CN111638345A - 检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的试剂盒及方法 - Google Patents
检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的试剂盒及方法 Download PDFInfo
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Abstract
本发明提供了检测非小细胞肺癌患者外周血循环肿瘤细胞E‑Cadherin基因突变的试剂盒及方法,属于分子生物学技术领域。该试剂盒包括稀释液、脱色液、染色液A、染色液B、鼠抗人E‑Cadherin单克隆抗体、山羊抗小鼠IgG/HRP、SABC、0.1%Triton X‑100、0.3%H2O2、试剂A、试剂B、6×PBS缓冲液。本发明提供的检测方法,不用穿刺活检获取组织标本即可检测到晚期或复发非小细胞肺癌患者E‑Cadherin表达情况。该技术属于微创,并能够实时检测。能够避免染色过程中可能产生的边缘效应导致的假阳性结果,稳定性好,降低细胞的损失,提高检测的准确性。
Description
技术领域
本发明提供了一种检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的试剂盒及检测方法,属于分子生物学技术领域。
背景技术
肺癌是导致癌症患者死亡的主要恶性肿瘤之一,在我国,肺癌的发病率和死亡率均居第一位。非小细胞肺癌(NSCLC)约占全部肺癌的85%,而这其中超过70%的NSCLC患者在确诊时已为晚期。尽管手术和放化疗等治疗技术不断提高,NSCLC患者5年生存率仍低于20%,主要死因包括局部复发与远处转移。
循环肿瘤细胞(Circulating tumor cell,CTC)是从实体肿瘤脱落进入外周血液循环的肿瘤细胞,自1989年被发现以来,目前已有多种方法用于外周血循环肿瘤细胞的检测。近期研究表明,其检测对于评估肿瘤患者尤其是晚期肿瘤患者的预后以及选择合适的个体化治疗具有重要的临床意义。因CTC检测具有微创、实时检测等特点,被称为肿瘤的“液态活检”。
目前,山东省第一医科大学、山东省药物研究院联合山东凯歌智能机器有限公司就循环肿瘤细胞检测鉴定关键技术、检测设备、试剂盒开发与生产进行合作,山东祺欣生物科技有限公司、山东喻晓生物科技有限公司、济南杏恩生物科技有限公司、山东发现生物技术有限公司等单位进行推广应用,本项目为山东省重大科技创新工程项目,以山东第一医科大学济南校区的山东省药物研究院为核心,落实注册人制度,依托循环肿瘤细胞检测鉴定核心诊断技术,进一步注册鉴定诊断试剂盒,以包括PD1、PD-L1、ER、PR、Her-2、GPC-3、VEGF、P53、Vimentin、TKI-EGFR、RAS、CK、ALK-D5F3、CD20、ALK/EML4、Beta-catenin、E-Cadherin、EP-CAM、HPV、IDH-1、PSA、PSMA、VEGF、GFAP、细胞角蛋白、AE1/AE3、雌激素受体、孕激素受体、BCA-225、CA 125、CEA、EMA、ERCC1、HPV、Ki-67、P53、TOP2A等作为CTCs表达的示踪剂,注册超灵敏、超快速、高覆盖、低成本、准确特异的鉴定诊断试剂盒,通过与在济南注册的山东凯歌智能机器有限公司、山东祺欣生物科技有限公司、山东喻晓生物科技有限公司、济南杏恩生物科技有限公司、山东发现生物技术有限公司合作进行研发与推广。
