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[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。
[Objective] To study the correction of high radiosensitivity of AT gene in AT5BIVA (AT cell) in patients with telangiectasia with ataxia (AT) by ATM gene in order to evaluate the radioprotective function of ATM gene. [Method] The eukaryotic expression vector PEBS7-YZ5 containing the cDNA of ATM gene was transfected into AT cells by electroporation, and screened by hygromycin to obtain stable cell lines. The transcription of ATM cDNA was detected by RT-PCR. Western blot The expression of ATM protein was verified by immunofluorescence staining. Cytoplasmic cleavage-promoting micronuclei (CBMN) were used to immunize PEBS7-YZ5-AT cells, PEBS7-AT cells and untransfected AT cells with 60COγ rays at 0,1,2,3,4Gy After observing and comparing the difference between the three micronucleus rate and the rate of micronuclei cells. [Result] PEBS7-YZ5 was successfully transfected into AT cells. ATM cDNA fragments were detected by RT-PCR and ATM protein was detected by Western blot. The rates of micronuclei and micronuclei in PEBS7-YZ5-AT cells were significantly lower than those in PEBS7-AT cells and AT cells at 0, 1, 2, 3 and 4 Gy doses (P <0.01) There was no significant difference between PEBS7-AT cells and AT cells (P> 0.05). [Conclusion] The high radiation sensitivity of AT cells is corrected by ATM gene. The ATM gene has the radioprotective function. The eukaryotic expression vector PEBS7-YZ5 containing ATM gene is expected to be a radioprotectant.