摘要
该研究采用溴化乙锭(ethidium bromide,Et Br)对人永生化角质形成细胞(HaCaT)进行诱导,采用RT-PCR法、MTT法和健那绿染色技术联合鉴定Et Br对HaCaT细胞线粒体DNA(mitochondrial DNA,mt DNA)拷贝数的影响,建立不同mt DNA拷贝数目的HaCaT细胞模型。结果显示,HaCaT细胞经100 ng/m L和50 ng/m L Et Br分别处理10 d后,细胞形态随着处理时间的延长发生改变,由规则铺路石形状逐渐变圆,某些细胞体积变小,成团生长,细胞膜边缘不光滑,且随着mt DNA数目的减少,细胞增殖速率明显下降。RT-PCR分析结果显示,与对照组相比,50 ng/m L和100 ng/m L Et Br处理10 d的HaCaT细胞中,mt DNA的拷贝数分别减少了52.9%和97.6%[以线粒体DNA脱氢酶1(mitochondrial NADH dehydrogenase 1 gene,ND1)与甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)的比值来表示mt DNA的相对拷贝数变化情况]。健那绿染色显示,对照组HaCaT细胞中可见分散存在的蓝绿色线粒体颗粒,100 ng/m L Et Br处理10 d后的HaCaT细胞中,显微镜下蓝绿色线粒体颗粒明显减少。综上所述,该研究证明了HaCaT细胞可通过Et Br诱导被成功培养成ρ–细胞,且Et Br浓度与HaCaT细胞mt DNA拷贝数的变化在一定的范围内存在着剂量–效应关系。
In this study, the HaCaT cell model with different mitochondrial DNA(mt DNA) numbers was established based on ethidium bromide(Et Br) induction. The copy number of mt DNA was identified by RT-PCR, MTT assay and Janus Green B staining technique. The results showed that the cell morphology changed significantly, for instance, the shape of the polygon was gradually rounded, some cells became smaller and the edge of cell were not smooth, after HaCaT cells were exposed to Et Br for 1 and 10 days in 100 ng/m L and 50 ng/m L dosage. Along with the decrease of mt DNA number, the cell proliferation efficiency decreased significantly. RT-PCR analysis showed that compared with the normal control group, there were 52.9% and 97.6% of the mt DNA copy numbers in the HaCaT cells which had treated with 50 ng/m L and 100 ng/m L Et Br for 10 days, respectively [the ratio of ND1(mitochondrial NADH dehydrogenase 1 gene)/GAPDH(glyceraldehyde-3-phosphate dehydrogenase) to the relative copy number of mt DNA changes]. Janus Green B staining showed that the blue-green mitochondrial granules existed in the control group, which meant the mitochondria remained normal activity in the control group cells. While there were no blue-green mitochondrial under the microscope in exposed cells, indicating that Et Br could induce HaCaT cells changed into ρ–HaCaT cells. It meant that HaCaT cells could be successfully cultured into ρ– cell lines under Et Br exposure, and a dose-dependent manner was occurred between the changes of mt DNA copy and Et Br exposure. In conclusion, HaCaT cells can be successfully cultured into ρ– cells by Et Br induction, and the changes of mt DNA copy number with Et Br are in a dose-dependent manner.
作者
倪一平
王小娟
孙一丹
张莉
马陈西南
刘纪廷
严锐
陶莎莎
张洁
安艳
Ni Yiping Wang Xiaojuan Sun Yidan Zhang Li Ma Chenxinan Liu Jiting Yan Rui Tao Shasha Zhang Jie An Yan(School of Public Health, Soochow University, Suzhou 215123, China)
出处
《中国细胞生物学学报》
CAS
CSCD
2017年第9期1188-1195,共8页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:81573173
81473008
81673203)
江苏省高校基金(批准号:16KJB330009)资助的课题~~