CN111638355A - 一种检测乳腺癌患者外周血循环肿瘤细胞E-Cadherin表达的试剂盒及检测方法 - Google Patents
一种检测乳腺癌患者外周血循环肿瘤细胞E-Cadherin表达的试剂盒及检测方法 Download PDFInfo
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Abstract
本发明涉及一种检测乳腺癌患者外周血循环肿瘤细胞E‑Cadherin表达的试剂盒及检测方法,属于分子生物学技术领域。所述的试剂盒,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一抗100μL、连接有辣根过氧化物酶的兔抗鼠IgG/HRP 100μL、0.1%Triton X‑100 100μL、0.3%H2O2100μL、试剂A 15ml、试剂B 50ml、6×PBS缓冲液60mL。本发明的检测方法是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乳腺癌患者外周血中的CTC,进一步运用免疫组化技术检测CTC的E‑Cadherin表达情况。
Description
技术领域
本发明涉及一种检测乳腺癌患者外周血循环肿瘤细胞E-Cadherin表达的试剂盒及检测方法,属于分子生物学技术领域。
背景技术
乳腺癌是严重威胁女性健康的恶性肿瘤之一,并且发病率呈逐年上升的趋势。乳腺癌术后复发转移导致的死亡位居中国和美国女性癌症死因的第6位和第2位,是乳腺癌治疗失败的主要原因。因此,对乳腺癌术后转移的治疗关键在于利用有效的检查技术及时准确的做出诊断。抗原肿瘤标志物检测具有简便快捷、对患者造成的创伤和损害小等优势,对乳腺癌的发生、发展、治疗及预后具有重要的临床意义。
E-钙粘蛋白(E—Cadherin)是上皮细胞表型最经典的标志物,表达于绝大多数上皮细胞表面,尤以细胞连接处最为集中,是上皮细胞表面最重要的细胞间粘附分子之一。它通过介导细胞间的粘附反应,维持上皮组织结构的完整性和细胞的极性,具有细胞接触性生长抑制功能,防止细胞的相互分离。E-Cadherin是典型的某些类型肿瘤形成/浸润抑制因子,E-Cadherin的检测对于检测乳腺癌转移及复发具有重要意义。而目前临床实践中,乳腺癌患者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。
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。
发明内容
为了克服多次或实时检测的问题,同时避免穿刺活检面临出血、胰瘘、感染等风险,本发明提供了一种检测乳腺癌患者外周血循环肿瘤细胞E-Cadherin表达的试剂盒及检测方法,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乳腺癌患者外周血中的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:
一种检测乳腺癌患者外周血循环肿瘤细胞E-Cadherin表达的试剂盒,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一抗100μL、连接有辣根过氧化物酶的兔抗鼠IgG/HRP 100μL、0.1% Triton X-100 100μL、0.3% H2O2 100μL、试剂A15ml、试剂B 50ml、6×PBS缓冲液 60mL。
所述稀释液包括:含量为0.5g/L的乙二胺四乙酸盐,含量为6g/L的硫酸钠;含量为4g/L的氯化钠,含量为2g/L的二羟基甲基脲,含量为6g/L的羟乙基哌嗪乙硫磺酸;含量为0.5g/L的十二烷基磺基甜菜碱,含量为0.5g/L的叠氮钠,含量为0.3g/L的聚氧乙烯聚氧丙烯醚嵌段共聚物,其余是水。
所述脱色液是乙醇与醋酸按体积比1:2组成。
所述染色液A为DAB染色液;所述染色液B为苏木素染色液。
所述试剂A为甲醛-二甲苯按照体积比1:3组成;所述试剂B为二甲苯。
任一项所述的试剂盒非诊断目的检测乳腺癌患者外周血循环肿瘤细胞E-Cadherin表达的方法,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乳腺癌患者外周血中的CTC:采集无法获取组织标本的晚期或复发乳腺癌患者外周血: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45ml进行稀释,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。
步骤(5)所述检测CTC的E-Cadherin表达的具体方法如下:
S1 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;
S2 滴加100μl 0.1% Triton X-100,室温孵育15min,DI水洗2min×3次;
S3 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一抗,室温孵育2h或4℃过夜,PBS洗2min×3次;
S4 滴加100μl连接有辣根过氧化物酶的兔抗鼠IgG/HRP,18~26℃温度下孵育20min,PBS洗2min×3次;
S5 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;
S6 显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;
S7 盐酸酒精分化8秒,自来水返蓝5min;
S8 将返蓝后的CTC采用75%乙醇,95%乙醇,100%乙醇梯度乙醇脱水各10 min,然后加入15 mL试剂A,振荡均匀后过滤;加入试剂B,脱色30 min,中性树脂封固;
