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[Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells].
- Source :
-
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences [Zhong Nan Da Xue Xue Bao Yi Xue Ban] 2006 Dec; Vol. 31 (6), pp. 843-7. - Publication Year :
- 2006
-
Abstract
- Objective: To investigate the effects of DNA hypomethylation on mRNA and protein expression of perforin promotor in T cells.<br />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.<br />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).<br />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.
Details
- Language :
- Chinese
- ISSN :
- 1672-7347
- Volume :
- 31
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences
- Publication Type :
- Academic Journal
- Accession number :
- 17213580