WO2021213310A1 - 一种通过外周血循环肿瘤细胞检测食道鳞癌患者pd-l1基因表达的免疫荧光试剂盒 - Google Patents

一种通过外周血循环肿瘤细胞检测食道鳞癌患者pd-l1基因表达的免疫荧光试剂盒 Download PDF

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WO2021213310A1
WO2021213310A1 PCT/CN2021/088061 CN2021088061W WO2021213310A1 WO 2021213310 A1 WO2021213310 A1 WO 2021213310A1 CN 2021088061 W CN2021088061 W CN 2021088061W WO 2021213310 A1 WO2021213310 A1 WO 2021213310A1
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peripheral blood
tumor cells
filter
labeled goat
cell carcinoma
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胡敏
李胜
李�浩
于冰
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山东第一医科大学(山东省医学科学院)
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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
    • G01N33/53—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
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0694—Cells of blood, e.g. leukemia cells, myeloma 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
    • 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
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

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  • the invention provides an immunofluorescence kit for expressing PD-L1 gene in circulating tumor cells of peripheral blood of patients with esophageal squamous cell carcinoma, and provides a detection method, belonging to the technical field of molecular biology.
  • Esophageal cancer is one of the most common malignant tumors of the digestive tract in the world, accounting for about 2% of all malignant tumors.
  • the pathological types of esophageal cancer mainly include esophageal squamous carcinoma ESCC and esophageal adenocarcinoma (EADC).
  • About 90% of esophageal cancer patients in China are esophageal squamous cell carcinoma.
  • the quality of life of patients with esophageal cancer has gradually been gradually improved to a certain extent. It has been improved, but on the whole, the effect of esophageal cancer treatment is still not satisfactory.
  • the overall 5-year survival rate of esophageal cancer patients in China after surgery is only 20%-30%.
  • the immunotherapy with PD-1/PD-L1 as an immune target has brought a new dawn to the treatment of esophageal squamous cell carcinoma.
  • Tumor cells can bind to PD-1 on the surface of immune cells and T cells through PD-L1 on their surface, and conduct inhibitory signals to make T cells The inability to recognize tumor cells and send out attack signals to tumor cells leads to immune escape of tumor cells.
  • literature search there is no report on the detection of circulating tumor cell PD-L1 of esophageal squamous cell carcinoma at home and abroad. Therefore, the detection of circulating tumor cell (CTC) PD-L1 expression is of great value for the prognosis of esophageal squamous cell carcinoma and the evaluation of immunotherapy efficacy.
  • CTC circulating tumor cell
  • Circulating tumor cells are tumor cells that fall off from solid tumors and enter the peripheral blood circulation. Since they were discovered in 1989, there have been a variety of methods for detecting circulating tumor cells in the peripheral blood. Recent studies have shown that its detection has important clinical significance for evaluating the prognosis of cancer patients, especially advanced cancer patients, and selecting appropriate individualized treatments. Because CTC detection has the characteristics of minimally invasive and real-time detection, it is called "liquid biopsy" of tumors.
  • Immunofluorescence analysis technology combines immunological methods (antigen-antibody specific binding) and fluorescent labeling technology to study the method of specific protein antigen distribution in cells. Because the fluorescence emitted by fluorescein can be detected under a fluorescence microscope, fluorescein emits bright fluorescence (yellow-green or orange-red) when irradiated by the excitation light, and the cells or tissues where the fluorescence is located can be seen. Quantitative techniques are used to determine the content. In order to carry out cell qualitative and localization analysis of the antigen.
