目的 :探讨大肠癌细胞 Fas配体 (Fas L ) m RNA和蛋白的表达及其功能。方法 :分别采用 RT- PCR和 Western印迹法以及氚释放细胞活性测定技术检测大肠癌细胞株中 Fas L m RNA和蛋白的表达及其功能。结果 :所检测的 6株大肠癌细胞(RPMI 4...目的 :探讨大肠癌细胞 Fas配体 (Fas L ) m RNA和蛋白的表达及其功能。方法 :分别采用 RT- PCR和 Western印迹法以及氚释放细胞活性测定技术检测大肠癌细胞株中 Fas L m RNA和蛋白的表达及其功能。结果 :所检测的 6株大肠癌细胞(RPMI 4788、HCT- 116、DL D- 1、L o Vo、Wi Dr和 COL O 32 0 DM)全部表达 Fas L m RNA和蛋白 ;DL D- 1,L o Vo和 Wi Dr等部分大肠癌细胞具有 Fas依赖的细胞毒活性。其中 DL D- 1细胞活性具有量效关系 ,并且乙酸肉豆蔻佛波醇 (PMA )和离子霉素(ionom ycine )刺激能显著增强 DL D- 1的细胞活性。结论 :DL D- 1、L o Vo和 Wi Dr等 3株大肠癌细胞表达功能性 Fas配体 ,并可能以此反击机体免疫系统 ,逃避免疫监督。展开更多
The regulator of expression of virion(Rev)protein binds specifically to the Rev-responsive element(RRE)RNA in order to regulate the expression of the human immunodeficiency virus(HIV)-1 genes.Fluorescence indicator di...The regulator of expression of virion(Rev)protein binds specifically to the Rev-responsive element(RRE)RNA in order to regulate the expression of the human immunodeficiency virus(HIV)-1 genes.Fluorescence indicator displacement assays have been used to identify ligands that can inhibit the ReveRRE interaction;however,the small fluorescence indicators cannot fully replace the Rev peptide or protein.As a result,a single rhodamine B labeled Rev(RB-Rev)model peptide was utilized in this study to develop a direct and efficient ReveRRE inhibitor screening model.Due to photon-induced electron transfer quenching of the tryptophan residue on the RB fluorophore,the fluorescence of RB in Rev was weakened and could be dramatically reactivated by interaction with RRE RNA in ammonium acetate buffer(approximately six times).The interaction could reduce the electron transfer between tryptophan and RB,and RRE could also increase RB fluorescence.The inhibitor screening model was evaluated using three known positive ReveRRE inhibitors,namely,proflavin,6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine(ICR 191),and neomycin,as well as a negative drug,arginine.With the addition of the positive drugs,the fluorescence of the ReveRRE decreased,indicating the displacement of RB-Rev.This was confirmed using atomic force microscopy(AFM)and the fluorescence was essentially unaffected by the addition of arginine.The results demonstrated that RB-Rev can be used as a fluorescent probe for recognizing small ligands that target RRE RNA.The ReveRRE inhibitor screening model offers a novel approach to evaluating and identifying long-acting Rev inhibitors.展开更多
目的:观察用RNA干扰的方法抑制大鼠成骨细胞核激活因子受体配体(Ligand of receptor activator ofNF-κB,RANKL)mRNA表达后,成骨细胞RANKL、OPG蛋白表达和I型胶原表达、细胞增殖能力的时间变化规律。方法:设计4条RANKL序列特异性小干扰R...目的:观察用RNA干扰的方法抑制大鼠成骨细胞核激活因子受体配体(Ligand of receptor activator ofNF-κB,RANKL)mRNA表达后,成骨细胞RANKL、OPG蛋白表达和I型胶原表达、细胞增殖能力的时间变化规律。方法:设计4条RANKL序列特异性小干扰RNA(siRNA),用Lipofectamin2000转染成骨细胞,采用荧光实时定量聚合酶链反应(PCR)检测RANKL的表达,筛选出最有效的干扰序列,并用Western blot检测转染后1、2、3、5、7天RANKL、OPG蛋白的表达,同时观察成骨I型胶原表达、细胞增殖能力在相同时间点的变化。结果:对siRNA中有一对可使大鼠成骨细胞的RANKLmRNA水平下降89%。用最佳siRNA转染后1、2、3、5、7天,与空白对照组相比,RANKL蛋白表达率分别为59.1%、39.5%、26.6%、40.0%、57.3%(P<0.05),OPG蛋白表达率较空白对照组降低,但差异不具有显著性(P>0.05)。成骨细胞的I型胶原表达、增殖能力在相同时间点较空白对照组降低,差异也不具有显著性(P>0.05)。结论:靶向siRNA可显著下调成骨细胞RANKL表达,同时对OPG表达及成骨细胞的主要功能无显著影响。展开更多
文摘目的 :探讨大肠癌细胞 Fas配体 (Fas L ) m RNA和蛋白的表达及其功能。方法 :分别采用 RT- PCR和 Western印迹法以及氚释放细胞活性测定技术检测大肠癌细胞株中 Fas L m RNA和蛋白的表达及其功能。结果 :所检测的 6株大肠癌细胞(RPMI 4788、HCT- 116、DL D- 1、L o Vo、Wi Dr和 COL O 32 0 DM)全部表达 Fas L m RNA和蛋白 ;DL D- 1,L o Vo和 Wi Dr等部分大肠癌细胞具有 Fas依赖的细胞毒活性。其中 DL D- 1细胞活性具有量效关系 ,并且乙酸肉豆蔻佛波醇 (PMA )和离子霉素(ionom ycine )刺激能显著增强 DL D- 1的细胞活性。结论 :DL D- 1、L o Vo和 Wi Dr等 3株大肠癌细胞表达功能性 Fas配体 ,并可能以此反击机体免疫系统 ,逃避免疫监督。
基金supported by the Natural Science Foundation of Shaanxi Province of China(Grant No.:202012119)the Start-up Funding of Shaanxi University of Science and Technology(Grant No.:2019BJ-48)the Innovation Capability Support Program of Shaanxi Province of China(Grant No.:2021PT-044).
文摘The regulator of expression of virion(Rev)protein binds specifically to the Rev-responsive element(RRE)RNA in order to regulate the expression of the human immunodeficiency virus(HIV)-1 genes.Fluorescence indicator displacement assays have been used to identify ligands that can inhibit the ReveRRE interaction;however,the small fluorescence indicators cannot fully replace the Rev peptide or protein.As a result,a single rhodamine B labeled Rev(RB-Rev)model peptide was utilized in this study to develop a direct and efficient ReveRRE inhibitor screening model.Due to photon-induced electron transfer quenching of the tryptophan residue on the RB fluorophore,the fluorescence of RB in Rev was weakened and could be dramatically reactivated by interaction with RRE RNA in ammonium acetate buffer(approximately six times).The interaction could reduce the electron transfer between tryptophan and RB,and RRE could also increase RB fluorescence.The inhibitor screening model was evaluated using three known positive ReveRRE inhibitors,namely,proflavin,6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine(ICR 191),and neomycin,as well as a negative drug,arginine.With the addition of the positive drugs,the fluorescence of the ReveRRE decreased,indicating the displacement of RB-Rev.This was confirmed using atomic force microscopy(AFM)and the fluorescence was essentially unaffected by the addition of arginine.The results demonstrated that RB-Rev can be used as a fluorescent probe for recognizing small ligands that target RRE RNA.The ReveRRE inhibitor screening model offers a novel approach to evaluating and identifying long-acting Rev inhibitors.
