CAJ | 학술논문

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60Co γ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 60Co γ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.60Co γ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60Co γ射线诱导的B16细胞区域性基因组不稳定性改变.
목적 응용록색형광단백(GFP)표기적기인조불은정성보고계통,검측60Co γ사선유도B16세포구역성기인조불은정성개변.방법 실험분위3조:미전염조、전염조급전염대조조.응용지질체전염법,장GFP표기목적질립급대조질립분별전입B16세포,경G418사선,유한희석법배양형성단극륭생장.급여0、2화4 Gy 60Co γ사선조사,공취초형광현미경기록GFP표체세포수,계산GFP표체솔.결과 건립료함유GFP표기적구역성기인조불은정성보고계통적GFP-B16세포주.60Co γ사선조사후,2화4 Gy조균가견GFP-B16세포표체GFP,병여조사제량、조사후시간밀절상관.GFP표체솔수조사제량(F =36.55、36.76,P＜0.05)화조사후시간적증가이증고(t=-3.27、-3.16、-4.26、-6.11、-7.17,P＜0.05).조사후제3천,GFP표체솔증고폭도최명현(2.46±0.24),제5천체도고봉(3.82±0.35),증고폭도추우은정.0 Gy조재조사후2주,자발록색형광단백표체솔위1/60만.결론 GFP표기적기인조불은정성보고계통가검측도60Co γ사선유도적B16세포구역성기인조불은정성개변.
Objective To detect the regional genomic instability of B16 cells treated with 60Co γ-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 60Co γ-rays at doses of 0,2 and 4 Gy.The regional genomic instability of B16 cellswas quantified by counting the number of cells with GFP expression.Results B-16 cell strain steadilyexpressing the GFP-based genomic instability reporting system was established successfully.GFP-positiveB16 cells were observed at 1 d after irradiation with 60Co γ-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 60Co γ-rays can be detected using a GFP-labelled genomic instability reporter system.