  • the specimens for PD-L1 gene detection in patients with esophageal squamous cell carcinoma are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to achieve multiple or real-time detection. Therefore, the detection of circulating tumor cell (CTC) PD-L1 gene expression is of great value for the prognosis of esophageal squamous cell carcinoma and the evaluation of the efficacy of immunotherapy.
  • CTC circulating tumor cell
  • Shandong First Medical University, Shandong Pharmaceutical Research Institute and Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd. and other units have Research on the industrialization of key technologies for tumor cell detection and identification.
  • This project is a major scientific and technological innovation project in Shandong Province. This project will take the Shandong Provincial Pharmaceutical Research Institute on the Jinan Campus of Shandong First Medical University as the core and implement the registrant system.
  • Circulating tumor cell detection and identification of core diagnostic technology, and further registration and identification of diagnostic kits to include 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, pregnancy Hormone receptors, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A, etc.
  • the identification and diagnosis kits are industrialized and promoted through cooperation with Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., and Shandong Discovery Biotechnology Co., Ltd., which are registered in Jinan.
  • the present invention provides an immunofluorescence kit and detection method for PD-L1 gene expression in circulating tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma, using a membrane filter device to separate and obtain circulating tumor cells (CTC) in the peripheral blood of patients with advanced esophageal squamous cell carcinoma , Further use immunofluorescence technology to detect the expression of PD-L1 gene on CTC.
  • CTC circulating tumor cells
  • An immunofluorescence kit for detecting PD-L1 gene expression in peripheral blood circulating tumor cells of patients with esophageal squamous cell carcinoma including diluent 45mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, 200 ⁇ l methanol, 200 ⁇ l 2% PFA, 100 ⁇ l of 10% goat serum, 100 ⁇ l of primary antibody suspension composed of mouse anti-CK, rat anti-CD45 and rabbit anti-PD-L1 genes, fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat 100 ⁇ L of anti-rabbit secondary antibody suspension, DAPI mounting plate;
  • mice anti-CK, rat anti-CD45 and rabbit anti-PD-L1 genes in the primary antibody suspension were diluted 1:100, 1:400, and 1:500 respectively, and the total volume was 100 ⁇ L;
  • Fluorescently labeled goat anti-mouse, fluorescently labeled goat anti-rat, and fluorescently labeled goat anti-rabbit in the secondary antibody suspension were diluted 1:500.
  • the diluent is composed of 1 mmol/L EDTA + 1 mmol moon silicate + 0.1% BSA + 0.2% poloxamer.
  • the decolorizing liquid is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
  • the staining solution A is DAB staining solution
  • the staining solution B is hematoxylin staining solution.
  • the method for detecting PD-L1 gene expression in circulating tumor cells in peripheral blood of patients with esophageal squamous cell carcinoma for non-diagnostic purposes of the above kit includes the following steps:
  • the specific method of using immunofluorescence to detect the expression of PD-L1 gene of peripheral blood CTC is as follows:
  • the membrane filtration device used in the present invention to separate circulating tumor cells 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.
  • the blood sample container is set on the upper part of the iron stand through the bracket.
  • Below the blood sample container is a filter, the filter is connected to the waste liquid tank through the infusion set, and the waste liquid tank is arranged on the base.
  • the filter includes a filter upper port, a filter membrane, a filter membrane platform and a filter lower port.
  • the filter membrane is placed on the filter membrane platform; the upper port of the filter is connected to the blood sample container, and the lower port of the filter is connected to the waste liquid tank through the infusion device.
