Selection And Preliminary Application Of Aptamers For Clinical Liver Cancer Tissue

Liver cancer has been one of the most fatal malignant tumors worldwide with very high morbidity and fatality rate. Developing efficient and high speed methods for biomarker identification, and exploiting novel molecular probes with admirable sensitivity and specificity have significant importance for the clinical dignosis, classification and prognosis of cancer.In recent years, based on protein-SELEX and cell-SELEX strategy, aptamers have emerged to be a new kind of molecular probe in many research fields including cancer research. Normaly, protein-SELEX utilizes prurified proteins as selection target, and cancer cell lines are used as selection target in cell-SELEX. However, these selection targets cannot imitate the real canceration site in vivo because tumour pathogenesis is affected by multiple factors in vivo, including the micro-environments of blood vessels and matrix cells surrounding tumour cells. Therefore, aptamers generated by these stratedies may have difficulties in the recognition of real clinical tumor tissues, which greatly limited their applications in clinical cancer research. A recently developed SELEX strategy, termed tissue-SELEX, utilizes clinical tumor tissues as selection target, and the aptamers developed by this method could be readily used in clinical application and biomarker identification. However, the report of aptamers generated by tissue-SELEX is still rare, this search field requires further development.In this thesis, an aptamer that can distinguish cancerous liver tissues from normal ones was successfully obtained by tissue-SELEX, and some properties of this aptamer were also studied.(1) Based on tissue-SELEX,using cancerous liver tissues as selection target and adjacent normal liver tissue as control, we have successfully generated an aptamer, termed SW1, after 11 round of selection. This aptamer can specificly bind to cancerous liver tissues rather than normal liver tissue. In addition, aptamer BC15, preiously developed by Shao and coworkers against breast cancer tissue, was used to block related binding site in tumor tissues in order to generate an aptamer that can bind to a new binding site.(2) Formaldehyde fixed cancer cells were used as substitution of tissue to charaterize the aptamer SW1. By using fluorescent co-location, the target molecule of SW1 was located in the cell nucleus, possibly a nucleic acid-binding protein. The dissociation constant of SW1 to liver cancer cell line was tested to be in the nanomolar range, indicating that SW1 has good affinity to liver cancer cells. Competetion experiment indicated that the binding site of aptamer SW1 is different from that of BC15. Moreover, SW1 was tested to have a recognition rate of 72.7% to a large number of clinical liver cancer tissues by using tissue microarray, and SW1 showed good binding ability to cancerous tissues derived from different human organs including colon, lung, cerix, etal.Taken together, aptamer SW1 possesses good recognition ability to cancerous tissues, which makes it a promissing probe in clinical screening of liver cancer. In addition, the binding site of SWl was proved to be different from that of BC15, indicating that the target molecule could be a novel cancer biomarker that can promote the early diagnosis and effective therapy of liver cancer.