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The Effects of5-azacytidine and Trichostatin a on the Expression of MMP-1and MMP-3in HaCaT Cells

Author: ZhaoRongXin
Tutor: HuangJinHua
School: Central South University
Course: Clinical
Keywords: HaCaT cells MMP1 MMP3 5-azacytidine Trichostatin A
CLC: R758.1
Type: Master's thesis
Year: 2013
Downloads: 6
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Abstract


Background:Photoaging refers to the premature skin aging due to repeated Exposure to ultraviolet, and its characteristic histological changes include the deposition of degenerative elastotic material and basophilic degeneration of collagen, accompanied with the degradation of types I and III procollagen. Although its mechanism is not yet fully understood, but the most important mechanism that causes skin photoaging is that the increased matrix metalloproteinases (MMPs) lead to the degradation of fibrous connective tissue. In the occurrence and development of the skin photoaging, as well as in the many recent studies, the MMP-1and MMP-3are considered to be the most important, and they are also the current research focus.Objectives:(1)To investigate whether the expression of MMP1, MMP3in HaCaT cells is regulated by DNA methylation or not;(2)To investigate whether the MMP1, MMP3expression in HaCaT cells is regulated by histone acetylation or not;(3)To investigate whether the MMP1, MMP3expression in HaCaT cells is regulated by the synergistic role of both DNA methylation and histone acetylation, and to provide a theoretical basis for the further study of epigenetic mechanisms of skin photoaging.Methods:(1) To culture conventionally in vitro the human keratinocytes cells (HaCaT cells);(2) To treat the HaCaT cells with5-azacytidine (5-azaC)(1μmol/L) or/and TrichostatinA (TSA)(1μmol/L); they are divided into:5-azaC treatment group, TSA treatment group and both5-azaC and TSA treatment group, who act respectively on the HaCaT cells Oh,24h,48h and72h, and then to extract the supernatant and mRNA;(3)To detect respectively the expression of MMP1/MMP3in the HaCaT cells of the different treatment groups from the levels of mRNA and protein with the reverse-transcription polymerase chain reaction (RT-PCR) and Elisa analysis. Results:(1)24h,48h and72h later treated by5-azaC (1μmol/L), in comparasion with the state of Oh (control group), in the HaCaT cells, the mRNA levels and protein expression of MMP-1and MMP-3were significantly increased (P <0.05);(2)24h,48h and72h later treated by TSA (1μmol/L), in comparasion with the state of Oh (control group), in the HaCaT cells, the mRNA levels and protein expression of MMP-1and MMP-3were significantly increased (P <0.05);(3)24h,48h and72h later treated by both5-azaC (1μmol/L) and TSA (1μmol/L), in comparasion with the state of Oh (control group), in the HaCaT cells, the mRNA levels and protein expression of MMP-1and MMP-3were significantly increased (P<0.05); Futhermore, compared with the different time points after the treatment of only5-azaC or only TSA, both the mRNA levels and protein expression of MMP1、MMP3in HaCaT cells of24h,48h and72h were higher, and the difference had statistical significance (P <0.05).Conclusions:(1) The expression of MMP1and MMP3in HaCaT cells is regulated by DNA methylation and histone acetylation;(2). DNA methylation and histone acetylation play a synergistic role on the expression of MMP1and MMP3in HaCaT cells。Figures:15, Tables:7, References:35.

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CLC: > Medicine, health > Dermatology and Venereology > Dermatology > Physical skin disease
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