过1000μm,深度不超过500μm,并使用另一面基材进行贴合,形成完整芯片。
具体地,微流控分选芯片包括基片和与之贴合的盖片;
如图1所示,所述基片的一面上设有主流道、侧流道A和侧流道B,两个侧流道分别靠近主流道的左、右两端部;所有流道宽度均为1000μm;
所述基片的另一面上设有C入口(细胞样品进液孔)、S入口(缓冲液进液孔)、N出口(非目标细胞储液孔)和T出口(目标细胞储液孔);四个口均贯穿到基片另一面与流道连通;且C入口的位置对应于主流道的左端部,S入口的位置对应于侧流道A端部,N出口的位置对应于主流道的右端部,T出口的位置对应于侧流道B端部;
C入口可通入待分选混合细胞样品、S入口可通入缓冲液、T出口为目标细胞收集,N出口为非目标细胞收集。
另外,所述主流道内还设有偏转电极装置用于细胞分选,偏转电极装置的具***置位于N出口与T出口汇合处;根据流动细胞信号的有无,可控制开启/关闭电极。带负电的细胞在电极形成的电磁场中发生偏转,进入指定的通向N出口或T出口的管道。
分选过程中,含有目标细胞的混合样品由C入口处流入芯片主流道,缓冲液于S入口进入芯片侧流道,二者在流道交汇处混合后继续沿主流道向同一方向流动。混合细胞流经偏转电极装置时,以电极开闭控制进入的管道,目标细胞被分选而到达T出口,而非目标细胞则沿主流道继续到达N出口,分选完成。
分选完成后,芯片随即弃用,每次分选前需要重新更换芯片,以保证分选环境洁净、可控和无交叉污染。
实施例2 基于微流控分选芯片从孕妇宫颈脱落细胞中分离筛选滋养层细胞
一、滋养层细胞的分选方法包括如下步骤:
1、将宫颈脱落细胞液样本制备成样本细胞悬浮液;具体方法包括如下文(1)-(5)所示;
2、向样本细胞悬浮液中加入特异性抗体进行孵育;具体方法包括如下文(6)-(14)所示;
3、利用实施例1的微流控分选芯片,将步骤2孵育完成的细胞重悬液进行荧光标记微流控细胞分选;具体方法包括如下文(15)-(18) 所示。
其中,特异性抗体的组合如表1所示:
表1:滋养层细胞上表达的抗原及抗体组合
二、具体地,滋养层细胞的分选方法包括如下步骤:
(1)将细胞保存液(宫颈脱落细胞)在震荡混匀器上混匀,5min;
(2)将保存液取出置于15ml离心管中,在保存液的瓶中再加入3ml的1×PBS,震荡混匀后,取出置于15ml离心管中;
(3)3000rpm离心10min,弃去上清;
(4)加入1ml的1×PBST,混匀后移入1.5mlEP管中,3000rpm离心5min后弃去上清;
(5)重复步骤4两次,制备出细胞悬浮液;
(6)加入200μl的0.3%的Triton X-100,混匀后室温通透20min;
(7)重复步骤4三次;
(8)加入一抗:分别加入按照比例稀释后的鼠抗人CK7单克隆抗体、鼠抗人CK18单克隆抗体、鼠抗人β-HCG单克隆抗体、鼠抗人MMP9单克隆抗体、鼠抗人CDH5单克隆抗体、鼠抗人P单克隆抗体、鼠抗人hPL单克隆抗体、兔抗人HLA-G单克隆抗体、兔抗人CD31单克隆抗体(abcam公司)200μl,混匀后,4℃,60min;
(9)重复步骤4三次;
(10)加入二抗-荧光标记复合物:按比例稀释的羊抗兔及羊抗鼠抗体200μl,混匀后,37℃,60min;
(11)重复步骤4三次后用200μl buffer(DPBS+0.1%BSA+2mM EDTA)重悬;
(12)2℃-8℃反应20min;
(13)加入1ml的1×PBST,混匀后移入1.5mlEP管中,3000rpm离心5min后弃去上清;
(14)重复步骤(13)两到三次,加入200μl 1×PBST重悬,得细胞重悬液;
(15)将获得的细胞重悬液通入实施例1的微流控分选芯片的C入口,并在S入口通入1×PBST;
(16)将微流控芯片放置于细胞分选仪(KPCS-001,凯普生物)载物台上固定,并开启电源设定指定程序,运行;
(17)程序结束后,搜集T出口的标本,获得分选的滋养层细胞。
(18)搜集N出口的标本,为宫颈脱落细胞中去除了滋养层细胞的剩余细胞。
实施例3 基于流式细胞仪从孕妇宫颈脱落细胞中分离筛选滋养层细胞的方法
滋养层细胞的分选方法包括如下步骤:
1、将宫颈脱落细胞液样本制备成样本细胞悬浮液;具体方法同实施例2中的(1)-(5) 所示;
2、向样本细胞悬浮液中加入特异性抗体进行孵育;具体方法同实施例2中的(6)-(14) 所示;其中,特异性抗体的组合同上表1所示;
