| Objective: 1. Evolution of DNA aptamers with high affinity and specificity for malignant brain tumor gliosarcoma cell recognition and clinical tissue imaging.2. To select aptamers against glioma stem cells via Cell-SELEX, which can be used as a molecular probe to identify and sort glioma stem cells.Methods: 1. The DNA library and primers were purchased from Sangon Biotech Co.,Ltd. China, comprised a randomized region of 45(N45) nucleotides flanked by 18 nt primer. Cell-SELEX was applied to generate DNA aptamers against human gliosarcoma cell line k308, normal human brain astroglia cell SVGp12 was used as counter selection. Enriched pool was amplified cloned and charactered. Flow cytometry was applied to monitor the process of the DNA aptamer selection and measure the K_d value. Afterward, the aptamer was further investigated the recognition capability of target cells from U251, U87, T98 G and other cancer cells. Furthermore,laser confocal fluorescence microscopy was used to evaluate the recognition ability of the aptamer to clinical gliosarcoma tissue.2. The DNA library and primers were synthesized, comprised a randomized region of 40(N40) nucleotides flanked by 20 nt primer. Cell-SELEX was applied to generate DNA aptamers against glioma stem cells. After MACS, glioma stem cells were by identified by immunocytochemical staining, Western-Blot and RT-PCR.Glioma cells U87 were used as counter selection. Enriched pool was amplified,cloned and charactered. Flow cytometry was applied to monitor the process of the DNA aptamer selection and measure the K_d value. Afterward, the aptamer was further investigated the recognition capability of target cells from U251, T98 G and other cancer cells. Furthermore, laser confocal fluorescence microscopy was used to evaluate the recognition ability of the aptamer to glioma stem cells. Flow cytometry assay of competition was performed between aptamer and CD133 antibody binding to glioma stem cells.Results: 1. Five DNA aptamers were successfully selected specifically targeting gliosarcoma cells after 16 rounds of selection. The K_d value of these DNA aptamers are all in the nanomolar range from 10 n M to 100 n M. These aptamers can selectively distinguish gliosarcoma cells from human normal astroglia cells. Among them,aptamer WQY-9 was found to have the highest affinity and best specificity against gliosarcoma cells. An optimized sequence WQY-9-B with a K_d value of 23 ± 5 n Mheld the same recognition ability to gliosarcoma cells as that of WQY-9, and the target of aptamer WQY-9-B has been preliminarily verified as extracellular membrane protein. More importantly, tissue imaging results demonstrated that aptamer WQY-9-B was highly specific to the corresponding tumor tissue and displayed as high as73.3% positive detection rate against gliosarcoma tissue.2. Glioma stem cells pose the property of forming sphere, differentiation and xenograft tumorigenicity in nude mice. Five DNA aptamers were successfully selected specifically targeting glioma stem cells after 20 rounds of selection. Among these aptamers, W5 with a K_d value of 4.9 ± 1.4 n M was found to have the highest affinity and best specificity against glioma stem cells. An optimized sequence W5-7with a K_d value of 23 ± 5 n M held the same recognition ability to glioma stem cells at4 oC and 37 oC. The target of aptamer W5-7 is not CD133 protein. Furthermore, W5-7can bind CD133 positive glioma stem cells.Conclusions: 1. We have identified a panel of aptamers with high affinity and specificity against gliosarcoma cells. Aptamer WQY-9-B shows the comparative recognition ability to aptamer WQY-9. Moreover, aptamer WQY-9-B shows excellent recognition capacity for tissue sections of clinical gliosarcoma samples. Therefore,aptamer WQY-9-B can be considered as a novel molecular probe for gliosarcoma diagnosis.2. We have generated several aptamers with high affinity and specificity against glioma stem cells. Aptamer W5 and W5-7 show high affinity and specificity targeting glioma stem cells. The target of aptamer W5-7 has been preliminarily verified as extracellular membrane protein. W5-7 can be considered as a novel molecular probe for identifying and sorting glioma stem cells, which would play a critical role in clinic diagnosis and discovering the biomark of glioma stem cell. |
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