喉癌中侧群细胞的分选及其体外生物学特性

Isolation and characterization in vitro of side population cells in laryngeal carcinoma cell line

目的:探讨喉癌细胞株Hep-2中干细胞样侧群(SP)细胞的分选方法及其干细胞特性。方法:制备喉癌Hep-2单细胞悬液,经Hoechst 33342染色后,用荧光激活细胞分选技术分选出人喉癌SP和非侧群(NSP)细胞,测定SP细胞亚群所占的比例。电子显微镜下观察SP及NSP细胞形态,检测两群细胞的细胞周期、CCK8检测细胞的增殖能力。荧光定量PCR检测SP及NSP细胞中的ABCG2mRNA的相对量,结果用2-ΔΔCt计算。结果:流式细胞仪分析结果表明,SP细胞在Hep-2细胞株中占(13.17±1.85)%。生长曲线(CCK8法)示,SP细胞较NSP细胞有更强的增殖能力;两者所处的细胞周期相似,S期细胞比例及增殖指数两者相比,t分别为0.678、0.614(P<0.05)。荧光定量PCR示SP细胞中ABCG2mRNA含量高于NSP细胞。结论:人Hep-2细胞株中存在一部分能将Hoechst33342排出细胞外的SP细胞。SP细胞较NSP有更强的增殖及体内成瘤能力,具有干细胞活性。SP细胞中ABCG2mRNA表达量明显高于NSP细胞。

Objective：To explore a feasible way to detect the tumor stem-like ceils in laryngeal carcinoma cell- line Hep-2,and to anlaIyze the properties of the side population （SP） cells being sorted. Method：After the single- cell suspension of Hep-2 was prepared,SP and non-SP （NSP） cells were sorted by fluorescence-active cell sorting （FACS） using Hoechst33342 staining. Then measured the percentage of SP cells, and the two sub-population cells were observed by electron microscopy. Their cell-cycle, ability of proliferation were detected after sorting. The ABCG2 mRNA in SP and NSp cells were detected using quantitive realtime polymerase chain reaction（qPCR）, then analysed with 2-AACto Result：SP cells were isolated from Hep-2 cells in a proportion of （13. 174-1.85）~ with re- spect to the whole cell population. Cell growth curve indicated that proliferative capacity of the SP cells was better than the NSP cells（P〈0.05）. The S-phase and proliferation index of the two sub-populations are similar. The qPCR shows that he ABCG2 had higher expression in SP cells than in NSP cells. Conclusion： Human Hep-2 cell line contains a subpopulation of cells excluding Hoechst33342 dye. Contained. The SP cells has better proliferative capacity in vitro and stronger tumorigenicity than the NSP cells. Meanwhile, the ABCG2 had higher expression in SP cells.