WO2022001826A1 - 一种检测胰腺癌患者外周血循环肿瘤细胞E-Cadherin表达的免疫荧光试剂盒 - Google Patents

一种检测胰腺癌患者外周血循环肿瘤细胞E-Cadherin表达的免疫荧光试剂盒 Download PDF

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WO2022001826A1
WO2022001826A1 PCT/CN2021/102099 CN2021102099W WO2022001826A1 WO 2022001826 A1 WO2022001826 A1 WO 2022001826A1 CN 2021102099 W CN2021102099 W CN 2021102099W WO 2022001826 A1 WO2022001826 A1 WO 2022001826A1
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peripheral blood
filter
pancreatic cancer
cadherin
tumor cells
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French (fr)
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郝继辉
李胜
任贺
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天津市肿瘤医院（天津医科大学肿瘤医院）
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D29/00—Filters with filtering elements stationary during filtration, e.g. pressure or suction filters, not covered by groups B01D24/00 - B01D27/00; Filtering elements therefor
    • B01D29/01—Filters with filtering elements stationary during filtration, e.g. pressure or suction filters, not covered by groups B01D24/00 - B01D27/00; Filtering elements therefor with flat filtering elements
    • B01D29/05—Filters with filtering elements stationary during filtration, e.g. pressure or suction filters, not covered by groups B01D24/00 - B01D27/00; Filtering elements therefor with flat filtering elements supported
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