3、利用流式细胞分选仪(BDFACSAria II型,美国)将步骤2孵育完成的细胞重悬液进行荧光标记分选,包括如下步骤:
1)打开流式细胞仪,按照操作说明进行每日开机操作;
2)对仪器进行液流调整,使液流断点位置位于窗口中上部;
3)分选液路调整,确认液滴延迟,调整上样速度到1000-2000 events/秒;
4)选择5ml流式管作为收集装置,分选细胞数目选择3000,方向为左,添加要分选的细胞群,依次设门,放入收集管;
5)将孵育完成的细胞悬浮液放入进样仓,设置5.0的收集速率,上样;
6)在300-500V范围内调整电压,使荧光染料之间补偿尽量小,调整设门位置,使阳性细胞位于中央,将门内细胞进行收集;收集管中的细胞即为选中的目的细胞。
按照给定标记组合获得目标细胞群,如图2、图3。其中图2为单荧光标记分选结果,图3为双荧光标记分选结果。
实施例4 方法的应用案例---人类STR鉴定
1、按照上述实施例2的方法,针对样本(宫颈脱落细胞)分选分离滋养层细胞。
2、将分离后的滋养层细胞与孕妇的宫颈脱落细胞进行DNA提取:
1)将分选后的滋养层细胞(按照实施例2方法进行细胞分选的第17步获得的滋养层细胞),与按照实施例2方法进行细胞分选的第18步获得的细胞(宫颈脱落细胞中去除了滋养层细胞的剩余细胞),分别12000rpm 离心3min。
2)弃去上清,加200μl溶液P重悬沉淀。
3)加入20μl的蛋白酶K、200μl溶液L,混匀。
4)56℃温浴20min,颠倒混匀。
5)加入200μl无水乙醇,充分混匀后,转入吸附柱,10000rpm离心1min,弃去收集管中废液。
6)加500μl的W1,10000rpm离心1min,弃去收集管中废液。
7)加500μl的W2,10000rpm离心1min,弃去收集管中废液。
8)加500μl的W2,10000rpm离心1min,弃去收集管中废液。
9)空管离心 12000rpm,3min,弃去收集管。
10)将吸附柱放入新的1.5ml离心管,开盖静置2min,加50μl的TE,静置5min后,12000rpm离心2min,弃去柱子。
11)测定提取的DNA浓度及纯度。
3、将分离后的滋养层细胞与孕妇的宫颈脱落细胞中的DNA提取后,使用阅微D-21人类STR鉴定试剂盒进行PCR扩增,使用3500xL测序仪对PCR产物进行毛细管电泳分析,并用G5-Matrix Standard对仪器进行荧光校准,同时编写了相应的Panels,bins文件,并用软件GeneMapper ID version 3.0来进行结果分析。
4、进行STR检测
1)PCR扩增,扩增体系如表2所示:
表2:PCR扩增体系
PCR反应程序:50℃,10min;96℃,4min;(94℃,5sec;60℃,1min10sec)×27cycles;60℃,30min;15℃,保存。
2)STR检测结果
按照说明书将Hidi 8.7μl 内参0.3μl 混匀,加入1ml扩增后产物,使用3500xL测序仪对PCR产物进行毛细管电泳分析,并用G5-Matrix Standard对仪器进行荧光校准,同时编写了相应的Panels,bins文件,并用软件GeneMapper ID version 3.0来进行结果分析,结果如图4~7所示,结果表明,标本中除孕妇本人的DNA外,确实存在另一独立个体DNA信息,且与孕妇本人具有较强的亲缘关系。
3)Y-STR检测
将STR中检测到Y染色体的标本使用阅微40Y试剂盒进行Y-STR检测,结果如图8所示,结果表明该标本中存在男性DNA。
实施例5 方法的应用案例---耳聋易感基因检测
使用实施例2所述方法和商品化试剂盒(耳聋易感基因检测试剂盒(PCR+导流杂交法),潮州凯普生物化学有限公司,国械注准20153401698)对孕妇的宫颈脱落细胞(样本来源:广州凯普医学检验所)进行耳聋易感基因检测,具体对耳聋相关基因(GJB2, GJB3,SLC26A4和mtDNA)的9个突变位点(mtDNA1494、mtDNA1555、SLC26A4-IVS7(-2)、SLC26A4-2168、GJB2-35、GJB2-176、GJB2-235、GJB2-299、GJB3-538)进行检测。
用实施例4中孕妇的宫颈脱落细胞中的DNA提取所述方法进行提取,之后用商品化试剂盒(耳聋易感基因检测试剂盒(PCR+导流杂交法),潮州凯普生物化学有限公司,国械注准20153401698)进行后续检测,具体操作详见说明书。原始结果如图9所示,结果分析见表3。
