^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

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4Gy^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4Gy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。0Gy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。

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 ceils induced by 60Coγ-rays can be detected using a GFP-labelled genomic instability reporter system.