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  • the invention relates to an immunofluorescence kit for detecting the expression of E-Cadherin in peripheral blood circulating tumor cells of pancreatic cancer patients, and belongs to the technical field of molecular biology.
  • Pancreatic cancer is a common malignant tumor of the digestive system and one of the malignant tumors with the worst prognosis. Among the deaths caused by malignant tumors, pancreatic cancer ranks fourth, and 90% of patients die within one year after diagnosis, with a 5-year survival rate. less than 5%. Because the early symptoms of pancreatic cancer are insidious, non-specific, highly invasive, early metastases, mostly in the middle and late stages when diagnosed, the surgical resection rate is low, only 15% at the time of diagnosis, so the selection of treatment methods for pancreatic cancer patients is particularly important. , which can make some patients avoid unnecessary surgical trauma and risks.
  • E-Cadherin is the most classic marker of epithelial cell phenotype.
  • the down-regulation of E-Cadherin expression marks the decline of the adhesion between cells. Therefore, E-Cadherin is used as a marker for the identification of EMT in pancreatic cancer.
  • E-Cadherein plays an important role in cell-cell adhesion and is involved in the maintenance of tissues and organs. In recent years, more and more attention has been paid to the relationship between EMT and tumor drug resistance in pancreatic cancer.
  • E-Cadherin detection specimens of pancreatic cancer patients are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to perform multiple or real-time detection.
  • Shandong First Medical University, Shandong Pharmaceutical Research Institute and Shandong Kaige Intelligent Machine Co., Ltd. are cooperating on the development and production of key technologies, detection equipment, and kits for the detection and identification of circulating tumor cells.
  • Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd. and other units have promoted and applied. This project is a major scientific and technological innovation project in Shandongzhou.
  • the research institute is the core, implements the registrant system, relies on the core diagnostic technology of circulating tumor cell detection and identification, and further registers the identification and diagnosis kits, including 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, cytokeratin , AE1/AE3, estrogen receptor, progesterone receptor, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A, etc.
  • the present invention provides an immunofluorescence kit and detection method for detecting the expression of E-Cadherin in peripheral blood circulating tumor cells of pancreatic cancer patients , using a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with advanced or recurrent pancreatic cancer for which no tissue specimens can be obtained, and further use immunofluorescence technology to detect the expression of E-Cadherin in CTCs.
  • An immunofluorescence kit for detecting the expression of E-Cadherin in peripheral blood circulating tumor cells of patients with pancreatic cancer including diluent 45mL, destaining solution 1mL, staining solution A 0.5mL, staining solution B 1mL, 200 ⁇ l methanol, 200 ⁇ l 2% PFA, 100 ⁇ l 10 % goat serum, 100 ⁇ l of primary antibody suspension consisting of mouse anti-CK, rat anti-CD45 and rabbit anti-E-Cadherin, fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, fluorescently labeled goat anti-rabbit Composition of secondary antibody suspension 100 ⁇ L, DAPI mounting medium;
  • mouse anti-CK, rat anti-CD45 and rabbit anti-E-Cadherin were diluted 1:100, 1:400 and 1:500, respectively, with a total volume of 100 ⁇ L;
  • Fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit were diluted 1:500 in the secondary antibody suspension.
  • the diluent is potassium chloride 19g/L, dipotassium ethylenediaminetetraacetate 4.8g/L, pyridinium sodium 2.6g/L, potassium dihydrogen phosphate 17g/L, sodium hydroxide 9g/L, 0.1g/L L trehalose, dihydroxymethyl urea 2g/L, and the balance is purified water.
  • the decolorizing solution is composed of 0.5% hydrochloric acid-70% ethanol solution.
  • the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.
  • the method for detecting the expression of E-Cadherin in peripheral blood circulating tumor cells of patients with pancreatic cancer for the non-diagnostic purpose of the above immunofluorescence kit includes the following steps:
  • peripheral blood samples were diluted with 45ml of diluent, and after dilution, paraformaldehyde was added to fix the peripheral blood samples for 10 minutes, and the final concentration was 0.25%;
  • S3 primary antibody incubation drop 100 ⁇ l of primary antibody suspension consisting of mouse anti-CK, rat anti-CD45 and rabbit anti-E-Cadherin onto the filter, incubate at 37°C for 1 h or 4°C overnight, and wash with PBS for 3 min ⁇ 3 times;