表3 耳聋结果分析
结果表明,该试验检测结果与临床检测结果一致。
实施例6 方法的应用案例---耳聋易感基因检测
使用实施例2所述方法和商品化试剂盒(α-和β-地中海贫血基因检测试剂盒(PCR+膜杂交法),潮州凯普生物化学有限公司,国食药监械(准)字2012第3400399号)对孕妇的宫颈脱落细胞(样本来源:凯普医学检验所检测人源标本的剩余核酸样本)进行耳聋易感基因检测检测,具体对常见的3种α-地贫缺失型(--SEA、-α3.7、-α4.2),2种α-地贫突变型(CS、QS)及11种β-地贫突变型(CD14-15、CD17、CD27-28、CD41-42、CD43、CD71-72、-28、-29、IVS-I-1、IVS-II-654、β EN)进行检测。
用实施例4中孕妇的宫颈脱落细胞中的DNA提取所述方法进行提取,之后用商品化试剂盒(α-和β-地中海贫血基因检测试剂盒(PCR+膜杂交法),潮州凯普生物化学有限公司,国食药监械(准)字2012第3400399号)进行后续检测,具体操作详见说明书。原始结果如图10所示,结果分析见表4。
表4 地贫结果分析
结果表明,该试验检测结果与临床检测结果一致。
实施例7 方法的应用案例---全外显子组基因测序
1、在已知的耳聋家系中(父、母(孕16周)、大儿子(聋儿)、小儿子(聋儿)),按照实施例2中所述方法,获得孕妇的宫颈脱落细胞标本和羊水标本,孕妇本人以及其他家系成员采集全血标本,并提取DNA(孕妇宫颈脱落细胞分选后标本按照实施例4所述提取方法获得,羊水和全血标本使用人类全血基因组DNA提取试剂盒(凯普生物,中国)进行提取(样本来源:凯普医学检验所检测人源标本的剩余核酸样本),获得的人基因组DNA使用人全外显子组检测试剂盒(艾吉泰康生物科技(北京)有限公司,货号T086V4)进行全外显子测序(WES):
将基因组DNA使用转座酶Tn5处理生成300bp片段建成DNA文库;两端加接头P5,P7, index1,2;选取适合长度DNA片段,并扩增纯化;与带有生物素的外显子探针库进行杂交;利用生物素biotin与链霉亲和素的强结合能力,将带有链霉亲和素的磁珠与已结合目标文库的探针结合起来;吸附磁珠,去除上清液;洗脱掉磁珠上的DNA,PCR扩增文库;鉴定文库质量;上机测序。
2、全外显子测序结果
使用高通量测序技术进行全外显子组检测,对检测到的致病或疑似致病位点使用Sanger测序进行验证。送检样本为孕16周胎儿样本,有两个哥哥为聋哑,父母均正常。样本检出CDH23基因c.8363T>C (p.Leu2788Pro)杂合突变,GJB2基因c.109G>A (p.Val27Ile)杂合突变。家系图谱见图11。
结果表明,分选后的脱落细胞标本可有效检出致病突变,与羊水标本相比完全一致。检测结果可发现,该家系的致病原因和致病突变的携带情况。
实施例8 方法的应用案例---全基因组DNA拷贝数变异(CNV)检测
1、采用Phalanx Biotech公司CytoOneArray染色体芯片及配套检测试剂盒,根据比较基因组杂交技术的原理(aCGH)检测全基因组DNA 拷贝数变异:
实施例2方法分选的滋养层细胞,用实施例4中孕妇的宫颈脱落细胞中的DNA提取所述方法进行提取,获得的人基因组DNA经片段化、扩增前处理、扩增及PCR产物纯化后,用两种不同的荧光染料进行标记(正常样品用Cy3标记呈现绿色,患者样品用Cy5标记呈现红色);
荧光产物经纯化后注入芯片,芯片经清洗后进行扫描并获得结果。
2、全基因组DNA拷贝数变异(CNV)检测结果
全染色体示意图见图12,异常染色体的示意图见图13。图13中横轴为染色体区带的示意图,纵轴为样本与标准样本间的信号比值(以log2 ratio表示)。当染色体拷贝数有显著差异时以不同颜色来表示。染色体发生扩增的区域(Gain)以蓝色表示,染色体发生缺失的区域(Loss)以红色表示。图中黑线的长度与数值分别代表每个区段(Segment)的大小与信号的平均值。
