Establishment of HaCaT Cell Model with Different Mitochondrial DNA Numbers

Abstract: In this study， the HaCaT cell model with different mitochondrial DNA (mtDNA) numbers was established based on ethidium bromide (EtBr) induction. The copy number of mtDNA 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 EtBr for 1 and 10 days in 100 ng/mL and 50 ng/mL dosage. Along with the decrease of mtDNA 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 mtDNA copy numbers in the HaCaT cells which had treated with 50 ng/mL and 100 ng/mL EtBr for 10 days， respectively [the ratio of ND1 (mitochondrial NADH dehydrogenase 1 gene)/GAPDH (glyceraldehyde-3-phosphate dehydrogenase) to the relative copy number of mtDNA 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 EtBr could induce HaCaT cells changed into ρ–HaCaT cells. It meant that HaCaT cells could be successfully cultured into ρ– cell lines under EtBr exposure， and a dose-dependent manner was occurred between the changes of mtDNA copy and EtBr exposure. In conclusion， HaCaT cells can be successfully cultured into ρ– cells by EtBr induction， and the changes of mtDNA copy number with EtBr are in a dose-dependent manner.

CSTR: 32200.14.cjcb.2017.09.0008