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目的:观察研究半边旗中二萜类化合物5F(11a-羟基-15-氧-16-烯-贝壳杉烷-19酸)诱导人胰腺癌细胞株AsPC-1凋亡的效果及其机制。方法:用脂质体转染法将P53E向凋亡之因子基因(PUMA)反义核酸(反义PUMAcDNA)的真核表达载体pcDNA3.1-PUMAAS和空载体pcDNA3.1-导入AsPC-1细胞,G418筛选阳性细胞,获得稳定转染的阳性克隆。将转染载体的AsPC-1阳性克隆细胞和未转染载体的AsPC-1细胞分别暴露于浓度为8.875,37.5,142μmol/L的5F中作用72h。RT-PCR和Western blotting检测不同组细胞经5F作用72h后的PUMA表达;MTT检测细胞生长抑制,流式细胞仪(FCM)检测细胞凋亡,Hoechst 33258荧光染色法和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法检测细胞的凋亡。结果:5F促进AsPC-1细胞凋亡,抑制细胞生长,并有明显的剂量依赖性,在细胞凋亡的同时伴有PUMA表达的上调;当细胞转染PUMA反义核酸抑制PUMA表达后,受5F作用的细胞出现PUMA蛋白表达明显降低,同时伴有细胞凋亡的抑制及细胞的明显增殖。结论:5F促进体外AsPC-1细胞凋亡,并抑制其生长,其诱导凋亡与上调PUMA有关。
Objective: To observe the effect and mechanism of apoptosis of AsPC-1 induced by diterpenoid 5F (11a-hydroxy-15-oxo-16-kauretic-19 acid) in Pancreaticus. Methods: The eukaryotic expression vector pcDNA3.1-PUMAAS of P53E was transfected into AsPC-1 cells by lipofectamine (antisense PUMA) and empty vector pcDNA3.1- Positive cells were screened by G418 to obtain stable transfected positive clones. AsPC-1 positive clones transfected with vector and AsPC-1 cells untransfected were exposed to 5F at concentrations of 8.875, 37.5 and 142μmol / L for 72h. The PUMA expression in different groups was detected by RT-PCR and Western blotting at 72h after treatment with 5F. The growth inhibition was detected by MTT assay. The apoptosis was detected by flow cytometry (FCM), Hoechst 33258 fluorescence staining and terminal deoxynucleotidyl transferase Mediated dUTP nick end labeling (TUNEL) method to detect cell apoptosis. Results: 5F promoted the apoptosis of AsPC-1 cells and inhibited the growth of the cells in a dose-dependent manner. In addition, 5F up-regulated the apoptosis of the cells while inhibiting the expression of PUMA. 5F-induced PUMA protein expression was significantly reduced, accompanied by inhibition of cell apoptosis and cell proliferation. CONCLUSION: 5F can promote the apoptosis of AsPC-1 cells and inhibit the growth of AsPC-1 cells in vitro. The induction of apoptosis is related to the up-regulation of PUMA.