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目的诱导建立线粒体DNA(mitochondrial DNA,mtDNA)缺失的肺癌A549细胞系(ρ0A549)以探究mtDNA与肿瘤发生和治疗敏感性的关系。方法将A549细胞培养于含50ng/ml溴化乙锭(ethidium bromide,EB)、100μg/ml丙酮酸钠和50μg/ml尿嘧啶1640细胞培养液中传代培养,利用光镜、电镜和PCR方法鉴定ρ0A549细胞系构建的真实性。结果 RT-PCR结果显示,未经EB等处理的A549细胞可同时扩增出定位于mtDNA的1528bp片段和位于核DNA上的18SrDNA的559bp片段,而经EB等处理后的A549细胞RNA反转录只能扩增出定位于mtDNA的1528bp片段。光镜下,ρ0A549细胞形态呈成拉长枝状,明显不同于EB未处理A549细胞的多角形状。ρ0A549细胞传代时间为5d。在诱导成功后继续传代约20代时,细胞生长开始出现迟滞,传代时间大于7d。结论成功建立缺乏mtDNA的人肺癌ρ0A549细胞系,为进一步研究线粒体和mtDNA与肿瘤发生之间的关系提供了方法。
Objective To establish a lung cancer cell line A549 (ρ0A549) with deletion of mitochondrial DNA (mtDNA) to explore the relationship between mtDNA and tumorigenesis and therapeutic sensitivity. Methods A549 cells were subcultured in culture solution containing 50ng / ml ethidium bromide (EB), 100μg / ml sodium pyruvate and 50μg / ml uracil 1640 cells and identified by light microscopy, electron microscopy and PCR ρ0A549 cell line construction of the authenticity. Results RT-PCR results showed that A549 cells without EB could amplify the 1528bp fragment located in mtDNA and the 559bp fragment of 18SrDNA located in nuclear DNA, while the RNA reverse transcription Only the 1528 bp fragment localized to mtDNA was amplified. Light microscope, ρ0A549 cells elongated elongated dendrites, significantly different from EB untreated A549 cells polygonal shape. ρ0A549 cell passage time of 5d. After the successful induction of the success of the passage of about 20 generations, cell growth began to appear delayed, passage time is greater than 7d. Conclusion The successful establishment of human lung cancer ρ0A549 cell line lacking mtDNA provides a method for further study on the relationship between mitochondria and mtDNA and tumorigenesis.