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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UMUC2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。
Objective To investigate the molecular mechanism of apoptosis induced by growth inhibitory factor (PML) in bladder tumor. Methods The recombinant eukaryotic expression vector PMEP4 / PML and the control PMEP4 empty vector were transfected into the bladder tumor cell line UMUC2 using lipofectamine 2000 respectively. The resistant clones stably expressing PML were screened by 300μg / ml hygromycin B . Laser confocal detection of PML protein expression. DNAladder assay for tumor cell apoptosis. Western blotting was used to detect the expression of apoptosis related proteins. Results After induced by CdSO4 5μmol / L, confocal microscopy showed that there were speckled spots of yellowish-green fluorescence in the nucleus of PMEP4 / PML transfected cells, but there was no specific Fluorescent blot expression. DNA ladder showed that the apoptotic ladder appeared in transfected PML cells 24 hours after the induction of PML expression. Western blotting was used to detect apoptosis related protein cysteine protease 3, activated PARP upregulation, and survivin expression downregulation. Conclusion The apoptosis of bladder tumor cells induced by PML may be related to upregulation of cysteine protease 3, activation of PARP protein and inhibition of survivin expression.