CN117448257A - 一种粘性体液中稀有细胞的分离鉴定方法 - Google Patents
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Abstract
本申请属于细胞分离技术领域,公开了一种粘性体液中稀有细胞的分离鉴定方法,包括步骤S1:粘性体液样本的采集;步骤S2:将蛋白还原剂加入粘性液体样本中进行消化;步骤S3:将消化后的单细胞悬液离心、重悬,并铺在微孔芯片或玻片上,待细胞沉降吸附后冲洗掉附着的细胞及杂质,获得细胞微孔芯片或玻片;步骤S4:将所述细胞微孔芯片或玻片根据目的细胞表达的蛋白进行免疫染色,荧光成像后,取出阳性细胞单细胞至EP管中;步骤S5:获取的阳性细胞单细胞进行全基因组扩增后,进行基因组测序,与标准DNA进行比对,即可鉴定稀有细胞的来源。该方法可高效的在粘性体液中尤其是妇女宫颈粘液样本分离出稀有单细胞悬液,鉴定出目的细胞。
Description
技术领域
本申请属于细胞分离技术领域,尤其涉及一种粘性体液中稀有细胞的分离鉴定方法。
背景技术
获得临床相关的稀有细胞,可从细胞水平上进行多个层次的研究,包括基因组和转录组分析、蛋白表达水平、细胞代谢及激素水平等研究。目前与临床相关的稀有细胞研究主要集中在对外周血中循环肿瘤细胞、尿液中脱落细胞、脑脊液中稀有细胞、胸水中肿瘤细胞等的研究,这些体液为非粘性体液,样本本身呈单细胞悬液状态,处理较为简单,可直接进行离心或富集等操作。然而,临床中相关的一些人体粘性液体中的细胞,也具有非常重要的研究意义,但是这些细胞被包裹在粘液中,需要将细胞从粘液中释放后,才能对其进行研究。因此,取样后如何快速高效的消化粘液,获得完整无损坏的单细胞、制备成均匀的单细胞悬液,是后续实验能否顺利进行的关键步骤。例如,数据表明,在孕早期,胎儿绒毛外滋养层细胞会脱落游移至孕妇宫颈管中,被宫颈粘液包裹,因此孕早期采集孕妇宫颈可获得胎儿细胞。完整的胎儿细胞含有胎儿的完整基因组,与孕妇外周血中的游离基因片段用于无创产前检测相比更有优势,孕早期无创获得胎儿细胞,可实现对染色体疾病、单基因病、复杂多基因病、新发基因病等的检测。从宫颈粘液中分离得到胎儿滋养细胞是比传统的侵入性方法更好的选择,因为它更安全,且在妊娠早期就可以检测到。此外,它含有更多的胎儿细胞和更少的母血细胞的干扰,为分离胎儿细胞提供了优势。
如何快速地完成从临床门诊或手术室样本收集到实验室单细胞悬液制备,以及后续目的细胞的分离鉴定,这个过程决定着细胞的活性状态及检测效率。因此,急需一种能够快速完成粘性体液中稀有细胞的分离鉴定方法。
发明内容
为了克服上述技术问题,本申请提供了一种粘性体液中稀有细胞的分离鉴定方法,该方法可高效的在粘性体液中分离出稀有单细胞,且对较复杂的妇女宫颈粘液样本,同样地可以高效获得单细胞悬液,分离鉴定出目的细胞。
本申请提供的一种粘性体液中稀有细胞的分离鉴定方法,包括以下步骤:
步骤S1:粘性体液样本的采集;
步骤S2:将蛋白还原剂加入粘性液体样本中,混匀,孵育15-20分钟,得到消化后的单细胞悬液;
步骤S3:将所述单细胞悬液离心、重悬,将重悬后的单细胞悬液铺在微孔芯片或玻片上,待细胞沉降吸附后冲洗掉附着的细胞及杂质,获得细胞微孔芯片或玻片;
步骤S4:将所述细胞微孔芯片或玻片根据目的细胞表达的蛋白进行免疫染色,荧光成像后,取出阳性细胞单细胞至EP管中;
步骤S5:将获取的阳性细胞单细胞进行全基因组扩增后,进行基因组测序,与标准DNA进行比对,即可鉴定稀有细胞的来源。
采用上述技术方案,通过使用蛋白还原剂对粘性体液样本进行消化,蛋白还原剂可以水解蛋白二硫键,实现粘液中单细胞之间的分散,并结合微孔芯片或玻片沉降附着,可以在短时间内快速实现稀有细胞的分离。
优选地,所述粘性体液为鼻腔粘液、痰液或妇女宫颈粘液。
本申请的方法针对鼻腔粘液、痰液或妇女宫颈粘液中的稀有细胞分离而言快速且高效。
