中文摘要：

肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis-inducing ligand,TRAIL）对癌细胞有独特的细胞毒性作用,而对正常细胞没有影响.但乳腺癌细胞耐受TRAIL诱导凋亡.本研究探索磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase,PI3K）信号通路对人乳腺癌MCF-7细胞耐受TRAIL的影响.采用MTT法、显微照相以及DAPI染色观察TRAIL对MCF-7细胞生长的抑制作用以及诱导细胞凋亡状况;流式细胞分析细胞凋亡的情况;激光共聚焦显微镜观察多聚ADP核糖多聚酶-1（poly（ADP-ribose）polymerase-1,PARP-1）的迁移和定位;Western印迹分析死亡受体、caspase-3/8、磷酸化的AKT[pAKT（Ser473）]、Src和PARP-1等蛋白质表达.结果显示,小剂量TRAIL（〈80 nmol/L）和Ly294002（〈40μmol/L）对MCF-7细胞生长没有显著的抑制作用,但是大剂量TRAIL（160 nmol/L）和Ly294002（80μmol/L）则能抑制MCF-7细胞生长;低剂量Ly294002协同TRAIL抑制MCF-7细胞生长,并诱导细胞凋亡;Ly294002和TRAIL共同作用能促进PARP-1从胞浆进入细胞核;蛋白质表达分析显示,MCF-7细胞均表达死亡受体DR4、DR5、诱骗受体DcR1和DcR2、以及caspase-8,但是不表达caspase-3;Ly294002和TRAIL共同作用也能抑制pAKT（Ser473）和Src的表达,并且导致PARP-1断裂.本研究结果提示,抑制PI3K信号可增加MCF-7细胞对TRAIL诱导的敏感性;MCF-7细胞通过PI3K/AKT途径促进Src的表达耐受TRAIL的细胞毒性作用;Ly294002联合TRAIL是一种新的药物组合方式治疗乳腺癌.

英文摘要：

Tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） is a member of the tumornecrosis factor （TNF） superfamily and has been shown to induce extrinsic pathway of apoptosis in manytypes of cancer cells, but in breast cancer MCF-7 cell it displayed resistance to cytotoxicity of TRAIL.This investigation aim to study the effect of phosphatidylinositol 3-kinase （PI3K） signal pathway on MCF-7 cells resisted the cytotoxicity of TRAIL. Human breast cancer MCF-7 cells were treated with TRAIL orLy294002 alone/in combination. MTT cell viability assay was used to evaluate growth of the ceils. Forshowing the change of morphology and nucleolus in MCF-7 cells, both microscopical photograph and 4,6-diamino-2-phenylindole（DAPI） stained detects were performed. Flow cytometry was applied to analyzethe apoptosis of the cells; Laser confocal microscopy was applied to observe location and migration of poly（ADP-ribose） polymerase -1 （ PARP-1 ） and Western blot analysis was conducted to evaluate DR4, DRS,DcR1, DcR2, caspase-3/8, Src and pAKT（Ser473） protein levels. The results indicated that sequentialtreatment of small dose of Ly294002 （〈40 μmol/L） or TRAIL （ 〈80 nmol/L） had little suppressiveimpact on the growth, but Ly294002 （80 μmol/L） or TRAIL（ 160 nmol/L） significant inhibited theproliferation of MCF-7 ceils. Pretreated with Ly294002 （20μmol/L） followed treated with TRAIL （40nmol/L） resulted in significant synergistic cytotoxicity and apoptosis, suggesting that MCF-7 ceils wereunder strong apoptotic stimuli. Western blot assay showed that MCF-7 expressed DR4, DR5, DcR1,DcR2, caspase-8. Pretreatment of MCF-7 cells with Ly294002 （20 μmol/L） and followed treat withTRAIL （40 nmol/L） not only promoted PARP-1 to enter cytoblast, but also suppressed the expression ofSrc and pAKT （Ser473） in the cancer cells. These findings strongly suggest that inhibition of PI3K/AKTsignal enhances sensitivity of MCF-7 ceils to TRAIL; MCF-7 cells resistance the apoptosis in