生长抑制因子诱导膀胱肿瘤细胞凋亡的研究

目的探讨生长抑制因子(PML)诱导膀胱肿瘤细胞凋亡的分子机制。方法应用脂质体Lipofectamine2000分别将重组可诱导表达的真核表达载体PMEP4／PML和对照PMEP4空载体转染到膀胱肿瘤细胞系UM-UC-2中,300μg／ml潮霉素B筛选稳定表达PML的抗性克隆。激光共聚焦检测PML蛋白表达。DNAladder法检测肿瘤细胞凋亡。Western印迹法检测凋亡相关蛋白表达情况。结果经5μmol／L的CdSO4诱导表达后,激光共聚焦显微镜观察发现转染PMEP4／PML组的细胞核内有散在的斑点样的黄绿色荧光亮点,而转染空载体PMEP4组细胞未见特异性的荧光斑点表达。DNA梯度法显示转染PML细胞在诱导PML表达后24h出现梯状凋亡条带。Western印迹法检测凋亡相关蛋白半胱氨蛋白水解酶3、活化PARP上调,而survivin的表达水平下调。结论PML诱导膀胱肿瘤细胞凋亡可能与上调半胱氨蛋白水解酶3、活化PARP蛋白,抑制survivin的表达有关。

The molecular mechanism of PML in inducing apoptosis of bladder cancer cell line

OBJECTIVE: Study the affection of inducing expression of PML in the apoptosis and the molecular mechanism of bladder cancer. METHODS: PMEP4/PML inducible expression vector was transfected into bladder cancer UM-UC-2 cells by lipofectamine 2000 system. The positive clone cells were selected by 300 microg/ml hygromycin B and confirmed by laser confocal imaging system. Then, using the in vitro DNA ladder apoptotic assay and Western blot, the affection of inducing expression of PML on apoptosis and its molecular mechanism of bladder cancer cell was studied. RESULTS: Comparing with the vector control group, PML specific nuclear speckle significantly increased in the PMEP4/PML bladder cells. DNA ladder assay demonstrated bladder cancer cell expressing PML occurred apoptosis while the control vector cells were not influenced. Overexpression of PML could reduce Survivin expression and upregulate caspase3 and cleaved PARP protein expression CONCLUSION: Our results demonstrated that overexpression of PML could induce bladder cancer cell apoptosis through the caspase dependent pathways.