  • the filter membrane is made of hydrophobic material, and the filter holes with a diameter of 8 microns are uniformly spread on it; 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, so when it contains CTC After the peripheral blood is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole, and the CTC is trapped on the filter membrane because the diameter is larger than the filter hole.
  • the detection method provided by the present invention can detect the PD-L1 gene expression of patients with advanced or recurrent esophageal squamous cell carcinoma without puncture biopsy to obtain tissue samples, and can realize real-time dynamic detection by using minimally invasive technology.
  • the method provided by the present invention has good separation of circulating tumor cells, can avoid the interference of blood cells, can avoid false positive results caused by the edge effect that may occur in the staining process, has good stability, reduces cell loss, and improves detection accuracy sex.
  • Figure 1 is a schematic diagram of the structure of the 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 a schematic diagram of the structure of the filter membrane of the membrane filtration device of the present invention.
  • Figure 4 is an image of circulating tumor cells isolated from peripheral blood of a patient with esophageal squamous cell carcinoma
  • Figure 5 shows the PD-L1 gene immunofluorescence staining image of circulating tumor cells in the peripheral blood of patients with advanced esophageal squamous cell carcinoma, where A is merge, B is the target gene expression (red), C is CK (green), and D is CD45 (blue).
  • Component content Diluent 45mL Decolorizing liquid (volume ratio of 95% alcohol and 100% xylene 1:1) 1mL Staining solution A (DAB staining solution) 0.5mL Staining solution B (hematoxylin staining solution) 1mL 10% goat serum (diluted in PBS) 100 ⁇ L Primary antibody suspension 100 ⁇ L Secondary antibody suspension 100 ⁇ L
  • the primary antibody suspension is composed of mouse anti-CK, rat anti-CD45, and rabbit anti-PD-L1 genes.
  • the mouse anti-CK, rat anti-CD45, and rabbit anti-PD-L1 genes are respectively BD wash buffer at 1:100 , 1:500 and 1:400 dilution, after dilution, take 10 ⁇ L mouse anti-CK, 50 ⁇ L rat anti-CD45 and 40 ⁇ L rabbit anti-PD-L1 gene to form the 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. They are respectively commercially available Alexa Fluor 546 goat Anti-mouse, Alexa Fluor 488 goat Anti -rat and Alexa Fluor 647 goat Anti-rabbit, take the same amount of the above three fluorescently labeled secondary antibodies, respectively dilute and mix 1:500 with BD wash buffer to obtain the secondary antibody suspension.
  • the membrane filtration 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, after the peripheral blood containing CTC is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, and the CTC is larger than the diameter.
  • the filter hole 10 is trapped on the filter membrane 7. What needs to be explained here is that the diluent plays the role of de-adhesion and dispersion. The laurate and poloxamer are used together to ensure that blood cells and CTC do not adhere and are fully dispersed in the diluent to effectively pass through the filter membrane. 7 was detained.
  • Figure 5 is an immunofluorescence staining image of circulating tumor cells in the peripheral blood of patients with advanced esophageal squamous cell carcinoma. According to immunological and morphological findings, it is found that the tumor cells are large in size, have abnormal nucleus-to-cytoplasmic ratio, and immunologically show typical CTCs.
  • the detected circulating tumor cells were confirmed by immunofluorescence to confirm PD-L1 and compared with the results of PD-L1 in gross specimens of esophageal squamous cell carcinoma, to observe the difference, mainly for patients with negative PD-L1 in gross specimens and positive expression of circulating tumor cells, instruct esophageal squamous cells
  • Targeted therapy of cancer provides new ideas for targeted therapy of esophageal squamous cell carcinoma.

