反义肽核酸阻抑线粒体基因表达诱导肺癌SPC细胞凋亡的实验研究  

The effect of blocking the expression of mtDNA on lung cancer SPC-A1 cell by antisense peptide nucleic acid

作　　者：王光辉[1] 黄桂君[1] 余时沧[2] 张志远[1] 钱桂生[1] 

机构地区：[1]第三军医大学新桥医院呼吸内科研究所,重庆400037 [2]第三军医大学西南医院病理研究所,重庆400038

出　　处：《重庆医科大学学报》2009年第11期1457-1461,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金项目(编号:30670919)

摘　　要：目的:探讨以反义肽核酸(Anti-sense peptide nucleic acid,asPNA)封闭肺癌SPC-A1细胞线粒体DNA(Mitochondrial DNA,mtDNA)转录启动子后,对其生物学特性的影响。方法:合成针对mtDNA重链转录启动子区的asPNA,构建asPNA-三苯磷复合物并鉴定,以该复合物转染人肺腺癌SPC-A1细胞系,激光共聚焦显微镜确定该复合物的亚细胞定位,RT-PCR检测转染48 h后对mtDNA编码基因转录的影响,流式细胞术评估细胞周期分布及凋亡/坏死情况,自发光荧光仪检测细胞内ATP浓度,以未转染asPNA-三苯磷复合物的肺腺癌SPC-A1细胞为对照。结果:成功获得asPNA-三苯磷复合物,激光共聚焦显微镜显示该复合物顺利进入SPC-A1细胞线粒体;同未转染组相比,转染组SPC-A1细胞mtDNA编码基因的转录水平有所下降,而细胞内ATP浓度明显降低(P<0.01);同时,细胞周期分析显示,转染组出现明显的亚二倍体峰,而AnnexinⅤ-PI双标结果进一步证实,转染组肺癌细胞凋亡率明显增加。结论:asPNA在电子移位亲脂性阳离子-三苯磷的运载下,能够进入SPC-A1细胞线粒体并阻抑其mtDNA编码基因的转录,进而影响肺癌细胞的能量合成并诱导其凋亡。Objective：To study the change of the physiological characteristic of lung cancer SPC-A1 after its mtDNA transcript promoter was blocked by antisense peptide nucleic acid （ asPNA ）. Methods： asPNA to the transcript promoter region on the heavy chain of mtDNA was synthesized,then asPNA-triphenyl phosphate complex was constructed and identified. This complex was transfected into SPC-A1. laser confocal microscope was used to detect the location of asPNA-triphenyl phosphate complex in SPC-A1. At 48 h behind transfection, the transcription of the gene encoded by mtDNA was detected by RT-PCR. Cell cycle and apoptosis/necrosis of SPC-A1 were detected by flow cytometry. The concentration of ATP in SPC-A1 was examined by spontaneous fluorescence analyzer.All of those results were compared with those of untransfected SPC-A1. Results：asPNA - triphenyl phosphate complex was successfully obtained. Confocal laser scanning microscope discovered that antisense peptide nucleic acid - triphenyl phosphate complex had been successfully transfected into the mitochondrion of SPC-A1. The transcriptional level of the gene encoded by mtDNA and concentration of ATP in the transfected SPC-A1 were lower significantly than the untransfected SPC-A1 （P〈O.O1）. The analysis of Cell cycle displayed that noticeable sbu-G1 peak appeared in the transfected group. Annexin V -PI double labeling confirmed the apoptosis rate of lung cancer cell in the transfected group significantly increased. Conclusion： Carried by electronic shift lipophilic cation-triphenyl phosphate,asPNA can enter the mitochondrion of SPC-A1 and block the transcript of gene encoded by mtDNA to impact the synthesis of energy and induce apoptosis of SPC-A1.

关 键 词：肺癌 反义肽核酸 线粒体 转录 凋亡 

分 类 号：R734.2[医药卫生—肿瘤]