CN111812324A - 一种检测肺癌循环肿瘤细胞的方法 - Google Patents
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Abstract
本发明公开了一种检测肺癌循环肿瘤细胞的方法，采集肺小结节患者外周血，加入红细胞裂解液合并离心的方法去除红细胞，再用连接磁珠的CD45抗体磁性分离去除白细胞。剩余的循环细胞经洗涤、沉淀，进行涂片。用CEP8荧光探针、EpCAM抗体和Pan‑CK抗体进行染色，并在激光共聚焦显微镜下进行扫描。采集信号，并进行读片，判定并计数CTCs。本发明的显著优点为所需标本量少，与单一marker检测法相比较，对CTCs的检出量多，检出率更高，检测效果好。
Description
技术领域
本发明涉及细胞的检测方法，特别涉及一种检测肺癌循环肿瘤细胞的方法。
背景技术
循环肿瘤细胞(Circulating Tumor Cells，CTCs)是肿瘤组织脱落的，进入血液循环的少量肿瘤细胞。研究表明，在肿瘤发生发展的各个阶段均可用不同的方法，在患者的外周血中检测到CTCs。目前，CellSearch是经美国FDA批准的CTCs收集和鉴定，进而辅助诊断恶性肿瘤的技术方法。该技术采用肿瘤细胞表面EpCAM和Cytokeratin蛋白表达增多的特点，利用磁珠连接识别并结合该蛋白的特异性的抗体，对采集自15ml人外周血中的CTCs进行富集，并进一步用免疫荧光的方法进行鉴定。该方法与其他方式联合应用，可以监控表面标志物EpCAM+/Cytokeratin+的肿瘤细胞，用于前列腺癌、乳腺癌、结直肠癌的辅助诊断。但是，对于这两个标志物阴性的循环肿瘤细胞则无法检出。存在漏检的问题。
发明内容
发明目的：本发明目的是提供一种避免漏检、检出量大、效果好的检测肺癌循环肿瘤细胞的方法。本发明方法应用CD45免疫荧光和CEP8荧光原位杂交、EpCAM和Pan-CK标记物免疫荧光联合检测肺小结节患者外周血样本。
技术方案：本发明提供一种检测肺癌循环肿瘤细胞的方法，采集外周血，加入红细胞裂解液合并离心的方法去除红细胞，再用连接磁珠的CD45抗体磁性分离去除白细胞，剩余的循环细胞经洗涤、沉淀，进行涂片，依次用CEP8荧光探针、EpCAM抗体和Pan-CK抗体进行染色、扫描、采集信号、读片、计数CTCs。
进一步地，所述方法，包括如下步骤：
(1)用ACD抗凝管收集外周静脉血；
(2)采用红细胞裂解缓冲液CS2(Cyttel)裂解红细胞，离心，去除红细胞；
(3)离心后的细胞沉淀在CS1(Cyttel)中重悬，然后与抗CD45抗体结合免疫磁珠孵育，梯度离心，去除白细胞；
(4)将含有CTCs的剩余溶液涂抹在载玻片上；
(5)载玻片用枸橼酸钠溶液(SSC)浸泡，然后用乙醇脱水；
(6)将CEP8添加到载玻片上，变性、杂交后用甲酰胺浸泡，用枸橼酸钠溶液(SSC)孵育2-3次；
(7)将载玻片在乙醇中再次脱水，将细胞与CD45在室温下孵育，然后用BSA洗涤，用含有DAPI的封固剂进行固定；
(8)将载玻片用PBS浸没，去掉盖玻片，并用CYP1洗载玻片；
(9)吸去多余液体，加稀释好的Pan-CK和EpCAM抗体，避光孵育；
(10)避光下，取CYP2洗片，吸去多余液体；
(11)加稀释好的荧光抗体DGG和DMO，避光孵育，CYP2洗片，吸去多余液体；
(16)复染：将DAPI管瞬时离心后，液面处取DAPI染液，加至标本区；
(17)盖片：盖上盖玻片，吸去周边液体，镜下观察，重新核对CD45阴性细胞，以确定CEP8、pan-CK和EpCAM的表达；
(18)阅片；
(19)结果判定：pan-CK+或EpCAM+或CEP8+且CD45-和DAPI+特征的细胞被认为是循环肿瘤细胞(CTCs)。
进一步地，所述步骤(5)中用75％、85％和100％乙醇脱水。
进一步地，所述步骤(7)中载玻片分别在75％、85％和100％乙醇中再次脱水。
进一步地，所述步骤(14)阅片采用奥林巴斯BX63显微镜观察载玻片。
进一步地，所述步骤(9)避光孵育1.5-2h。
进一步地，所述步骤(11)避光孵育25～30min。
有益效果：本发明将CEP8+、EpCAM+和pan-CK+联合用以检测CTCs，极大提高了CTCs的检出量，对CTCs检出的阳性率更高。
附图说明
图1为本发明方法检测捕获的循环肿瘤细胞(CEP8+/CD45-/DAPI+)，该细胞未被CD45染红色，同时核中含有多个荧光原位杂交信号，该细胞为循环肿瘤细胞；
图2为本发明方法检测捕获的循环肿瘤细胞(EpCAM+/CD45-/DAPI+)，该细胞未被CD45染红色，同时marker染绿色，该细胞为循环肿瘤细胞；
图3为本发明方法检测捕获的循环肿瘤细胞(pan CKs+/CD45-/DAPI+)，该细胞未被CD45染红色，同时marker染橙黄色，该细胞为循环肿瘤细胞。
具体实施方式
本实施例的方法包括如下步骤：
(1)收集样本：采用ACD抗凝管收集每个受试者的外周静脉血3.2ml；