  • S4 secondary antibody incubation drop 100 ⁇ l of a secondary antibody suspension consisting of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit onto the filter membrane, incubate at room temperature for 30 minutes, and wash with PBS after completion 2min ⁇ 3 times;
  • S5 uses DAPI-containing mounting medium to mount, read and capture images
  • the membrane filtration device for separating tumor cells used in the present invention includes a filter, a blood sample container, a waste liquid tank and an iron stand, the iron stand is provided with a base, a stand and a bracket, and the blood sample container is arranged on the upper part of the iron stand through the stand, Below the blood sample container is a filter, and the filter is connected to a waste liquid tank through an infusion set, and the waste liquid tank is arranged on the base.
  • the filter includes an upper filter port, a filter membrane, a filter-carrying membrane platform and a filter lower port, the filter membrane is placed on the filter-carrying membrane platform; the upper filter port is connected to the blood sample container, and the lower port of the filter is connected to a waste liquid tank through an infusion device.
  • the filter membrane is made of hydrophobic material, and is evenly covered with filter holes with a diameter of 8 microns.
  • the expression of E-Cadherin in patients with advanced or recurrent pancreatic cancer can be detected without obtaining tissue specimens by needle biopsy.
  • the technique is minimally invasive and enables real-time detection.
  • the method provided by the present invention can avoid false positive results caused by edge effects that may occur in the dyeing process, has good stability, reduces cell loss, and improves detection accuracy.
  • FIG. 1 is a schematic structural diagram of a membrane filtration device of the present invention
  • FIG. 2 is a schematic cross-sectional view of the structure of the filter of the membrane filtration device of the present invention
  • Fig. 3 is the structural representation of the filter membrane of the membrane filtration device of the present invention.
  • Figure 4 is an image of immunofluorescence staining of circulating tumor cells in peripheral blood of a patient with recurrent pancreatic cancer.
  • the primary antibody suspension is composed of mouse anti-CK, rat anti-CD45 and rabbit anti-E-Cadherin.
  • the mouse anti-CK, rat anti-CD45 and rabbit anti-E-Cadherin are respectively 1:100, 1 with BD wash buffer. :500 and 1:400 dilution, after dilution, take 10 ⁇ L mouse anti-CK, 50 ⁇ L rat anti-CD45 and 40 ⁇ L rabbit anti-E-Cadherin to form a primary antibody suspension;
  • the secondary antibody suspension is composed of fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit, which are commercially available Alexa Fluor 546 goat Anti-mouse, Alexa Fluor 488 goat Anti-mouse, respectively.
  • -rat and Alexa Fluor 647 goat Anti-rabbit take equal amounts of the above three fluorescently labeled secondary antibodies, dilute them with BD wash buffer at 1:500 and mix well to obtain a secondary antibody suspension.
  • the configuration of the diluent potassium chloride 19g, dipotassium EDTA 4.8g, sodium pyridinium 2.6g, potassium dihydrogen phosphate 17g, sodium hydroxide 9g, trehalose 0.1g, dihydroxymethyl urea 2g , purified water 945.5g.
  • the filter device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;
  • the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. Therefore, when peripheral blood containing CTCs is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, while the diameter of CTCs is larger than The filter pores 10 are trapped on the filter membrane 7 .
  • the filter was dried and observed under a microscope to determine the presence of CTCs.
  • the prepared blood samples have poor stability, and some blood samples will form layers, and blood cells are prone to aggregation and adhesion, which affects the final detection effect.
  • the decolorizing solution prepared from 0.5% hydrochloric acid-70% ethanol solution has better decolorizing effect than the decolorizing solution composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
  • Figure 4 shows the immunofluorescence staining images of circulating tumor cells in the peripheral blood of a patient with recurrent pancreatic cancer. According to the immunological and morphological manifestations, it was found that the tumor cells were large in size and abnormal in nuclear-cytoplasmic ratio, and the immunological manifestations were typical CTCs.
  • E-Cadherin in the detected circulating tumor cells was confirmed by immunofluorescence and compared with the results of E-Cadherin in pancreatic cancer surgical or puncture tissue specimens to observe the differences.
  • the expression has great potential and provides an important basis for evaluating the prognosis of pancreatic cancer chemotherapy combined with immunotherapy.

