中文摘要：

目的分离鉴定胰腺癌中肿瘤干细胞样的侧群（SP）细胞亚群并探讨磷脂酰肌醇3-激酶／雷帕霉素靶蛋白（PBK／mTOR）信号通路对其生存与增殖的调控。方法应用流式分析检测5个胰腺癌细胞系中SP细胞的含量。观察加入PI3K／mTOR信号通路特异性抑制剂LY294002或雷帕霉素培养后胰腺癌细胞PANC-1中SP细胞的含量变化。通过平板集落形成试验，NOD—SCID小鼠异种移植成瘤试验和移植瘤的SP再次分析评价PANC-1 SP细胞的自我更新和分化潜能。采用MTr试验和集落形成试验检测LY294002或雷帕霉素对分选的SP细胞和非SP细胞的抑制作用。结果除了BXPC-3，其他胰腺癌细胞系都存在维拉帕米敏感的SP细胞。SP细胞具有较高的集落形成能力（SP细胞：43．7％±3．1％，非SP细胞8．3％±1．6％，P=0．000）和成瘤能力（至少100倍于非SP细胞），并能够发生不对称分裂生成非SP细胞。加入LY294002或雷帕霉素培养后PANC-1中SP细胞的含量明显降低（LY294002，7．60％±0．27％ vs 1．90％±0．22％，P=0．000；雷帕霉素，7．60％±0．27％ vs 1．14％±0．20％，P=0．000）。与非SP细胞相比，LY294002及雷帕霉素均优先抑制SP细胞。结论SP细胞富集胰腺癌肿瘤干细胞。PBK／mTOR信号通路参与对其生长增殖的调控，可能成为根治胰腺癌的治疗新靶点。

英文摘要：

Objective To isolate and identify the side population （SP） cells in pancreatic cancer and investigate the role of phosphatidylinositol 3-kinase/mammalian target of rapamycin （PI3K/mTOR） pathway in the survival and proliferation of them. Methods Pancreatic cancer cell of the lines PANC-1, BXPC-3, ASPC-1, PC-3, and SW-1990 were cultured. Hoechst 33432 staining and fluorescence-activated cell sorter （FACS） were used to sort the SP cells. Verapamil, inhibitor of ATP binding cassette （ABC） transporter,was added before the Hoechst 33342 inoculation to observe its influence on the SP proportion. Media with LY294002, specific inhibitor of P13K, or rapamycin, specific inhibitor of mammalian target of rapamycin （mTOR）, were used to culture PANC-1 cells to observe the survival of cells. Twenty-one NOD- SCID mice were randomly divided into 7 equal groups. Four groups were inoculated subcutaneously with SP cells of the concentrations of 5 × 10^5, 5 × 10^4, 5 × 10^3, or 1 × 10^3 at the right axillary fossa and with non-SP ceils at the left then Hoechst 33342 staining, flow cytometric sorting were used to detect the content of SP cells at the left axillary fossa. The other 3 groups were injected subcutaneously with non-Hoechst 33342 treated cells of the concentrations of 5 × 10^5, 5 × 10^4, 5 × 10^3, or 1 × 10^3 at the right axillary fossa and PBS at the left axillary fossa. Ten weeks later the mice were killed to undergo pathological examination. Results All cell lines were found to exhibit verapamil-sensitive SP cells except BXPC-3 cells. The SP cell proportion of the PANC-1 cells was 7.84%. No SP cell was found in the cells treated with verapamil. The colonyformation ability of the SP cells was （43.7 ± 3.1 ） %, significantly higher than those of the non-SP cells and cells without Hoechst 33342 cells [ （8.3 ±1.6） % and （ 10.2 ± 1.9） % respectively, both P = 0. 000 ]. The tumorigenic ability of the SP cells was 100 times as those of the non-SP cells and Hoechst 33342 untreated cells.