【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。更多还原

【Abstract】 Objective:To study the change of the physiological characteristic of lung cancer SPC-A1 after its mtDNA transcriptpromoter was blocked by antisense peptide nucleic acid(asPNA).Methods:asPNA to the transcript promoter region on the heavychain of mtDNA was synthesized,then asPNA-triphenyl phosphate complex was constructed and identified.This complex wastransfected into SPC-A1.laser confocal microscope was used to detect the location of asPNA-triphenyl phosphate complex inSPC-A1.At 48 h behind transfection,the transcription of the gene encoded by mtDNA was detected by RT-PCR.Cell cycle andapoptosis/necrosis of SPC-A1 were detected by flow cytometry.The concentration of ATP in SPC-A1 was examined by spontaneousfluorescence analyzer.All of those results were compared with those of untransfected SPC-A1.Results:asPNA-triphenyl phosphatecomplex was successfully obtained.Confocal laser scanning microscope discovered that antisense peptide nucleic acid-triphenylphosphate complex had been successfully transfected into the mitochondrion of SPC-A1.The transcriptional level of the gene encodedby mtDNA and concentration of ATP in the transfected SPC-A1 were lower significantly than the untransfected SPC-A1(P<0.01).Theanalysis of Cell cycle displayed that noticeable sbu-G1 peak appeared in the transfected group.Annexin Ⅴ-PI double labelingconfirmed the apoptosis rate of lung cancer cell in the transfected group significantly increased.Conclusion:Carried by electronicshift lipophilic cation-triphenyl phosphate,asPNA can enter the mitochondrion of SPC-A1 and block the transcript of gene encoded bymtDNA to impact the synthesis of energy and induce apoptosis of SPC-A1.更多还原