CN111679079A - 通过外周血循环肿瘤细胞检测结直肠癌患者cea基因表达的免疫荧光试剂盒及检测方法 - Google Patents
通过外周血循环肿瘤细胞检测结直肠癌患者cea基因表达的免疫荧光试剂盒及检测方法 Download PDFInfo
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Abstract
本发明提供一种结直肠癌患者外周血循环肿瘤细胞CEA基因表达的免疫荧光试剂盒及检测方法,属于分子生物学领域,具体的,试剂盒主要包括:山羊血清,小鼠抗CK、大鼠抗CD45和兔抗CEA基因组成的一抗混悬液,荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成的二抗混悬液。检测方法主要包括采集外周血,处理外周血,过滤富集循环肿瘤细胞,免疫荧光方法检测循环肿瘤细胞CEA基因表达情况。
Description
技术领域
本发明提供了通过外周血循环肿瘤细胞检测结直肠癌患者CEA基因表达的免疫荧光试剂盒及检测方法,属于分子生物学技术领域。
背景技术
结直肠癌是世界上最常见的恶性肿瘤之一,2018年死亡率居恶性肿瘤第四位。在美国,结直肠癌是第三大最常见的恶性肿瘤。在国内,2018年中国全年流行病学数据:结直肠癌的发病率和死亡率均为第5位,年新发病例37.63万,25%被确诊时已发生转移。结直肠癌早期多无症状,出现症状时多为中晚期且有转移。尽管外科手术和化疗方案不断革新,进展期结直肠癌患者的生存率依旧很差。由于结直肠癌尚缺乏较好的预防方法,所以改善结直肠癌预后并提高生存率的最有效的措施就是早期诊断并治疗结直肠早期癌及癌前病变。
CEA(carcinoembryonic antigen)是指癌胚抗原,是一种酸性糖蛋白,45%为蛋白质,含有岩藻糖、甘露糖、半乳糖及唾液酸等成分,分子量为150-300KDa,其编码基因位19号染色体。既往研究表明,结肠癌、胃癌、胰腺癌等消化道肿瘤患者血清中CEA均呈现不同程度的升高,CEA被广泛用作各种消化系统肿瘤的诊断及检测指标。随后研究发现,在肝癌、乳腺癌、卵巢癌、子宫癌、泌尿系肿瘤等其他恶性肿瘤中也有不同程度的阳性率,在恶性肿瘤的鉴别诊断、病情监测、疗效评价等方面仍有重要作用。而目前临床实践中,结直肠癌患者CEA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。
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”。
免疫荧光分析技术即将免疫学方法(抗原抗体特异结合)与荧光标记技术结合起来用以研究特异蛋白抗原在细胞内分布的方法。由于荧光素所发出的荧光可在荧光显微镜下检出,荧光素受激发光的照射而发出明亮的荧光(黄绿色或橘红色),可以看见荧光所在的细胞或组织,利用定量技术测定含量,从而对抗原进行细胞定性和定位分析。
针对目前临床实践中,结直肠癌患者CEA基因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。因此,检测循环肿瘤细胞(CTC)CEA基因表达情况对结直肠癌预后及免疫治疗疗效评估具有重要价值。
发明内容
针对现有技术中的检测肿瘤晚期或复发结直肠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CEA基因实时动态状态,及现有检测方法容易出现假阳性和假阴性的缺点,本发明提供了一种结直肠癌患者外周血循环肿瘤细胞CEA基因表达的免疫荧光试剂盒及检测方法,利用膜过滤装置分离获得晚期结直肠癌患者外周血中的循环肿瘤细胞(CTC),进一步运用免疫荧光技术检测CTC上CEA基因表达情况。
本发明通过以下技术方案实现:
一种检测结直肠癌患者外周血循环肿瘤细胞CEA基因表达的免疫荧光试剂盒,包括稀释液45mL,脱色液1mL,染色液A 0.5mL,染色液B 1mL,200μl 甲醇、200μl 2%PFA,100μl10%山羊血清,小鼠抗CK、大鼠抗CD45和兔抗CEA基因组成的一抗混悬液100μl,荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成的二抗混悬液100μL,DAPI封片剂;
一抗混悬液中小鼠抗CK、大鼠抗CD45和兔抗CEA基因分别按1:100、1:400和1:500稀释,总体积为100μL;
二抗混悬液中荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔按1:500稀释。
其中,所述稀释液是由1mmol/L EDTA+1mmol月硅酸盐+0.1% BSA+0.2%泊洛沙姆组成。
其中,所述脱色液是由95%酒精与100%二甲苯按体积比1:1组成。
其中,所述染色液A为DAB染色液;所述染色液B为苏木素染色液。
一种利用上述的试剂盒检测结直肠癌患者外周血循环肿瘤细胞CEA基因表达情况的方法,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结直肠癌患者外周血:采集无法获取组织标本的晚期或复发结直肠癌患者肘正中静脉外周血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;
(5)向滤器中加入200μl 2%PFA,室温固定5min,完成后0.5ml PBS漂洗3次,每次2min;
(6)再向滤器中加入200μl预冷的甲醇,4℃固定15min,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;
(7)运用免疫荧光方法检测外周血CTC的CEA基因表达情况。
其中,所述的步骤(7)运用免疫荧光方法检测外周血CTC的CEA基因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,PBS洗2min×3次;
(2)封闭:向滤膜上滴加100μl 10%山羊血清,室温放置30min,完成后吸去多余的血清(注:羊血清用PBS稀释);
(3) 一抗孵育:向滤膜上滴加100μl小鼠抗CK、大鼠抗CD45和兔抗CEA基因组成的一抗混悬液,37℃孵育1h或4℃过夜,完成后PBS洗3min×3次;
(4)二抗孵育:向滤膜上滴加100μl荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成的二抗混悬液,室温孵育30min,完成后PBS洗2min×3次;
(5)使用含DAPI的封片剂封片,阅片,采图;
(6)采照完成后,脱片后进行瑞氏吉姆萨染色,与IF结果进行对比。
本发明所使用的膜过滤分离循环肿瘤细胞装置,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;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的能够被滤过,而CTC因直径大于滤孔的被截留在滤膜上。
