目的探讨NF-κB与放射线诱导的人食管癌细胞上皮间质转化(EMT)间的关系。方法累积照射食管癌Eca109细胞60 Gy,得到Eca109R60细胞,克隆形成实验检测其放射抵抗性,显微镜下观察细胞形态学变化。Real-time PCR和免疫细胞化学检测Eca109和Eca109R60细胞NF-κB、E-cadherin和Vimentin的表达情况。结果 Eca109R60细胞具备了EMT样表型且放射抵抗性更强。Eca109R60细胞中E-cadherin m RNA表达明显下调(P=0.003),Vimentin、NF-κB m RNA表达升高(P=0.004,P=0.004)。相关性分析显示NF-κB与E-cadherin m RNA表达呈负相关,与Vimentin未见有明显相关性,但已显示出正相关的趋势。免疫细胞化学法检测Eca109R60细胞E-cadherin阳性染色程度明显减弱,Vimentin、NF-κB p65阳性染色程度明显增强。结论 Eca109R60细胞放射抗性可能由EMT与NF-κB信号通路相互作用调控。
Background and Objective: The mRNA levels of 59 genes, detected by cDNA microarray, were up-regulated in the radioresistant human esophageal cacinoma cell line TE13R120 as compared with its parental cell line TE13 before and after radiation, and the expression of NRAGE gene showed a gradually up-regulating tendency. This study aimed to further detect the differences of NRAGE gene and protein expression and apoptosis between TE13R120 and TE13 cells, and to investigate the relationship between the NRAGE and the radioresistance of TE13R120 cells and its mechanism. Methods: The two cell lines were irradiated by 60 Co γ-ray at different conditions. Reverse transcription- polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry were used to detect the expression of NRAGE. Flow cytometry (FCM) was used to detect the cell apoptosis before and after irradiation. Results: The mRNA level of NRAGE was higher in TE13R120 cells than in TE13 cells before and after irradiation (before radiation: 0.25 ± 0.03 vs. 0.49 ± 0.03; 4 Gy 4 h: 0.31 ± 0.03 vs. 0.53 ± 0.02; 4 Gy 16 h: 0.32 ± 0.04 vs. 0.59 ± 0.04; 4 Gy 24 h: 0.36 ± 0.05 vs. 0.72 ± 0.04; 2 Gy 12 h: 0.32 ± 0.02 vs. 0.64 ± 0.04; 6 Gy 12 h: 0.36 ± 0.02 vs. 0.79 ± 0.05; 10 Gy 12 h: 0.46 ± 0.04 vs. 0.85 ± 0.01; P < 0.01), and the mRNA level of NRAGE was increased gradually with the increase of radiation dose and time in the two cell lines (P < 0.05 and P < 0.01). Western blot results showed no difference of NRAGE protein level in cytoplasm between TE13R120 cells and TE13 cells before and after irradiation, but its level in nuclei was higher in TE13R120 cells than in TE13 cells at different radiation time and dosages. Immunocytochemistry showed similar results as Western blot. FCM showed no significant difference in apoptosis rate between TE13R120 and TE13 cells before and after radiation. Conclusion: NRAGE may play an important role in the radiation responses of the two cell lines, and may participate in the formation of radioresistance of TE13R120
目的探讨射线反复照射方法建立食管癌放射抗性细胞系的重复性及稳定性,并观察辐射抗性细胞与其亲代细胞之间的放射敏感性的差异。方法应用射线对人食管鳞状细胞癌细胞TE13进行反复照射,累计剂量120 Gy,建立具有放射抗性的细胞系TE13R120。采用细胞克隆形成实验测定2种细胞的放射生物学参数,检测其辐射抗性,采用单击多靶模型拟合存活曲线。经连续8 d培养细胞,绘制2种细胞的生长曲线并用Logrank检验计算群体倍增时间。比较此次实验结果与初次建系时结果的相似性。结果接受120 Gy总剂量照射后,TE13R120较TE13表现出明显的放射抗性,TE13R120的放射生物学参数D_0、Dq、N均高于TE13(2.36、2.17、2.50 vs 1.90、1.11、1.80),SF_2、α/β均低于TE13(0.53、2.67 vs 0.73、8.00)。TE13R120的细胞群体倍增时间为20.70 h,长于亲代TE13(17.67 h)。该结果与此前初次建系时结果相似。结论应用射线反复照射并逐步筛选建立辐射抗性细胞系的方法可靠,能建立具有稳定辐射抗性表型的细胞系。
Micro RNAs(miRNAs), approximately 21 to 23 nucleotides(nt) in length, belong to a set of small non-coding RNA molecules that were not thought to be functional until the recent decades. miRNAs play important roles in many diseases such as various kinds of cancers and immune disorders. Many studies have focused on the relationship between miRNAs and diseases. miRNAs are significant mediators in human growth and development and in the genesis and development of diseases. Almost 30% of the activity of protein-coding genes is forecasted to be regulated by miRNAs in mammals, and some miRNAs are regarded as potential therapeutic targets for various diseases. In this review, we outline some functions of miRNAs, especially those related to diseases.