样本检测到1处异常,为22q11.21缺失,异常片段起始-结束位置[UCSC hg19]为arr22q11.21 (19006943_21461068)x1,异常片段大小2.454 Mb。相关疾病区域为Pathogenic(致病性,ACMG分类),此段异常涵盖TBX1、CRKL、GP1BB、SLC25A1、DGCR10、TSSK1A、GSC2、CLTCL1等106个ISCA基因。此区域异常位于22q11.2 recurrent (DGS/VCFS)region (includes TBX1)。22q11.2近端(A-D)区域的缺失与DiGeorge /Velocardioffacial (DGS/VCFS)综合征相关,临床表现通常为先天性心脏疾病、心脏异常、特征性面部特征、DD/ID、行为问题、免疫缺陷和低钙血症(PMID 25217958)。此区域异常位于22q11.2 recurrent region (central, B/C-D) (includes CRKL),此区域缺失可能导致的临床表型包括:畸形的面部特征,生长受限/身材矮小,中枢神经***异常/发作,发育迟缓,智力障碍,骨骼异常,心血管缺陷,泌尿生殖***异常和免疫缺陷/反复感染(PMID25123976)。
结果表明,该分选细胞标本可有效的进行染色体结构变异检测并检出相应的突变。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (9)
1.一种分离滋养层细胞的方法,其特征在于,包括如下步骤:
(1)将宫颈脱落细胞液样本制备成样本细胞悬浮液;
(2)向样本细胞悬浮液中加入特异性抗体进行孵育;
所述特异性抗体是对应滋养层细胞表面或胞内表达的特异性抗原的抗体组合,即HLA-G+CK7、HLA-G+CK18、HLA-G+β-HCG、CD31+HPL、MMP9+CD31、HLA-G+HPL、HLA-G+MMP9、HLA-G+CD31、HLA-G+P、CD31+P、HLA-G+CDH5、CD31+CDH5、CD31+CK7+HLA-G、HLA-G+CK18+CD31、HLA-G+β-HCG+CD31、CD31+HPL+HLA-G、MMP9+CD31+HLA-G、CD31+P+HLA-G或HLA-G+CDH5+CD31的特异性抗体组合;
(3)利用流式细胞仪将步骤(2)孵育完成的细胞重悬液进行荧光标记分选,获得分离纯化的胎盘滋养层细胞;
或利用微流控分选芯片,将步骤(2)孵育完成的细胞重悬液进行荧光标记微流控细胞分选,获得分离纯化的胎盘滋养层细胞。
2.根据权利要求1所述的方法,其特征在于,所述微流控分选芯片包括基片和与之贴合的盖片;
所述基片的一面上设有主流道、侧流道A和侧流道B,两个侧流道分别靠近主流道的左、右两端部;
所述基片的另一面上设有C入口、S入口、N出口和T出口;四个口均贯穿到基片另一面与流道连通;且C入口的位置对应于主流道的左端部,S入口的位置对应于侧流道A端部,N出口的位置对应于主流道的右端部,T出口的位置对应于侧流道B端部;
所述主流道内、在N出口与T出口汇合处,还设有偏转电极装置。
3.根据权利要求2所述的方法,其特征在于,所有主流道、侧流道A和侧流道B的流道宽度均不超过1000μm,深度均不超过500μm。
4.根据权利要求1所述的方法,其特征在于,步骤(2)中一抗孵育条件为:4℃反应30-90min;二抗-荧光标记复合物孵育条件为2℃-8℃反应20min。
5.根据权利要求1所述的方法,其特征在于,步骤(2)的具体方法是:通过孵育将一抗、二抗-荧光标记复合物先后分步与目标抗原进行特异性连接,中间使用洗涤和离心分离技术避免交叉污染。
6.根据权利要求3所述的方法,其特征在于,步骤(3)的具体方法是:孵育完成的细胞重悬液通入微流控分选芯片的C入口,并在S入口通入缓冲液;然后将微流控分选芯片放置于细胞分选仪并运行分选程序,程序结束后,搜集T出口的标本,获得分选的滋养层细胞。
7.根据权利要求1所述的方法,其特征在于,步骤(3)中细胞分选液相体系为0.2%-0.4%Triton-X-100。
8.根据权利要求1所述的方法,其特征在于,步骤(1)中细胞悬浮液的最佳体系为含0.2%-0.4% FBS 的1xPBS。
9.权利要求1-8任一所述方法在构建人类STR鉴定、人类染色体倍性检测、地中海贫血基因检测、耳聋基因检测、全外显子组基因测序、染色体微缺失/重复检测、或染色体结构变异检测的产品方面的应用。
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US11796443B2 (en) | 2023-10-24 |
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