CN114480634A - 半乳糖凝集素-1作为一种生物标志物在诊断肿瘤耐药中的应用 - Google Patents
半乳糖凝集素-1作为一种生物标志物在诊断肿瘤耐药中的应用 Download PDFInfo
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Abstract
本发明涉及半乳糖凝集素‑1（Galectin‑1）作为一种标志物在肿瘤耐药诊断中的应用。本发明经过细胞实验发现在肝癌顺铂耐药细胞系中Galectin‑1的mRNA水平及蛋白水平明显高于亲本细胞；免疫组织化学实验发现动物肝癌顺铂耐药组织中Galectin‑1蛋白表达水平高于对照组。本发明提示Galectin‑1在早期发现肿瘤细胞耐药方面具有很大的潜力，为早期评估抗肿瘤药物疗效，肿瘤耐药监测提供了一种标志物。
Description
技术领域
本发明涉及疾病诊断领域，具体的说，涉及半乳糖凝集素-1作为一种生物标志物在肿瘤耐药诊断中的应用。
背景技术
恶性肿瘤已经严重危害人类健康。耐药是癌症患者复发及死亡的重要原因之一。因此，恶性肿瘤耐药的分子机制是目前研究的热点，旨在开发新的检测方法对其进行早期检测，则有助于耐药事件的及时发现和尽早干预，有助于延长患者寿命和提高生活质量。
到目前为止，肿瘤耐药诊断主要依赖于影像学上的变化。然而，传统的结构影像滞后于分子改变。肿瘤耐药是一个复杂的过程，在这个复杂过程中，肿瘤细胞往往需要大量表达某些特异性蛋白来逃避抗肿瘤药物的攻击及促进细胞耐药可塑性。某些分泌蛋白可能进入循环系统，在血液中被检测到，成为肿瘤耐药的有效生物标志物，对这些生物标志物进行检测可以帮助肿瘤耐药的及早诊断，监测抗肿瘤药物的有效性，成为肿瘤耐药预测的重要手段。
半乳糖凝集素-1（Galectin-1）是β-半乳糖凝集素家族成员之一，其是一种重要的生物标记物，在肿瘤细胞的耐药、增殖、侵袭、转移和免疫逃逸等方面发挥重要作用。大量研究表明，Galectin-1在多种肿瘤（肺癌、肝癌、胃癌、宫颈癌、乳腺癌、神经胶质瘤、黑色素瘤等）中均有过表达，其表达水平和正常组织相比明显的升高。同时，作为一种过表达的物质，Galectin-1会分泌到人体血液中。因此，Galectin-1可成为早期监测肿瘤耐药，抗肿瘤药物疗效的重要生物标志物。
而目前缺乏Galectin-1在肿瘤耐药诊断中的应用。
发明内容
本发明的目的是针对现有技术中的不足，提供Galectin-1作为一种生物标志物在肿瘤耐药诊断中的应用。
第一方面，本发明提供了检测Galectin-1蛋白及其编码基因表达水平的试剂在制备肿瘤耐药诊断试剂盒中的应用。
作为本发明的一个优选例，所述试剂盒用于肿瘤耐药的早期诊断。
第二方面，本发明提供了检测Galectin-1蛋白或其编码基因表达水平的试剂在制备监测抗肿瘤药物疗效的试剂盒中的应用。
作为本发明的一个优选例，所述监测抗肿瘤药物的疗效。
针对以上个技术方案，作为其中的一个优选例，所述试剂盒用于监测血清、血浆、全血或肿瘤细胞中Galectin-1蛋白或其编码基因的表达水平。
作为其中的另一优选例，所述检测Galectin-1蛋白或其编码基因表达的试剂是特异性扩增半乳糖凝集素-1蛋白的编码基因的引物；或特异性识别Galectin-1蛋白的编码基因或其转录本的探针；或特异性结合Galectin-1蛋白的结合分子；或特异性识别Galectin-1蛋白的编码基因或其转录本的芯片。
更优选的，所述试剂盒还包括：核酸提取试剂；聚合酶链反应试剂；蛋白免疫印迹试剂；蛋白免疫组化试剂；酶联免疫吸附试剂；或使用说明书。
本发明优点在于：
肿瘤耐药诊断主要依赖于影像学上的变化。然而，传统的结构影像滞后于分子改变。因此，生物标志物被提出作为肿瘤耐药诊断的指标。在本研究中，细胞实验发现在肝癌顺铂耐药细胞系中Galectin-1的mRNA水平及蛋白水平明显高于亲本细胞；免疫组织化学实验发现动物肝癌顺铂耐药组织中Galectin-1蛋白表达水平高于对照组。
方法：成功构建肝癌顺铂耐药细胞系Hep 3B-DR，通过qPCR和蛋白免疫印迹实验检测Galectin-1在Hep 3B-DR与其亲本细胞（Hep 3B）中mRNA和蛋白表达。成功构建肝癌耐药小鼠模型，通过免疫组化检测Galectin-1在耐药小鼠模型与对照组中表达。
细胞实验发现在肝癌顺铂耐药细胞系中Galectin-1的mRNA水平及蛋白水平明显高于亲本细胞；免疫组织化学实验发现动物肝癌顺铂耐药组织中Galectin-1蛋白表达水平高于对照组。
结论：肿瘤耐药与Galectin-1表达水平升高有关。我们研究表明，Galectin-1在早期诊断肿瘤耐药，监测抗肿瘤药物疗效等方面具有巨大的潜力。
附图说明
图1：肝癌细胞系中Galectin-1 qPCR与蛋白质免疫印迹检测结果图；
