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.
Subject(s)
Adult , Humans , Azacitidine , Pharmacology , Cells, Cultured , DNA Methylation , Perforin , Genetics , Promoter Regions, Genetic , Genetics , T-Lymphocyte Subsets , MetabolismABSTRACT
Objective To investigate the changes in hydrogen sulfide(H_2S) level in plasma and myocardial tissues of doxorubicin-induced heart failure rats,and to study the role of H_2S in the development of doxorubicin-induced heart failure.Methods Male Wistar rats were randomly divided into 2 groups as follows:the doxorubicin group(n=12),in which 2.5 mg/kg of doxorubicin was injected intraperitoneal once a week for 10 weeks(total dose of 25 mg/kg);the control group(n=9),in which an(equivalent) volume of physiological saline was administered weekly for a total of 10 weeks.The observation of behavior was taken at the same time.Hemodynamic and echocardiographic measurements were obtained 10 weeks after treatment.Meanwhile,H_2S concentrations in serum and myocardial tissues were evaluated by modified sulfide electrode method.The changes of H_2S level between 2 groups were analyzed.Results The rats treated with doxorubicin showed inanimate behavior,decrease of the body temperature,activities and food intake,faster breathing,significant loss of weight,the cumulative mortality was 33%.Left ventricular systolic pressure(LVSP),difference of left ventricular pressure[△LVP=LVSP-left ventricular diastolic pressure(LVDP)] and left ventricular peak rate of contraction(+LV dp/dtmax),left ventricular peak rate of relaxation(-LV dp/dtmax)were significantly reduced in the group of doxorubicin rats(P
ABSTRACT
Objective To investigate the changes of hydrogen sulfide (H_2S) level in plasma in order to explore the role of H_2S in the development of pulmonary hypertension (PH) secondary to congenital heart disease (CHD).Methods There were 9 CHD patients and 9 normal children in this study. The plasma concentration of H_2S and pulmonary artery pressure (PAP) of each child were measured. Meanwhile, the relationship between H_2S level and PAP was analyzed.Results The plasma level of H_2S in the group of CHD significantly decreased compared with control group (32.13?2.25) ?mol/L vs [(43.69?2.05)?mol/L, P