荧光探针及流式分选法追踪胰腺癌细胞增殖的研究  

Research on Tracing the Proliferation of Pancreatic Cancer Cells with the Fluorescence Probe Following FASC

作　　者：米蕊芳[1,2] 潘春晓[1,2] 张俊文[1] 刘福生[1] 金贵善[1] 

机构地区：[1]首都医科大学附属北京天坛医院、北京市神经外科研究所脑肿瘤研究中心,100050 [2]中国医学科学院基础医学研究所/清华大学医学部北京协和医学院基础学院病理学系,100005

出　　处：《医学研究杂志》2016年第6期29-32,共4页Journal of Medical Research

基　　金：国家自然科学基金资助项目(81302186,81372354);北京市自然科学基金资助项目(7151002);北京市神经外科研究所创新基金资助项目(所青年-2014-003);北京市医院管理局青年人才培养“青苗”计划(QML20150505)

摘　　要：目的应用羟基荧光素二醋酸盐琥珀酰亚胺脂(5,6-carboxyfluorescein diacetate,succinimidyl ester,CFSE)荧光探针及流式分选的方法追踪胰腺癌细胞增殖情况。方法应用低血清培养法(0.5%)对细胞进行细胞周期同步化;利用CFSE染色追踪胰腺癌全基因组突变文库细胞增殖;并于一定的时间点,应用流式分选法分选增殖速度减慢细胞群;通过CCK-8法检测分选前后的细胞增殖曲线。结果 CFSE探针可均匀染色胰腺癌细胞,呈现较强的绿色荧光;在CFSE荧光探针染色后的不同时间点(0、48、96及144h),胰腺癌细胞荧光强度呈现逐级衰减趋势;应用细胞饥饿法对细胞进行同步化后,细胞呈现更好的同步化衰减趋势;在一定的荧光衰减时间点(144h),通过流式分选从细胞突变文库中获得了胰腺癌增殖减慢细胞群;CCK-8实验显示,相对于分选前的细胞,分选出的荧光强度强的胰腺癌细胞群增殖速度明显减慢(P=0.006)。结论 CFSE荧光探针可很好追踪胰腺癌细胞增殖,结合流式分选法可获得肿瘤突变文库中的增殖减慢细胞群。Objective To trace the proliferation of pancreatic cancer cell using the CFSE fluorescent dye and obtain the cells with low proliferation rate by fluorescence activated cell sorting （FACS）.Methods We synthronized the cell cycles of pancreatic cancer cells through culturing them in the low serum medium （0.5％）.We traced the proliferation rate of pancreatic cancer cells in the mutation library using the CFSE probe.We selected the cells with low proliferation rate in the pancreatic cancer library by FACS at the selected time point.We detected the cell proliferation rate using CCK-8 method.Results CFSE dyed the pancreatic cancer cells evenly with high green fluorescence intensity.The fluorescence intensity was gradually attenuated at the time point of 0,48,96 and 144h.The cell cycles of the pancreatic cancer cells were synthronized when starved in the low serum medium for 48 h.And the cells with low proliferation rate were successfully selected following FACS after tracing for 144 hours with CFSE in vitro.The proliferation rate of the selected cells was significantly decreased compared with the original pancreatic cancer cells（P =0.006）.Conclusion CFSE fluorescent dye can be used to trace the proliferation of the pancreatic cancer cells,and the cell with low proliferation rate is successfully selected with CFSE dye following FACS.

关 键 词：胰腺癌 荧光探针 流式分选 ASPC-1细胞 

分 类 号：R73[医药卫生—肿瘤]