人参皂苷Rg3对人胰腺癌细胞Pim-3及Bad凋亡蛋白表达的影响

背景与目的：人参皂苷Rg3是一种抗肿瘤中药单体，有研究表明人参皂苷Rg3对肝癌、肺癌、结肠癌、胰腺癌等肿瘤细胞的浸润具有明显抑制作用。本实验目的是研究人参皂苷Rg3对人胰腺癌细胞株PANC-1中原癌基因Pim-3及磷酸化Bad蛋白pBad（Ser112）、Bad（Serl36）表达的影响。方法：用浓度为0、10、20、40和80μmol／L的人参皂苷Rg3处理PANC-1细胞24h后，用四甲基偶氮唑盐（MTF）法检测人参皂苷Rg3对PANC-1细胞增殖的抑制作用；用倒置显微镜和流式细胞术观察人参皂苷Rg3对PANC-1细胞凋亡的诱导作用；用Western blot法检测经不同浓度人参皂苷Rg3处理后的PANC-1细胞中pim-3、Bad、pBad（Ser112）和pBad（Ser136）的表达；定向克隆Pim-3的发夹状寡核苷酸（shorthairpinRNA，shRNA）到真核表达载体形成重组质粒pSilencer3．1-HINeo—Pim-3shRNA．将重组质粒通过脂质体介导转染PANC-1细胞，采用AnnexinV／PI流式细胞分析法检测转染后PANC-1细胞的凋亡．用Westernblot法检测转染后PANC-1细胞的pim-3及pBad（Serl12）的表达。结果：10、20、40和80btmol／L的人参皂苷Rg3对PANC-1细胞增殖的抑制率分别为20．2％、33．4％、52．8％、65．3％；经10～80μmol／L的人参皂苷Rg3处理后PANC-1细胞呈现明显的凋亡形态学改变，80μmol／L人参皂苷Rg3处理PANC-1细胞24h后，与正常对照组细胞凋亡率（3．3±2．1）％比较，处理组细胞凋亡率（12．2±1．3）％增加（P〈0．05）；人参皂苷Rg3处理PANC-1细胞24h后，Bad总蛋白表达没有明显变化。pim-3和pBad（Ser112）的表达均随人参皂苷Rg3浓度的增加而逐渐减弱。PANC-1细胞中pBad（Ser136）不表达；Pim-3shRNA转染PANC-1细胞后．与正常对照组比较，转染组早期凋亡率和总凋亡率均增加（11．5±3．7）％VS．（5．8±2．2）％，P〈0．01；（20．8±2．6）％VS．（13．0±4．1）％，P〈O．05]，转染组pim-3及pBad（S

Effect of ginsenoside Rg3 on Pim-3 and Bad proteins in human pancreatic cancer cell line PANC-1

Background and Objective： Ginsenoside Rg3 is a traditional Chinese medicine monomer which possesses anticancer effects. This study was to investigate the effects of ginsenoside Rg3 on Pim-3 and phosphorylated Bad （pBad） proteins, pBad （Ser112） and pBad （Ser136） in human pancreatic cancer cell line PANC-I. Methods= PANC-1 cells were exposed to 10, 20, 40 and 80 pmol/L ginsenoside Rg3 for 24 h. A short hairpin RNA （shRNA） of Pim-3 was cloned and inserted into a eukaryotic expression vector pSilencer 3.1-H1 Neo to construct pSilencer 3.1-H1 Neo- Pim-3. pSilencer 3.1-H1 Neo-Pim-3 was then transfected into PANC-1 cells. Cell proliferation was measured by MTT- assay; cell apoptosis was observed under an invert microscope and measured by flow cytometry with Annexin V/ PI staining; protein expressions of Pim-3, Bad, pBad （Ser112） and pBad （Ser136） were measured by Western blot. Results： The inhibitory rates of 10, 20, 40 and 80μmol/L ginsenoside Rg3 on PANC-1 cells were 20.2%, 33.4%, 52.8% and 65.3%, respectively. Typical morphological changes in apoptosis were induced by ginsenoside Rg3. The apoptotic rate of PANC-1 cells was significantly higher in the ginsenoside Rg3 treatment group （80 μmol/L） than in the control group （12.2% vs. 3.3%, P〈0.05）. Ginsenoside Rg3 had no influence on the total Bad protein expression, but decreased both Pim-3 and pBad （Ser112） expressions in a dose-dependent manner, pBad （Ser136） was not expressed in PANC-1 cells. Compared with the control group, the percentages of early and total apoptotic cells were significantly increased in PANC-1 cells transfected with pim-3-shRNA E （11.5±3.7）% vs. （5.8±2.2）%, P〈0.01;（20.8±2.6）% vs.（13.0±4.1）%,P〈0.05], while the expressions of pim-3 and pBad （Ser112） were both decreased. Conclusion： The anti- tumor effect of ginsenoside Rg3 may be associated with the decrease of Pim- 3 and pBad （Ser112）.