【摘要】 目的建立高表达跨膜型肿瘤坏死因子-α胞内段(TNF-intracellular domain,TNF-ICD)基序的人乳腺癌MCF-7细胞株,为研究TM-TNF-α的反向信号提供工具。方法采用脂质体介入法将含人TNF-ICD的pIRES2-EGFP病毒载体导入MCF-7中,经鉴定、筛选获得能稳定表达TNF-ICD细胞株。采用MTT法检测转染细胞对TNF-α的敏感度,比色法检测NO含量,ELISA方法检测NF-κB的活性。结果TNF-ICD基因转染入MCF-7细胞后表达在细胞膜上,并能提高该细胞NF-κB活性,抵抗TNF-α的杀伤,增加其NO产生;应用NF-κB抑制剂PDTC处理后能恢复该细胞对TNF的敏感性。结论获得稳定表达TNF-ICD的MCF-7细胞株,TNF-ICD可能作为TM-TNF-α细胞内的一段活化基序参与TM-TNF-α的反向信号,并通过活化NF-κB,抵抗可溶性TNF-α介导的细胞毒作用,增加NO的产生。更多还原

【Abstract】 Objective To establish the MCF-7 cell line which express transmembrane (TM)-TNF-α intracellular domain (TNF-ICD) protein stably and highly, and to investigate the reverse signaling of TM-TNF-α. Methods The vector of pIRES2-EGFP, which contained human TNF-ICD cDNA, was transduced into a human breast tumor cell line MCF-7 with Lipofectamine 2000. The positive cell clone expressing TNF-ICD protein was detected with indirect immunofluorescence method and flow cytometry. The cytotoxicity of TNF-α against the MCF-7 cells expressing TNF-ICD was determined by bioassay. NO production and NF-κB activity were detected by ELISA method. Results The MCF-7 cells transfected with the TNF-ICD gene could express high lever of TNF-ICD protein, which was confirmed by immunofluorescence and FACS. Bioassay shown that the MCF-7 cells expressing TNF-ICD was resisted to the cytotoxicity of TNF-α. The NO production and NF-κB activity of the transfected cells increased significantly. PDTC, a NF-κB inhibitor, could restore efficiently the sensibility of TNF-ICD-transfected MCF-7 cells to TNF-α. Conclusion The establishment of tumor cell line MCF-7 expressing TNF-ICD gene provide a good model for further studying reverse signaling of TM-TNF-α. TM-TNF-α intracellular domain (-76-1) may be an active domain which involved in the reverse signaling of TM-TNF-α, stimulating NF-κB, and resisting to the cytotoxicity of TNF-α.更多还原