摘要
目的研究吉西他滨体外对人胰腺癌AsPC-1细胞生长的作用机制。方法用脂质体转染法将p53正向凋亡调控因子(PUMA)反义核酸(反义PUMA cDNA)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1、5、10和15μmol/ml的吉西他滨中作用72 h。RT-PCR和Western blotting检测不同组细胞经吉西他滨作用72 h后的PUMA表达;MTT检测细胞生长抑制,流式细胞仪(FCM)、Hoechst 33258荧光染色法和TUNEL法检测细胞凋亡。结果吉西他滨促进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,受吉西他滨作用的细胞出现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。结论吉西他滨促进体外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 investigate the effect of proliferation and apoptosis in pancreatic cancer patients treated by gemcitabine. Methods pcDNA3.1-PUMAAS and pcDNA3.1- was transfected into AsPC-1 cells by Lipofectamine 2000 methods and stable transfected clony was chosen through G418. AsPC-1 cells were divided into transfected pcDNA3. 1- PUMAAS group, transfected pcDNA3. 1- group and control group without transfection. All AsPC-1 cells in three groups were treated with 10 μl efial concentrations （ 1, 5, 10 and 15 μmol/ml） of gemcitabine. MTT assay was used to observe the inhibitory actions of gemcitabine on AsPC-1 cells. The apoptotic rate was detected by using flow cytometry. The apoptosis was assessed by means of Hoechst 33258 dye staining and TUNEL staining. The expression of PUMA protein was detected by using westem blot and RT-PCR. Results The inhibitory action of cell growth was seen in AsPC-1 control cells and pcDNA3.1- cells dealing with gemcitabine as well as the promoting of apoptosis. Gemcitabine inhibited the proliferation of AsPC-1 cells in a concentration-dependent manner. The apoptosis induced by gemeitabine was accompanied with the upregulation of PUMA. In pcDNA3.1-PUMAAS cells treated with gemcitabine, the apoptosis rate was significantly decreased and the proliferation rate was increased compared with the AsPC-1 control cells and pcDNA3.1- cells. The expression of PUMA was significantly decreased in pcDNA3.1-PUMAAS cells than that of pcDNA3.1- cells. Conclusion Gemcitabine can depress the proliferation of AsPC-1 cells in vitro mainly through the induction of apoptosis and the mechanism is probably related to its effect on the regulation of PUMA.
出处
《山东医药》
CAS
北大核心
2009年第32期13-16,共4页
Shandong Medical Journal
基金
江苏省科学研究基金资助项目(JS2007-021)
