【摘要】： 目的創(chuàng)新性應(yīng)用GFP標(biāo)記的基因組非穩(wěn)定性報(bào)告系統(tǒng),進(jìn)一步探索電離輻射誘導(dǎo)B16細(xì)胞系基因組非穩(wěn)定性改變。方法采用脂質(zhì)體轉(zhuǎn)染的方法,將帶有GFP標(biāo)記的基因組非穩(wěn)定報(bào)告系統(tǒng)轉(zhuǎn)入B16細(xì)胞。G418篩選,選取單克隆并擴(kuò)增后,給予電離輻射(60Coγ射線)0Gy、2Gy、4Gy劑量照射,利用共聚焦熒光顯微鏡定性、定量分析電離輻射誘導(dǎo)的基因組非穩(wěn)定性改變。結(jié)果在共聚焦顯微鏡下觀察:①照射后第1d,2Gy、4Gy即出現(xiàn)綠色熒光表達(dá),第1-3d綠色熒光表達(dá)量多,第3d達(dá)到高峰,第5d綠色熒光表達(dá)量開始減少;②綠色熒光表達(dá)量與輻射劑量呈正相關(guān):4Gy2Gy0Gy組;③0Gy在第1-5d未出現(xiàn)綠色熒光蛋白的表達(dá)。④繼續(xù)培養(yǎng)0Gy組2周后,觀察到自發(fā)GFP的表達(dá),約占總細(xì)胞數(shù)的0.00016%(2/1200000)。結(jié)論①GFP標(biāo)記的區(qū)域性染色體基因組非穩(wěn)定報(bào)告系統(tǒng)成功轉(zhuǎn)染B16細(xì)胞。②GFP標(biāo)記基因組非穩(wěn)定報(bào)告系統(tǒng)轉(zhuǎn)染B16細(xì)胞系的G418篩選終濃度為500ug/ml,培養(yǎng)維持濃度為250ug/ml。③應(yīng)用有限稀釋法可以獲得成功轉(zhuǎn)染GFP標(biāo)記的基因組非穩(wěn)定性報(bào)告系統(tǒng)的B16克隆生長(zhǎng)。96孔板中可獲得率為13.3%(8/60孔)。④60Coγ射線可以成功誘導(dǎo)B16細(xì)胞區(qū)域性基因非穩(wěn)定性表達(dá),并被GFP標(biāo)記報(bào)告系統(tǒng)檢測(cè)。⑤GFP表達(dá)量與60Coγ射線照射劑量、照射后時(shí)間成正比。⑥電離輻射誘發(fā)區(qū)域性染色體基因組非穩(wěn)定性,可在照射后的子代細(xì)胞中觀察到。
[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 緯 ray) of 0 Gy, 2 Gy, 4 Gy, respectively, and the cells were screened by G418, then irradiated with ionizing radiation (緯 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 緯 -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 緯 -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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R363

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