CN115197914A - 一种分离和检测循环肿瘤细胞的方法 - Google Patents
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Abstract
本发明属于细胞检测领域,具体涉及一种循环肿瘤细胞的分离和检测方法。本发明所述的一种分离和检测循环肿瘤细胞的方法,包括如下步骤:采用Ficoll密度梯度离心法对血液样本进行离心,吸取单个核细胞层洗涤后加入红细胞裂解液离心、洗涤得到外周血单个核细胞;分为第一部分细胞与第二部分细胞;对所述第一部分细胞,应用免疫磁珠分选技术(MACS)进行阳性分选富集,然后采用荧光标记抗体对富集的细胞染色后进行流式细胞分选检测;对所述第二部分细胞,应用实时荧光定量PCR法检测以下标记物的表达:Ep‑CAM、CK8、CK18、CK19、CD44v6、CD45、CXCR4、SDF‑1;综合步骤C和步骤D的检测结果,确定所述血液样本的循环肿瘤细胞的分离结果。
Description
技术领域
本发明属于细胞检测领域,具体涉及一种循环肿瘤细胞的分离和检测方法。
背景技术
循环肿瘤细胞的概念于澳大利亚学者Ashworth在1896年率先提出,它是癌细胞从原发肿瘤或转移肿瘤中逐渐分化脱落而进入血液循环。具体来说,原发性肿瘤在血管中生长到一定的阶段,会直接侵袭周围的血管,肿瘤细胞首先通过整联蛋白附着于血管的基底膜上而生长;当肿瘤细胞的数量逐渐增多,其分泌的基质金属蛋白酶也逐渐增加,通过逐步地消化去除掉IV型胶原蛋白,突破基底膜屏障,进入血液,即被人们称为循环肿瘤细胞;循环肿瘤细胞在进入人体血液后,会随着肿瘤的血流循环而游走到全身,形成复发转移。因此,有必要确定一种有效的方法来分离和检测循环肿瘤细胞。
近些年,越来越多的研究人员通过“液体活检”方法来识别和评估循环肿瘤细胞,分析循环肿瘤细胞对于复发风险较大的患者,在其进行特殊辅助医学治疗的分层和监测其治疗反应等方面都有帮助。该技术可及早发现复发高危人群,降低肿瘤复发风险,显著提高生存率。与传统的影像学诊断、内窥镜检查以及病理学诊断相比,循环肿瘤细胞计数更早于发现肿瘤或者发现肿瘤复发,可以高频度的监测,达到实时监测疾病进展及更换治疗方案的目的,而且只需抽取患者少量外周血,对患者没有副作用,易接受。
现有技术中循环肿瘤细胞直接分离、鉴定的方法有两大类:一类是基于循环肿瘤细胞物理尺寸的粗分离,即通过过滤方法筛选循环肿瘤细胞。使用孔径8μm的滤膜,但很多白细胞粒径大于8μm,导致白细胞堵塞滤膜;不同时期的循环肿瘤细胞大小不同,如进入休眠期的循环肿瘤细胞,可以轻易穿过8μm孔径滤膜,就会造成循环肿瘤细胞漏检。另一类是基于细胞表面抗原标志物的分离,该方法最具代表性产品是强生公司的CellSearch全自动循环肿瘤细胞检测仪,是唯一被FDA和CFDA批准的用于循环肿瘤细胞临床检测的技术,但是该方法单纯使用免疫磁珠,分离出的循环肿瘤细胞活性较差,存在检测灵敏度较差、漏检等问题。
发明内容
为了解决现有技术的问题,本发明的目的在于提供一种分离和检测循环肿瘤细胞的方法,可从血液样品中有效分离循环肿瘤细胞,提高循环肿瘤细胞检测的灵敏度和特异度,有效降低漏检率。
本发明所述的一种分离和检测循环肿瘤细胞的方法,包括如下步骤:
A.血液样本处理:采用Ficoll密度梯度离心法对血液样本进行离心,离心后体系至少分为四层,分别为血浆血小板层、单个核细胞层、分层液层、多形核白细胞及红细胞层;吸取单个核细胞层洗涤后加入红细胞裂解液离心、洗涤得到外周血单个核细胞;
B.将步骤A获得的外周血单个核细胞分为第一部分细胞与第二部分细胞;
