RNA干扰下调NF-κB p65基因表达诱导胰腺癌细胞株PANC-1的凋亡【荐】.docVIP

RNA干扰下调NF-κB p65基因表达诱导胰腺癌细胞株PANC-1的凋亡 作者：王殿忠 作者单位：南京医科大学鼓楼临床医学院 普通外科，江苏 南京 210008 【摘要】 目的 运用RNA干扰(RNA interference，RNAi)技术下调胰腺癌细胞株PANC-1中NF-ΚB p65基因表达，并评价其对肿瘤细胞凋亡的影响。方法 利用阳离子脂质体LipofectamineTM 2000将化学合成的人NF-ΚB p65的小干扰RNA(small interference RNA，siRNA)转染入胰腺癌细胞株PANC-1中。采用RT-PCR法测定细胞内NF-ΚB p65mRNA的表达，酶联免疫吸附测定法(ELISA)检测NF-ΚB亚单位p65的DNA结合活性的改变，运用Hoechst33258核染色、流式细胞仪和透射电子显微镜检测p65基因表达下调对PANC-1细胞凋亡的影响。结果 化学合成的人NF-ΚB p65 siRNA能有效地抑制PANC-1细胞中NF-ΚB p65mRNA的表达(P0.01)，同时ELISA结果显示，RelA siRNA组的p65亚单位与DNA结合活性明显低于对照组(P0.05)。Hoechst33258核染色、流式细胞仪和透射电子显微镜检测发现下调p65基因表达能够诱导PANC-1细胞的凋亡。结论 体外实验初步证明了NF-ΚB p65基因在胰腺癌细胞凋亡调控方面扮演重要角色，通过沉默其表达可诱导胰腺癌细胞的凋亡。 【关键词】 RNA干扰 -ΚB PANC-1细胞 细胞凋亡 胰腺肿瘤 Apoptosis of pancreatic carcinoma cell strain PANC-1 induced by knockdown of NF-ΚB p65 expression with RNA interference WANG Dianzhong， XIE Min， PAN Yiming， et al. Department of General Surgery， Drum Tower Hospital， Nanjing Medical University， Nanjing 210008 Abstract Objective To observe the apoptotic effects of RNAi-mediated NF-ΚB p65 gene on silencing pancreatic carcinoma cell strain PANC-1. Methods Chemically synthesized small interference RNA (siRNA) directed against human NF-ΚB p65 was transfected into pancreatic carcinoma cell PANC-1 using cationic liposome LipofectamineTM 2000 as the transfecting agent. The expression of NF-ΚB p65 gene was detected by RT-PCR technique. The DNA binding activity of NF-ΚB was detected by the chemicon non-radioactive NF-ΚB p65 transcription factor assay kit. Nuclear DNA staining by Hoechst 33258， flow cytometry， transmission electron microscope was used to ascertain whether cell apoptosis could be induced by down-regulating the expression of p65 gene in PANC-1 cell. Results The results of RT-PCR indicated that chemically synthesized siRNA directed against human NF-ΚB p65 could knock down the transcription and expression of NF-ΚB p65 gene. The difference between the RelA siRNA group and control groups was significant (P0.01). Aft