E-钙粘蛋白(E—Cadherin)是上皮细胞表型最经典的标志物,E-Cadherin表达的下调标志着细胞之间粘附能力的下降,因此E-Cadherin被当做鉴定非小细胞肺癌EMT发生的主要手段之一,作为一类细胞表面糖蛋白,E-Cadherein在细胞-细胞之间的粘附中发挥重要作用,并参与组织器官的维持。近年来有关于非小细胞肺癌EMT和肿瘤耐药之间的关系日益受到重视,大量研究显示在不同的肿瘤当中由于EMT的发生而同时耐药性的升高,并且都伴随着E-Cadherin表达的改变。目前,国内外NSCLC诊治指南均认为:NSCLC患者E-Cadherin突变检测的时间点包括初诊时及争取在疾病进展时再次检测,应通过重复检测减少假阳性和假阴性。而目前临床实践中,NSCLC患者E-Cadherin检测的标本主要为肿瘤组织,来源于手术或穿刺活检,很难做到多次或实时检测。
发明内容
为了克服晚期或复发非小细胞肺癌患者无法实时或反复穿刺获取组织标本、进而不能评估患者E-Cadherin状态的不足,本发明提供了一种非小细胞肺癌患者外周血循环肿瘤细胞ALK基因突变非诊断目的的检测方法:利用膜过滤装置分离获得无法获取组织标本的晚期或复发非小细胞肺癌患者外周血中的CTC,进一步运用免疫组化技术检测CTC的E-Cadherin表达情况。
本发明采用的技术方案如下:
本发明提供了一种检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的试剂盒,包括稀释液45mL、脱色液1mL、染色液A 0.5mL、染色液B 1mL、鼠抗人E-Cadherin单克隆抗体100μL、山羊抗小鼠 IgG/HRP 100μL、SABC 100μL、0.1% Triton X-100 100μL、0.3% H2O2 100μL、试剂A 0.5mL、试剂B 1mL、6×PBS缓冲液 60mL;所述PBS缓冲液的pH值为7.4。
进一步的,所述稀释液是由1mmol/L EDTA+0.1% BSA+0.1%海藻糖+0.2%聚氧乙烯聚氧丙烯醚嵌段共聚物组成,所述基础液为Tris-HCl缓冲剂。
进一步的,所述脱色液是由95%酒精与100%二甲苯按容积比1:1组成。
进一步的,所述染色液A为DAB染色液;所述染色液B为苏木素染色液。
进一步的,所述试剂A为质量分数为0.6%的羟丙基甲基纤维素二甲苯混合液;所述试剂B为乙醇和1,2-丙二醇按照体积比3:1组成。
本发明还提供了一种利用上述试剂盒非诊断目的检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的方法,包括以下步骤:
(1)利用膜过滤装置分离获取无法获得组织标本的晚期或复发非小细胞肺癌患者外周血中的CTC:采集无法获取组织标本的晚期或复发非小细胞肺癌患者外周血:肘正中静脉外周血5ml;
(2)外周血样预处理:将采集的外周血样采用稀释液进行10倍稀释,稀释后加多聚甲醛固定外周血样10分钟,固定终浓度为0.25%;
(3)利用膜过滤分离肿瘤细胞装置过滤外周血样,分离获得外周血CTC:将预处理的外周血样加入到膜过滤分离肿瘤细胞装置的血样容器中,使其依靠重力自然过滤;
(4)过滤结束后,从膜过滤分离肿瘤细胞装置中取下滤器,将循环肿瘤细胞染色液A液0.5ml加入到滤器中,染色3min,PBS缓冲液冲洗干净;滤液过滤完全后加入染色液B液1ml,染色2min,纯水1ml冲洗2次,取下滤膜,放置在载玻片上,干燥后在显微镜下观察,确定是否存在CTC;
(5)运用免疫组化技术检测CTC的E-Cadherin表达情况。
本发明检测CTC的E-Cadherin表达的具体方法如下:
(1)脱色:将带有CTC的滤膜从载玻片上取下,置于脱色液中浸泡4-6小时,脱去CTC染色液;
(2)滴加100μl 0.1% Triton X-100,室温孵育15min,DI水洗2min×3次;
(3)滴加100μl 0.3% H2O2,室温孵育10min,PBS洗2min×3次;(4)滴加100μl鼠抗人E-Cadherin单克隆抗体一抗,室温孵育2h或4℃过夜,PBS洗2min×3次;
