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[目的]鉴定放射抗拒性食管癌细胞株KYSE-150R获得上皮—间质转化(EMT)表型。[方法]食管鳞状细胞癌细胞株KYSE-150经分次反复照射、单克隆培养,筛选出具有放射抗拒性细胞株KYSE-150R。克隆形成实验计算细胞存活率,拟合生存曲线并计算放射生物学参数。高倍显微镜下观察细胞形态学变化。采用RT-PCR、Western Blot和免疫荧光检测上皮表型标志物E钙黏蛋白(E-cad)、间质表型标志物波形蛋白(VIM)和转录因子Slug、Snail与Twist的表达。[结果]构建的食管癌细胞株KYSE-150R比其亲本对射线更加抗拒,经传代50代后仍保留放射抗拒性。放射抗拒的食管癌细胞株KYSE-150R发生EMT相符的形态学改变:细胞呈纺锤体状,极性消失。与母株相比,KYSE-150R中E-cad表达下降,VIM、Slug及Snail表达上调,Twist表达未见明显变化。免疫荧光染色证实细胞膜E-cad蛋白的丢失,上调的β-catenin移位至细胞核。[结论]放射抗拒的食管癌细胞株KYSE-150R获得了EMT表型,其与食管癌放射抗拒的形成有关。
[Objective] To identify the epithelial-mesenchymal transition (EMT) phenotype of radiation-resistant esophageal cancer cell line KYSE-150R. [Method] The esophageal squamous cell carcinoma cell line KYSE-150 was screened out with the radiation-resistant cell line KYSE-150R by repeated irradiation and monoclonal culture. Clonal formation experiments were used to calculate cell viability, fit survival curves and calculate radiobiological parameters. Morphology changes were observed under high magnification microscope. The expressions of epithelial phenotype markers E cadherin, interstitial phenotype vimentin (VIM) and transcription factors Slug, Snail and Twist were detected by RT-PCR, Western Blot and immunofluorescence. [Results] The constructed esophageal cancer cell line KYSE-150R was more resistant to radiation than its parent, and remained radioresistant after 50 passages. Radiation-resistant esophageal cancer cell line KYSE-150R EMT consistent morphological changes occur: cells were spindle-shaped, the polarity disappears. Compared with the parental strain, the expression of E-cadherin in KYSE-150R was down-regulated and the expression of VIM, Slug and Snail was up-regulated, but there was no significant change in Twist expression. Immunofluorescence staining confirmed the loss of E-cad protein in the cell membrane, and upregulated β-catenin translocated to the nucleus. [Conclusion] The EMT phenotype of the radiation-resistant esophageal cancer cell line KYSE-150R is related to the formation of radioresistance in esophageal cancer.