图2：肝癌小鼠模型组织中Galectin-1免疫组织化学检测结果图。
具体实施方式
实施例1
材料与方法：
细胞
肝癌细胞系Hep 3B（购买于ATCC）。构建肝癌耐药细胞系（Hep 3B-DR）：采用低剂量顺铂持续刺激，直到细胞在含1 μg/mL顺铂的培养基中存活。
qPCR技术
使用Total RNA Kit I（Omega, 货号R6834-02），按照说明书方案从培养细胞中分离总RNA。随后以1 μg RNA为模板，使用PrimeScriptTM RT reagent Kit （Takara,RR047A）生成cDNA。根据生产说明书使用TB Green n -R Premix Ex TaqTMⅡ（Takara, 货号RR820A）和qPCR分析系统（BIO-RAD, CFX Maestro）进行qPCR。相对表达按ΔΔCT相对定量方法进行分析，并与ACTB（内参基因）表达归一化。在三个重复中进行mRNA的表达测量。
蛋白质印迹技术
细胞溶解在RIPA裂解缓冲（Beyotime, 货号P0013C）含有蛋白酶抑制剂（ThermolFisher, 货号78430）30分钟。整个细胞溶解产物在13000转离心10分钟, BCA测定上清蛋白浓度，用loading buffer（EpiZyme, 货号LT101S）在100°C下煮沸5分钟。来自不同处理组的等量蛋白质通过SDS-PAGE分离并电转移到PVDF膜（BioTrace, 货号66485）。封膜后，在4℃下用一抗过夜。然后用过氧化物酶标记的山羊抗兔（或小鼠）IgG (ZSGB-BIO, ZB-2301,ZB-2305)在室温下孵育1 h。ECL检测试剂孵育后，用ChemiDoc/XPS+对信号进行可视化。本研究使用的主要抗体是来自Cell Signaling Technology的 Galectin-1/LGALS1(12936S)。GAPDH (60004-1-Ig)来源于Proteintech。
动物模型
雌性Balb/c/nu小鼠（广东省医学实验动物中心，中国，广东），构建肝癌顺铂耐药动物模型，采用顺铂（腹腔注射，10 mg/kg/周）诱导异种移植模型，治疗4周后体积较大的肿瘤再次移植到新的受体，连续进行2次。
免疫组织化学实验
肝癌动物组织切片，人肝癌组织切片均经过中山大学附属第五医院动物伦理委员会、医学伦理委员会批准。将4 μm福尔马林固定石蜡切片在60℃孵育2小时后进行免疫组化染色，对Galetin-1进行免疫组化染色。切片经二甲苯和分级酒精脱水，然后在pH 6.0的柠檬酸缓冲液中进行热诱导抗原修复（HIER）。HIER在压力锅(110℃)中进行。然后，用3% H2O2孵育10分钟阻断内源性过氧化物酶活性。用10%山羊血清在PBS中室温阻断30分钟。然后用一抗Galectin-1 （CST, 13888）在4℃孵育过夜，室温下用二抗（Max Vision, KIT-0014180）孵育30 min。检测方法:将样品置于DAB底物（Max Vision, KIT-0014180）下，苏木精复染。进行半定量(评分:1-低强度，2-中间强度，3-高强度)表达分析。
结论
图例1：细胞实验证实在Hep 3B-DR中Galectin-1的mRNA水平（图1 A）及蛋白水平明显高于Hep 3B（图1 B）。
图例2：免疫组织化学实验证实动物肝癌顺铂耐药组织中Galectin-1蛋白表达水平高于对照组（图2）。
Claims (7)
1.检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂在制备肿瘤耐药诊断试剂盒中的应用。
2.根据权利要求1所述的应用，其特征在于，所述试剂盒用于肿瘤耐药的早期诊断。
3.检测半乳糖凝集素-1蛋白或其编码基因表达水平的试剂在制备监测抗肿瘤药物疗效的试剂盒的应用。
4.根据权利要求3所述的应用，其特征在于，所述试剂盒用于监测抗肿瘤药物疗效。
5.根据权利要求1-4任一所述的应用，其特征在于，所述试剂盒用于检测血清、血浆、全血、肿瘤细胞中半乳糖凝集素-1蛋白或其编码基因的表达水平。
6.根据权利要求1-4任一所述的应用，其特征在于，所述检测半乳糖凝集素-1蛋白或其编码基因表达的试剂是特异性扩增半乳糖凝集素-1蛋白的编码基因的引物；或特异性识别半乳糖凝集素-1蛋白的编码基因或其转录本的探针；或特异性结合半乳糖凝集素-1蛋白的结合分子；或特异性识别半乳糖凝集素-1蛋白的编码基因或其转录本的芯片。
7.根据权利要求6所述的应用，其特征在于，所述试剂盒还包括：核酸提取试剂；聚合酶链反应试剂；蛋白免疫印迹试剂；蛋白免疫组化试剂；酶联免疫吸附试剂；或使用说明书。
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