Sonic hedgehog信号通路与食管癌放射抗拒的相关性研究

Relationship between Sonic hedgehog signaling pathways and radioresistance of esophageal cancer

目的:研究Sonic hedgehog(Shh)信号通路在食管癌放射抗拒中的作用及机制。方法:以人食管癌细胞Eca109为亲本株,通过X射线反复照射(累计照射剂量60 Gy)诱导形成放射抗拒细胞株Eca109R。分别采用集落形成实验检测细胞放射敏感性,CCK-8实验检测细胞活力的变化,来证实放射抗拒细胞株Eca109R的放射抗拒性。免疫荧光及Western blot检测Eca109R和亲本细胞Eca109内Gli1蛋白与Shh蛋白的表达。结果:集落形成实验显示2 Gy的X线照射后,Eca109和Eca109R细胞的存活分数(SF_2)分别为0.499±0.042和0.937±0.013;同时,CCK-8实验显示,Eca109R细胞的活力抑制率明显低于Eca109细胞(P<0.05),证实Eca109R细胞有明显的放射抗拒性,诱导形成放射抗拒株成功。免疫荧光结果显示Eca109R细胞中Gli1表达明显高于Eca109细胞(P<0.05)。Western blot实验结果显示Eca109R细胞中的Gli1与Shh蛋白表达明显高于Eca109细胞(P<0.05)。结论:食管癌放射抗拒性的产生可能与Shh信号通路相关蛋白的过表达有关。

AIM： To investigate the potential role of Sonic hedgehog （Shh） signaling pathways in the radiore-sistance of esophageal cancer. METHODS： Radioresistant cell line Ecal09R was established by repeating X-ray irradia-tion at dose of 60 Gy in total using Ecal09 cells as parental cells. The radiosensitivity of the parental and radioresistant cells was confirmed by colony formation assay. The cell viability was detected by CCK-8 assay. The intracellular protein levels of Shh and Glil were determined by Western blot and immunofluorescence. RESULTS ： The survival fractions at dose of 2 Gy for Ecal09R cells and Ecal09 cells were 0. 937 ±0. 013 and 0. 499 ± 0. 042,respectively. The inhibitory rate of cell viability decreased gradually in the Ecal09R cells （P 〈0. 05） , suggesting that the radioresistant cell line was success-fully established. The results of Western blot indicated that the protein expression of Shh and Glil was much higher in the Ecal09 R cells than that in the Ecal09 cells （ P 〈 0. 05 ） . Immunofluorescence staining showed that Glil was expressed in the cytoplasm and nucleus, and presented nuclear clustering in the Ecal09R cells. The positive rate of Gill expression in Ecal09cellswas 52.3% ± 0.035% , wliiletliatinE cal09R cellswas8 7 .6% ±0.021%） （ P 〈0 .05 ） .CONCLUSION：The radioresistance of esophageal cancer may be related to the activation of Shh signaling pathways with over-expression of Glil and other related proteins.