Studies On Aptamer Mediated Hematologic Malignancies Cells Recognition And Its Stability

Hematologic malignancies, as one group of common malignant tumors, is a big threat to human health. In order to make the early diagnosis and treatment of hematologic malignancies possible, and therefore to increase the cure rate, it is of great importance to identify hematologic malignancies cells correctly. Aptamers, selected by SELEX, not only have high affinity and specificity as antibody, but also have many advantages over antibody in many respects. Expected as a new type of"diagnosis antibodies", aptamers have many potential applications in the diagnosis and treatment of tumor. Aiming at this advanced research direction, three parts of work on the recognition of hematologic malignancies cells by aptamers, selected by cell-SELEX, are discussed in this thesis. They are as follows:1. Study on recognition of Burkitt's lymphoma cells in different conditions using cell-SELEX selected aptamersThe recognition of Ramos (Burkitt's lymphoma cell line) cells and primary Ramos cells under different conditions by using aptamer TD05, which was selected against Ramos cells through cell-SELEX, has been investigated using Flow Cytometry. The results were compared with the recognition of specific CD19 antibody towards Ramos cells. The experiments results show that TD05 could effectively identify the Ramos cells from different sources in different conditions, and shows a superior adaptability to the reaction condition. As a new candidate of"diagnosis antibodies", TD05 shows huge potential application in early diagnosis and testing of hematologic malignancies.2. Study on the stability of cell-SELEX selected aptamers in bloodThe stability of aptamer TD05 in blood has been investigated using 20% denaturing PAGE and Flow Cytometry. TD05 was then modified with 3′-3′dT and Locked Nucleic Acid (LNA), respectively. The changes of the affinity and specificity of modified TD05 were analyzed. The results indicated that 3′-3′dT and LNA modification could improve the stability of TD05 in blood. The halflife of TD05 in serum could be prolonged from 0.5 hour to more than 3 hours. The results also showed that the modification has no influence on TD05's affinity and specificity. These results provided technical support for further application of TD05 in vivo detection.3. Preliminary study on the three-dimensional conformation of cell-SELEX selected aptamer in target recognitionSgc8c, which was selected against CCRF-CEM (T cell acute lymphoblastic leukemia (ALL) cell line) cells through cell-SELEX, was modified with fluorescence quenching group, fluorescence resonance energy transfer group, PEG and a part of complementary sequence, respectively. The binding of these modified aptamers with CCRF-CEM cells was tested by Flow Cytometry. Results indicated that 3′and 5′of sgc8c might hybridize into double-strand, and this part of sgc8c is very important to recognize target. The results afford reference for further application of sgc8c in tumor diagnosis.