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Abstract

一种检测胰腺癌患者外周血循环肿瘤细胞E‑Cadherin表达的免疫荧光试剂盒，属于分子生物学技术领域。一种检测胰腺癌患者外周血循环肿瘤细胞E‑Cadherin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无法获取组织标本的晚期或复发胰腺癌患者外周血中的CTC，进一步运用免疫荧光技术检测CTC的E‑Cadherin表达情况。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胰腺癌患者E‑Cadherin表达情况。取样过程属于微创，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

一种检测胰腺癌患者外周血循环肿瘤细胞E-Cadherin表达的免疫荧光试剂盒 技术领域
本发明涉及一种检测胰腺癌患者外周血循环肿瘤细胞E-Cadherin表达的免疫荧光试剂盒，属于分子生物学技术领域。
背景技术
胰腺癌是消化系统常见的恶性肿瘤，也是预后最差的恶性肿瘤之一，在恶性肿瘤所导致的死亡中，胰腺癌排第四位，90％患者在确诊后一年内死亡，5年生存率不足5％。由于胰腺癌早期症状隐匿，且没有特异性，侵袭性强，转移早，确诊时多处于中晚期，手术切除率低，确诊时候仅15％，因此对胰腺癌患者治疗方法的选定显得尤为重要，可以使部分患者避免不必要的手术创伤及风险。
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被当做鉴定胰腺癌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。近年来有关于胰腺癌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。而目前临床实践中，胰腺癌患者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胰腺癌患者外周血循环肿瘤细胞E-Cadherin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无法获取组织标本的晚期或复发胰腺癌患者外周血中的CTC，进一步运用免疫荧光技术检测CTC的E-Cadherin表达情况。
本发明采用的技术方案如下：
一种检测胰腺癌患者外周血循环肿瘤细胞E-Cadherin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，100μl 10％山羊血清，小鼠抗CK、大鼠抗CD45和兔抗E-Cadherin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E-Cadherin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。
所述稀释液为氯化钾19g/L，乙二胺四乙酸二钾4.8g/L，吡啶鎓钠2.6g/L，磷酸二氢钾17g/L，氢氧化钠9g/L，0.1g/L海藻糖，二羟基甲基脲2g/L，余量为纯化水。
所述脱色液是由0.5％盐酸-70％乙醇溶液组成。
所述染色液A为DAB染色液；所述染色液B为苏木素染色液。
上述免疫荧光试剂盒非诊断目的检测胰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胰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胰腺癌患者外周血：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；
(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E-Cadherin表达情况。
步骤(7)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)；
S3一抗孵育：向滤膜上滴加100μl小鼠抗CK、大鼠抗CD45和兔抗E-Cadherin组成的一抗混悬液，37℃孵育1h或4℃过夜，完成后PBS洗3min×3次；
S4二抗孵育：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；
S5使用含DAPI的封片剂封片，阅片，采图；
S6采图完成后，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胰腺癌患者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为复发胰腺癌患者外周血循环肿瘤细胞免疫荧光染色图像。
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。
本发明所使用的免疫荧光试剂盒具体规格如表1所示：
表1试剂盒具体规格
组分 含量
稀释液 45mL
脱色液 1mL
染色液A 0.5mL
染色液B 1mL
甲醇 200μl
2％PFA 200μl
10％山羊血清(PBS稀释) 100μL
一抗混悬液 100μL
二抗混悬液 100μL
DAPI封片剂 H-1200
所述的一抗混悬液由鼠抗CK、大鼠抗CD45和兔抗E-Cadherin组成，鼠抗CK、大鼠抗CD45和兔抗E-Cadherin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E-Cadherin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 wash buffer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10例胰腺癌患者(同时检测10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：氯化钾19g，乙二胺四乙酸二钾4.8g，吡啶鎓钠2.6g，磷酸二氢 钾17g，氢氧化钠9g，海藻糖0.1g，二羟基甲基脲2g，纯化水945.5g。
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胰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。
通过观察，10例健康志愿者均未查到CTC；除2例复发胰腺癌患者和1例晚期胰腺癌患者未检测到CTC外，其余4例复发胰腺癌患者和3例晚期胰腺癌患者均检测到CTC(表2)，本次检测阳性率为70％。
值得注意的是，当稀释液不添加二羟基甲基脲或者海藻糖时，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
Figure PCTCN2021102099-appb-000001
二、运用免疫荧光技术检测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0.5％盐酸-70％乙醇溶液的脱色液中浸泡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。
由0.5％盐酸-70％乙醇溶液制备的的脱色液效果比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。
图4为复发胰腺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E-Cadherin的表达并与胰腺癌手术或穿刺组织标本E-Cadherin结果对比，观察其差异性，验证了CTCs作为无创活检用于实时评估胰腺癌E-Cadherin表达有巨大潜力，为评估胰腺癌化疗联合免疫治疗预后提供重要依据。

Claims (6)

  1. 一种检测胰腺癌患者外周血循环肿瘤细胞E-Cadherin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，100μl 10％山羊血清，小鼠抗CK、大鼠抗CD45和兔抗E-Cadherin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E-Cadherin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所述的免疫荧光试剂盒，其特征在于，所述稀释液为氯化钾19g/L，乙二胺四乙酸二钾4.8g/L，吡啶鎓钠2.6g/L，磷酸二氢钾17g/L，氢氧化钠9g/L，0.1g/L海藻糖，二羟基甲基脲2g/L，余量为纯化水。
  3. 根据权利要求1所述的免疫荧光试剂盒，其特征在于，所述脱色液是由0.5％盐酸-70％乙醇溶液组成。
  4. 根据权利要求1所述的免疫荧光试剂盒，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任一项所述的免疫荧光试剂盒非诊断目的检测胰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胰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胰腺癌患者外周血：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；
    (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E-Cadherin表达情况。
  6. 根据权利要求5所述的检测方法，其特征在于，步骤(7)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)；
    S3一抗孵育：向滤膜上滴加100μl小鼠抗CK、大鼠抗CD45和兔抗E-Cadherin组成的一抗混悬液，37℃孵育1h或4℃过夜，完成后PBS洗3min×3次；
    S4二抗孵育：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；
    S5使用含DAPI的封片剂封片，阅片，采图；
    S6采图完成后，脱片后进行瑞氏吉姆萨染色，与IF结果进行对比。
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