CN104122392B - 检测ccl15趋化因子的试剂在制备筛选甲状腺滤泡癌试剂中的应用 - Google Patents

检测ccl15趋化因子的试剂在制备筛选甲状腺滤泡癌试剂中的应用 Download PDF

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CN104122392B
CN104122392B CN201410286443.1A CN201410286443A CN104122392B CN 104122392 B CN104122392 B CN 104122392B CN 201410286443 A CN201410286443 A CN 201410286443A CN 104122392 B CN104122392 B CN 104122392B
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ccl15
thyroid
reagent
follcular carcinoma
thyroid follcular
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CN104122392A (zh
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宁光
黄凤娇
王曙
叶蕾
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Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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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
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

本发明属于生物医药领域，特别涉及CCL15趋化因子在制备筛选甲状腺滤泡癌试剂中的应用。本发明以确诊为甲状腺滤泡癌的新鲜甲状腺滤泡肿瘤组织标本为研究对象，对其进行了蛋白提取、细胞因子抗体芯片检测、细胞培养、细胞趋化实验和抗体中和实验等，验证了甲状腺滤泡癌中CCL15高表达，而且CCL15高表达组的巨噬细胞浸润密度显著大于CCL15低表达组，FTC培养上清可促进Thp-1单核细胞株的迁移。所以，CCL15可作为潜在的生物标记物，应用于甲状腺滤泡癌的筛选试剂中，同时也为CCL15应用在甲状腺滤泡癌的筛选试剂中提供了实验数据和理论基础。

