Selection Of Adipocyte Specific Aptamer, Its Molecular Recognition And Initial Application

Objective:To construct differentiation model of3T3-L1cell line and apply the Cell-based systematic evolution of ligands by exponential enrichment (SELEX) to this cell line aim to obtain the aptamers for the membrane proteins on the mature3T3-L1cells.Methods:Designed and synthesized random sequence DNA library, primers were labeled with fluorescein isothiocyanate (FITC) and biotin, respectively. Preadipocyte were differentiated into mature adipocyte thought classic cocktail method. Chose mature differentiated3T3-L1fat cells as positive cell, while its precursor cell, preadipocyte and HepG2as a negative cell. Thorough repeated incubation, separation steps with positive and negative cells, the enriched pool were amplified by polymerase chain amplification and used for the next round of screening or monitoring. The pool enrichment was monitored by flow cytometry. The enriched pools were then sequenced by454sequencing technology. Individual aptamer candidate were then selected, synthesized and their binding ability were identified by performing binding test with flow cytometry.Results:the cell-SELEX technology were applied on differentiated mature adipocyte, we have successfully enriched the ssDNA pool through nineteen rounds of positive and negative selection processes. After sequencing analysis, four aptamer, named Adipo-2, Adipo-4, Adipo-5and Adipo-8, were capable of binding to mature differentiated3T3-L1adipocyte. Conclusion:By applying cell-SELEX technology to3T3-L1adipocyte selection, high affinity aptamers can be generated. Objective:To investigate the specificity and affinity of aptamers obtained in the first part of the experiment, determine their dissociation constant and different incubation condition on the binding ability of aptamers to adipocyte.Methods:Aptamers were synthesized and test for their binding ability to cells from different system by using flow cytometry; Aptamer were diluted to different concentrations and incubate with differentiated adipocyte, the binding fluorescence were obtained at each point by using flow cytometry and used for Kd determination by using sigmaplot software; The binding properties of aptamers to adipocyte were characterized by test the binding ability at varied temperature, insulin stimulation on adipocyte, different truncated strategy of adipo-8.Results:Both aptamer Adipo1and Adipo8can bind with differentiated adipocyte, while no fluorescence can be observed when incubated with cells from other system. Dissociation constant determination demonstrated that aptamer adipo-8, adipo-1can bind to adipocyte with high affinity and specificity, with Kd values of17.8±5.1nM and97.5±13.5nM respectively.4℃and37℃incubation conditions did not affect the binding of the two measured aptamer. The binding ability of adipo8to mature adipocyte was lost when mature adipocyte were treated with trypsin and proteinase K before incubation, no such phenomonom can be observed in adipo-1.500nM Insulin stimulation on adipocyte before experiment did not affect the binding of adipo-8to differented adipocyte.Conclusion:Aptamer adipo8can specifically recognise differentiated adipocyte with high specificity and avidity. The target of adipo8could probably a protein that unrelated to insulin action, but further investigations are needed to elucidate the target. Objective:Given the fact that aptamer adipo8can specifically recognize differentiated3T3-L1cell, this part of the experiment design to explore the binding of adipo-8to adipose tissure and primary isolated adipocyte, and also the possible application such as adipo-8based adipogenesis monitoring, flow sorting of differentiated fat cells and possible in vivo applications as well.Methods:Male SD rats were sacrificed by cervical dislocation, the epididymal adipose tissue, liver, skeletal muscle and pancreatic tissue were then take out and used for n paraffin slide sections or frozen sections preparation. For frozen slide, fresh tissue were removed and immediately embedded with OCT and rapidly frozened at-20℃, tissues then cut into thin slices of10μm thick and mounted on glass slide, stabilized with ice-cold acetone at room temperature of4℃. After fixed for15min, the tissure slide then washed with PBS and used for H&E staining or aptamer staining. Fluorescence from both tissue treatment method were visulized by fluorescence microscopy; To explore the correlation between relative fluorescence intensity and time course of differentiation, we detect the binding of adipo-8and control ssDNA on adipocyte at different differentiation stage, the use of flow cytometry for sorting of mature adipocyte have also been performed; adipo-8were cy5-labled and injected via tail vein, different tissue were collected and used for fluorescence detection.Results:Frozen epididymal adipose tissue slices show strong fluorescence signal under microscopy when incubated with adipo-8while no such phenomenon can be noticed either when incubated with lib control or adipo-8incubate with frozen liver, skeletal muscle, pancreatic tissue. There is a positive relationship between the relative fluorescence intensity of the adipo-8and control library and differentiation stage. No specific fluorescence signal can be seen in different tissue under microscopy when adipo-8were injected into tail vein of SD rats.Conclusion:The aptamer Adipo-8can specifically recognize frozen section of adipose tissue and primary separated rat adipocytes. Aptamers adipo-8could be used to monitor and quantify3T3-L1cell differentiation, and used for mature fat cells separation. Adipo8can be used to discover specific biomarkers in the fat cell membrane and the possible development of a fat tissue-specific targeted drug therapy vector.