人穿孔素N端肽段的放射诱导表达研究  

Radiation-inducible expression of human perforin N-terminal in lung cancer cells

作　　者：张蕾[1] 李芳秋[2] 韩艳玲[2] 朱锡旭[3] 

机构地区：[1]南京师范大学生命科学学院,南京210002 [2]南京军区南京总医院解放军临床检验医学研究所,南京210002 [3]南京军区南京总医院放射治疗科,南京210002

出　　处：《中国免疫学杂志》2008年第5期403-406,408,共5页Chinese Journal of Immunology

基　　金：南京市科技发展计划项目(批准号:200401070-5)

摘　　要：目的:构建人穿孔素N端(hPFN-N)的真核放射诱导表达载体,并在放射诱导条件的刺激下研究hPFN-N的表达产物(rhPFN-N)在肺癌细胞SPC-A1中的分布及其对该细胞的细胞毒性作用。方法:以克隆有hPFN全长cDNA序列的载体pcDNA3·1(+)/hPFN为模板,用PCR扩增hPFN-N,将该基因片段克隆到含放射诱导启动子的真核表达载体pcDNA3·1(+)/Egr-1中,以重组体稳定转染SPC-A1细胞,经过X射线的放射诱导后,应用RT-PCR、细胞免疫化学法检测hPFN-N蛋白的表达,用MTT法测定该蛋白对靶细胞的杀伤活性。结果:成功构建了真核放射诱导表达载体pcDNA3·1(+)/Egr-hPFN-N。以重组体转染SPC-A1细胞后,用RT-PCR检测到hPFN-N mRNA的表达。细胞免疫化学法检测结果呈阳性反应,MTT法检测结果为rhPFN-N对靶细胞的杀伤活力为29·2%。结论:构建了pcDNA3·1(+)/Egr-hPFN-N真核放射诱导表达载体,在经过X射线的放射诱导后,hPFN-N能够在SPC-A1细胞的细胞膜上大量表达,表达产物rhPFN-N对靶细胞有明显杀伤活力。Objective： To construct an eukaryotic radiation-inducible expressing vector of the human perforin N-tenninal（hPFN-N） ,and to investigate the distribution and the killing effect of human perforin N-terminal mmcat ed 118 amino acid polypeptide （rhPFN-N, 22-139aa） on tumor cells.Methods：The gene hPFN-N was amplified by PCR from the plasmid pcDNA3.1（ ＋ ）/hPFN and an enkaryotic radiation-inducible expression vector pcDNA3.1（ ＋ ）/Egr-hPFN-N was constructed after DNA recombination.After transfecting SPC-A1 cells with this recombination vector via liposome mediation, the expression of the hPFN-N protein was detected by RT-PCR and Immunocytochemical method and the killing effect of hPFN-N protein was assessed by standard MTT chromatometry. Results： DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic radiation-inducible expressing vector pcDNA3.1 （ ＋ ）/ Egr-hPFN-N had been constructed successfully. After the recombinant plasmid being transfected into SPC-A1 cells and being irradiated by X ray, RT-PCR verified the expression of hPFNN mRNA.The result of Immunocytocbemical assay was positive and in MTT assay the killing activity of rhPFN-N on target cells was 29.2%. Conclusion：After being irradiated the hPFN-N gene is expressed on the cell membrane and the killing activity of rhPFN-N on target cells is 29.2%.

关 键 词：穿孔素 EGR-1启动子 放射诱导 杀伤活性 SIC—A1细胞 

分 类 号：R733[医药卫生—肿瘤] Q789[医药卫生—临床医学]