| 刘静,王亚婷,林海,韩倩,张春晓,白欧.60Coγ射线诱导GFP转染的B16细胞区域性基因组不稳定性改变的研究[J].中华放射医学与防护杂志,2012,32(5):465-468 |
| 60Coγ射线诱导GFP转染的B16细胞区域性基因组不稳定性改变的研究 |
| The regional genomic instability induced by 60Coγ-rays in B16 cells transfected by GFP |
| 投稿时间：2012-02-07 |
| DOI：10.3760/cma.j.issn.0254-5098.2012.05.004 |
| 中文关键词: 基因组不稳定性 60Co γ射线 B16细胞 |
| 英文关键词:Genomic instability 60Coγ-rays B16 cells |
| 基金项目:国家自然科学基金(31070759);吉林省科技厅国际合作课题(20070729-6) |
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| 中文摘要: |
| 目的 应用绿色荧光蛋白(GFP)标记的基因组不稳定性报告系统,检测60Coγ射线诱导B16细胞区域性基因组不稳定性改变。方法 实验分为3组:未转染组、转染组及转染对照组。应用脂质体转染法,将GFP标记目的质粒及对照质粒分别转入B16细胞,经G418筛选,有限稀释法培养形成单克隆生长。给予0、2和4 Gy 60Coγ 射线照射,共聚焦荧光显微镜记录GFP表达细胞数,计算GFP表达率。结果 建立了含有GFP标记的区域性基因组不稳定性报告系统的GFP-B16细胞株。60Coγ射线照射后,2和4 Gy组均可见GFP-B16细胞表达GFP,并与照射剂量、照射后时间密切相关。GFP表达率随照射剂量(F=36.55、36.76,P<0.05)和照射后时间的增加而增高(t=-3.27、-3.16、-4.26、-6.11、-7.17, P<0.05)。照射后第3天,GFP表达率增高幅度最明显(2.46±0.24),第5天达到高峰(3.82±0.35),增高幅度趋于稳定。0 Gy组在照射后2周,自发绿色荧光蛋白表达率为1/60万。结论 GFP标记的基因组不稳定性报告系统可检测到60Coγ射线诱导的B16细胞区域性基因组不稳定性改变。 |
| 英文摘要: |
| Objective To detect the regional genomic instability of B16 cells treated with 60Coγ-rays by a green fluorescence protein (GFP)-based genomic instability reporting system. Methods Three groups were employed as non-transfection group, vector control group and transfection group. The GFP-marked reporter construct pCMV-EGFP2XhoI for regional genomic instability was successfully transfected into B16 cells using liposome. B16 cells were selected by screening of G418 with a series of concentrations and limiting dilution cultures to yield a single colony. B16 cells with the genomic instability report system were then irradiated by 60Coγ-rays at doses of 0, 2 and 4 Gy. The regional genomic instability of B16 cells was quantified by counting the number of cells with GFP expression. Results B-16 cell strain steadily expressing the GFP-based genomic instability reporting system was established successfully. GFP-positive B16 cells were observed at 1 d after irradiation with 60Coγ-rays at doses of 2 and 4 Gy. Positive correlations between fluorescence intensity and dose and fluorescence intensity and time were also observed. The positive expression rate of GFP followed the increased of dose (F=36.55,36.76,P<0.05) and time(t=-3.27, -3.16, -4.26, -6.11, -7.17, P<0.05), and differences between groups were significant. The positive expression rate of GFP increased significantly at 3 d, and maximum expression was observed at 5 d(2.46 ± 0.24 and 3.82 ± 0.35). The level was tending towards stability. Spontaneous GFP expression at a ratio of 1/600 000 was observed in 0 Gy group after 2 weeks of culture. Conclusions The regional genomic instability of B16 cells induced by 60Coγ-rays can be detected using a GFP-labelled genomic instability reporter system. |
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