摘要
[背景与目的]喉癌是头颈部常见的恶性肿瘤,在头颈部恶性肿瘤中发病率仅次于鼻咽癌居第二位,占20%左右；喉癌90%以上为鳞状细胞癌。早期喉癌治疗效果较好,喉功能可得以保留,5年生存率可达80-95%；晚期喉癌由于浸润和转移,治疗效果差,5年生存率在60%以下。临床上约40%喉癌为Ⅲ-Ⅳ期的晚期患者。因此,喉癌治疗的关键在于有效的早期诊断,而喉癌中尤其以声门上型和声门下型喉癌早期诊断困难,临床上亟待寻找相关的肿瘤标志物以辅助早期诊断。
[方法]收集喉鳞状细胞癌患者的手术切除新鲜肿瘤组织和对应的正常喉粘膜组织进行体外原代培养,收获其条件培养基。条件培养基所含总蛋白以10%聚丙烯酰胺凝胶电泳分离,随后进行质谱分析,以此构建喉鳞癌相关游离蛋白数据库,并对数据库中的蛋白利用BinGo系统进行生物信息学分析。继而,从上述喉鳞癌相关蛋白数据库中选取KLK6、DJ-1、14-3-3sigma三个蛋白,在肿瘤组织和外周血样品中进行验证。通过酶联免疫吸附分析方法测定喉鳞癌患者、头颈部良性肿瘤及正常健康人血浆KLK6蛋白水平,分析各组之间及手术前后血浆中KLK6蛋白浓度变化；采用免疫组织化学染色的方法,观察KLK6、DJ-1、14-3-3sigma蛋白在喉鳞癌组织及正常喉粘膜上皮中表达变化,并分析三种蛋白表达变化与临床病理因素的关系。
[结果]在原代组织培养的条件培养基中共鉴定出982个蛋白质,其中在正常喉粘膜组织条件培养基中鉴定出的蛋白为213个,在喉鳞癌组织中鉴定出的特异蛋白为48个。通过对蛋白数据库进行生物信息学分析发现：条件培养基蛋白参与了蛋白水解、糖代谢、免疫反应、应激反应等重要细胞功能过程。对差异蛋白进行聚类分析表明,在正常喉粘膜组织条件培养基中鉴定出的蛋白主要参与免疫防御以及细胞骨架相关的生物学过程,在喉鳞癌组织条件培养基中鉴定出的蛋白则主要以正向调节上皮细胞增殖、细胞粘附及疾病相关的生物学过程。酶联免疫吸附实验结果显示：喉鳞癌患者血浆KLK6的浓度明显高于头颈部良性肿瘤组和健康对照组(P<0.001)；喉鳞癌患者手术前后血浆KLK6蛋白浓度差异有统计学意义(P<0.001)；伴有淋巴结转移的喉鳞癌患者血浆KLK6蛋白浓度高于无淋巴结转移者(P<0.05)。免疫组织化学染色结果表明：在喉鳞癌组织中,KLK6蛋白阳性表达率明显高于正常喉粘膜组织(P<0.001)；DJ-1蛋白阳性表达率显著高于正常喉粘膜组织(P<0.001)；14-3-3sigma蛋白阳性表达率显著低于正常喉粘膜组织,两组之间差异显著(P<0.001)。
[结论]体外培养喉鳞癌组织并进行蛋白质组学分析是一种新的标志物筛选方法。此培养体系为寻找血液中新的喉鳞状细胞癌相关分子标志物、为辅助喉鳞癌诊断和指导治疗提供了新的线索。KLK6、DJ-1、14-3-3sigma蛋白在喉鳞癌发生发展过程中可能发挥重要作用。
[Background and objectives! Laryngeal carcrinoma is the second morbidity malignant tumor of head and neck, which accounts for about 20%of all head and neck cancers. Pathologically, over 90% is laryngeal squamous cell carcinoma (LSCC). The early laryngeal carcinomas have relatively good therapeutic effects, with a 5-year survival rate of 80%-95%, and the function of larynx can be conserved:while the advanced cancers have poor therapeutic effects, and the 5-year survival rate is less than 60%. In clinical practice,40%of patients presented with advanced stageⅢorⅣ. Therefore, the effective early diagnosis is crucial to cure the laryngeal carcinoma. So it is an essential task to screen and identify specific biomarkers for detection or diagnosis of LSCC.
[Methods] In this study, proteomic methods were used to compare four pairs of the tumor and normal laryngeal tissue samples derived from patients with LSCC. With a chemically defined serum-free medium, the primary organ cultures were established from the paired tissue samples first. The conditional medium (CM) were then collected from the tissue cultures. Total proteins in the CM samples were separated by 10% SDS-PAGE, and then mass spectrometry (MS) was used to identify the proteins. A protein database of the CM for LSCC and normal laryngeal tissue was set up and analyzed by Biological Network Gene Ontology tool. Then we selected three proteins from the CM proteins identified in blood plasma samples and tissues. Enzyme linked immunosorbent assay (ELISA) technique was applied to detected plasma concentration of human kallikerin 6 (KLK6) protein. Immunohistochemisry (IHC) was used to detect the expression of KLK6, DJ-1 and 14-3-3sigma protein in LSCC tissues and adjacent normal laryngeal tissues.
[Results] Totally,982 proteins were identified in the CM. Among them,213 proteins were only detected in the CM of normal laryngeal tissues,48 proteins were only detected in the CM of LSCC. By Biological Network Gene Ontology tool analysis, it showed that the CM proteins participate the processing of proteolysis, extracellular matrix organization, glucose metabolic process and immune response. Function analysis showed that in the CM of normal laryngeal tissues, proteins mainly take part in immune response and cytoskeleton constitution. These secreted proteins may play a role in the immune reaction and serine proteinase inhibitor. Whereas, the CM proteins of LSCC involved in epithelia cell proliferation and transforming growth factor-beta production. ELISA results showed that the plasma level of KLK6 protein in LSCC patients was higher than that of head and neck benign tumor patients and normal control groups (P<0.001).There was significant difference of plasma concentration of KLK6 between patients in pre-operative and post-operative LSCC groups (P<0.001). Comparing with groups of lymph node metastasis and groups of without lymph node metastasis, there was also difference (P<0.05). IHC results demonstrated that the positive expression rate of KLK.6 and DJ-1 protein in LSCC were higher than that of normal tissues (P<0.001). The 14-3-3sigma protein expression in LSCC were much lower than that in normal tissues (P<0.001).
[Conclusions] We discovered a novel method to detect free proteins released into blood by laryngeal carcinoma. These data provided a new way to analyze tumor markers of LSCC in the peripheral blood for early detection and therapy. KLK6, DJ-1,14-3-3sigma proteins may play an important role in the pathogensis and development of LSCC.
引文
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