反义寡核苷酸阻断NF-кB对胰腺癌细胞增殖活性影响的研究

Effect of NF-кB antisense oligodeoxynucleotides on proliferation of pancreatic carcinoma cells

目的:研究核因子-кB(NF-кB)亚单位p65反义寡核苷酸(ASODN)对人胰腺癌细胞增殖活性的影响,探讨p65 ASODN治疗胰腺癌的可行性。方法:设计合成p65ASODN,体外转染人胰腺癌细胞株PANC-1,用流式细胞仪和荧光显微镜检测转染效率,用RT-PCR分析p65 mRNA的表达,用四甲基偶氮唑盐(MTT)法和克隆形成试验分析细胞增殖能力。结果:流式细胞仪和荧光显微镜检测表明,p65 ASODN可成功转染PANC-1细胞;转染后,RT-PCR结果显示反义治疗组、空脂质体组和空白对照组p65 mRNA相对表达量分别为0.24±0.05、0.37±0.04和0.39±0.03,反义治疗组p65基因表达显著下降,P=0.000;MTT检测结果显示,在转染后24、48和72h反义治疗组吸光度(OD)值均降低,细胞增殖受到抑制,抑制作用在转染后48h最为明显,P<0.01;克隆形成试验结果显示,反义治疗组、空脂质体组和空白对照组的克隆形成率分别为(14.9±3.3)%、(23.3±2.0)%和(25.8±2.5)%,反义治疗组克隆形成率显著下降,P=0.000。结论:脂质体介导的p65ASODN能有效抑制胰腺癌PANC-1细胞株p65基因的表达,从而降低细胞增殖活性,可能对胰腺癌有治疗作用。

OBJECTIVE： To investigate the effect of NF-κB subunit p65 antisense oligodeoxynucleotides（ASODN） on the proliferation of pancreatic carcinoma cells and the therapy effects of p65 ASODN on pancreatic carcinoma. METHODS： p65 ASODN were designed and constructed and were transfected into human pancreatic carcinoma cell line PANC-1. The rate of transfection was detected by flow cytometry and fluorescence microscopy. RT-PCR, MTT and clone assay were used to detect the expression level of p65 mRNA and proliferation of PANC-1 cells. RESULTS： p65 ASODN was successfully transfected into PANC-1 cells. After the transfection, RT-PCR revealed that the expressions of p65 mRNA of antisense group, liposomes control group and blank control group were 0. 24±0. 05, 0. 37±0.04 and 0. 39±0.03, respectively. The expression of p65 mRNA of the antisense group was significantly lower than those of the other groups, P= 0. 000. The resuits from the MTT assay indicated that 24 hours, 48 hours and 72 hours after the transfection, the cell proliferation of the antisense group was inhibited. The inhibition peaked at 48 hours after the transfection, P〈0. 01. The clone assay revealed that the cloning efficiencies of the antisense group, liposomes control group and blank control group were （14.9±3.3）%, （23.3±2.0）% and （25.8±2.5）%. The cloning efficiency of the antisense group was reduced, P = 0. 000. CONCLUSIONS： The transfection of p65 ASODN could effectively down-regulate the expression of p65 mRNA and reduce cell proliferation of PANC-1 cells. The therapeutic prospect of p65 ASODN on pancreatic carcinoma is worthy of further investigation.