Study Of The Relationship Between Telomere Abnormality And Aplastic Anemia

ObjectiveTo identify changes in telomere length and gene expression of shelterin (which is composed of six core components:TRF1, TRF2, POT1, TIN2, TPP1and RAP1) in severe aplastic anemia(SAA). Looking for the reason why telomere getting short in SAA patients and the pathway that abnormal telomeres cause AA.Methods15normal controls and27cases of SAA who were diagnosed by TianJin General hospital between2011, May and2012, May were enrolled in this study. There were9untreated and18recovering patients. CD3+T cells were sorted by immunomagnetic separation. Telomere length was tested by southern blot and gene expression of TRF1, TRF2, POT1, TIN2, TPP1and RAP1were detected by RT-PCR.Bone marrow mononuclear cells (BMMNC) from normal controls were cultured in the culture medium that contain AA serum, TNF-α and IFN-γ respectively. The apoptosis of these cells were detected by flowcytometry. The mRNA expression of POT1were tested by RT-PCR and ATR, phosphorylated ATR, and phosphorylated ATM/ATR substrate were detected by western blot.ResultsFirst Section Telomeres length of CD3+T cells were found significantly shorter in SAA untreated group (4.42±1.13) kb and recovering group (5.82±0.97) kb than control group(9.18±3.31)kb. Telomeres Length of CD3+T cells turned shortening in accordance with TH/s (Thelper/Tsuppressor) decreasing(r=0.564, p=0.029). There was no relationship between Telomeres Length of CD3+T cells and hemoglobin, count of WBC and platelet, proportion of reticulocyte, the recovery time of white blood cells (WBCs) and at least one lineage of bone marrow hematopoietic cells.Telomeres of peripheral white blood cells(PWBCs) were found significantly shorter in SAA untreated group (4.89±1.66) kb and recovering group (7.04±1.47) kb than control group (11.65±5.55) kb. There was no difference between SAA untreated group and recovering group. Telomeres Length of PWBCs also turned shortening in accordance with TH/s decreasing(r=0.593, p=0.007). There was no relationship between Telomere Length of PWBCs and hemoglobin, count of white WBC and platelet, proportion of reticulocyte, the recovery time of WBC and at least of one lineage of bone marrow hematopoietic cells.Second Section The mRNA expression of POT1decreased in untreated SAA patients (0.16(0.02-0.29)) and over expressed in recovering patients (1.17(0.82-1.86))(P<0.05) in CD3+T cells. The mRNA expression of RAP1was significantly higher in untreated patients(4.14(0.29～2.83)) than that in recovering group (0.87(0.30～1.73)) and controls (0.62(0.45～4.07))(P<0.05) in CD3+T cells. There were no differences among SAA untreated group, recovering group and controls about the expression of TRF1、TRF2. TIN2～TPP1in CD3+T cells. The mRNA expression of POT1decreased in SAA untreated group(0.29(0.09-0.58)) than in recovering group(0.78(0.22-2.69)) and controls (1.88(0.93-3.81)),(P<0.05). The mRNA expression of TRF1、TRF2、TIN2、TPP1and RAP1were no differences among SAA untreated group, recovering group and controls in PWBCs.Third Section The apoptosis rate increased in cells that cultured in SAA sersum (13.88±4.55) than in fetal calf serum (9.85±2.67). The mRNA expression of POT1was significantly declined in cells that sorted from untreated SAA patient (0.45±0.29) and in cells that cultured by SAA sersum (0.59±0.49) and in the culture medium containing IFN-y (0.40±0.35) than the cells that cultured in fetal calf serum(1.19±0.62). The expression of the protein ATR, phosphorylated ATR, and phosphorylated ATM/ATR substrate were detected significantly increased in cells from SAA sersum group, TNF-α group and IFN-y group. And the expression of the protein above increased with thickening of TNF-α.ConclusionFirst Section Short telomeres were found in both CD3+T cells and PWBCs of SAA patient. The telomere length of both CD3+T cells and PWBC was shorter in accordance with decreasing of TH/s. Suggesting telomeres length strongly linked to the immunological function status.Second Section High expression of RAP1mRNA in CD3+T cells sorted from untreated SAA patients may related to T cells differentiation. The mRNA expression of POT1decreased in both CD3+T cells and PWBCs from untreated SAA should be explained by activating apoptosis.Third Section TNF-a and IFN-y are the effective component of SAA sersum, which can induce cell apoptosis through POT1and the pathway of ATR, and at last cause AA.