人穿孔素C端肽段的放射诱导表达  

Radiation-inducible expression of human perforin C-terminal

作　　者：韩艳玲[1] 李芳秋[2] 张蕾[2] 朱锡旭[3] 

机构地区：[1]南京师范大学生命科学学院,南京210097 [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：《临床检验杂志》2008年第2期118-120,共3页Chinese Journal of Clinical Laboratory Science

基　　金：南京市科技计划项目(20040107-5)

摘　　要：目的观察克隆的人穿孔素(perforin,PFN)羧基端肽段在肺癌细胞SPC-A1中的放射诱导表达及其对该细胞的杀伤活性。方法用PCR方法获取hPFN-C基因片段,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,脂质体介导转染SPC-A1细胞。放射诱导表达后,RT-PCR、免疫细胞化学方法检测该基因片段在SPC-A1中的表达。MTT法检测该基因片段的表达产物对SPC-A1细胞的杀伤活性。结果成功获取hPFN-C基因片段,构建了放射诱导表达的真核表达载体,转染SPC-A1细胞后,免疫细胞化学法证实目的蛋白出现在细胞质中,而未照射的细胞则没有hPFN-C基因表达。MTT法检测显示,与对照组相比,有hPFN-C表达的SPC-A1细胞存活率仅为0.657。结论成功构建hPFN-C基因片段的放射诱导表达载体,它在SPC-A1细胞中获得可控性表达,其表达产物对该细胞具有较强的杀伤活性。Objective To observe the expression and killing effect of human perforin C-terminal induced by radiation on SPC-Al cell line. Methods Human perforin C-terminal gene segment was obtained by PCR, then it was cloned into vector pcDNA3. 1 （ ＋ ） which contained Egr-1 promoter induced by radiation. The recombinant plasmid was transfected into SPC-Al cells by lipofectamine regent. After inducing by radiation, reverse transcription polymerase chain reaction （RT-PCR） and immunocytochemistry analysis were used to detecte Sp17 transcript and protein respectively. Killing effect was detected by colorimetric MTT assay. Results Human PFN-C gene segment was cloned and inserted into pcDNA3.1 （ ＋ ） /Egr-1 vector correctly. By immunocytochemistry, hPFN-C protein could be detected in the cytoplasm of SPC-Al ceils induced by radiation, but not in the ceils without induction. In MTT assay, the cell viability of the test groups reduced to 0. 657 significantly when compared with that in control groups （P 〈 0.01 ）. Conclusion hPFP-C gene segment could be controllably expressed in SPC-Al ceils and the expressed protein had killing effect.

关 键 词：穿孔素 SPC—Al细胞 EGR-1启动子 放射诱导表达 

分 类 号：Q786[生物学—分子生物学]