一种新的GFP/DNA双标记流式细胞技术  

A new GFP/DNA multi-parameter flow cytometry

作　　者：董波[1] 饶亚岚[1] 鲁茁壮[1] 王澜[1] 从玉文[1] 陈肖华[1] 张军权[1] 高荣莲[1] 王治东[1] 毛秉智[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850

出　　处：《军事医学科学院院刊》2004年第6期565-568,597,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：全军"十五"指令性基金课题 (0 1L0 18)

摘　　要：目的 :为了检测细胞转染了带绿色荧光蛋白 (GFP)标记的融合基因后细胞周期的改变 ,以研究目的基因与细胞周期的关系 ,建立GFP/DNA双标记流式细胞技术。方法 :①以小鼠脾细胞为模型 ,以不同浓度的多聚甲醛(PFA)预固定细胞不同时间 ,再换用 70 %乙醇固定细胞 2 4h以上 ,观察不同固定方法对细胞周期检测的影响 ;②以Ad EGFP感染的Hep 2细胞为模型 ,观察不同固定方法对细胞周期、GFP阳性细胞检出率和GFP的荧光强度的影响 ;③用最佳固定方法观察转染p2 1/WAF1/CIP1 EGFP融合基因的 2 93细胞的细胞周期 ,以验证方法的可靠性。结果 :0 .5 %PFA预固定 6 0min ,再换用 70 %乙醇固定细胞 ,不影响细胞周期检测 ,而且GFP荧光强度和转染效率的检测最佳。以此方法固定细胞 ,GFP/DNA双标记流式细胞技术检测了转染 p2 1/WAF1/CIP1 EGFP融合基因的2 93细胞的细胞周期 ,可观察到p2 1表达阳性的细胞发生了G0 /G1期阻滞。结论 :优化的细胞固定方法可以有效地阻止GFP从细胞内流出 ,建立的GFP/NDA双标记流式细胞技术可以用于研究带GFP标签的基因表达蛋白与细胞周期的关系。Objective:To investigate a new method of GFP/DNA multi-parameter flow cytometry in order to analyze the role of the target gene in cell cycle regulation. Methods: ①For finding the desirable method of fixation which has little effect on cell cycle,the murine spleen cells were fixated with different concentrations of paraformaldehyde(PFA) for different periods of times,and then with 70% ethanol for 24 h. The fixated cells were then stained with propidium iodide(PI)and analyzed by flow cytometry. ②Hep-2 cells infected with Ad-GFP recombinant adenovirus carrying green fluorescent protein were used to examine the effects of some of above fixation methods on the GFP fluorescence. ③As an example, the role of p21/WAF1/CZP/1-EGFP in cell cycle regulation was investigated in transfected 293 cells fixated with the above method. Results:It was found that one of the methods, which included fixation with 0.5%PFA for 60 min and then with 70% ethanol for 24 h, had little influence on cell cycle progression, fluorescent intensity and infection ratio of GFP. Using this method, the expression of p21 fusion protein was observed to induce arrest of cell cycle at G 0/G 1 phase in transfected 293 cells,which was consistent with the data reported before. Conclusion:A new fixation method of GFP/DNA multi-parameter flow cytometry was established,which is easy and reliable for cell cycle analysis.

关 键 词：细胞周期 流式细胞术 绿色荧光蛋白 基因表达 转染 

分 类 号：Q253[生物学—细胞生物学]