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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 investigate the method for the selection of stem cell-like population (SP) cells and the characteristics of stem cells in laryngeal carcinoma cell line Hep-2. Methods: Hep-2 single cell suspension of laryngeal cancer was prepared. After stained with Hoechst 33342, human laryngeal squamous cell carcinoma SP and non-side cell (NSP) cells were sorted by fluorescence-activated cell sorting. proportion. The morphology of SP and NSP cells was observed under an electron microscope. The cell cycle of the two groups of cells was examined. The proliferation of cells was detected by CCK8. The relative amount of ABCG2 mRNA in SP and NSP cells was detected by fluorescence quantitative PCR and the result was calculated by 2-ΔΔCt. Results: The results of flow cytometry analysis showed that the percentage of SP cells in Hep-2 cell line was (13.17 ± 1.85)%. Growth curve (CCK8 method) showed that SP cells had stronger proliferative ability than NSP cells. The cell cycle of the two cells was similar, the proportion of cells in S phase and proliferation index were 0.678,0.614 (P < 0.05). Fluorescent quantitative PCR showed that ABCG2 mRNA content in SP cells was higher than that in NSP cells. Conclusion: There is a part of human Hep-2 cell line which can excrete Hoechst33342 into extracellular SP cells. SP cells have stronger proliferation and in vivo tumorigenicity than NSP and have stem cell activity. The expression of ABCG2 mRNA in SP cells was significantly higher than that in NSP cells.