目的 从人前列腺癌PC-3细胞系中分离、富集并鉴定前列腺癌干细胞,探讨具有肿瘤干细胞特性的侧群(SP)细胞与未分选的总体细胞(NST)之间的生物学行为差异.方法 采用无血清培养液(SFM)悬浮细胞聚球法体外培养PC-3 细胞系.以PC-3悬浮成球细胞为实验组,常规以含血清培养液(SSM)中单层贴壁培养的PC-3细胞为对照组,通过流式细胞技术检测两种细胞中CD44+、CD133+细胞及SP细胞比例.通过生物学功能实验比较两种细胞的增殖、血管生成、迁移、侵袭能力的差异.结果 PC-3成球细胞可以在SFM中生存并形成悬浮细胞球.PC-3悬浮成球细胞具有自我更新和分化能力,在体外可以连续、稳定传代,在SFM中滴加血清后可以分化,交替培养于SSM和SFM中可以实现与贴壁细胞之间的相互转化.PC-3悬浮成球细胞中CD44+、CD133+细胞及SP细胞比例均显著高于常规培养的PC-3贴壁细胞(P<0.05).相对于NST细胞,SP细胞具有更强的迁移、侵袭和血管生成能力,而SP细胞增殖能力则相对较弱.RT-qPCR和Western blot实验发现SP细胞中缺氧诱导因子2α(HIF-2α)、血管生成因子(VEGF)和基质金属蛋白酶(MMP)基因和蛋白水平均高于NST细胞.结论 PC-3悬浮成球细胞具有肿瘤干细胞特性,采用无血清悬浮细胞聚球培养法可以从PC-3 细胞系中简便、高效地富集前列腺癌干细胞.从人前列腺癌PC3细胞系分离出的SP细胞的血管生成、迁移、侵袭能力均强于NST细胞,而增殖能力却弱于NST细胞.%Objective To isolate, enrich and identify prostate cancer stem cells in human prostate cancer PC-3 cells, and investigate the difference in biological behaviors between side population (SP) cells with tumor stem cell characteristics and non-sorted total (NST) cells. Methods PC-3 cells were cultured in vitro by SFM suspension cell agglutination. The suspension sphere-forming PC-3 cells were set as the experimental groups and the single-layer adherent PC-3 cells cultured with SSM conventionally were set as the control groups. The percentages of CD44+CD133+cells and side population(SP) cells in the two kinds of cells were detected with Flow Cytometry (FCM). Differences in proliferation, angiogenesis, migration, and invasiveness between the two cell types were compared by biological functional experiments. Results The PC-3 sphere-forming cells could survive in SFM and form floating cell spheres successfully. The PC-3 sphere-forming cells exhibited the abilities of self-renewal and differentiation, and they grew well in vitro with continuous and stable passage. They can differentiate after adding serum in SFM, and achieved with the transformation of adherent cells by alternating culture in SSM and SFM. The percentages of CD44+CD133+cells and SP cells in the PC-3 floating sphere-forming cells were higher significantly than that of the PC-3 adherent cells (P<0.05). Compared with NST cells, SP cells presented stronger abilities of migration, invasion and angiogenesis. However, SP cells had a relatively weaker proliferative capacity. RT-qPCR and Western blot showed that the gene and protein levels of hypoxia-inducible factor 2α (HIF-2α), angiogenic factor (VEGF) and matrix metalloproteinases (MMPs) in SP cells were higher than those in NST cells. Conclusion The PC-3 sphere-forming cells possessed some properties of cancer stem cells. Using the suspension culture method with SFM, we isolated and enriched conveniently and effectively prostate cancer stem cells in PC-3 cells. The SP cells isolated from PC3 cells had stronger ability of angiogenesis, migration and invasion than NST cells, but their proliferative capacity was weaker.
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