放射抗拒性食管癌细胞系的建立及基因表达差异分析

目的观察食管癌细胞经射线反复照射后放射敏感性的变化，应用基因芯片技术分析放射抗拒性食管癌细胞基因表达变化。方法食管鳞状细胞癌细胞株TE13经反复γ射线照射（累积剂量120Cy），逐步筛选出具有放射抗拒性的细胞TE13R120。相差显微镜下观察TE13及TE13R120的形态学差异；应用细胞克隆形成实验，验证TE13及TE13R120两种细胞的不同放射敏感性，用流式细胞术检测它们的细胞周期分布特征；基因芯片分析两种细胞基因表达差异。结果TE13R120的细胞群体倍增时间为39．93h，长于亲代TE13（33．94h）。TE13及TE13R120的放射敏感性明显不同（仇值分别为1．63和2．85Gy，SF2值分别为0．55和0．64，Dq值分别为1．38和1．15Gy，n值分别为2．33和1．49）。两种细胞经4Gy放射线照射后，出现不同的细胞周期分布改变，12～48h TE13R120细胞周期变化不明显，而其亲代细胞TE13发生明显G1期阻滞。应用DNA芯片对2159个基因进行了筛选，TE13R120与TE13相比，上调基因96个，下调基因80个。结论新的食管癌细胞系TE13R120比其亲本对射线更加抗拒，并且在基因水平上发生明显变化。4Gy照射后12～48hTE13R120细胞周期变化不明显，而其亲代细胞TE13发生明显G1期阻滞。

Isolation and characterization of radioresistant human esophageal cancer cells and the differential gene expression by cDNA microarray analysis

Objective To observe the differential gene expression in esophageal cancer cell with different radiosensitivity by cDNA microarray analysis. Methods A radioresistant cell line, TE13R120, was isolated and characterized in vitro from a human esophageal cancer cell line TE13 by repeated ~ 60 Co gamma-ray irradiation, 120 Gy in total. Morphological changes from TE13 to TE13R120 were observed by phase-contrast microscopy. The relative radiosensitivities of tumor cells were assessed by standard colony formation assays. The time-course of cell cycle distribution after irradiation was investigated by flow cytometry (FCM). The differential gene expression was identified using cDNA microarray technique. Results The population doubling time of TE13 and TE13R120 was 33.94 h and 39.93 h, respectively. For TE13R120 cells, D_0 of radiation sensitivity was 2.85 Gy, while D_0 was 1.63 Gy for TE13 cells. The surviving fractions at 2 Gy (SF_2) were 0.64 for TE13R120 cells and 0.55 for TE13 cells. Cell cycle distributions were different for TE13 cells and TE13R120 cells 12-48 h after irradiation. A marked accumulation of G_1 phase was observed after 4 Gy irradiation for TE13 cells, and reached a peak at 12 h after irradiation. However, no apparent change of cell cycle distribution for TE13R120 cells was observed after irradiation and only a slight accumulation of cells in G_2/M phase was observed during the period from 12 h to 24 h after irradiation. By the use of cDNA microarray analysis of differential gene expression between TE13R120 cell line and its parental TE13 cell line, 96 upregulated genes in TE13R120 cells and 80 down-regulated genes were found. Conclusions A radioresistant variant cell line, denoted by TE13R120, from the TE13 cell line was successfully isolated by repeated gamma-radiation exposures. Significant changes in gene expressions were observed in this variant cell line. A marked arrest of cells in the G_1 phase was observed in TE13 cells after 4 Gy irradiation with, but no apparent change of cell cycle distribution for TE13R120 cells. ;