放射抗拒性食管癌细胞株KYSE-150R上皮—间质转化特性鉴定

Identification of Epithelial-mesenchymal Transition Phenotype in Radioresistant Esophageal Carcinoma Cell Line KYSE-150R

[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。

[Purpose] To identify the acquisition of epithelial-mesenchymal transition phenotype in radioresistant esophageal carcinoma cell line KYSE-150R.[Methods] The radioresistant cell line KYSE-150R was established by fractionated irradiation（FIR） and monoclone culture.Cell survival was accounted by clonogenic assays and the morphological changes were observed by microscopy.RT-PCR,Western Blot and immunofluorescence were performed to examine expression of E-cadherin,vimentin,Slug,Snail and Twist.[Results] The established esophageal carcinoma cell line KYSE-150R showed higher radioresistance than parental cell line.The KYSE-150R cell line maintained radioresistant phenotype for at least 5 months with 50 passages after the cessation of radiation.KYSE-150R cells showed phenotypic changes consistent with EMT：spindle-cell shape,and loss of polarity.Compared with mother cell line the expression of E-cadherin downregulated and the expression of vimentin,Snail and Slug upregulated in KYSE-150R cells.However,the expression of Twist did not show marked changes.[Conclusion] Radioresistant KYSE-150R cells acquire phenotype of EMT,and it might relate to radioresistance for esophageal carcinoma.