电离辐射诱导B16细胞基因组非稳定性的实验研究

发布时间：2019-02-22 18:42

【摘要】： 目的创新性应用GFP标记的基因组非稳定性报告系统,进一步探索电离辐射诱导B16细胞系基因组非稳定性改变。方法采用脂质体转染的方法,将带有GFP标记的基因组非稳定报告系统转入B16细胞。G418筛选,选取单克隆并扩增后,给予电离辐射(60Coγ射线)0Gy、2Gy、4Gy剂量照射,利用共聚焦荧光显微镜定性、定量分析电离辐射诱导的基因组非稳定性改变。结果在共聚焦显微镜下观察:①照射后第1d,2Gy、4Gy即出现绿色荧光表达,第1-3d绿色荧光表达量多,第3d达到高峰,第5d绿色荧光表达量开始减少;②绿色荧光表达量与辐射剂量呈正相关:4Gy2Gy0Gy组;③0Gy在第1-5d未出现绿色荧光蛋白的表达。④继续培养0Gy组2周后,观察到自发GFP的表达,约占总细胞数的0.00016%(2/1200000)。结论①GFP标记的区域性染色体基因组非稳定报告系统成功转染B16细胞。②GFP标记基因组非稳定报告系统转染B16细胞系的G418筛选终浓度为500ug/ml,培养维持浓度为250ug/ml。③应用有限稀释法可以获得成功转染GFP标记的基因组非稳定性报告系统的B16克隆生长。96孔板中可获得率为13.3%(8/60孔)。④60Coγ射线可以成功诱导B16细胞区域性基因非稳定性表达,并被GFP标记报告系统检测。⑤GFP表达量与60Coγ射线照射剂量、照射后时间成正比。⑥电离辐射诱发区域性染色体基因组非稳定性,可在照射后的子代细胞中观察到。
[Abstract]:Objective to explore the genomic instability of B16 cell line induced by ionizing radiation using GFP labeled genomic instability report system. Methods the genomic unstable reporting system labeled with GFP was transfected into B16 cells by liposome transfection. The clones were selected and amplified, and then irradiated with ionizing radiation (60Co 纬 ray) of 0 Gy, 2 Gy, 4 Gy, respectively, and the cells were screened by G418, then irradiated with ionizing radiation (纬 ray) of 0 Gy, 2 Gy, 4 Gy, respectively. Quantitative analysis of genomic instability induced by ionizing radiation was carried out by confocal fluorescence microscopy. Results under confocal microscope: 1 on the 1st day after irradiation, 2Gy, 4Gy appeared green fluorescence expression, the amount of green fluorescence expression reached the peak at the 1st day after irradiation, reached the peak at the 3rd day, and began to decrease at the 5th day; 2the expression of green fluorescence was positively correlated with the dose of radiation: 4Gy2Gy0Gy group; The expression of green fluorescent protein (GFP) was not found in 30Gy at the 1st to 5th day. 4 the spontaneous GFP expression was observed in the 0Gy group for 2 weeks, accounting for 0.00016% (2 / 1200000) of the total number of cells. Conclusion B16 cells were successfully transfected with 1GFP labeled regional chromosomal genomic instability report system. The final concentration of G418 screening for B16 cell line transfected with 2GFP labeled genomic instability report system was 500ugr / ml. The growth of B16 clones successfully transfected with GFP labeled genomic instability report system could be obtained by using limited dilution method at culture maintenance concentration of 250ug/ml.3. The yield of B16 clones in 96 well plates was 13. 3% (8 / 60 holes). 4. 60Co 纬 -rays can successfully induce regional gene instability in B16 cells. The expression of 5GFP was detected by GFP marker report system. The expression of 5GFP was directly proportional to the dose of 60Co 纬 -ray and the time of exposure was 1.6%. The genomic instability of regional chromosomes was induced by ionizing radiation. The results showed that the expression of 5GFP was observed in the progeny cells after irradiation.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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