Abstract

一种通过外周血循环肿瘤细胞检测食道鳞癌患者PD-L1基因表达的免疫荧光试剂盒，该试剂盒主要包括：山羊血清，小鼠抗CK、大鼠抗CD45和兔抗PD-L1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PD-L1基因表达情况。

Description

一种通过外周血循环肿瘤细胞检测食道鳞癌患者PD-L1基因表达的免疫荧光试剂盒 技术领域
本发明提供了一种食道鳞癌患者外周血循环肿瘤细胞PD-L1基因表达的免疫荧光试剂盒，并提供检测方法，属于分子生物学技术领域。
背景技术
食管癌是世界上最常见的消化道恶性肿瘤之一，约占所有恶性肿瘤的2％。食管癌的病理类型主要包括食管鳞状细胞癌(esophageal squamous carcinoma ESCC)和食管腺癌(esophageal adenocarcinoma EADC)，在我国的食管癌患者中约90％以上均为食管鳞状细胞癌。近年来随着食管癌诊断技术的不断提高，手术操作技能的不断改进，术前新辅助化疗的应用，再加上术后规律放、化疗的施行，食管癌患者的生活质量在一定程度上逐步得到了改善，但是从整体上而言，食管癌治疗的效果仍然不尽如人意，我国食管癌患者术后的5年总体生存率仅为20％一30％。
PD-1/PD-L1为免疫靶点的免疫疗法为食道鳞癌治疗带来了新的曙光。研究发现，免疫抑制与免疫逃逸和肿瘤细胞PD-L1的过表达密切相关，肿瘤细胞可通过其表面的PD-L1与免疫细胞T细胞表面的PD-1结合，传导抑制性信号，使得T细胞不能识别肿瘤细胞和向肿瘤细胞发出攻击信号，导致了肿瘤细胞免疫逃逸。经文献检索发现，目前食道鳞癌循环肿瘤细胞PD-L1的检测国内外均未见报道。因此，检测循环肿瘤细胞(CTC)PD-L1表达情况对食道鳞癌预后及免疫治疗疗效评估具有重要价值。
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，食道鳞癌患者PD-L1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)PD-L1基因表达 情况对食道鳞癌预后及免疫治疗疗效评估具有重要价值。
目前，山东省第一医科大学、山东省药物研究院联合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，对于循环肿瘤细胞检测鉴定关键技术进行产业化推广的研究，本项目为山东省重大科技创新工程项目，本项目将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产业化推广。
发明内容
针对现有技术中的检测肿瘤晚期或复发食道鳞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PD-L1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，本发明提供了一种食道鳞癌患者外周血循环肿瘤细胞PD-L1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食道鳞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PD-L1基因表达情况。
本发明通过以下技术方案实现：
一种检测食道鳞癌患者外周血循环肿瘤细胞PD-L1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PD-L1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PD-L1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。
上述的试剂盒非诊断目的检测食道鳞癌患者外周血循环肿瘤细胞PD-L1基因表达的方法，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食道鳞癌患者外周血：采集无法获取组织标本的晚期或复发食道鳞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PD-L1基因表达情况。
其中，所述的步骤(7)运用免疫荧光检测外周血CTC的PD-L1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PD-L1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食道鳞癌患者PD-L1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为食道鳞癌患者外周血分离获取的循环肿瘤细胞影像图；
图5为晚期食道鳞癌患者外周血循环肿瘤细胞PD-L1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)。
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。
本发明实施例所使用的免疫荧光试剂盒具体规格如表1所示：
表1
组分 含量
稀释液 45mL
脱色液(95％酒精与100％二甲苯体积比1∶1) 1mL
染色液A(DAB染色液) 0.5mL
染色液B(苏木素染色液) 1mL
10％山羊血清(PBS稀释) 100μL
一抗混悬液 100μL
二抗混悬液 100μL
所述的一抗混悬液由鼠抗CK、大鼠抗CD45和兔抗PD-L1基因组成，鼠抗CK、大鼠抗CD45和兔抗PD-L1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PD-L1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 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例食道鳞癌患者(同时检测15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食道鳞癌患者外周血中的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，15例健康志愿者均未查到CTC；除2例食道鳞癌患者未检测到CTC外(1例晚期食道鳞癌患者+1例复发食道鳞癌患者)，其余8例均检测到CTC(表1)，本次检测阳性率为80％。
表2实施例CTC检测结果
Figure PCTCN2021088061-appb-000001
二、运用免疫荧光技术检测CTC的PD-L1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为晚期食道鳞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PD-L1并与食道鳞癌大体标本PD-L1结果对比，观察其差异，主要针对大体标本PD-L1阴性而循环肿瘤细胞表达阳性的患者，指导食道鳞癌的靶向治疗，为食道鳞癌靶向治疗提供新的思路。

Claims (6)

  1. 一种检测食道鳞癌患者外周血循环肿瘤细胞PD-L1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，100μl 10％山羊血清，小鼠抗CK、大鼠抗CD45和兔抗PD-L1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PD-L1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食道鳞癌患者外周血循环肿瘤细胞PD-L1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食道鳞癌患者外周血：采集无法获取组织标本的晚期或复发食道鳞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PD-L1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)运用免疫荧光检测外周血CTC的PD-L1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PD-L1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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