S9 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乳腺癌患者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为乳腺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 试剂盒具体规格
运用此技术方法分离获取并鉴定15例乳腺癌患者(同时检测5例正常人样本做阴性对照)外周血循环肿瘤细胞的实施例。
所述稀释液的配置:0.5g乙二胺四乙酸盐,6g硫酸钠;4g氯化钠,2g二羟基甲基脲,6g羟乙基哌嗪乙硫磺酸; 0.5g十二烷基磺基甜菜碱,0.5g叠氮钠,0.3g聚氧乙烯聚氧丙烯醚嵌段共聚物,980.5g水。
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乳腺癌患者外周血中的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。
图4为乳腺癌患者外周血分离获取的循环肿瘤细胞影像图,从图中可以看出,其细胞核较大,细胞核形状不规则,细胞直径(长端)大于15um;高核质比,核质比大于0.8;细胞核染色质边移,巨大核仁,异常核分裂。
通过观察,5例健康志愿者均未查到CTC;除4例复发乳腺癌患者未检测到CTC外,其余6例复发乳腺癌患者和6例晚期乳腺癌均检测到CTC(表2),本次检测阳性率为80 %。
值得注意的是,当稀释液不添加羟乙基哌嗪乙硫磺酸或者聚氧乙烯聚氧丙烯醚嵌段共聚物时,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,置于乙醇与醋酸按容积比1:2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一抗,室温孵育2h(或4℃过夜),PBS洗2min×3次;滴加100μl连接有辣根过氧化物酶的兔抗鼠IgG/HRP,室温(18~26℃)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,然后加入体积比1:3的甲醛和二甲苯,振荡均匀后过滤,二甲苯脱色,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。
由乙醇与醋酸组成的脱色液效果比95%酒精与100%二甲苯按容积比1:1组成的脱色液,脱色效果好。
当不采用试剂A只用二甲苯脱色,检测结果的准确率为85%,采用试剂A和试剂B,即本发明的细胞集中,且形态清晰,分散均匀性好,准确率可达到99%。
所检测的循环肿瘤细胞应用免疫组化证实E-Cadherin的表达并与乳腺癌手术或穿刺组织标本E-Cadherin结果对比,观察其差异,验证了CTCs作为无创活检用于实时评估乳腺癌E-Cadherin表达的潜力,为评估乳腺癌化疗联合免疫治疗预后提供重要依据。
Claims (7)
1.一种检测乳腺癌患者外周血循环肿瘤细胞E-Cadherin表达的试剂盒,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一抗100μL、连接有辣根过氧化物酶的兔抗鼠IgG/HRP 100μL、0.1% Triton X-100 100μL、0.3% H2O2100μL、试剂A 15ml、试剂B 50ml、6×PBS缓冲液 60mL。
2.根据权利要求1所述的试剂盒,其特征在于,所述稀释液包括:含量为0.5g/L的乙二胺四乙酸盐,含量为6g/L的硫酸钠;含量为4g/L的氯化钠,含量为2g/L的二羟基甲基脲,含量为6g/L的羟乙基哌嗪乙硫磺酸;含量为0.5g/L的十二烷基磺基甜菜碱,含量为0.5g/L的叠氮钠,含量为0.3g/L的聚氧乙烯聚氧丙烯醚嵌段共聚物,其余是水。
3.根据权利要求1所述的试剂盒,其特征在于,所述脱色液是乙醇与醋酸按体积比1:2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为甲醛-二甲苯按照体积比1:3组成;所述试剂B为二甲苯。
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乳腺癌患者外周血循环肿瘤细胞E-Cadherin表达的方法,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乳腺癌患者外周血中的CTC:采集无法获取组织标本的晚期或复发乳腺癌患者外周血: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45ml进行稀释,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,步骤(5)所述检测CTC的E-Cadherin表达的具体方法如下:
S1 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;
S2 滴加100μl 0.1% Triton X-100,室温孵育15min,DI水洗2min×3次;
S3 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一抗,室温孵育2h或4℃过夜,PBS洗2min×3次;
S4 滴加100μl连接有辣根过氧化物酶的兔抗鼠IgG/HRP,18~26℃温度下孵育20min,PBS洗2min×3次;
S5 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;
S6 显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;
S7 盐酸酒精分化8秒,自来水返蓝5min;
S8 将返蓝后的CTC采用75%乙醇,95%乙醇,100%乙醇梯度乙醇脱水各10 min,然后加入15 mL试剂A,振荡均匀后过滤;加入试剂B,脱色30 min,中性树脂封固;
S9 光学显微镜下镜检。
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