促凋亡因子Smac过表达增强胰腺癌Panc-1细胞化疗敏感性研究

目的探讨胞浆过表达促凋亡因子Sm ac对胰腺癌细胞Panc-1化疗敏感性的影响。方法应用RT-PCR获得Sm accDNA(不含线粒体定位序列),定向克隆入pEGFP-N1质粒载体中,经限制性酶切、PCR及测序鉴定。利用脂质体将pEGFP-N1/Sm ac重组体转染胰腺癌细胞株Panc-1,应用荧光显微镜检测Sm ac-EGFP绿色荧光融合蛋白表达;流式细胞仪测定顺铂、5-FU诱导的转染细胞凋亡率;四甲基偶氮唑蓝(MTT)法检测细胞生长抑制率。结果荧光显微镜下可见转染pEGFP-N1/Sm ac的细胞胞浆自发绿色荧光;在顺铂、5-FU作用下,N1/Sm ac转染组细胞凋亡率分别为(36.8±5.5)%、(47.6±6.9)%,明显高于未转染组(12.3±3.2)%、(19.2±4.7)%及空载体转染组(16.9±3.7)%、(22.5±5.8)%,其差异有统计学意义(P<0.01)。N1/Sm ac转染组细胞的生长抑制率亦明显高于未转染组及空载体转染组(P<0.01)。结论胞浆过表达Sm ac活性分子可明显促进顺铂、5-FU诱导的Panc-1细胞凋亡,增强其对化疗药物的敏感性,为探索基于凋亡的胰腺癌治疗新策略提供实验依据。

Cytosolic over-expression of Second Mitochondria-derived Activator of Caspases (Smac) increase chemotherapeutic sensitivity of pancreatic carcinoma cell line Panc-1

Objective To investigate the effects of second mitochondria-derived activator of caspases(Smac/DIABLO) on chemotherapeutic sensitivity of pancreatic carcinoma cell line Panc-1.Methods The cDNA fragment of Smac(without Mitochondrial targeting sequence) obtained by RT-PCR was inserted into a plasmid vector named pEGFP-N1 in the correct direction.The pEGFP-N1/Smac vector was confirmed using restriction enzyme digestion,PCR and DNA sequencing.The recombinant plasmid was transfected into Panc-1 cells using Lipofectamine reagent,and the expression of green fluorescence fusion proteins Smac-EGFP was determined by fluorescence microscopy.The cell apoptosis induced by cisplatin or 5-fluorouracil(5-FU) was analyzed by flow cytometry.Cellular proliferation was assayed by tetrazolium bromide(MTT) colorimetry.Results After the transfection of pEGFP-N1/Smac to Panc-1 cells,the cytosolic expression of green fluorescence protein was identified by fluorescence microscopy.Then Panc-1 cells were exposed to cisplatin or 5-FU.The apoptosis rate of pEGFP-N1/Smac transfected cells was significantly increased compared to that of untreated cells and control cells transfected with empty vector pEGFP-N1(P<0.01).The proliferation of N1/Smac-transfected Panc-1 cells was also significantly reduced(P<0.01).Conclusion Cytosolic overexpression of Smac/DIABLO in Panc 1 cells could markedly promote cisplatin or 5-FU induced apoptosis and increase its chemotherapeutic sensitivity.These results suggest that Smac may have potential therapeutic applications in pancreatic carcinoma based on its modulation of apoptosis.