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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 investigate the biological characteristics of SPC-A1 cell lung cancer cells after blockade of mitochondrial DNA (mtDNA) transcriptional promoter with anti-sense peptide nucleic acid (asPNA). METHODS: The asPNA targeting to the mtDNA heavy chain transcriptional promoter region was synthesized, and the asPNA-triphenyl phosphate complex was constructed and identified. The complex was transfected into human lung adenocarcinoma cell line SPC-A1. Confocal laser scanning microscopy The effect of subcellular localization and RT-PCR on the transcription of mtDNA encoding gene was detected 48 h after transfection. The cell cycle distribution and apoptosis / necrosis were evaluated by flow cytometry. The intracellular ATP concentration was measured by luminescence fluorometer. AsPNA-triphenyl phosphate complexes of lung adenocarcinoma SPC-A1 cells as a control. Results: AspNA-triphenyl phosphate complex was successfully obtained and the complex was successfully expressed in the mitochondria of SPC-A1 cells by laser confocal microscopy. Compared with the untransfected group, the transcription level of mtDNA encoding gene in SPC-A1 cells (P <0.01). At the same time, the cell cycle analysis showed that the sub-diploid peak appeared in the transfection group, while the AnnexinⅤ-PI double-labeled results further confirmed that the transfected lung cancer cells Apoptosis rate increased significantly. CONCLUSION: asPNA can enter the mitochondria of SPC-A1 cells and inhibit the transcription of mtDNA encoding gene under the transport of lipophilic cation-triphenylphosphine, thereby affecting the energy synthesis and inducing apoptosis of lung cancer cells.