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NF-κB P65亚基siRNA增强吉西他宾诱导胰腺癌细胞凋亡作用的实验研究

An experimental study of gemcitabine inducing pancreatic cancer cell apoptosis potentiated by nuclear factor-kappa B P65 siRNA

摘要:

目的 探讨NF-κB P65亚基siRNA(NF-κB P65 siRNA)在体内外增强吉西他宾诱导胰腺癌细胞凋亡的作用及机制.方法 培养人胰腺癌细胞株BxPC-3和PANC-1,分为空白对照组、阴性干扰序列对照组、吉西他宾组、NF-κB P65 siRNA组和联合治疗组.MTT方法检测细胞增殖情况;Western blot方法检测NF-κB P65及凋亡相关蛋白的表达水平;Annexin V-FITC/PI染色流式细胞仪和激光共聚焦显微镜检测胰腺癌细胞凋亡情况;电泳凝胶迁移实验检测NF-κB的DNA结合活性.BxPC-3接种裸鼠皮下建立胰腺癌移植瘤模型.治疗后监测肿瘤体积;TUNEL染色检测移植瘤组织细胞凋亡指数.结果 转染后72 h,与其他组相比,联合治疗组显著降低了细胞活力指数(P<0.05),下调了Bel-2和proeaspase-3的表达水平,同时上调了Bax的表达水平;流式细胞仪检测结果显示,联合治疗组细胞凋亡率高于其他组(P<0.05);EMSA实验结果证实,NF-κB P65 siRNA组和联合治疗组NF-κB的DNA结合活性低于对照组(P<0.05).联合治疗能够通过诱导凋亡而抑制裸鼠移植瘤生长(P<0.01).结论 NF-κB P65 siRNA可以通过抑制NF-κB的DNA结合活性,调控凋亡相关蛋白的表达水平,激活线粒体凋亡途径,从而增强吉西他宾对胰腺癌细胞的促凋亡作用.

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abstracts:

Objective To investigate the effect and mechanism of NF-kB P65 gene silencing by small interference RNA on the apoptosis of human pancreatic cancer cells induced by gemcitabine in vitro and in vivo. Methods Human pancreatic cancer cells ( BxPC-3 and PANC-1 ) were cultured and respectively divided into five groups; blank control group,negative control siRNA group,gemcitabine group, NF-κB P65 siRNA group and gemcitabine + P65 siRNA group. The ability of cell proliferation was analyzed by MTT; the expression of NF-κB P65 and the apoptosis related proteins were examined by Westrn blot assay; the apoptosis was evaluated by the flow cytometry and laser scanning confocal microscopy analysis stained with Annexin V-FITC/PI; the DNA binding activity of NF-κB was examined by electrophoretic mobility shift assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors.The tumor volume was monitored and TUNEL assay was used to assess the apoptosis index in tumor tissue after treatment Results At 72 h after transfection, the combination with gemcitabine and p65 siRNA significantly decreased the cell viability index (P <0. 05) ,and down-regulated the expression of Bcl-2 and procaspase-3 and up-regulated the expression of Bax compared with other groups. The combinded treatment significantly increased the rate of apoptosis compared with other groups (P < 0. 05). EMSA assay indicated that the DNA binding activity of NF-κB significantly decreased in NF-κB P65 siRNA group and gemcitabine + P65 siRNA group compared with Control group. The combinded therapy inhibited the growth of pancreatic xenograft tumors by apoptosis induction in nude mice (P < 0. 01 ). Conclusions The effect of gemcitabine inducing cell apoptosis may be potentiated through inhibiting the DNA binding activity of NF-κBand regulating the expression of apoptosis related proteins by NF-?B P65 siRNA, which can activate the mitochondria apoptosis pathway in pancreatic cancer in vitro and in vivo.

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作者: 孔瑞 [1] 孙备 [1] 王双佳 [1] 潘尚哈 [1] 王刚 [1] 陈华 [1] 薛东波 [1] 姜洪池 [1]
第一作者: 孔瑞
期刊: 《中华外科杂志》2010年48卷2期 128-133页 MEDLINEISTICPKUCSCD
分类号: R73
栏目名称: 论著
DOI: 10.3760/cma.j.issn.0529-5815.2010.02.013
发布时间: 2010-04-06
基金项目:
国家自然科学基金 教育部新世纪优秀人才支持计划 黑龙江省杰出青年科学基金
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