Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells / 中南大学学报(医学版)
Journal of Central South University(Medical Sciences)
;
(12): 843-847, 2006.
Article
in Chinese
| WPRIM
| ID: wpr-813589
ABSTRACT
OBJECTIVE@#To investigate the effects of DNA hypomethylation on mRNA and protein expression of perforin promotor in T cells.@*METHODS@#T cells were isolated from the peripheral venous blood of healthy donors by density gradient centrifugation. CD4(+) and CD8(+) subsets were isolated using Miltenyi beads and protocols provided by the manufacturer. Where indicated the T cells were stimulated with PHA for 24 h, then treated with 5-azaC for an additional 72 h. Genomic DNA, mRNA, and protein were isolated from untreated and 5-azaC-treated T cells. Purified DNA was treated with sodium bisulfite, the desired sequences were amplified in sequential fragments using nested PCR. The amplified fragments were cloned into bacteria DH5 alpha and 5 independent clones for each of the amplified fragments were sequenced. The expression of perforin was determined using real time RT-PCR and Western blot.@*RESULTS@#The perforin mRNA and protein in the CD4(+) and CD8(+) subsets treated with 5-azaC were significantly higher than those in the untreated subsets (P<0.05). The results of bisulfite genomic sequencing showed that the methylation of perforin promotor was significantly reduced in the treated cells compared with the untreated cells (P<0.05).@*CONCLUSION@#The mRNA and protein expression of perforin significantly increases in the CD4(+) and CD8(+) T cells treated with 5-azaC,which is associated with DNA hypomethylation of perforin promoter in T cells.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pharmacology
/
Azacitidine
/
Cells, Cultured
/
T-Lymphocyte Subsets
/
Promoter Regions, Genetic
/
DNA Methylation
/
Perforin
/
Genetics
/
Metabolism
Limits:
Adult
/
Humans
Language:
Chinese
Journal:
Journal of Central South University(Medical Sciences)
Year:
2006
Type:
Article
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