C.对所述第一部分细胞,应用免疫磁珠分选技术(MACS)进行阳性分选富集,然后采用荧光标记抗体对富集的细胞染色后进行流式细胞分选检测;
D.对所述第二部分细胞,应用实时荧光定量PCR法检测以下标记物的表达:Ep-CAM、CK8、CK18、CK19、CD44v6、CD45、CXCR4、SDF-1;
E.综合步骤C和步骤D的检测结果,确定所述血液样本的循环肿瘤细胞的分离结果。
根据本发明所述的分离和检测循环肿瘤细胞的方法的进一步特征,所述步骤A中,血液样本离心前加入HBSS缓冲液按1:1进行稀释。
根据本发明所述的分离和检测循环肿瘤细胞的方法的进一步特征,所述步骤A中,所述Ficoll密度梯度离心法所采用的离心液为淋巴细胞分离液,体积为血液样本的2倍。
根据本发明所述的分离和检测循环肿瘤细胞的方法的进一步特征,所述步骤C中,所述阳性细胞分选磁珠为CD326(Ep-CAM)磁珠,用量为20ul/107个细胞。
根据本发明所述的分离和检测循环肿瘤细胞的方法的进一步特征,所述步骤C中,所述荧光标记抗体是选自:采用荧光染料PE标记的白细胞表面共同抗原CD45抗体、采用荧光染料FITC标记的人细胞角蛋白CK(8/18)抗体、采用荧光染料APC标记的上皮细胞粘附分子CD326/Ep-CAM抗体。
本发明所述的分离和检测循环肿瘤细胞的方法具有以下优势:
(1)联合免疫磁珠分选技术(magnetic activated cell sorting,MACS)和流式细胞分选技术(fluorescence activated cell sorting,FACS)检测外周血循环肿瘤细胞数量。该方法主要依赖于上皮细胞特异性标志物的表达,如上皮细胞中的细胞角蛋白(cytokeratin,CK),这些特异性标志物在上皮细胞上有明显的表达,而在白细胞上表达不明显。细胞角蛋白是由一个角蛋白的中间丝穿过胞浆内部和细胞骨架所形成的蛋白质;其表达主要依赖于上皮细胞的类型、终末分化过程的时点和生长阶段。上皮细胞粘附分子(epithelial cell adhesion molecule,Ep-CAM),又叫CD326,是一种常见的用于细胞群阳性选择的表面标记物,而一种特殊的白细胞表面标记物(CD45)用于阴性选择。因此,将循环肿瘤细胞定义为CD45-CK+CD326+细胞。一般来说,在每7.5mL外周血内,正常人的循环肿瘤细胞数量为0~1个;把含有1~5个循环肿瘤细胞定义为具有复发或转移的低风险;若循环肿瘤细胞数量大于5个,则该患者具备复发或转移的高风险,提示预后不好。
(2)应用实时荧光定量PCR法检测循环肿瘤细胞相关表面标记物的表达,包括Ep-CAM、CK8、CK18、CK19、CD44v6、CD45、CXCR4、SDF-1。
(3)可提高循环肿瘤细胞检测的灵敏度和特异度,有效降低漏检率,便于临床推广应用。
附图说明
图1为从一名58岁男性晚期肺腺癌患者分离的循环肿瘤细胞的检测结果。
图2为同一名58岁男性晚期肺腺癌患者的循环肿瘤细胞表面标记物的实时荧光定量PCR法表达结果。
具体实施方法
下面结合具体实施例对本发明做进一步阐述。
实施例1:临床中晚期非小细胞肺癌患者循环肿瘤细胞数量的检测
(1)样本制备
本组临床诊断中晚期非小细胞肺癌病例共30例,共采集外周血样本30份。
(2)样本检测
1)将7.5mL上述外周血样本轻柔颠倒数次混匀;
2)将上述血液与7.5mL HBSS混合,缓慢加至含有15mL淋巴细胞分离液的50mL离心管的液面上,1,200rpm室温离心20min,用5mL移液管吸出雾状单个核细胞层,并将其均分成两部分(一部分用于实施例1,另一部分用于实施例2);