(5)滴加100μl山羊抗小鼠 IgG/HRP,18~26℃温度下孵育20min,PBS洗2min×3次;
(6)滴加100μlSABC,在18~26℃温度下孵育20min,PBS洗2min×3次;
(7)滴加100μl DAB显色液,18~26℃孵育并随时在显微镜下观察显色情况,观察时间为3~10min;
(8)显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;
(9)盐酸酒精分化8秒,自来水返蓝5min;
(10)将返蓝后的CTC采用75%乙醇(1min),95%乙醇(1min),100%乙醇(1min)梯度乙醇脱水,然后加入0.5mL试剂A,振荡均匀后,加入1mL试剂B,摇动混合均匀后,离心沉淀,将沉淀物采用中性树脂封固;
(11)光学显微镜下镜检。
本发明所使用的膜过滤分离肿瘤细胞装置,包括滤器、血样容器、废液缸和铁架台,所述铁架台设有底座、立架和支架,所述血样容器通过支架设置于铁架台上部,血样容器的下方为滤器,滤器通过输液器联通至废液缸,废液缸设置于底座上。
所述滤器包括滤器上口、滤膜、载滤膜平台和滤器下口,滤膜置于载滤膜平台上;滤器上口接血样容器,滤器下口通过输液器接废液缸。
所述滤膜为疏水材料制成,其上均匀布满口径为8微米的滤孔。
本发明的有益效果是:
(1)本发明提供的检测方法,不用穿刺活检获取组织标本即可检测到晚期或复发非小细胞肺癌患者E-Cadherin表达情况。该技术属于微创,并能够实时检测。
(2)本发明提供的方法,能够避免染色过程中可能产生的边缘效应导致的假阳性结果,稳定性好,降低细胞的损失,提高检测的准确性。
附图说明
图1为本发明的膜过滤装置结构示意图;
图2为本发明膜过滤装置的滤器的结构示意剖视图;
图3为本发明膜过滤装置的滤器滤膜的结构示意图;
图4为肺癌患者外周血分离获取的循环肿瘤细胞影像图;
图中:1铁架台、2血样容器、3滤器、4输液器、5废液缸、6滤器上口、7滤膜、8载滤膜平台、9滤器下口、10滤孔、11底座、12立架、13支架。
具体实施方式
下面结合附图和实施例对本发明阐述如下。
本发明所使用的试剂盒具体规格如表1所示:
表1
运用此技术方法分离获取并鉴定8例非小细胞肺癌患者(同时检测8例正常人样本做阴性对照)外周血循环肿瘤细胞的实施例。
实施例1
一、利用膜过滤装置分离获取无法获得组织标本的晚期或复发非小细胞肺癌患者外周血中的CTC,确定CTC是否存在:
自肘正中静脉采集空腹8-12小时的空腹血5ml,用45ml稀释液(成分:1mmol/L EDTA+0.1% BSA+0.1%海藻糖+0.2%聚氧乙烯聚氧丙烯醚嵌段共聚物)稀释外周血,然后加入3ml的4%多聚甲醛固定稀释后的血样10分钟;
在固定的间期,组装膜过滤装置:如附图1、图2、图3所示,该过滤装置由滤器3、滤膜7、血样容器2、废液缸5、铁架台1构成;
用10mlPBS润湿滤器3,然后将固定好的外周血样加入到膜过滤装置的血样容器2中,使其依靠重力自然过滤,CTC被截留在滤膜7上;
肿瘤细胞直径一般大于15微米,而血细胞(包括红细胞、白细胞)直径一般小于8微米,因此当含有CTC的外周血经过滤后,血细胞因直径小于滤孔10能够被滤过,而CTC因直径大于滤孔10被截留在滤膜7上。
过滤结束后,从过滤装置中取下滤器3,打开并移走滤器上口6,将将循环肿瘤细胞染色液A液0.5ml加入到滤器中,染色3min,PBS缓冲液冲洗干净;滤液过滤完全后加入B液,1ml,染色2min,纯水1ml,PBS缓冲液将滤器3冲洗干净,用眼科镊子取下滤膜7,细胞面朝上,放置在载玻片上;
将滤膜干燥后在显微镜下观察,确定是否存在CTC。
通过观察,从表2中看出,8例健康志愿者均未查到CTC;复发非小细胞肺癌患者及晚期非小细胞肺癌患者均检测到CTC,本次检测阳性率为100%,值得注意的是,当稀释液不添加0.1%海藻糖或者不添加0.2%聚氧乙烯聚氧丙烯醚嵌段共聚物时,单一的采用0.3%海藻糖或者0.3%聚氧乙烯聚氧丙烯醚嵌段共聚物,制备的血样稳定性差,部分血样还会形成分层,血液细胞容易发生聚集和粘连,影响最终的检测效果。
