甲状腺癌中类干细胞的侧群细胞的分离及鉴定

Isolation and characterization of a stem-like side population in thyroid cancer cell lines

目的:研究不同甲状腺肿瘤细胞中是否存在具备干细胞特性的侧群(side population,SP)细胞,同时比较侧群细胞和非侧群(non-SP)细胞生长、成瘤及侵袭性的差异。方法:以多种人甲状腺未分化癌细胞株为对象,利用干细胞高表达ABCG2转运体,可将荧光染料Hoechst泵出胞外的特性,运用双波长流式细胞仪从不同的甲状腺肿瘤细胞株中分选出具有干细胞特性的侧群细胞,进而通过半定量PCR及免疫荧光染色比较侧群和非侧群细胞的干细胞标志OCT4及肿瘤耐药基因ABCG2、MDR1表达的情况,采用克隆形成率及细胞侵袭迁移实验比较两组细胞群的克隆形成能力和侵袭转移能力。结果:人甲状腺未分化癌细胞株中存在具有干细胞特性的侧群细胞,该群细胞高表达干细胞标志OCT4及肿瘤耐药基因ABCG2和MDR1;同时在体外培养中,此侧群细胞可产生侧群细胞和非侧群细胞;侧群细胞比非侧群细胞具有更强的体外克隆形成能力和侵袭转移能力。结论:甲状腺肿瘤细胞中存在具有干细胞特性的侧群细胞,此群细胞可生成普通的瘤体细胞,提示该群细胞可能是肿瘤耐药和复发的根源。

Objective： To identify the presence of cancer stem-like cells（side population,SP） in thyroid cancer and compare the growth pattern,clonogenity and invasive potential between cancer SP and non-SP cells.Methods： Three different human anaplastic thyroid cancer cell lines（including HTh74,SW1736,and C643） were stained with Hoechst 33342 and sorted for a small fraction of SP by fluorescence-activated cell sorting（FACS）.The gene expression of stem cell marker-Oct4（octamer-binding transcription factor 4） and genes related to cancer resistance and relapse-ABCG2（ATP-binding cassette superfamily G member 2） and MDR1（multidrug resistance 1） in SP and non-SP cells was compared by performing RT-PCR and immunofluorescent staining.Colony formation assay was performed to evaluate the self-renewal potential of SP cells,and cell invasive potential was measured by using the Matrigel invasion assay.Results： Cancer SP cells possessed some intrinsic stem cell properties as they preferentially expressed stem cell marker Oct4 and ABC transporter genes-ABCG2 and MDR1,and can generate both SP and non-SP cells in vitro.Colony formation analysis and Matrigel invasion assay revealed that these cancer SP cells were more clonogenic and displayed higher invasive potential than non-SP cells.Conclusion： These findings suggest that the SP in thyroid cancer cell lines is enriched with stem-like cancer cells that may be the source of tumor drug resistance and recurrence.