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

RNA干扰下调NF-κB p65基因表达诱导胰腺癌细胞株PANC-1凋亡 　【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(Plt;0.01)，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(Plt;0.05)。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 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 (Plt;0.01). After transfection， the DNA binding activity of NF-κB p65 in RelA siRNA group was much lower than that of the control groups (Plt;0.05). Nuclear DNA staining by Hoechst 33258， flow cytometry， transmission electron microscopy showed that knockdown of p65 expression by RNAi induced apoptosis in PANC-1 cell. Conclusion The study provides basis