| Telomeres are distinctive DNA-protein structures that cap the ends of linear chromosomes. It is very important to keep the chromosomes stabilization. Telomerase is a ribonucleoprotein enzyme that replaces repetitive (TTAGGG) sequences on the ends of chromosomes that would otherwise be lost during successive cell divisions. Telomerase activity is closely linked to attainment of cellular immortality, a step in carcinogenesis, while lack of such activity contributes to cellular senescence. Telomerase is activated in more than 85% of malignant tumors.Human telomeric repeat binding factor 1(TRF1) is a telomere associated proteins. It participates in a physiological homeostatic mechanism controlling cellular proliferative potential.TRF1 is involved in negative feedback mechanism that allows telomere shortening by inhibiting the activity of telomerase. Down-regulation of TRF1 expression results in telomere elongation and may be involved in cell immortalization and cancerogenesis. Yamada have found expression of TRF1 and TRF2 mRNAs was higher level in the normal cells than in human malignant hematopoietic cell lines and/or in patients with acute leukemia. When differentiationof the malignant hematopoietic cell line HL-60 was induced using tumor-necrosis-factor 471 and all-trans retinoic acid (ATRA), telomerase activity decreased gradually during differentiation and initially low expression of TRF1 and TRF2 mRNAs increased during differentiation. 2000,Aragona's group has reported to determine the TRF1 expression by immunohistochemistry (IHC) in gastrointestinal tumors with polyclonal antibody anti-TRFl. The results suggest the expression level of TRF1 in malignant tumor is lower than that of normal tissue, inflammation and benign tumor.2001, they used same way to study brain tumors , the result is similar. But the quantitative analysis of TRF1 in acute leukemia and kidney cancer, the correlativity between TRF1 and activity of telomerase are not available.There is no report about monoclonal antibody for TRF1 yet.In this study, based the monoclonal anti-TRFl33-277 antibody, we will identification of the monoclonal antibody TRF133-277 and application in acute leukemia and kidney malignant tumor, in order to explore the relationship between the TRF1 and tumor.1. Identification of the monoclonal antibody against human telometric repeat binding factor 1(TRF1) protein: In ascites fluid, the monoclonal antibody liter is 1:3200, hybridoma culture supernatant belongs to IgG. By Western-blot , the antibody identified a specific located at about 60KD,which was consistent with the TRF1 protein as control. With the antibody, immunohistochemical staining showed that TRF1 protein located in the cytoplasm of epithelial cell and bone marrow cell. The anti-TRFl33-27 monoclonal antibody is useful for detecting of TRF1 protein in tissue specimens with high specificity.2. Quantitative analysis of the expression level of TRF1 protein in human acute leukemia and relationship between expression level of TRF1 protein and telomerase: a quantitative western-blotting technique was developed using anti-TRFl33-27 monoclonal antibody and GST-TRF1 fusion protein as a standard to further determine the expression level of TRF1 protein in total proteins extracted fromclinical specimens. 20 acute leukemia was studied , 11 normal volunteer bone marrow as control.The expression level of TRF1 protein is significantly higher(p<0.01) in normal bone marrow (2.217+0.461g/ul)than that of acute leukemia(0.754+0.343g/l). There is no remarkable difference of expression level of TRF1 protein between ALL and ANLL(0.628+0.281g/l vs 0.844+0.360g/l, p>0.05).After chemotherapy, TRF1 expression level of patients with complete remeision raised(0.772+0.307g/l vs 1.683+0.344g/l, p<0.01),but lower than that of normal(2.217+0.461g/l ,p<0.01). TRF1 expression level of patients without complete remession wasn't remarkable difference after chemotherapy(0.726 +0.443g/l vs 0.894+0.338g/l, p>0.05). TRF1 expression level of patients with complete remession... |
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