优选地,步骤S1中所述采集为将粘性体液样本置于缓冲液HBSS或PBS中。
优选地,步骤S2与步骤S1的间隔时间不超过1h。
采用上述技术方案,在粘性体液样本采集后1h内进行消化,此时的消化效率最高,如果样本放置较长时间后再消化,易出现难以完全消化、出现絮状物等现象,且影响细胞完整性。
优选地,步骤S2中所述蛋白还原剂为二硫苏糖醇(DTT)、β-巯基乙醇(BME)、三-(2-甲酰乙基)膦盐酸盐(TCEP-HCl)。
优选地,步骤S2中所述蛋白还原剂的终浓度为0.6%。
优选地,步骤S2中所述消化的体系体积为10-15mL,消化的时间为15-20min。
采用上述技术方案,消化的时间过短则可观察到消化效果较差,消化体系体积在4-8mL之间,消化时间15-20分钟之间,得到的单细胞悬液分散性最好,且无明显细胞碎片出现。
优选地,步骤S3中所述离心条件为300g/min,离心5min,所述重悬为用缓冲液HBSS或PBS重悬。
本申请相对于现有技术而言,提供了一种可高效、快速处理临床粘性体液及目的细胞的分离鉴定方法,并以孕早期孕妇宫颈粘液样本为例,提供了完整的样本采集、处理、铺片、目的细胞鉴定及分离流程,此流程高效、快速,且分离到的单细胞状态佳,可成功的进行基因组胎源性鉴定。
附图说明
图1本申请粘性体液中稀有细胞的分离鉴定方法流程图。
图2粘液样本在采集后30min消化15min得到的单细胞悬液图。
图3粘液样本在采集后1.5h后消化15min得到的单细胞悬液图。
图4步骤S3的操作图,其中,图4A为将细胞悬液铺在微孔芯片上,图4B为将细胞悬液铺在玻片上。
图5为微孔芯片上免疫染色后,荧光成像结果。
图6为玻片上免疫染色后,荧光成像后系统自动筛选结果。
图7为分离到的目的细胞单细胞基因座毛细电泳图谱。
图8为胎儿绒毛组织细胞基因座毛细电泳图谱。
具体实施方式
以下结合具体实施例和附图来进一步描述本申请,本申请的优点和特点将会随着描述而更为清楚。予以特别说明的是:以下实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行,以下实施例中所用原料除特殊说明外均可来源于普通市售。
实施例1孕早期孕妇宫颈粘液样本稀有细胞分离
(1)粘性体液样本的采集:用细胞刷刷取孕妇宫颈粘液,刷取宫颈粘液时,刷头轻轻伸入宫颈管约2cm,刷柄顺时针旋转10周使刷头缠绕宫颈粘液。刷头伸入不可过深,过深会引起出血;也不可伸入太浅,太浅时刷取的主要为外口鳞状上皮,粘液较少。
带有宫颈粘液的细胞刷放入含有2mL HBSS的15mL离心管中,液面要没过整个刷头,轻轻晃动细胞刷,使刷头与HBSS充分接触,放置4℃冰盒,并于30min内带回实验室。
(2)称取0.03g TCEP放至离心管底部,加入2.5mL HBSS充分溶解,用氢氧化钠调pH至7-8之间,加入细胞刷样本管,调整体系体积至5mL,TCEP终浓度为0.6%,枪轻轻吹打混匀,,消化15分钟,获得单细胞悬液,如图2所示,得到的单细胞分散性好,无粘液残渣,无明显细胞碎片。
(3)对上述得到的单细胞悬液以300g/min离心5min,并用缓冲液HBSS或PBS重悬至500uL,将得到的悬液铺至微孔芯片,如图4A所示,待细胞落入微孔芯片孔中,冲洗掉附着的细胞及杂质,方便后续免疫染色。
实施例2单细胞的免疫染色
对上述准备好的细胞微孔芯片,用anti-HK2抗体对滋养层细胞进行染色鉴定,具体流程如下:
1)取已经铺片完成的芯片,吸去芯片表面的HBSS,加入400μL 2% PFA,用枪轻轻吹吸混匀,室温固定15min;
2)吸去芯片表面的PFA,用400μL的PBS清洗芯片,重复洗4次后,取200μL0.5%Triton-100加载芯片表面,用枪轻轻吹吸混匀,对细胞进行透膜,室温下透膜15min,用400μL的PBS清洗芯片,重复洗4次;
3)清洗后的芯片用400μL 3% BSA和2% Fc受体封闭剂封闭细胞的非特异性结合位点,室温封闭1h;
