半边旗中二萜类化合物5F诱导胰腺癌细胞凋亡机制的探讨

The Mechanism of Apoptosis of Human Pancreatic Cancer PC-3 Cell Line Induced by Diterpenoid Compound 5F Isolated from Pteris Semipinnata L

目的:观察研究半边旗中二萜类化合物5F(1la-羟基-15-氧-16-烯-ent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PUMA/siRNA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8.875μmol·L^(-1)、37.5μmol·L^(-1)、142μmol·L^(-1)的5F中作用72h。RT-PCR检测不同组细胞经5F作用24h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,Hoechst33258和PI双染色染色了解细胞凋亡的变化。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PUMA表达抑制后,受5F作用的细胞出现PUMA表达的降低,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。

This study aimed to reveal the effects of diterpenoid compound 5F （ent-lla-hydroxy-15-oxo-kaur-16-en- 19-oic acid） isolated from Pteris Semipinnata L. on apoptosis of pancreatic cancer cell line PC-3, and its working mechanism. PUMA/siRNA was transfected into AsPC-1 cells by Lipofectamine 2000 methods. Stably transfeeted colony was chosen through the G418.AsPC-1 cells treated with 10μL 5F at serial concentrations of 8.875, 37.5,142 μmol·L^-1. The MTT assay was used to observe the inhibitory actions of 5F on AsPC-1 cells. The apoptotic rate of the cells was detected by flow cytometry. The apoptosis was assessed by Hoechst 33258 and PI dye staining. The expression of PUMA mRNA was detected by RT-PCR. Cell growth, as well as apoptosis, was observed in the AsPC-1 control cells dealt with 5F. 5F inhibited the proliferation of AsPC-1 cells in a concentration-dependent manner. The apoptosis induced by 5F was accompanied by the up-regulation of PUMA. In PUMAAS cells treated with 5F, the apoptosis rate decreased greatly and the proliferation rate increased compared those in the AsPC-1 control cells. The expression of PUMA was decreased greatly compared with the control cells treated with 5F. The results showed 5F depresses the proliferation of AsPC-1 cells in vitro, mainly through the induction of apoptosis, which makes it a potential agent for pancreatic cancer therapy. The mechanism is probably related to its effect on the regulation of PUMA a expression