有益效果
(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结直肠癌患者CEA基因表达情况,利用微创技术,能够实现实时动态检测。
(2)本发明提供的方法,循环肿瘤细胞分离好,能够避免血细胞的干扰,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为结直肠癌患者外周血分离获取的循环肿瘤细胞影像图;
图5为晚期结直肠癌患者外周血循环肿瘤细胞CEA基因免疫荧光染色图像;其中,A为merge,B显色目标基因表达(红),C显色为CK(绿),D显色为CD45(蓝)。
其中,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。
本发明实施例所使用的免疫荧光试剂盒具体规格如表1所示:
所述的一抗混悬液由鼠抗CK、大鼠抗CD45和兔抗CEA基因组成,鼠抗CK、大鼠抗CD45和兔抗CEA基因分别用BD wash buffer按1:100、1:500和1:400稀释,稀释后取 10μL小鼠抗CK、50μL大鼠抗CD45和40μL兔抗CEA基因组成一抗混悬液;
所述的二抗混悬液由荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成,分别为市售Alexa Fluor 546 goat Anti-mouse,Alexa Fluor 488 goat Anti-rat和Alexa Fluor 647 goat Anti-rabbit,取等量的上述三种荧光标记二抗,分别用BD washbuffer分别按1:500稀释并混匀得二抗混悬液。
运用此技术方法分离获取并鉴定7例结直肠癌患者(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结直肠癌患者外周血中的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。此处需要说明的是,稀释液起到了去黏连分散的作用,月桂酸盐和泊洛沙姆配合使用,保证血细胞和CTC不黏连,并充分分散在稀释液中,从而有效的通过滤膜7被截留。
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,检测结果如表2所示。
通过观察,8例健康志愿者均未查到CTC;除2例结直肠癌患者未检测到CTC外(1例复发+1例晚期),其余5例均检测到CTC(表1),本次检测阳性率为71%。表1 实施例CTC
二、运用免疫荧光技术检测CTC的CEA基因表达情况:
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,PBS洗2min×3次;滴加100μl 10%山羊血清,室温放置30min,完成后吸去多余的血清,向滤膜上滴加100μl一抗混悬液,37℃孵育1h,完成后PBS洗3min×3次;然后向滤膜上滴加100μl二抗混悬液,室温孵育30min,完成后PBS洗2min×3次;使用含DAPI的封片剂封片,阅片,采图;采照完成后,脱片后进行瑞氏吉姆萨染色,与IF结果进行对比。
图5为晚期肝细胞癌患者外周血循环肿瘤细胞免疫荧光染色图像,根据免疫学及形态学表现,发现肿瘤细胞细胞体积大,核质比异常,免疫学表现为典型的CTCs。
所检测的循环肿瘤细胞应用免疫荧光证实CEA基因的表达并与结直肠癌大体标本CEA基因结果对比,观察其差异,主要针对大体标本CEA基因表达阴性而循环肿瘤细胞表达阳性的患者,指导结直肠癌的靶向治疗,为结直肠癌靶向治疗提供新的思路。
Claims (6)
1.一种检测结直肠癌患者外周血循环肿瘤细胞CEA基因表达的免疫荧光试剂盒,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,200μl 甲醇、200μl 2%PFA,100μl10%山羊血清,小鼠抗CK、大鼠抗CD45和兔抗CEA基因组成的一抗混悬液100μl,荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成的二抗混悬液100μL,DAPI封片剂;
一抗混悬液中小鼠抗CK、大鼠抗CD45和兔抗CEA基因分别按1:100、1:400和1:500稀释,总体积为100μL;
二抗混悬液中荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔按1:500稀释。
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+1mmol月硅酸盐+0.1% BSA+0.2%泊洛沙姆组成。
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%二甲苯按体积比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。
5.一种利用权利要求1-4任一项所述的试剂盒非诊断目的检测结直肠癌患者外周血循环肿瘤细胞CEA基因表达的方法,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结直肠癌患者外周血:采集无法获取组织标本的晚期或复发结直肠癌患者肘正中静脉外周血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;
(5)向滤器中加入200μl 2%PFA,室温固定5min,完成后0.5ml PBS漂洗3次,每次2min;
(6)再向滤器中加入200μl预冷的甲醇,4℃固定15min,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;
(7)运用免疫荧光方法检测外周血CTC的CEA基因表达情况。
6.根据权利要求5所述的检测方法,其特征在于,所述的步骤(7)运用免疫荧光方法检测外周血CTC的CEA基因表达情况的具体方法如下:
(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,PBS洗2min×3次;
(2)封闭:向滤膜上滴加100μl 10%山羊血清,室温放置30min,完成后吸去多余的血清(注:羊血清用PBS稀释);
(3) 一抗孵育:向滤膜上滴加100μl小鼠抗CK、大鼠抗CD45和兔抗CEA基因组成的一抗混悬液,37℃孵育1h或4℃过夜,完成后PBS洗3min×3次;
(4)二抗孵育:向滤膜上滴加100μl荧光标记的羊抗小鼠、荧光标记的羊抗大鼠、荧光标记的羊抗兔组成的二抗混悬液,室温孵育30min,完成后PBS洗2min×3次;
(5)使用含DAPI的封片剂封片,阅片,采图;
(6)采照完成后,脱片后进行瑞氏吉姆萨染色,与IF结果进行对比。
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