甲狀腺惡性潛能未定的高分化腫瘤差異表達蛋白的蛋白質組學分析

唐銘 楊慧 劉鵬杰 徐敏潔 張迎紅 陳天星

[摘要] 目的 篩選與鑒定甲狀腺惡性潛能未定的高分化腫瘤（WT-UMP）差異表達蛋白質分子，尋找新的蛋白質標志物。 方法 收集2015年1月～2016年12月云南省第一人民醫院（以下簡稱“我院”）手術切除新鮮標本甲狀腺WT-UMP 20例及其周圍正常甲狀腺組織20例，另收集2016年1～12月我院病理科甲狀腺乳頭狀癌（PTC）蠟塊30例。從冰凍新鮮樣本中分別提取總蛋白進行雙向凝膠電泳（2-DE），經PDQuest 7.3圖像分析軟件檢測表達差異點，利用基質輔助激光解吸電離串聯飛行時間質譜（MALDI-TOF-MS）和SWISS-PROT數據庫對差異蛋白進行鑒定，通過PANTHER分析對差異蛋白進行分類，選擇其中5種差異表達蛋白通過免疫組織化學染色進行驗證。 結果 共鑒定出27種差異蛋白，在甲狀腺WT-UMP中呈高表達的25個，呈低表達的2個。免疫組化染色顯示5種蛋白Cytosolic non-specific dipeptidase（CNDP2）、Cytokeratin18（CK18）、Cathepsin B（CTSB）、Calreticulin（CRT）及Annexin A5（ANXA5）均定位于細胞漿。CK18、CTSB、CRT及ANXA5這4種蛋白的表達在WT-UMP中均高于正常甲狀腺，差異有統計學意義（P < 0.05），在PTC中均高于WT-UMP，差異有統計學意義（P < 0.05）。 結論 成功篩選了甲狀腺WT-UMP差異表達蛋白質分子，這些蛋白質表達的改變，可能參與了WT-UMP的發生和發展。同時通過免疫組化的驗證，有利于WT-UMP的早期診斷。

[關鍵詞] 甲狀腺惡性潛能未定的高分化腫瘤；差異表達蛋白；蛋白質組學；蛋白質標志物

[中圖分類號] R365 [文獻標識碼] A [文章編號] 1673-7210（2018）03（a）-0060-06

[Abstract] Objective To identify the differential expression proteins in thyroid well-differentiated tumour of uncertain malignant potential （WT-UMP） and to look for new protein biomarkers. Methods A total of 20 thyroid WT-UMP resection samples and 20 normal thyroid tissues adjacent to thyroid WT-UMP were obtained from January 2015 to December 2016 in the First People's Hospital of Yunnan Province （hereinafter referred to as "our hospital"）. In addition， 30 blocks papillary thyroid carcinoma （PTC） were obtained from the pathology department of our hospital from January to December 2016. The total proteins which were extracted from frozen thyroid samples in each group were profiled with two-dimensional electrophoresis （2-DE）. The differential protein spots were identified by PDQuest 7.3 software and identified by matrix-assisted laser desorption ionization time-of-fight mass spectrometry （MALDI-TOF-MS） and SWISS-PROT database. The different proteins were classified by PANTHER analysis and five differentially expressed proteins of these spots were further validated through immunohistochemistry （IHC）. Results 27 proteins were identified with MS. There were 25 high expression and 2 low level expression proteins in thyroid WT-UMP. IHC revealed the following five proteins located in the cytoplasm： cytosolic non-specific dipeptidase （CNDP2）， cytokeratin 18 （CK18）， cathepsin B （CTSB）， calreticulin （CRT） and annexin A5 （ANXA5）. Four kinds of proteins include CK18， CTSB， CRT and ANXA5 were over-expressed in thyroid WT-UMP compared with normal tissues， the difference was statistically significant （P < 0.05）； Meanwhile， the four proteins were over-expressed in PTC compared with thyroid WT-UMP （P < 0.05）. Conclusion The differentially expressed protein molecules were successfully screened and identified in thyroid WT-UMP. The changes of these proteins expression may be involved in the genesis and development of thyroid WT-UMP. The newly identified protein biomarkers can positively contribute to early thyroid WT-UMP diagnosis by using IHC.

[Key words] Thyroid well-differentiated tumour of uncertain malignant potential； Differentially expressed protein； Proteomics； Protein markers

2017年第四版內分泌腫瘤WHO分類提出了甲狀腺交界性腫瘤的概念。這類腫瘤具有非常惰性的生物學行為，而且治愈率高，無需追加RAI治療[1]。準確診斷這類腫瘤將減少對患者侵襲性的治療、減輕其心理壓力并降低社會經濟負擔。惡性潛能未定的高分化腫瘤（WT-UMP）屬于甲狀腺交界性腫瘤的其中一種類型，是一種全由或部分具有乳頭狀甲狀腺癌核改變和可疑包膜或血管侵犯的、高分化濾泡細胞組成的有包膜或境界清楚的腫瘤。……