摘要
目的确认肿瘤干细胞(CSC)发生β-连环素(0-catenin)核易位能否分化出钠碘转运体(NIS)膜表达缺失、对治疗抵抗的甲状腺癌细胞。方法从人甲状腺滤泡癌细胞KrC 133中分选侧群干细胞(SP),并进行细胞“干性”鉴定;对FTC133来源的SP细胞进行β-catenin转染,再将其促分化培养,并收集分化细胞。应用Western印迹、Transwell、MTT方法,验证分化细胞的上皮细胞间质转化(EMT)属性及体外增殖侵袭能力的变化;应用免疫荧光染色和细胞摄碘试验,观察分化细胞的N1S功能性表达及摄碘能力的变化;应用分化细胞建立严重联合免疫缺陷(SCID)小鼠皮下瘤模型,观察瘤体对"T体内治疗的反应,并应用免疫组化技术初步检测治疗相关功能蛋白的表达变化。结果FTC133中侧群干细胞约0.03%。应用3-catenin转染FTC133源SP细胞后.其分化细胞中出现了典型EMT转化,其中上皮属性蛋白(E钙黏素和细胞角蛋白18)表达缺失,间质属性蛋白(波形蛋白、纤连蛋白、尿激酶型纤溶酶原激活因子)反常表达;与未转染组和空质粒转染组相比,转染组SP细胞分化细胞体外增殖能力分别增强了约85.4%和81.0%(均P<0.01),侵袭能力则上升了78.8%和84.4%(均戸<0.01)0免疫荧光染色显示,β-catenin转染SP细胞后,其分化细胞出现明显的B-catenin核易位,同时伴随NIS蛋白由膜表达向浆表达的显著转变;体外摄碘率则分别下降约52.8%和45.2%(均P<0.01)。动物实验进一步显示,β-catenin转染SP细胞后,其分化细胞体现了很强的肿瘤生长促进能力和对抗治疗的能力(均P<0.05)。结论干细胞发生β-catenin核易位可能分化为对治疗抵抗的甲状腺癌细胞。
Objective To confirm whether β-catenin nuclear translocation in thyroid cancer stem cells can differentiate into thyroid cancer cells without functional membrane expression of sodium-iodine transporter (NIS) and be resistant to iodide 131 treatment. Methods Thyroid cancer stem cells were firstly isolated as a side population (SP) from human thyroid cancer cell line FTC 133. The SP cells from FTC 133 were transfected with P-catenin, and then differentiated. The cells were further collected for Western blot, Trans well and MTT assay to investigate the epithelial-mesenchymal transition (EMT) characteristics, tumor growth, invasion, and iodine uptake potency in vitro. Functional NIS expression and iodide uptake in differentiated cells were detected with immunofluorescent staining and iodide uptake assay,respectively. Subcutaneous severe combined immunodeficient (SCID) mice tumor model was induced with differentiated cancer cells to explore the in vivo effect of radioiodine treatment. Further immunohistochemical staining was performed to reveal the changes of functional proteins involved in tumor radioiodine treatment. Results Side population was isolated from FTC 133 accounting for about 0.03%, with high expression of stem cell markers and decreased expression of differentiated cell markers. Western blot showed prominent EMT phenotype in the differentiated cells from β-catenin transfected stem cell model, with absence of epithelial expression of E-cadherin and cytokeratin 18, as well as abnormal expression of vimentin,fibronectin and urokinase-type plasminogen activator. Moreover,compared with cells differentiated from untransfected or empty plasmid transfected stem cells, in vitro proliferation markedly increased 85.4% and 81.0%, respectively (both P<0.01);while in vitro invasion augmented 78.8% and 84.4%, respectively (both PvO.Ol). Immunofluorescent staining identified that, after transfected with P-catenin, differentiated cells underwent 3-catenin nuclear translocation and NIS localization transferred from memb
作者
兰玲
邓微
崔岱
陈海翎
霍丽丽
左庆瑶
李伟
张国英
罗勇
Lan Ling;Deng Wei;Cui Dai;Chen Hailing;Huo Lili;Zuo Qingyao;Li Wei;Zhang Guoying;Luo Yong(Department of Endocrinology, Beijing Jishuitan Hospital, Beijing 100035, China;Department of Endocrinology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China;Department of Urology, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China)
出处
《中华医学杂志》
CAS
CSCD
北大核心
2019年第24期1904-1910,共7页
National Medical Journal of China
基金
国家自然科学基金面上项目(81372858).
