The Study Of The Expression Of P16 Gene On CD4~+ And CD8~+ T Cells From Peripheral Blood With SLE And Lympholeukocyte Apoptosis

ObjectiveTo study the expressions of p16 gene (mRNA and proteins) and lymphocytes apopotosis of CD4+ and CD8+ T cells from peripheral blood with SLE patients. To analyze the correlations between apoptosis and the expressions of p16 gene. To detect the activities of p16 gene promoter after transfecting the hypermethylated p16 promoter into CD4+ T cell in vitro and to reveal the effect of p16 gene methylation and expressions on lymphocytes apoptosis in SLE patients.Methods40 definitive SLE patients and 30 healthy controls were collected. CD4+ and CD8+ T cells were separated from the peripheral blood using immunomagnetic beads and counted. The Reverse transcription-polymerase chain reaction(RT-PCR) technique was applied to quantitatively analyze the expression of p16 mRNA of CD4+ and CD8+ cells, and p16 protein and apoptosis rate of CD4+ and CD8+ cells were detected by flow cytometry. p16 gene promoter with hypermethylation in vitro was transfected into CD4+ T cells in vitro and detected the activity using luciferase Reporter Gene Assay Kit.ResultsThe quantities of CD4+ and CD8+ T cells were significantly decreased in active SLE patients as compared with inactive and healthy controls. Levels of p16 mRNA expressions in CD4+ and CD8+ T cells were significantly increased in active patients with SLE as compared with inactive and healthy controls. They also have correlations with SLEDAI. In CD4+ T cells, the levels of p16 mRNA have correlation with photaesthesia,arthrositis,Nephros damage,decreased complement and ANA series. In CD8+ Tcells, it has correlation with arthrositis,Nephros damage, ANA series.Levels of p16 protein in CD4+ and CD8+ cells were significantly increased in active patients with SLE as compared with inactive and controls. CD4+ and CD8+ cells apoptosis rates were significantly positive increased in active patients with SLE as compared with inactive and controls. CD4+ and CD8+ cells apoptosis rates were positive correlated with the SLEDAI, so were with the expressions of p16 proteins.The activity of p16 promoter with hypermethylation in vitro transfected CD4+ T cells was significantly decreased than that without hypermethylation.ConclusionsThe quantities of CD4+ and CD8+ cells was significantly decreased. Levels of p16 mRNA and proteins expressions in CD4+ and CD8+ T cells was significantly increased. The apoptosis rate of CD4+ and CD8+T cells were increased and positive correlated with the SLEDAI,so were with the expressions of p16. It suggests that abnormal p16 expressions may be correlated with the apoptosis of CD4+ and CD8+ T cells.The increasing of p16 expression may inhibit the generation of CD4+ and CD8+ T cells in SLE patients and promote the apoptosis of CD4+ and CD8+ T cells. Previous studies which showed that p16 promoter regions of peripheral blood CD4+ T cells with SLE were hypermethylated, were not coherent with this study which presented high expressions of p16 gene on peripheral blood CD4+ T cells with SLE. The study showed the activities of p16 promoter region after hypermethylated in vitro transfected CD4+T cells were decreased, that may inhabit the expressions of downstream sequence.This discordance may correlate with the instability of heredity and epigenesis.