肿瘤干细胞在食管癌放射线抵抗中的作用及分子机制研究

目的：探究肿瘤干细胞在食管癌放射线抵抗中的作用及其分子机理，为食管癌的放疗提供理论参考。方法采用8 GyX线照射食管癌细胞系TE1，建立并筛选出放射线抵抗的食管癌细胞系TE1－res。利用细胞计数法比较增殖情况，采用流式细胞术比较CD44（ high） CD24（－） CD133（＋）分子表达及其凋亡情况，克隆形成实验比较克隆形成率及细胞存活曲线，BSP方法比较抑癌基因甲基化程度。成组t 检验或方差分析差异。结果 TE1－res 细胞比 TE1细胞增殖能力增强（平均值20．84×105∶4．46×105／d，P＝0．008），CD44（high） CD24（－）CD133（＋）细胞比例升高（38．0±2．9）％∶（10．1±1．3）％，P＝0．001]，抗凋亡能力增强（平均值33．23％∶10．50％，P＝0．003），8 Gy照射后克隆形成率升高（14．3±2．6）％∶（0．9±0．3）％，P＝0．011]，D0值升高（3．28 Gy ∶2．19 Gy，P＝0．125）， SPINT2、CDKN1B、DKK1、TP53、PPP2R1B抑癌基因启动子区甲基化水平升高（89．7±4．9）％∶（5．0±0．5）％（P＝0．001）、（92．3±4．7）％∶（10．4±0．7）％（P＝0．001）、（90．7±3．7）％∶（7．9±0．4）％（P＝0．001）、（83．4±5．7）％∶（17．2±1．2）％（P＝0．002）、（90．2±6．7）％∶（4．4±1．2）％（P＝0．002）]。结论肿瘤干细胞在食管癌放射线抵抗中具有重要作用，放射线抵抗与SPINT2、CDKN1B、DKK1、TP53及PPP2R1B等抑癌基因启动子区的高甲基化水平密切相关。

Cancer stem cells in radiation resistance of esophageal cancer:role and molecular mechanism

Objective To investigate the role of cancer stem cells in radiation resistance of esophageal cancer and its molecular mechanism, and to provide a theoretical basis for radiotherapy for esophageal cancer.Methods Esophageal cancer cell line TE1 was treated with 8 Gy of radiation. Esophageal cancer cell line with resistance to radiation, TE1-res, was established and screened.Cell counting was used to evaluate cell proliferation.Flow cytometry was used to determine the expression of CD44 (high) CD24(-) CD133(+) and apoptosis in cells.The colony formation assay was used to determine the colony-forming rate and cell survival curve.Bisulfite sequencing PCR was used to determine the methylation status of cancer suppressor genes.Comparison of the data was made by group t test or analysis of variance. Results Compared with TE1 cells, TE1-res cells had significantly enhanced proliferation, a significantly higher proportion of CD44( high) CD24(-) CD133(+) cells, and significantly enhanced resistance to apoptosis (mean value 20.84×105 vs.4.46×105/day, P=0.008;(38.0±2.9)%vs.(10.1±1.3)%, P=0.001;mean value 33.23% vs.10.50%, P=0.003 ) .After treatment with 8 Gy of radiation, TE1-res cells had significantly higher colony-forming rate and D0 value than TE1 cells ((14.3±2.6)%vs.(0.9±0.3)%, P=0.011;3.28 vs.2.19 Gy, P=0.125 ) .Moreover, the promoter methylation in cancer suppressor genes including SPINT2, CDKN1B, DKK1, TP53, and PPP2R1B was significantly enhanced in TE1-res cells than in TE1 cells ((89.7±4.9)%vs.(5.0±0.5)%, P=0.001;(92.3±4.7)%vs.(10.4±0.7)%, P=0.001;(90.7±3.7)%vs.(7.9±0.4)%, P=0.001;(83.4±5.7)%vs.(17.2±1.2)%, P=0.002;(90.2±
6.7)%vs.(4.4±1.2)%, P=0.002).Conclusions Cancer stem cells play an important role in radiation resistance of esophageal cancer. The resistance to radiation is closely associated with promoter hypermethylation in cancer suppressor genes including SPINT2, CDKN1B, DKK1, TP53, and PPP2R1B.