胰腺癌干细胞的体外增殖与鉴定

目的 建立胰腺癌干细胞体外增殖体系并鉴定所获细胞.方法 ASPC-1胰腺癌细胞以1000细胞/ml的密度培养于含碱性成纤维生长因子、表皮生长因子、胰岛素、转铁蛋白、硒及牛血清白蛋白的DMEM-F12培养基中.待培养10～14 d,部分ASPC-1细胞增殖为细胞球后,收集细胞球进行鉴定.鉴定方法包括克隆形成实验、Hoechst 33342染料染色、CD44抗体免疫细胞化学染色以及实时定量逆转录PCR检测CD44 mRNA水平.结果 小部分ASPC-1细胞可在干细胞体外增值体系中增殖为细胞球.此体系增殖所获细胞中能够排出Hoechst 33342染料的细胞比例远高于普通培养体系中的细胞(>95%v8 2%～3%),且具更高的克隆形成能力[(10.92±3.38)%比(10.92±3.38)%,P<0.01].此外,此增值体系中所获细胞CD44表达率更高[(86.00 ±5.85)%比(18.71 ±3.64)%,P<0.01].结论 部分ASPC-1细胞可在本研究所建立的干细胞体外增殖体系中增殖,增殖所获细胞具有肿瘤干细胞特性.

Propagation and identification of pancreatic cancer stem cells in vitro

Objective To establish an assay in which a subset of pancreatic adenocarcinoma cell line ASPC-1 cells with stem cell properties can propagate, and identify cells obtained from this assay. Methods ASPC-1 cells were cultured in DMEM-F12 supplemented with EGF,bFGF,iusulin,transferrin, selenium and BSA at a density of 1000 cells/ml for 10 to 14 days to form spheres. Clone formation assay was used to identify the ability of propagation in vitro. Cells and spheres were stained by Hoechst 33342 dye to evaluate their capacity of excluding Hoechst dye. The expression of CD44 mRNA and protein was detected by using Real time PCR and immunocytochemsitry respectively. Results A subpopulation of AS-PC-1 cells could porpagte to form spheres in this assay. Almost all cells of obtained spheres had the hall-mark of excluding Hcechst 33342 dye,whereas only 2%-3% ASPC-1 cells cultured in normal condition.Colony forming efficiency of PANC-1 spheres was higher than that of ceils cultured in normal condition in vitro [(10.92 ± 3.38) % vs (10.92 ± 3.38) %, P < 0.01]. The CD44 protein expression of cells in AS-PC-1 spheres was higher than in those cultured in normal condition [(86.00±5.85)% vs (18.71 ±3.64) % ,P < 0.01]. Conclusion A subpopulation of ASPC-1 cells can propagate to form spheres with properties of stem cells in this assay.