抑制c-raf-1基因对人宫颈癌细胞放射敏感性影响的实验研究

Effect of inhibiting c-raf-1 on radiosensitivity in human cervical carcinoma cell line HeLa

目的:探讨用反义技术抑制癌基因c-raf-1对宫颈癌细胞(HeLa)放射敏感性的影响。方法:设立空白对照组、c-raf-1正义寡核苷酸组和脂质体组(仅加入等量的脂质体)作对照,用RT-PCR检测宫颈癌细胞处理前后c-raf-1表达水平的变化。用集落形成试验检测各实验组照射后人宫颈癌HeLa细胞株集落形成率,AO/EB荧光染色法检测细胞凋亡率的变化,流式细胞仪测定Bcl-2蛋白的表达。结果:c-raf-1反义寡核苷酸能有效抑制c-raf-1癌基因的表达,经脂质体介导的c-raf-1反义寡核苷酸作用的宫颈癌细胞经60Coγ射线照射后其集落形成率较正义寡核苷酸组及单纯照射组明显下降,而凋亡率明显增加,Bcl-2蛋白的表达显著下调,与对照各组比较有显著差异(P<0.01)。结论:c-raf-1反义寡核苷酸通过抑制c-raf-1基因降低人宫颈癌HeLa细胞放射抗性。其辐射增敏作用可能与c-raf-1反义寡核苷酸抑制了辐射抵抗信号转导途径。

Objective：To explore the effect of inhibiting c-raf-1 by antisense technology on radiosensitivity in human cervical carcinoma cell line HeLa. Methods：There were four groups in the study：control group,lipofectin group, sense oligodexynucleotides（S-ODN） group,antisense oligodexynucleotides（AS-ODN） group. The expression of c-raf-1 was detected by means of RT-PCR,Cellular response to irradiation was evaluated by the colony forming test,Apoptosis was observed by fluorescent staining. Bcl-2 protein expression was determined by flow cytometry. Results：AS-ODN group could significantly decreased the proliferation rate and increasing the apoptosis rate,significantly downregulating the expression of Bcl-2 of irradiated human cervical carcinoma cells, （vs lipofectin group or S-ODN group,P〈0.01）. Conclusion：c-raf-1 antisense oligodexynucleotides sensitized the HeLa to ionizing irradiation through decreasing the expression of c-raf-l,which might be related to inhibiting the celluar radiation-resistant signal transduction pathway and increasing the apoptosis rate of HeLa cells.