3)在上述溶液中继续加入10mL HBSS,1,200rpm离心5min后再次去除上清,洗两遍,加入5mL红细胞裂解液(1:10=浓缩液:灭菌水),然后溶液在室温下避光放置10min,1,200rpm离心5min后去上清;
4)加入10mL HBSS重悬细胞,然后1,200rpm离心5min,接着去除上清,又加入1mLHBSS,混匀计数;加入5mL磁珠分选缓冲液(buffer)重悬细胞,1,200rpm再次离心5min,去上清;
5)用余液重悬细胞,然后加入CD326磁珠,接着4℃遮光孵育30min,再用MACS缓冲液洗涤细胞,1,200rpm离心5min,去上清;
6)取LS型的磁珠分离管,用3mL MACS缓冲液润滑清洗磁珠分离柱,用500uLMACS缓冲液(≤50×106)将润洗分离柱液体中的细胞重悬后再加入分离柱,等到分离液体完全均匀流出;
7)使用3mL MACS缓冲液清洗管壁后再次重新加入一个分离柱(1次),3mL MACS缓冲液r加入一个分离柱(2次);
8)在管中均匀加入5mL MACS缓冲液,然后迅速地从中取出分离柱放置于一个流式管上,用电动推压器把这个分离管中的所有分离液体全部均匀推入到一个流式管中,1,200rpm离心5min,去除上清;
9)加入1mL HBSS后重悬上清细胞,1,200rpm离心5min,去掉上清;
10)依次加入抗体,CD326、CK、CD45各10μL(10μL/106),混匀,室温下避光后再孵育12min,用4mL PBS离心清洗1次,去上清,继续加入500μLPBS,最后上流式细胞仪;
11)收集循环肿瘤细胞(CD45-CK+CD326+细胞)数量,关闭流式细胞仪。
(3)检测结果分析
循环肿瘤细胞在外周血中的检出率为46.67%(14例),检测到循环肿瘤细胞个数0-28,平均7.97个/mL。图1为从一名58岁男性晚期肺腺癌患者分离的循环肿瘤细胞的检测结果,显示循环肿瘤细胞数量为21个。
实施例2:临床中晚期非小细胞肺癌患者循环肿瘤细胞相关基因表达的检测
(1)样本检测
1)计数106个LC-5细胞(一种肺鳞癌细胞株),逐级进行10倍稀释,获取105、104、103、102、10、1个LC-5细胞;
2)将提取自健康志愿者血液样本中外周血单个核细胞的RNA分别与提取自1、10、102、103、104、105个LC-5细胞的RNA混合;
3)将混合后的样本进行实时荧光定量PCR检测,扩增效率为96%~104%,斜率设置为-3.1~-3.5和-3.113,R2≥0.99。检测阈值设定在最低样本浓度情况下循环肿瘤细胞相关基因Ep-CAM、CK8、CK18、CK19、CD44v6、CD45、CXCR4、SDF-1同时出现扩增信号时的Ct值,任意一个基因的扩增Ct值大于该阈值即为阳性。
4)将实施例1中的得到雾状单个核细胞层5,000rpm离心5min,去掉上清;
5)加入1mL Trizol,4℃孵育10min,缓慢吹打细胞,使细胞裂解;
6)加入200μL氯仿,涡旋混匀;
7)4℃孵育10min,然后4℃、13,500rpm离心15min,取上层水相300~500μL到新的EP管;
8)加入300~500μL异丙醇(与水相体积比1:1),轻摇,4℃孵育10min,然后4℃、13,500rpm离心10min,去掉上清;
9)加入1mL预冷的75%乙醇(DEPC水),轻轻吹打,然后4℃、13,500rpm离心5min,去掉上清;
10)加入30~50μL DEPC水,室温放置10min,即得到总RNA反应液。