(2)去除样本红细胞：采用红细胞裂解缓冲液CS2(Cyttel)裂解红细胞，650g离心5min，；
(3)去除样本白细胞：离心后的细胞沉淀在CS1(Cyttel)中重悬，然后与抗CD45抗体结合免疫磁珠孵育，300g梯度离心5min；
(4)制片：剩余含有CTCs的溶液涂抹在载玻片上进行后续分析；
(5)脱水：载玻片用2×枸橼酸钠溶液(SSC)37℃浸泡15min，然后分别用75％、85％和100％乙醇脱水3min；
(6)杂交孵育：将CEP8添加到载玻片上，载玻片在76℃变性5min，杂交30min，然后用甲酰胺浸泡15min，用2×SSC孵育2次，每次5min；
(7)封片：接着将载玻片分别在75％、85％和100％乙醇中再次脱水3min，将细胞与Alexa Fluor 594共轭抗人CD45在室温下孵育1h，然后用0.2％BSA洗涤两次，使用含有DAPI的封固剂进行固定
(8)洗涤：将载玻片用1×PBS浸没，小心甩去盖玻片，并用CYP1洗载玻片1次，3min；
(9)再次孵育：吸去多余液体，加100μL稀释好的Pan-CK和EpCAM抗体，室温湿盒内避光孵育1.5-2h；
(10)洗涤：避光，取CYP2100～150μL/片，洗片3次，每次3min，吸去多余液体；
(11)加100μL稀释好的荧光抗体DGG和DMO，37℃湿盒内避光孵育25～30min，CYP24次，每次3min，吸去多余液体；
(12)复染：将DAPI管瞬时离心后，液面处取10μL DAPI染液，加至标本区；
(13)盖片：盖上盖玻片，轻轻按压盖玻片，并吸去周边液体，镜下观察，重新核对CD45阴性细胞，以确定CEP8、pan-CK和EpCAM的表达；
(14)阅片：采用奥林巴斯BX63显微镜观察载玻片。
(15)结果判定：pan-CK+或EpCAM+或CEP8+且CD45-和DAPI+特征的细胞是循环肿瘤细胞(CTCs)。
在22例肺小结节患者的3.5ml外周血中，采用CEP8、EpCAM和pan-CK三染色进行CTCs的鉴定，计数CEP8+或EpCAM+或pan-CK+且CD45-和DAPI+的细胞数量，由表1的计数结果显示单一marker染色量均较少，说明本发明联合三种marker检测CTCs的方法漏检率小，检测效果好。
表1应用本发明检测肺小结节患者外周血样本CTCs染色结果
Claims (7)
1.一种检测肺癌循环肿瘤细胞的方法，其特征在于：采集外周血，加入红细胞裂解液合并离心的方法去除红细胞，再用连接磁珠的CD45抗体磁性分离去除白细胞，剩余的循环细胞经洗涤、沉淀、涂片，依次用CEP8荧光探针、EpCAM抗体和Pan-CK抗体进行染色、扫描、采集信号、读片、计数。
2.根据权利要求1所述的检测肺癌循环肿瘤细胞的方法，其特征在于：包括如下步骤：
(1)用ACD抗凝管收集外周静脉血；
(2)采用红细胞裂解缓冲液CS2(Cyttel)裂解红细胞，离心，去除红细胞；
(3)离心后的细胞沉淀在CS1(Cyttel)中重悬，然后与抗CD45抗体结合免疫磁珠孵育，梯度离心，去除白细胞；
(4)将含有CTCs的剩余溶液涂抹在载玻片上；
(5)载玻片用枸橼酸钠溶液(SSC)浸泡，然后用乙醇脱水；
(6)将CEP8添加到载玻片上，变性、杂交后用甲酰胺浸泡，用枸橼酸钠溶液(SSC)孵育2-3次；
(7)将载玻片在乙醇中再次脱水，将细胞与CD45在室温下孵育，然后用BSA洗涤，用含有DAPI的封固剂进行固定；
(8)将载玻片用PBS浸没，去掉盖玻片，并用CYP1洗载玻片；
(9)吸去多余液体，加稀释好的Pan-CK和EpCAM抗体，避光孵育；
(10)避光下，取CYP2洗片，吸去多余液体；
(11)加稀释好的荧光抗体DGG和DMO，避光孵育，CYP2洗片，吸去多余液体；
(12)复染：将DAPI管瞬时离心后，液面处取DAPI染液，加至标本区；
(13)盖片：盖上盖玻片，吸去周边液体，镜下观察，重新核对CD45阴性细胞，以确定CEP8、pan-CK和EpCAM的表达；
(14)阅片；
(15)结果判定：pan-CK+或EpCAM+或CEP8+且CD45-和DAPI+特征的细胞被是循环肿瘤细胞(CTCs)。
3.根据权利要求2所述的检测肺癌循环肿瘤细胞的方法，其特征在于：所述步骤(5)中用75％、85％和100％乙醇脱水。
4.根据权利要求2所述的检测肺癌循环肿瘤细胞的方法，其特征在于：所述步骤(7)中载玻片分别在75％、85％和100％乙醇中再次脱水。
5.根据权利要求2所述的检测肺癌循环肿瘤细胞的方法，其特征在于：所述步骤(14)阅片采用奥林巴斯BX63显微镜观察载玻片。
6.根据权利要求2所述的检测肺癌循环肿瘤细胞的方法，其特征在于：所述步骤(9)避光孵育1.5-2h。
7.根据权利要求2所述的检测肺癌循环肿瘤细胞的方法，其特征在于：所述步骤(11)避光孵育25～30min。
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