选取31头经检测猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)抗原和抗体均为阴性的5周龄断奶仔猪,随机分为对照组16头和试验组15头,对照组仔猪每头滴鼻4 mL PBS,试验组仔猪每头滴鼻4 mL 5×105TCID50.mL-1PCV2悬液。于PCV2...选取31头经检测猪圆环病毒2型(PCV2)、猪繁殖与呼吸综合征病毒(PRRSV)抗原和抗体均为阴性的5周龄断奶仔猪,随机分为对照组16头和试验组15头,对照组仔猪每头滴鼻4 mL PBS,试验组仔猪每头滴鼻4 mL 5×105TCID50.mL-1PCV2悬液。于PCV2接种当天剖杀4头仔猪作为对照组,分别于14、21和35 d剖杀4头对照组和5头试验组仔猪,采集肝脏。用激光共聚焦显微镜检测核因子κB/P65(NF-κB/P65)蛋白的核易位变化;蛋白提取法分别提取肝脏细胞核蛋白和胞浆蛋白,Western blot定量检测细胞核中NF-κB/P65和细胞浆中p-IκBα、MyD88蛋白含量的变化;电泳迁移率(EMSA)法检测细胞核中NF-κB DNA的结合率变化。检测结果显示,PCV2接种仔猪,肝脏中NF-κB/P65蛋白核易位逐渐增多,到21 d达到峰值;p-IκBα、MyD88、NF-κB/P65 DNA结合率在接种后均先升高后趋于恢复,并于21 d达到高峰。与对照组仔猪相比,肝脏中MyD88和NF-κB/P65蛋白含量以及NF-κB DNA结合率在14和21 d试验组均显著升高(P<0.05),35 d含量变化不显著;21 d时试验组p-IκBα蛋白含量显著升高。相关性分析显示,NF-κB/P65蛋白含量与MyD88含量、NF-κB DNA结合率之间存在显著正相关,相关系数分别是0.566和0.528。结果表明,PCV2可通过激活MyD88启动NF-κB信号途径;通过IκBα的磷酸化降解激活NF-κB,并促进其进行核易位,使NF-κB与DNA发生结合,调控相关炎性细胞因子的转录和表达。展开更多
AIM: To explore the expression effect of mutated IκBα transfection on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells by inhibiting the activity of nuclear transcription factor-κB (NF-κB). METH...AIM: To explore the expression effect of mutated IκBα transfection on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells by inhibiting the activity of nuclear transcription factor-κB (NF-κB). METHODS: We used the mutated IicBa plasmid to transfect QBC939HCVC+ cells and QBC939 cells, and electrophoretic gel mobility shift assay (EMSA) to detect the binding activity of NF-κB DNA and the effect of the transfrecting mutated IκBα plasmid on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells and its expression protein (P-GP). RESULTS: Plasmid DNA was digested by restriction enzymes Xbal and Hand III, and its product after electrophoresis showed two bands with a big difference in molecular weight, with a size of 4.9 kb and 1.55 kb respectively, which indicated that the carrier was successfully constructed and digested with enzymes. The radioactivity accumulation of QBC939HCVC+ and QBC939 cells transfected with mutated IκBα plasmid was significantly lower than that of the control group not transfected with mutated IκBα plasmid. Double densimeter scanning showed that the relative signal density between the tansfection group and non-transfection group was significantly different, which proved that the mutated IκBα plasmid could inhibit the binding activity of NF-KB DNA in hilar cholangiocarcinoma cells. Compared to control group not transfected with m IκBα plasmid, the expression level of MDR-1mRNA in the QBC939 and QBC939HCVC+ cells transfected with mutated IκBα plasmid was lower. The expression intensity of P-GP protein in QBC939 and QBC939HCVC+ cells transfected with mutated IκBα was significantly lower than that of the control group not transfected with mutated IκBα plasmid. CONCLUSION: The mutated IκBα plasmid transfection can markedly reverse the multidrug resistance of hilar cholangiocarcinoma cells. Interruption of NF-κB activity may become a new target in gene therapy for hilar cholangiocar-cinogenesic carcinoma.展开更多
基金Supported by China Postdoctoral Science Foundation ,No. 2002031291
文摘AIM: To explore the expression effect of mutated IκBα transfection on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells by inhibiting the activity of nuclear transcription factor-κB (NF-κB). METHODS: We used the mutated IicBa plasmid to transfect QBC939HCVC+ cells and QBC939 cells, and electrophoretic gel mobility shift assay (EMSA) to detect the binding activity of NF-κB DNA and the effect of the transfrecting mutated IκBα plasmid on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells and its expression protein (P-GP). RESULTS: Plasmid DNA was digested by restriction enzymes Xbal and Hand III, and its product after electrophoresis showed two bands with a big difference in molecular weight, with a size of 4.9 kb and 1.55 kb respectively, which indicated that the carrier was successfully constructed and digested with enzymes. The radioactivity accumulation of QBC939HCVC+ and QBC939 cells transfected with mutated IκBα plasmid was significantly lower than that of the control group not transfected with mutated IκBα plasmid. Double densimeter scanning showed that the relative signal density between the tansfection group and non-transfection group was significantly different, which proved that the mutated IκBα plasmid could inhibit the binding activity of NF-KB DNA in hilar cholangiocarcinoma cells. Compared to control group not transfected with m IκBα plasmid, the expression level of MDR-1mRNA in the QBC939 and QBC939HCVC+ cells transfected with mutated IκBα plasmid was lower. The expression intensity of P-GP protein in QBC939 and QBC939HCVC+ cells transfected with mutated IκBα was significantly lower than that of the control group not transfected with mutated IκBα plasmid. CONCLUSION: The mutated IκBα plasmid transfection can markedly reverse the multidrug resistance of hilar cholangiocarcinoma cells. Interruption of NF-κB activity may become a new target in gene therapy for hilar cholangiocar-cinogenesic carcinoma.