11)冰上配制如下反应液(一次反应用量):总RNA反应液2μL,2×One StepRT-PCRBuffer 410μL,50×ROX Reference Dye or Dye II 0.4μL,PrimeScript 1StepEnzyme Mix 2μL,10μmol/L上下游引物各0.8μL,RNase Free dH2O 5.2μL,总共20μL。
12)上荧光定量PCR仪(ABI 7500)检测Ep-CAM、CK8、CK18、CK19、CD44v6、CD45、CXCR4、SDF-1、GAPDH基因的表达。扩增条件为:42℃5min、95℃10s、95℃5s、60℃34s、95℃15s、60℃1min、95℃15s、4℃59min 59s,40个循环。
(2)检测结果分析
循环肿瘤细胞相关基因在外周血中的检出率为43.33%(13例)。图2为同一名58岁男性晚期肺腺癌患者的循环肿瘤细胞相关细胞标记物的实时荧光定量PCR表达情况。
实施例3:联合MACS、FACS及实时荧光定量PCR法分离和检测循环肿瘤细胞
选择30名晚期肺腺癌患者,联合MACS、FACS(参见实施例1的方法)以及实时荧光定量PCR法(参见实施例2的方法),对得到的数据进行合并分析,对循环肿瘤细胞的检出率为63.33%(19例)。附表1为30例患者循环肿瘤细胞的检出情况。
附表1
注:n.s未检出。
应用免疫磁珠分选MACS技术进行阳性分选富集后,循环肿瘤细胞得到聚集,提供集中度,方便检测,再结合FACS,可有效减少漏检可能性。
应用实时荧光定量PCR可以在基因组水平上检测循环肿瘤细胞,具有高特异性。
由上表可知,联合MACS、FACS及实时荧光定量PCR法,可更高效地提高循环肿瘤细胞检测的灵敏度和特异度,有效降低漏检率。
Claims (5)
1.一种分离和检测循环肿瘤细胞的方法,其特征在于,包括如下步骤:
A.血液样本处理:采用Ficoll密度梯度离心法对血液样本进行离心,离心后体系至少分为四层,分别为血浆血小板层、单个核细胞层、分层液层、多形核白细胞及红细胞层;吸取单个核细胞层洗涤后加入红细胞裂解液离心、洗涤得到外周血单个核细胞;
B.将步骤A获得的外周血单个核细胞分为第一部分细胞与第二部分细胞;
C.对所述第一部分细胞,应用免疫磁珠分选技术(MACS)进行阳性分选富集,然后采用荧光标记抗体对富集的细胞染色后进行流式细胞分选检测;
D.对所述第二部分细胞,应用实时荧光定量PCR法检测以下标记物的表达:Ep-CAM、CK8、CK18、CK19、CD44v6、CD45、CXCR4、SDF-1;
E.综合步骤C和步骤D的检测结果,确定所述血液样本的循环肿瘤细胞的分离结果。
2.根据权利要求1所述的分离和检测循环肿瘤细胞的方法,其特征在于:所述步骤A中,血液样本离心前加入HBSS缓冲液按1:1进行稀释。
3.根据权利要求1所述的分离和检测循环肿瘤细胞的方法,其特征在于:所述步骤A中,所述Ficoll密度梯度离心法所采用的离心液为淋巴细胞分离液,体积为血液样本的2倍。
4.根据权利要求1所述的分离和检测循环肿瘤细胞的方法,其特征在于:所述步骤C中,所述阳性细胞分选磁珠为CD326(Ep-CAM)磁珠,用量为20ul/107个细胞。
5.根据权利要求1所述的分离和检测循环肿瘤细胞的方法,其特征在于:所述步骤C中,所述荧光标记抗体是选自:采用荧光染料PE标记的白细胞表面共同抗原CD45抗体、采用荧光染料FITC标记的人细胞角蛋白CK(8/18)抗体、采用荧光染料APC标记的上皮细胞粘附分子CD326/Ep-CAM抗体。
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