流式细胞仪分选侧群细胞条件的探索与优化  

Optimization of the conditions for sorting side population cells by flow cytometry

作　　者：叶惠慧[1] 贾浩源[1] 张蕾蕾[1] 薛建国[1] 祝源[1] 蒋留留[1] 李里明[1] 许文荣[1] 钱晖[1] 

机构地区：[1]江苏大学基础医学与医学技术学院,镇江市检验医学重点实验室,江苏镇江212013

出　　处：《临床检验杂志》2014年第1期30-33,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(91129718;81302119);江苏省自然科学基金(BK2012709;BK20130540);江苏省医学领军人才与创新团队(LJ201117);国家博士点基金项目(20113227110011)

摘　　要：目的探讨不同浓度的荧光染料(Hoechst 33342)和抑制剂维拉帕米(verapamil)对流式细胞仪分选侧群(SP)细胞的影响。方法实验分为Hoechst 33342组(Hoechst 33342浓度分别为2.5、5.0和7.5μg/mL)和抑制组(Hoechst 33342+verapamil,verapamil浓度分别为50、100和150μmol/L),用流式细胞仪分选人肺腺癌细胞系A549中SP细胞,摸索Hoechst 33342和verapamil的最佳浓度,并用本实验室构建的一株肿瘤细胞系K1进行验证。结果在verapamil和Hoechst 33342浓度分别为150μmol/L和7.5μg/mL时,A549细胞染色充分,SP细胞亚群与非SP(non-SP)细胞亚群分群明显,SP细胞约为2.2%。K1细胞在verapamil为50μmol/L和Hoechst 33342为5μg/mL时,SP细胞亚群与non-SP细胞亚群分群明显,SP细胞亚群可被verapamil抑制,其比例约为1.3%。分选后的SP细胞和non-SP细胞可进一步扩增培养。结论 Hoechst 33342和verapamil可影响肿瘤细胞中SP细胞分选效率。Objective：To investigate the impact of different concentrations of Hoechst 33342 and verapamil on the side population （SP） cells sorted by flow cytometry. Methods：The human lung adenocarcinoma A549 cells were treated by different concentrations of Hoechst 33342 （2.5, 5.0 and 7.5 μg/mL） and the Hoechst 33342 combined with 50, 100 or 150 μmol/L of verapamil, respectively, and sorted the SP cells by flow cytometry in order to obtain the optimal concentration of Hoechst 33342 and verapamil. Then, the tumor cell line K1 was used to verify the optimal concentration of Hoechst 33342 and verapamil. Results：When the concentrations of verapamil and Hoechst 33342 were 150 μmol/L and 7.5 μg/mL, respectively, the A549 cells were stained completely, and SP cells and non-SP cells were classified obviously. The SP cells accounted for about 2.2%. When the concentrations of verapamil and Hoechst 33342 were 50 μmol/L and 5.0 μg/mL, respectively, the SP cells and non-SP cells of K1 cells were classified obviously, and the SP cells accounted for about 1.3%. The sorted SP cells and non-SP cells could be further cultured and passaged. Conclusion：The concentration of Hoechst 33342 and verapamil may influence the sorting efficiency of SP cells in tumor cells.

关 键 词：侧群细胞 流式细胞术 肿瘤干细胞 人肺腺癌细胞 

分 类 号：R734.2[医药卫生—肿瘤]