靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖  

iRNA targeting Survivin coupling with gemcitabine suppressing proliferation of human pancreatic cancer cells

作　　者：刘文松[1] 燕海姣[2] 秦仁义[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院普外科,武汉430030 [2]中山大学肿瘤防治中心

出　　处：《中华实验外科杂志》2009年第7期849-851,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30471693）

摘　　要：目的构建靶向Survivin基因的siRNA真核表达载体，观察其对吉西他滨化疗抑制胰腺癌Panc-1细胞增殖的影响。方法构建靶向Survivin基因的siRNA真核表达载体psiRNA—Survivin，行酶切和测序鉴定。用重组质粒转染Panc-1细胞并筛选稳定转染的细胞株，绘制细胞生长曲线。采用逆转录-聚合酶链反应（RT—PCR）、Westernblot检测Survivin的mRNA和蛋白表达变化。以吉西他滨分别作用于对照组和转染组Panc-1细胞24h，噻唑蓝（MTr）比色法检测细胞的增殖，流式细胞仪检测细胞的凋亡。结果酶切和测序鉴定表明，成功构建了psiRNA-Survivin重组质粒。重组质粒稳定转染胰腺癌细胞株后，Survivin的mRNA和蛋白表达分别下调了79．2％和83．6％（P〈0．05），生长曲线明显变平缓，并能显著增强吉西他滨对Panc-1细胞的增殖抑制[（24．6±4．5）％／（38．7±5．2）％]和凋亡诱导作用[（16．7±2．5）％／（26．8±3．4）％，P〈0．05）。结论构建Survivin的siRNA表达载体可明显下调Survivin的表达，抑制Panc-1细胞增殖，并能提高细胞对吉西他滨的化疗敏感性。Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h, and then the growth inhibition rate was measured by MTT assay, and cell apoptosis rate was measured by flow cytometry. Resuits The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ （24.6 ± 4.5 ） % / （38.7 ± 5.2） % ] and apoptosis rate of these cells [ （ 16.7 ± 2.5 ） % / （26.8 ± 3.4） % ] were significantly increased after treatment by gemcitabine （P 〈 0.05）. Conclusion The constructed siRNA eukaryotic expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly, and increase the chemotherapy sensitivity to gemcitabine.

关 键 词：胰腺肿瘤 SURVIVIN SIRNA 

分 类 号：R735.9[医药卫生—肿瘤]