缺失线粒体DNA的人喉癌细胞系的建立

Establishment of Mitochondrial DNA-depleted Laryngeal Carcinoma Cell line

目的为了研究线粒体DNA在人喉癌细胞的增殖、分化及凋亡中的影响及作用,培养线粒体DNA缺失的喉癌细胞系,为构建融合喉癌细胞,研究线粒体DNA突变和线粒体功能改变与喉癌发生之间的关系奠定基础。方法人喉癌细胞系JHUo11加入10%FBS的RPMI1640培养基中,然后在培养基中加入丙酮酸、尿苷、溴化乙锭(EtBr)以去除线粒体DNA,不加溴化乙锭的o11细胞作为对照。经过90天培养,线粒体DNA缺失的喉癌细胞通过健那绿染色后光镜观察和PCR法鉴定。结果 o11细胞经过75ng/μl溴化乙锭持续作用90天后可见细胞肿胀变圆,透光度增加,线粒体DNA PCR扩增未见目的条带,细胞可扩增出定位于mtDNA的500bp片段,健那绿染色对照细胞,可见散在蓝绿色线粒体,而加EB的细胞未见线粒体。结论喉癌细胞o11经过溴化乙锭持续作用90天培养出线粒体DNA缺失的ρ0o11喉癌细胞,这种缺失线粒体DNA的细胞系需补充外源性丙酮酸和尿苷来维持其生存,否则细胞很快死亡。通过观察细胞的增殖和分化发现,ρ0o11细胞生长速度比未经EB处理的对照组慢的多。

Objective In order to investigate the role of mitochondrial DNA（mtDNA） in human laryngeal carcinoma cell proliferation and apoptosis,and provide the cell model in the following study.Mitochondrial DNA depleted laryngeal carcinoma cell lines was generated.Methods Laryngeal carcinoma cell line JHUo11 were maintained in RMPI1640 cell culture medium,supplemented with 10% FBS.To develop ρ0 cells,wild-type cells were chronically exposed to EB in medium additionally supplemented with pyruvate and uridine,cells without EB was as a control.The mtDNA status of cells was determined by PCR analysis and Janus＇green staining.Results After 90 days＇ treatment of 75 ng/ml EB,the cells became swoln and round under microscope.PCR amplification of mtDNA didn＇t show target band and Janus＇ green staining found nothing.The control cell was on the contrary.Conclusion we successfully established a novel ρ0 o11 laryngocarcinoma cell line after 90-day continuous treatment with EtBr.The resultant ρ0 cells displayed dependence upon uridine and pyruvate for growth,a common feature for mtDNA loss cell lines.The results from cell proliferation and generation assays showed that ρ0 o11 cells had a slower growth rate than parental cells.