胞质分裂阻滞微核法研究ATM基因辐射防护功能

A Study on Radiation Protective Function of ATM Gene Using Cytokinesis-block Micronucleus Method

[目的]研究ATM基因对毛细血管扩张性共济失调症(AT)患者皮肤成纤维细胞系AT5BIVA(AT细胞)高辐射敏感性的纠正情况,以评价ATM基因的辐射防护功能。[方法]利用电穿孔技术,将含有ATM基因cDNA的真核表达载体PEBS7-YZ5转染到AT细胞,用潮霉素筛选以获得稳定表达细胞株,RT-PCR检测ATM cDNA的转录,Western blot验证ATM蛋白的表达;用胞质分裂阻滞微核法(CBMN),在PEBS7-YZ5-AT细胞、PEBS7-AT细胞和未转染AT细胞经60COγ射线0、1、2、3、4Gy照射后,观察比较三者间微核率及微核细胞率的差异。[结果]PEBS7-YZ5成功转进AT细胞,RT-PCR检测到ATM cDNA片段,Westernblot检测到ATM蛋白的表达。在0、1、2、3、4Gy剂量下,PEBS7-YZ5-AT细胞微核率及微核细胞率明显低于PEBS7-AT细胞和AT细胞,其差异具有显著统计学意义(P<0.01);而PEBS7-AT细胞和AT细胞间无明显差异(P>0.05)。[结论]AT细胞高辐射敏感性被ATM基因纠正,ATM基因具有辐射防护功能,含有ATM基因的真核表达载体PEBS7-YZ5有望成为辐射防护剂。

[Purpose] To investigate the role of ATM gene in rectifing the high radiosensitivity of fibroblast AT5BIVA cell line which established from the skin of patients with ataxia-telangiectasia（AT）,and to evaluate the radiation protection function.[Methods] PEBS7-YZ5 containing ATM gene cDNA was transfected into AT cells by electroporation.Hygromicin was used to screen the cells with ATM protein stably expressed.RT-PCR was used to detect the expression level of ATM.Western blot was used to detect the expression of ATM protein.Using cytokinesis-block micronucleus method,the difference of micronucleus frequencies（MNF） and micronucleus cell frequencies（MNCF） of PEBS7-YZ5-AT cells,PEBS7-AT cells and AT cells treated with 0,1,2,3 and 4Gy exposures to 60CO γ-ray were observed.[Results] PEBS7-YZ5 was transfected into AT cells successfully.Fragment of ATM cDNA and ATM protein were detected by RT-PCR and Western blot respectively.After exposed to 1,2,3 and 4Gy 60CO γ-ray,the radiation-induced level of MNF and MNCF were significantly lower in the PEBS7-YZ5-AT cells,compared with those in PEBS7-AT cells and AT cells（P0.01）,and there was no significant difference between PEBS7-AT cells and AT cells（P0.05）.[Conclusion] ATM gene rectifies the high radiosensitivity of AT cells.ATM gene pocesses the function of radiaiton protection,and PEBS7-YZ5 plasmid is potential be radiation protection agent.