Description

检测CCL15趋化因子的试剂在制备筛选甲状腺滤泡癌试剂中的应用
技术领域
本发明属于生物医药领域，特别涉及CCL15趋化因子在制备筛选甲状腺滤泡癌试剂中的应用。
背景技术
甲状腺滤泡癌(follicularthyroidcarcinoma,FTC)作为分化型甲状腺癌的类型之一，其发病率仅次于甲状腺***状癌。大多数分化好的FTC和甲状腺滤泡性腺瘤(follicularadenoma,FA)无论在临床表现还是影像学特点上几乎无法鉴别；在细胞学形态上，细针穿刺(fineneedleaspiration,FNA)不能可靠地判断滤泡性肿瘤是否浸润包膜外或侵犯包膜血管，因此不能用于区分滤泡性癌或腺瘤。
同样，在术中行冷冻切片诊断时，也不可能对包膜做彻底检查，因此只有在多取材后才能作出明确诊断。在组织病理学上，这两种肿瘤的实质成分基本相同，唯一能区别的是包膜外和(或)血管侵犯。由于甲状腺滤泡性肿瘤大多为孤立性、有包膜，即使石蜡切片证实包膜外有微小浸润，术中行甲状腺腺叶切除术已能达到治疗目的，预后很好，10年存活率为70％～100％。而广泛浸润的甲状腺滤泡癌通常在大体和光镜下容易证实浸润性生长，肿瘤可侵犯到甲状腺外，手术切除后易复发和血道转移，预后较差，10年存活率仅25％～45％。
但是，对于冷冻切片病理报告诊断为“甲状腺滤泡性肿瘤，须等石蜡切片确定肿瘤有无包膜和/或血管侵犯”的患者，面临着二次手术的可能。正是由于甲状腺滤泡癌和甲状腺腺瘤的鉴别困难，外科医生难以制定合适的手术方案。
因此，如果能对甲状腺滤泡癌进行有效地检测和筛选，对病人的及时治疗有着至关重要的作用。
发明内容
本发明的目的是研究CCL15细胞因子在甲状腺滤泡癌(FTC)中的作用，为临床上治疗和检测甲状腺滤泡癌提供理论依据和实验数据。
甲状腺滤泡癌的病理特征是显示侵袭性的滤泡细胞肿瘤，同时缺少***状癌诊断性的核特征。肿瘤微环境是指除肿瘤实质细胞以外的其他所有细胞总称，其中数量最多的是经血液运输迁移至肿瘤部位的巨噬细胞，为肿瘤微环境中的主要成分。巨噬细胞来源于骨髓CD34+祖细胞，首先在骨髓中增殖***，再以单核细胞的形式经血液运输迁移。
本研究发现，巨噬细胞浸润散在甲状腺滤泡性肿瘤的滤泡间质，在癌旁正常甲状腺组织中几乎不存在。在甲状腺滤泡癌中，巨噬细胞的浸润密度显著高于其在甲状腺瘤中的密度。甲状腺滤泡癌中的巨噬细胞表型倾向于M2型。
在本课题中，我们分析了甲状腺滤泡癌(FTC)和甲状腺滤泡性腺瘤(FA)的细胞因子谱，二者均分泌MCP-1、CSF-1、VEGF等细胞因子，其中CCL15的表达差异最为显著。
CCL15是巨噬细胞炎症蛋白家族的一员，对单核细胞、T淋巴细胞及内皮细胞具有趋化作用。在我们的研究中，甲状腺滤泡癌细胞上CCL15的表达显著高于其在FA中的表达，并与巨噬细胞的浸润密度显著相关。在细胞功能实验中，CCL15中和抗体可抑制FTC133的上清对Thp-1的趋化作用。结合组织芯片上FTC浸润的巨噬细胞倾向于M2表型，显示甲状腺滤泡癌细胞通过分泌CCL15招募血液中的单核细胞迁移至肿瘤中心，促进肿瘤的发生发展。
基于CCL15在FTC中的高表达，CCL15可作为潜在的生物标记物，应用于FTC患者血清或甲状腺结节细针穿刺细胞盥洗液的检验，为鉴别FTC奠定一定的理论基础。
与现有技术相比，本发明的有益效果在于：1、本发明验证了甲状腺滤泡癌中CCL15高表达，甲状腺滤泡癌细胞通过分泌CCL15招募血液中的单核细胞迁移至肿瘤中心，促进肿瘤的发生发展。所以，CCL15可作为潜在的生物标记物，应用于甲状腺滤泡癌的检查。2、本发明为CCL15应用在甲状腺滤泡癌的筛选试剂中提供了实验数据和理论基础。
附图说明
图1为CCL15在甲状腺滤泡癌中的表达图,其中，图中的A为CCL15高表达组，图中的B为CCL15低表达组。
图2为CCL15对Thp-1细胞株的趋化作用图。
具体实施方式
下面结合实施例，对本发明作进一步说明：
实施例1
将确诊为甲状腺滤泡癌的新鲜手术组织标本，迅速放入组织冻存管，保存于液氮罐中备用。
1、甲状腺滤泡癌分泌细胞因子谱分析
本组实验中，我们提取了FTC和FA的液氮冷冻标本的蛋白，进行细胞因子谱分析。购自RayBio公司的人细胞因子抗体芯片G5含80种细胞因子。共对6个样品进行了芯片分析，4例来自FTC，2例FA。从细胞因子芯片原始检测图中可以看到无论是甲状腺滤泡癌还是甲状腺腺瘤，都表达CCL2、CSF-1、VEGF等趋化因子，但是表达差异最显著的是巨噬细胞炎症蛋白1δ(macrophageinflammatoryprotein1δ,MIP-1δ)，又名CCL15(chemokineC-Cmotifligand15,CCL15)。
2、CCL15在FTC中的表达
CCL15是一种单核细胞趋化因子。在观察到FTC和FA细胞因子谱中CCL15的表达差异后，我们在甲状腺滤泡性肿瘤组织芯片上进行免疫组化染色验证发现，76.7％的FTC病例高表达CCL15,具体如图1所述，图中的A为CCL15高表达组，图中的B为CCL15低表达组。此外,通过分析FTC和FA中CCL15的表达与CD68阳性的巨噬细胞浸润密度的关系发现，CCL15高表达组的巨噬细胞浸润密度显著大于CCL15低表达组。
3、CCL15促进甲状腺滤泡癌细胞对巨噬细胞的招募
基于上述CCL15高表达与巨噬细胞的密度显著相关，我们进一步验证了FTC细胞是否通过分泌CCL15招募单核细胞。FTC133是人的甲状腺滤泡癌细胞株，Thp-1是人的单核细胞株，细胞表面表达CCR1受体。我们收集FTC133的条件培养基(conditionalmedium,CM)，与Thp-1共培养24h后，观察Thp-1生物学行为的改变。如图2所示，与空白对照组相比FTC133的细胞培养上清能促进Thp-1细胞的迁移。在FTC133-CM的刺激下，Thp-1穿过细胞小室上层粘附在鼠尾胶上，说明FTC133可以通过其分泌的细胞因子直接作用于单核细胞。
综上所述，甲状腺滤泡癌中CCL15高表达，而且CCL15高表达组的巨噬细胞浸润密度显著大于CCL15低表达组，FTC133培养上清可促进Thp-1单核细胞株的迁移。所以，CCL15可作为潜在的生物标记物，应用于甲状腺滤泡癌的筛选试剂中，同时也为CCL15应用在甲状腺滤泡癌的筛选试剂中提供了实验数据和理论基础。

Claims (1)

1.检测CCL15趋化因子的试剂在制备筛选甲状腺滤泡癌试剂中的应用。
CN201410286443.1A 2014-06-24 2014-06-24 检测ccl15趋化因子的试剂在制备筛选甲状腺滤泡癌试剂中的应用 Active CN104122392B (zh)

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