In Vivo Imaging And Identification Of Target Protein Using Aptamer Against The Human Glioma Cell

Objective: The imaging diagnosis of glioma mainly relies on computed tomography and magnetic resonance imaging,but some non-neoplastic diseases are easily confused with it,resulting in misdiagnosis problems.Molecular imaging technology can achieve biological imaging at the cellular or molecular level through molecular probes with targeted recognition capabilities,providing a powerful tool for solving the misdiagnosis problems of current imaging technology.As a novel target recognition molecule,aptamers have the advantages of strong affinity and good biocompatibility,which are widely used in the in vitro and in vivo imaging of tumor cells.This study aims to improve in vivo stability of aptamer targeting glioma cells by chemical modification,and then evaluate the target recognition ability and in vivo stability of the aptamer after modification respectively.The feasibility of using the aptamer for imaging glioma in vivo was confirmed by the test of tumor-bearing mice model.Finally,the target molecule of aptamer are identified and verified by mass spectrometry and other techniques.This study would select the aptamer targeting glioma cells as the starting point,and be expected to lay a foundation for the in vivo imaging of gliomas based on aptamers and provide new candidates for the diagnosis of glioma.Methods: 1.To evaluate the targeted recognition ability and in vivo stability of aptamer after chemical modification,the binding ability of the modified aptamer to glioma cells was detected by flow cytometry.The stability of the modified aptamer in serum was detected by agarose gel electrophoresis.2.To test whether the aptamer could retain its recognition ability in vivo,the glioma tumor xenograft model was established and labeling probes were constructed by directly modifying the near-infrared fluorescent dye Cy5.Then an IVIS Lumina II in vivo imaging system monitored the in vivo distribution of aptamer and random sequences.3.The target of aptamer was identified by aptamer-based separation and enrichment,combined with mass spectrometry.Then the target was also validated by western blot,surface plasmon resonance(SPR),small interfering RNA transfection and other techniques.4.Immunohistochemistry was used to detect the expression of target protein in 10 normal brain tissues,44 astrocytomas(WHO II-III),and 22 glioblastoma(WHO IV).Results: 1.After chemical modification,the aptamer miantained the ability to detect glioma cells and could be stably presented in serum for up to 24 hours,which was 12 hours longer than that had not been chemically modified,indicating that the chemically modified aptamer retained the target recognition ability and had good serum stability.2.The in vivo fluorescence imaging results showed that the aptamer could be specifically enriched in the tumor site of mice.After 1 hour of injection,the fluorescence signal of the tumor site was the strongest.After 4 hours of injection,obvious signals still could be observed in the tumor site.The in vivo imaging of glioma tumor-bearing mice based on aptamer was preliminarily achieved,indicating that the aptamer targeting glioma cells was feasible for in vivo imaging.3.Polyacrylamide gel electrophoresis results showed that near the 260 kDa,the lanes of the aptamer group had an obvious differential band,mass spectrometry results showed that the target of the aptamer might be fibronectin,and the results of western blot showed that only in aptamer group detected the presence of fibronectin,SPR results showed that the equilibrium dissociation constant of the aptamer and fibronectin was 125 nM,and the results of small interfering RNA transfection showed that the binding ability of the aptamer to glioma cells was weakened after knockdown of fibronectin.The results of these assay showed that the target protein of the aptamer was fibronectin.4.Immunohistochemical staining showed that the mean histochemical scores of fibronectin in normal brain tissue,astrocytoma(WHO II-III)and glioblastoma(WHO IV)were 5.62,56.99,and 95.93 respectively,and its expression level increased when the grade of malignancy of the glioma increased.Conclusion: 1.The chemically modified aptamer had good serum stability and maintained the ability of targeted recognition.2.The in vivo imaging of tumor-bearing mice based on the aptamer was preliminarily achieved,indicating that the aptamer had the potential to be applied in the field of in vivo glioma imaging.3.The target protein of the aptamer was fibronectin and the expression of the target protein was aberrant in glioma tissues,when compared with normal brain tissues.