TLR4 shRNA质粒的构建及稳定转染人胰腺癌PANC1细胞株的筛选  

Construction of TLR4 shRNA plasmid and screening of human pancreatic cancer PANC1 cell line with stable transfection

作　　者：张建军[1,2] 王博[2] 田元[2] 张景辉[2] 吴河水[2] 

机构地区：[1]广东医学院附属南山医院胃肠外科,深圳518052 [2]华中科技大学同济医学院附属协和医院胰腺外科中心

出　　处：《中华胰腺病杂志》2012年第3期181-183,共3页Chinese Journal of Pancreatology

基　　金：国家自然科学基金（30972898）

摘　　要：目的构建靶向人TLR4基因的短发夹RNA（shRNA）真核表达质粒，转染胰腺癌细胞，并筛选出稳定转染的克隆细胞株。方法采用小分子干扰RNA（siRNA）软件设计3个靶向TLR4基因的shRNA，构建3个质粒表达载体，分别命名为TLR4-1、TLR4-2、TLR4—3；选取抑制效率最高的shRNA质粒，采用脂质体法转染胰腺癌PANC1细胞；实时定量PCR和流式细胞技术检测细胞TLR4shRNA基因沉默效率和转染效率；G418抗性筛选和有限稀释单克隆形成法挑选培育稳定转染TLR4shRNA的单克隆细胞系。结果转染48h后PANC1细胞瞬时转染效率为（46．72±5．06）％。转染_rLR4．1、TLR4-2、TLR4—3的细胞的TLR4mRNA表达水平分别为0．025±0．004、0．027±0．003、0．019±0．006，均显著低于未转染组的0．061±0．018和阴性对照转染组的0．057±0．015（P值均〈0．05）。以沉默效果最佳的TLR4—3转染细胞所获得的稳转细胞的转染效率为（82．79±8．16）％，较瞬转细胞显著提高（P=0．001）；TLR4mRNA表达水平为0．010±0．002，较瞬转细胞显著下调（P：0．001）；TLR4蛋白表达率为（0．54±0．32）％，显著低于未转染细胞的（87．42±5．00）％和转染阴性对照细胞的（82．90±5．00）％（P=0．000）。结论成功筛选到TLR4基因沉默的稳转PANC1细胞。Objective To construct the eukaryotic plasmid expression vector mediated short hairpin RNA（shRNA） interference targeting TLR4 gene, and transfect it into pancreatic adenocarcinoma cell line PANC1, then screen stably transfected clonal cell line. Methods Three shRNA interference expression plasmid vectors targeting the TLR4 gene were constructed, named TLR4-1, TLR4-2, TLR4-3. The shRNA plasmid with highest inhibitory efficiency was selected and transfected into PANC1 cells with liposome. The silencing efficiency and transfection efficiency of TLR4-shRNA was assayed with real-time quantitative PCR and flow cytometry analysis. Monoclonal cell with stable transfection of TLR4-shRNA were selected by geneticin 418 （G418） and limiting dilution analysis. Results Transient transfection efficiency of PANC1 was （46.72 ＋ 5.06） %. TLR4 mRNA expressions were 0. 025 ± 0. 004,0. 027 ±0. 003,0.019 ± 0. 006 in cells transfected with TLR4-1, TLR4-2, TLR4-3, respectively, which were significantly lower than that in untransfected group （0. 061± 0.018） and negative control group （0. 057 ± 0.015, P 〈 0.05 ）. The transfection efficiency of TLR4-3 vector in stably transfected clones [ （82.79 ± 8.16）% ] was significantly higher than that of transient transfection （P =0. 001 ）. The expression of TLR4 mRNA was decreased to 0.010 ± 0. 002, which was significantly lower than that of transient transfection （ P = 0.001 ）. The expression of TLR4 protein was （0.54 ±0.32） %, which was significantly lower than that negative control [ （82.9 ±5.00 ） %, P = 0.000 ]. TLR4 gene silencing are successfully identified. of untransfected cells [ （ 87.42 ± 5.00） % ] and that of Conclusions Stable transfection PANC1 cell lines with

关 键 词：胰腺肿瘤 TOLL样受体4 转染 RNA干扰 

分 类 号：R735.9[医药卫生—肿瘤]