表2 实施例CTC检测结果
二、运用免疫组化技术检测CTC的E-Cadherin表达情况:
将载玻片上载有CTC的滤膜7从载玻片上取下,置于95%酒精与100%二甲苯按容积比1:1混匀的脱色液中浸泡4-6小时,脱去CTC染色液;滴加100μl 0.1% Triton X-100,室温孵育15min,DI水洗2min×3次;滴加100μl 0.3% H2O2,室温孵育10min,PBS洗2min×3次;滴加100μl 鼠抗人E-Cadherin单克隆抗体一抗,室温孵育2h(或4℃过夜),PBS洗2min×3次;滴加100μl山羊抗小鼠 IgG/HRP,室温(18~26℃)孵育20min,PBS洗2min×3次;滴加100μlSABC,在18~26℃温度下孵育20min,PBS洗2min×3次;滴加100μl DAB显色液,室温(18~26℃)孵育并随时在显微镜下观察显色情况(一般为3~10min,时间不能超过10min);显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;盐酸酒精分化8秒,自来水返蓝5min;75%乙醇(1min),95%乙醇(1min),100%乙醇(1min)梯度乙醇脱水,然后加入0.6%的羟丙基甲基纤维素二甲苯混合液,振荡均匀后,加入乙醇和1,2-丙二醇混合溶剂(V:V=3:1),摇动混合均匀后,离心沉淀,将沉淀物晾干,中性树脂封固;光学显微镜下镜检,细胞病理学专家阅片,根据细胞膜和细胞浆着色程度判定E-Cadherin表达情况。
当将试剂B采用单一的乙醇或者1,2-丙二醇时,采用中性树脂固封后,本发明乙醇和1,2-丙二醇混合溶剂,固封后的检测准确率能够达到100%,而采用单一的乙醇的准确率为85%,而采用单一的1,2-丙二醇的准确率则仅为70%,能够避免染色过程中可能产生的边缘效应导致的假阳性结果,稳定性好,降低细胞的损失,提高检测的准确性。
图4为非小细胞肺癌患者外周血分离获取的循环肿瘤细胞影像图,其为2个CTC细胞,其细胞核异型性,核质比大于0.8,呈现高核质比。
所检测的循环肿瘤细胞应用免疫组化证实E-Cadherin的表达并与非小细胞肺癌大体标本E-Cadherin结果对比,观察其差异,主要针对大体标本E-Cadherin表达阴性而循环肿瘤细胞表达阳性的患者,指导非小细胞肺癌的靶向治疗,为非小细胞肺癌靶向治疗提供新的思路。
Claims (7)
1.一种检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的试剂盒,其特征在于,包括稀释液45mL、脱色液1mL、染色液A 0.5mL、染色液B 1mL、鼠抗人E-Cadherin单克隆抗体100μL、山羊抗小鼠 IgG/HRP 100μL、SABC 100μL、0.1% Triton X-100 100μL、0.3% H2O2 100μL、试剂A 0.5mL、试剂B 1mL、6×PBS缓冲液 60mL;所述PBS缓冲液的pH值为7.4。
2.根据权利要求1所述的试剂盒,其特征在于,所述稀释液是由1mmol/L EDTA+0.1%BSA+0.1%海藻糖+0.2%聚氧乙烯聚氧丙烯醚嵌段共聚物组成,所述基础液为Tris-HCl缓冲剂。
3.根据权利要求1所述的试剂盒,其特征在于,所述脱色液是由95%酒精与100%二甲苯按容积比1:1组成。
4.根据权利要求1所述的试剂盒,其特征在于,所述染色液A为DAB染色液;所述染色液B为苏木素染色液。
5.根据权利要求1所述的试剂盒,其特征在于,所述试剂A为质量分数为0.6%的羟丙基甲基纤维素二甲苯混合液;所述试剂B为乙醇和1,2-丙二醇按照体积比3:1组成。
