The Aptamers Selection Of Human Hepatocarcinoma Cell Bel-7404and Characterization Of The Aptamers

Liver cancer is one of the most common and malignant tumors. It can beclassified into hepatocellular carcinoma, cholangiocarcinoma and mixed cell canceraccording to different tissue origins of tumorigenesis, among which hepatocellularcarcinoma accounts for85~90%. Bile duct cancer originates at the extrahepatic bileduct from lower hepatic portal area to the common bile duct. Currently the clinicaldiagnosis of liver cancer is mainly based on morphology imaging of tissue and cells,and its difficult to achieve early diagnosis; drugs for cancer therapy lacks specificityfor target, and thereby the side effect is of great concern. In recent years, cell-SELEXtechnology obtained fast development because of its unique advantages, and providesa new idea to solve the abovementioned problem. As the product of cell-SELEX,nucleic acid aptamers has many advantages including low molecular weight, easysynthesis and modification, low toxicity, good stability, high affinity and specificity.It provides a new, ideal molecular tool for the diagnosis and treatment of malignanttumor and other serious diseases, and has a good application prospect in biomedicaland clinical research.In this work, we adopted cell-SELEX strategy to abtain nucleic acid aptamersthat can specifically recognize human hepatoma cell line Bel-7404, and examined theproperties of selected aptamers and applied them into preliminary application.Research contents include:(1) selection of Bel-7404aptamers for human hepatocellular carcinoma cell lineCell-SELEX was performed with human liver cancer cell line Bel-7404as targetcells and human cholangiocarcinoma cell line QBC-939as control cells. We fistlyoptimized the selection conditions including the annealing temperature for PCR andso on. Then, with the increasing rounds of screening, a negative selection wasintroduced to the process of selection, and the stringency of screening conditionsgradually increased. Finally, the selection reached the affinity plateau after15roundsof selection. The15th selected pool was subjected to cloning and sequencing, and thesequencs were aligned by homology. After subsequent verification, we abtained4aptamer sequences that can identify human hepatocellular carcinoma cell lineBel-7404with high affinity.(2) properties investigation for Bel-7404specific aptamer and preliminaryapplication In order to make the nucleic acid aptamers more suitable for future liver canc ercell detection and analysis. Flow cytometry was used to examine the aptamer bindingaffinity, cell specificity, temperature stability and protease digestion effect. On theother hand, in order to make the aptamers more applicable for application in complexbiological environment, we further optimized of the sequence and structure of theaptamer and verified its target recognition capacity in hepatocellular carcinoma tissuesections. The experimental results show that the aptamer can recognize humanhepatoma cell line Bel-7404with high affinity and high specificity; it demonstratesgood temperature stability, and is not affected by the proteinase effect. Theseinformation laid the foundation for the future application of these aptamers. Moreover,by sequence optimization, the nucleotides of aptamer is shortened from79nt to37ntwith an improvement on binding affinity. More importantly, the optimized aptamer isable to specifically identify Bel-7404tumor tissue sections.