Study On The Application Of Nucleolin Aptamer For The Targeted Treatment Of Pancreatic Cancer

[Objective]In this study,we analyzed the expression of nuclein on the surface of pancreatic cancer cells,investigated the targeting and anti-tumor effects of nuclein specific aptamer AS1411 in vivo and in vitro,and explored the possible mechanisms of diagnosis,targeted imaging and anti-tumor mechanism of pancreatic cancer,providing new methods and ideas for the early diagnosis and targeted therapy of pancreatic cancer.[Methods]1.The serum levels of nuclein in pancreatic cancer patients and healthy controls were detected by enzyme-linked immunosorbent assays,and the expression of nuclein in pancreatic cancer tissues and adjacent tissues was detected by immunohistochemistry.Statistical differences in pancreatic cancer were analyzed using Graphpad Prism and SPSS.2.Using the artificially synthesized G-rich Strand A(-TAMRA-5')and C-rich Strand I sequence(-BHQ2-3'),in0.01M PBS solution,annealing forms the imotif based activatable aptamer probe(I-AAP)that triggers the change of configuration.The imaging effect of the"signal-off"type I-AAP probe panc-1tumor cells was investigated by using the biresponsiveness of pH-acidic environment of nuclein and i-motif on the surface of panc-1 cell membrane targeted by AS1411 in the I-AAP probe.3.To investigate the tumor inhibition effect of cells in vitro,the adaptive ligand AS1411 was studied through the time-dependent experiment of cells,the CCK8method to detect cell proliferation,the Transwell invasion and migration experiment,and the flow cytometry to detect cell cycle.4.To investigate the tumor inhibition effect in vivo,Panc-1 cells were injected subcutaneously to build the model of pancreatic cancer in nude mice.After the model was successfully constructed,the tumor volume changes and relevant immunohistochemical staining differences were observed in the experimental control group and the AS1411 injection group after 10 days of injection treatment.[Results]1.The level of nuclein in pancreatic cancer patients were 6.93±2.06(ng/mL)higher than that of the healthy control group 5.16±2.01(ng/mL).The difference was statistically significant(P=0.023).The expression level of nucleolus in pancreatic cancer is higher than that in adjacent tissues(P=0.015).2.The"signal-off"I-AAP probe constructed in this study showed fluorescence effect in pancreatic cancer cells with pH 6.0 and 6.5 solutions,no fluorescence effect in pH 7.0 and pH7.5 solutions,and had imaging effect targeting pancreatic cancer and acid-sensitive response.3.The cell observation experiment of AS1411 showed that after 30min,AS1411could be absorbed into Panc-1 cells,and could be completely absorbed within 2h.Different concentrations of 25umol/L and AS1411 have a certain inhibitory effect on panc-1 in pancreatic cancer cells and a certain time dependence.Panc-1 cells treated with AS1411 showed decreased invasion compared with the control group(P=0.0467).Compared with the control group,the percentage of cell migration and healing after 72h culture,which were lower than the control group(P=0.015),and the s-phase cells in the AS1411 group were(24.57±1.33)% higher than that of the control group(18.43±1.28)%,and the cell cycle was blocked in s-phase.4.The nude mice was injected with 0.2 mL 5×10~6 cell/mL panc-1 cells in subcutaneous tissue of the right axilla,the tumor mass was formed 20 days later,and the tissue HE staining showed low differentiation adenocarcinoma.After 10 days of treatment with AS1411 injection,the tumor volume of the AS1411injection group was(240.2±36.5)mm~3 significantly smaller than that of the control group with saline injection(315.9±39.2)mm~3.[Conclusions]1.The expression level of nucleolus in serum and tissue of pancreatic cancer patients is higher,and nucleolus has certain auxiliary diagnostic efficacy for pancreatic cancer.2.The"signal-off"I-AAP probe is expected to develop into a molecular pathologic tracer method with low background,high specificity and applicability by improving the ability of targeted imaging by using the dual-pH response and nuclein on the surface of pancreatic cancer cells.3.AS1411 has a certain inhibitory effect on pancreatic cancer in vitro and in vivo.