| Prostate cancer is one of the most common malignancies that seriously threaten men’s health, and the incidence of this disease ranked the second among all male tumors, and has sharply global increasing tendency in recent years. Early detection and early treatment of prostate cancer could greatly improve the cure rate, and effectively extend the life of patients. However, the current early diagnosis of prostate cancer is mainly based on digital rectal examination, imaging, tumor markers and biopsy examination. Which have the disadvantages of can not be found in time, limitation of the detection ranges, causing damage to normal tissue, and easily confusing with other diseases. In the wa y, it is difficult to achieve early diagnosis of prostate cancer based on these techniques. Therefore, screening and achieving novel and specific molecular probes for prostate cancer cells is significant for establishing a simple, rapid, sensitive and specific early diagnosis method, and it also has very important sense to early diagnosis and treatment of prostate cancer.Cell-SELEX is an in vitro selection technology using the whole live cell as the target, which has achieved rapid development in the last few years. Cell-SELEX can produce aptamers that specifically recognize a certain cell line, and even distinguish different types of tumor cells. What’s more, in the selection, no prior knowledge of cell-surface marker proteins is required. Especially aptam er generated from Cell-SELEX can bind to the target molecule at its natural state, and it have the ability to be directly used in recognizing and detecting cells. Furthermore, Cell-SELEX will play a very important role in biomedical field, such as, the ear ly diagnosis and classification of cancer, the discovery of tumor biomarker and the mechanism of tumor occurrence and development.Therefore, in this thesis, we selected highly metastatic human prostatic cancer cell line as the positive cell, and the low m etastatic prostatic cancer cell line as the negative cell based on Cell-SELEX. Finally, with no prior knowledge of cell surface proteins, we selected a series of aptamers which could bind to prostate cancer cell surface with high specificity and high affin ity. Details of these works have been performed as follows:1. Screening aptamers for the recognizing human prostate cancer cell line PC-3M-1E8.Based on the Cell-SELEX, we chose the highly metastatic human prostate carcinoma cell line PC-3M-1E8 as target cells, and the low metastatic human prostate carcinoma cell line PC-3M-2B4 as control cells. In order to achieve the aptamer with high specificity and high affinity, we progressively increased the strength of the washing and other selection conditions to r emove the unbounded aptamers, and to improve the efficiency of screening. Finally, after 12 rounds of screening, the enrichment of the selection library reached a platform. After cloning and sequencing, a homology analysis was corried out on these sequences,and four sequences were obtained which are capable of binding PC-3M-1E8 and PC-3M-2B4 two cell lines aptamers simultaneously.2. Characterization and primary application of the aptamers for human prostate cancer cell.We analyzed and characterized the aptamers acquired by Cell-SELEX. Several main properties of the aptamers were performed here, such as, cell specificity, equilibrium dissociation constant and temperature stability, etc. The experimental results suggest that these aptamers have the advantage s of high specificity, high affinity, and high temperature stability. In addition, we analyzed the secondary structures of these aptamers by the software Mfold, and optimized the sequence structure based on the analysis of secondary structure. Finally, a n aptamer C1 with 51 nts were achieved, with equilibrium dissociation constant as low as 10 n M. According to the results of the treatment of protease, we speculated that the target molecule on the cell surface is likely to be a membrane protein, which provid e a strong foundation to discover specific tumor marker of prostate cancer cell. Most importantly, these newly generated aptamers is expected to play a key role in the early diagnosis and clinical treatment of prostate cancer. |
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