Characterization Of The Aptamer Against Uveal Melanoma Cell

Uveal melanoma(UM)is the most common malignant tumor of eye in adults,and it occurs in the eye uvea,including iris,ciliary body and choroid coat.Different from the skin melanoma can metastasize to the lung,lymph glands and other organs through lymphatic channel and blood vessel,most of UM metastasize to the liver through blood vessel.Metastasis to liver is the major cause of mortality in UM patients,more than 95%of deaths are related with it.The mean survival time(MST)is 6 to 8 months after the UM patients were diagnosed with metastasis to liver.Therefore,it is urgent to develop new methods for the diagnosis and treatment of UM to prolong the patients’ lifetimes.Aptamers are some ssRNA(single-strand RNA)or ssDNA(single-strand DNA),which have been screened through repeated rounds of in vitro selection(SELEX),they specifically recognize and bind target molecules after forming particular 3-dimention structures.Aptamers hold several attractive properties,such as high affinity and specificity,low immunogenicity,easy and reproducible synthesis,high stability,and so on.It is promising to apply aptamers in biomedical and clinical area in recent years.In this thesis,we selected an aptamer against uveal melanoma OCM-1 and analysed the characteristic of it.The aptamer selected in this thesis is expected to be a new biomarker for uveal melanoma diagnosis and treatment.The research content mainly concludes:(1)We used the uveal melanoma OCM-1 cell line as target cell,and the uveal melanocyte UC cell line as control cell.In order to obtain a specific identification of uveal melanoma aptamer,we screened 28 ssDNA in our laboratory by flow cytometry,and finded the XQ-2d can bind specifically to the uveal melanoma OCM-1 cell line.(2)To make good use of the aptamer XQ-2d in UM diagnosis and treatment,we investigated different properties of aptamer XQ-2d.By flow cytometry and laser scanning confocal microscope,we finded XQ-2d can bind specifically to the charoidal melanoma OCM-1 cell line and cannot bind to UC control cell line.The dissociation constant is 40.4± 12.9nM which is in nanomolar scale,showing high binding affinity towards OCM-1 cells.It also demonsrates high affinity.When dealing OCM-1 cells with different proteinase,we found that the targets of XQ-2d are some cell membrane proteins.The aptamer can enter into cells in physiological state,so it is a good candidate as a molecular probe for targeted drug delivery and targeted therapy.(3)Targeted drug delivery is particularly important in cancer treatment because many antitumor drugs are nonspecific and highly toxic to both cancerous and normal cells.Based on former research findings,we developed an aptamer-based drug delivery system with XQ-2d as the carrier of the antitumor drug doxorubicin(Dox).Using flow cytometry,we found that the interaction between XQ-2d with Dox did not affect the binding ability of XQ-2d,XQ2d-DOX and XQ-2d bound to the same site in UM.Additionally,a competition assay was performed by first incubating the OCM-1 cells with the XQ2d-Dox complex and then treating the cells with FITC-labeled XQ-2d.The results revealed that the XQ2d-Dox complex competitively inhibited the binding of XQ-2d to the target OCM-1 cells,which confirmed that the binding between the XQ2d-Dox complex and OCM-1 cells was dependent on the specific recognition of aptamer XQ-2d.At the end,using flow cytometry,we preliminarily checked the internalization of XQ2d-DOX,there were no significant fluorescence signal changes in the OCM-1 cells treated with the XQ2d-Dox complex compared to those treated with Dox alone,whereas in the UC cells,the fluorescence intensity from the XQ2d-Dox complex was much lower than that from the free Dox.Overall,these results preliminarily revealed that our aptamer-mediated drug delivery system has the ability to deliver drugs.