4)吸去芯片表面的封闭剂,用400μL PBS洗1次,加入400μL anti-HK2抗体(1:100),用枪轻轻吹吸混匀,室温孵育1h;
5)吸去芯片表面的抗体,用400μL PBS洗4次后,用400μL 3% BSA和5%羊血清封闭细胞的非特异性结合位点,室温封闭1h;
6)吸去芯片表面的封闭剂,用400μL PBS洗1次,加入400μL羊抗兔-FITC荧光二抗(1:400),用枪轻轻吹吸混匀,室温孵育1h;
7)吸去芯片表面的荧光抗体,用400μL PBS洗4次后,加入200μLDAPI细胞核染色液,室温孵育10min;
8)400μL PBS清洗芯片,重复洗8次后,芯片表面加盖新鲜的PBS,荧光成像系统对芯片进行扫描,白光通道曝光5ms,FITC通道曝光200ms,DAPI通道曝光50ms。
染色结果如图5所示,荧光成像后,取出阳性细胞单细胞至EP管中。
实施例3单细胞的鉴定
对上述获取的单细胞进行全基因组扩增后,进行基因测序,与胎儿绒毛组织细胞DNA进行比对,分离到的目的细胞单细胞基因座及胎儿绒毛组织细胞基因座的毛细电泳图谱分别如7、8所示,毛细电泳图谱比对结果如表1所示。
表1男胎样本Y-STR分型结果
经过统计分析,表2中展示了男胎样本STR分型匹配度统计结果,可以看出,计算出胎儿细胞与绒毛组织的匹配度为80%,匹配度较好,证明此方法分离得到的目的细胞为胎儿细胞,且细胞状态佳。
表2男胎样本STR分型匹配度统计
检材 | 出峰数 | 分型相同峰数 | 匹配度 |
绒毛提取DNA | 30 | ||
胎儿细胞WGA产物 | 35 | 26 | 80% |
对比例
与实施例1不同的是,步骤(1)样本采集后1.5h带回实验室进行蛋白还原剂消化,获得单细胞悬液,如图3所示,粘液难以完全消化(延长消化时间无明显改善),且消化后会出现絮状物,单细胞分散性不好。
本具体实施例仅仅是对本申请的解释,其并不是对本申请的限制,本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改,但只要在本申请的权利要求范围内都受到专利法的保护。
Claims (8)
1.一种粘性体液中稀有细胞的分离鉴定方法,其特征在于,所述方法包括以下步骤:
步骤S1:粘性体液样本的采集;
步骤S2:将蛋白还原剂加入粘性液体样本中,混匀,孵育15-20分钟,得到消化后的单细胞悬液;
步骤S3:将所述单细胞悬液离心、重悬,将重悬后的单细胞悬液铺在微孔芯片或玻片上,待细胞沉降吸附后冲洗掉附着的细胞及杂质,获得细胞微孔芯片或玻片;
步骤S4:将所述细胞微孔芯片或玻片根据目的细胞表达的蛋白进行免疫染色,荧光成像后,取出阳性细胞单细胞至EP管中;
步骤S5:获取的阳性细胞单细胞进行全基因组扩增后,进行基因组测序,与标准DNA进行比对,即可鉴定稀有细胞的来源。
2.根据权利要求1所述的分离鉴定方法,其特征在于,所述粘性体液为鼻腔粘液、痰液或妇女宫颈粘液。
3.根据权利要求1所述的分离鉴定方法,其特征在于,步骤S1中所述采集为将粘性体液样本置于缓冲液HBSS或PBS中。
4.根据权利要求1所述的分离鉴定方法,其特征在于,步骤S2与步骤S1的间隔时间不超过1h。
5.根据权利要求1所述的分离鉴定方法,其特征在于,步骤S2中所述蛋白还原剂为二硫苏糖醇(DTT)、β-巯基乙醇(BME)、三-(2-甲酰乙基)膦盐酸盐(TCEP-HCl)。
6.根据权利要求1所述的分离鉴定方法,其特征在于,步骤S2中所述蛋白还原剂的终浓度为0.6%。
7.根据权利要求1所述的分离鉴定方法,其特征在于,步骤S2中所述消化体系的体积为4-8 mL,消化的时间为15-20min。
8.根据权利要求1所述的分离鉴定方法,其特征在于,步骤S3中所述离心条件为300g/min,离心5min,所述重悬为用缓冲液HBSS或PBS重悬。
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