6.一种利用权利要求1-5任一项所述的试剂盒非诊断目的检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的方法,其特征在于,包括以下步骤:
(1)利用膜过滤装置分离获取无法获得组织标本的晚期或复发非小细胞肺癌患者外周血中的CTC:采集无法获取组织标本的晚期或复发非小细胞肺癌患者外周血:肘正中静脉外周血5ml;
(2)外周血样预处理:将采集的外周血样采用稀释液进行10倍稀释,稀释后加多聚甲醛固定外周血样10分钟,固定终浓度为0.25%;
(3)利用膜过滤分离肿瘤细胞装置过滤外周血样,分离获得外周血CTC:将预处理的外周血样加入到膜过滤分离肿瘤细胞装置的血样容器中,使其依靠重力自然过滤;
(4)过滤结束后,从膜过滤分离肿瘤细胞装置中取下滤器,将循环肿瘤细胞染色液A液0.5ml加入到滤器中,染色3min,PBS缓冲液冲洗干净;滤液过滤完全后加入染色液B液1ml,染色2min,纯水1ml冲洗2次,取下滤膜,放置在载玻片上,干燥后在显微镜下观察,确定是否存在CTC;
(5)运用免疫组化技术检测CTC的E-Cadherin表达情况。
7.根据权利要求6所述的检测方法,其特征在于,所述检测CTC的E-Cadherin表达的具体方法如下:
(1)脱色:将带有CTC的滤膜从载玻片上取下,置于脱色液中浸泡4-6小时,脱去CTC染色液;
(2)滴加100μl 0.1% Triton X-100,室温孵育15min,DI水洗2min×3次;
(3)滴加100μl 0.3% H2O2,室温孵育10min,PBS洗2min×3次;(4)滴加100μl鼠抗人E-Cadherin单克隆抗体一抗,室温孵育2h或4℃过夜,PBS洗2min×3次;
(5)滴加100μl山羊抗小鼠 IgG/HRP,18~26℃温度下孵育20min,PBS洗2min×3次;
(6)滴加100μlSABC,在18~26℃温度下孵育20min,PBS洗2min×3次;
(7)滴加100μl DAB显色液,18~26℃孵育并随时在显微镜下观察显色情况,观察时间为3~10min;
(8)显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;
(9)盐酸酒精分化8秒,自来水返蓝5min;
(10)将返蓝后的CTC采用75%乙醇(1min),95%乙醇(1min),100%乙醇(1min)梯度乙醇脱水,然后加入0.5mL试剂A,振荡均匀后,加入1mL试剂B,摇动混合均匀后,离心沉淀,将沉淀物采用中性树脂封固;
(11)光学显微镜下镜检。
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CN111638357A (zh) * | 2020-07-01 | 2020-09-08 | 山东凯歌智能机器有限公司 | 非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin突变的免疫荧光试剂盒及方法 |
CN111638345A (zh) * | 2020-07-01 | 2020-09-08 | 山东凯歌智能机器有限公司 | 检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的试剂盒及方法 |
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WO2022001823A1 (zh) * | 2020-07-01 | 2022-01-06 | 山东第一医科大学第二附属医院 | 检测非小细胞肺癌患者外周血循环肿瘤细胞E-Cadherin基因突变的试剂盒及方法 |
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