Aptamers Selected From HepG2 Cells And Their Application

Cancer is the second deadliest diseases nowadays all around the world,and the liver cancer is the second cause of cancer death,China occupies about half of the liver cancer cases and deaths in the total numbers.Liver cancer is difficult to cure and its postoperative survival rate is low as well,only early diagnosis and timely therapy can increase the survival rate of the patients.The differences between the cancer cells and the normal cells can be used for the early diagnosis of liver cancer.However,the specificity and sensitivity of markers used for early diagnosis of liver cancer are not high enough,which would result in misdiagnosis and omission diagnosis.Therefore,the HepG2 cells are used as target cells to select the aptamers with strong affinities and high specificities in this paper.The concrete research content is as follows:1.The selection of two aptamersThe liver cancer HepG2 cells(hepatoma carcinoma cell)are chosen as target cells and the L02 cells(human normal hepatocyte)are the control cells.By increasing the selection pressure,19 rounds of cell-SELEX are done to obtain two aptamers,named apt_A and apt_B.The secondary structure of the two aptamers is simulated by software.(Chapter 2)2.The investigation of the properties of the aptamersThe specificity,affinity and the binding ability of the selected aptamers with the tumor tissue sections are investigated respectively.(1)Both the flow cytometry and the confocal microscopy are used to investigate the specificity of apatmers.The FAM-labelled aptamers are incubated with different cells,and then the mixtures are detected by the flow cytometry and the confocal microscopy respectively.Both the results from flow cytometry and confocal microscopy confirm the high specificity of the two apatmers.(2)The equilibrium dissociation constants(Kd values)are used to measure the affinity of aptamers.Serial concentrations of aptamers are prepared to incubate with HepG2 cells.After the incubation,the average fluorescence intensity is detected by the flow cytometry.After fitting by the software,the Kd value of apt_A is 46.3±4.5 nM and the Kd value of apt_B is 101.8± 15.1 nM,which are lower than the Kd value of aptamers selected by others.(3)The xenograft tumors of HepG2 cells in nude mice are cultivated into tissue sections.The immunofluorescence assay is performed by incubating the tissue section with the FAM-labelled aptamers.Finally,we find that the two aptamers can bind with the xenograft in vivo specifically.(Chapter 3)3.The specific killing effect of drugs modified with aptamersAfter the drugs are combined with different aptamers,the cell proliferation toxicity experiments are done for HepG2 cells and L02 cells respectively.The result shows that the drugs modified with aptamers do kill a lot of HepG2 cells,but do not affect the survival rate of L02 cells.Meantime,the effects of the drugs modified with apt_A or apt_B are almost the same,the fatality rate of HepG2 cells is 50%,however,as for the drugs modified with apt_out(selected by others),the fatality rate is only 30%.The result confirms that the specific killing effects of apt_A and apt_B are stronger than apt out.(Chapter 4)...