The Feasibility Of Long-term Monitoring Transplanted Cells By Reporter Gene Imaging And GLP-1 Receptor Imaging

Objective:Recently,cell therapy has developed rapidly and brought novel treatments for clinical disease.However,how to monitor the long-term fate of transplanted therapeutic cells in vivo is still a challenge in cell therapy and molecular imaging.In this study,we prepared a novel hybrid baculovirus-“sleeping beauty”virus,and investigated the feasibility of this hybrid viral vector-mediated radionuclide reporter gene（Bac-NIS-SB-NeoR）in long-term monitoring transplanted therapeutic cells.In addition,to seek other imaging models of monitoring transplanted therapeutic cells,we as well investigated the feasibility of long-term and real-time monitoring transplanted isletβcells by GLP-1 analogues labeled with radionuclides.Methods:We introduced the transposase（SB100X）and two IR/DR sequences into the homologous recombination arms Tn7L and Tn7R of baculovirus backbone to construct novel plasmids of pFastBac-eGFP-SB-NeoR and pFastBac-NIS-SB-NeoR.The eGFP and NIS bacmids,obtained from constructed plasmids transformed into DH10bac,were transfected into sf-9 insect cells by liposomal transfection to produce recombinant baculovirus Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR.After U87 glioma cells infected by two recombinant baculovirus,G418 screening was used to obtain stable cells（Bac-eGFP-SB-NeoR-U87 and Bac-NIS-SB-NeoR-u87）.The eGFP positive proportion in Bac-eGFP-SB-NeoR-U87 and control cells was measured by flow cytometry.Meanwhile,the function of NIS protein in Bac-NIS-SB-NeoR-U87 cells was determined by dynamic iodide uptake and perchlorate inhibition assays.Then the long-term NIS gene expression in Bac-NIS-SB-NeoR-U87 cells was determined by RT-PCR,western blotting and radioiodine uptake assay at various weeks.Finally,long-term radionuclide imaging monitoring transplanted Bac-NIS-SB-NeoR-U87 cells which had been cultured for approximately 28 weeks in vitro was performed by SPECT.In addition,Liraglutide was labelled with ¹²⁵I by the Iodogen method and the labelled ¹²⁵I-Liraglutide was then used for in vitro saturation and competitive binding experiments with INS-1 cells.Finally,¹²⁵I-Liraglutide was used for monitoring transplanted INS-1 insulinoma by SPECT/CT.To explore more effective imaging probes,Irisin was labelled with ¹²⁵I by the Iodogen method,and then the C57/B16 mice was used for SPECT/CT imaging at various time by an injection of ¹²⁵I-Irisin,as well as radioactive distribution in various organs.Results:We have produced two recombinant baculovirus with titers of 8.68í10⁸ and4.01í10⁸ pfu/mL,and established two stable cell lines（Bac-eGFP-SB-NeoR-U87 and Bac-NIS-SB-NeoR-U87）.The eGFP positive proportion in Bac-eGFP-SB-NeoR-U87 cells was 64.27±3.64%,96.03±0.21%,97.50±1.00%and 97.43±0.81%at the1^st,18^th,53^thh and196^th day after infection,whereas a significant decline to 0 at the 18^th day in the control cells.The Bac-NIS-SB-NeoR-U87 cells could actively accumulate radioiodine and reach a peak at 45 min.Then the radioiodine uptake could be inhibited by perchlorate（ClO₄^-）in vitro.The in vitro assay revealed no obvious decline in NIS mRNA,protein level and radioiodine uptake in Bac-NIS-SB-NeoR-U87 cells during 28 weeks.In vivo,transplanted Bac-NIS-SB-NeoR-U87 cells were effectively monitored by SPECT imaging at 20^th,28^thh and 43^th day after cell transplantation.Furthermore,the labelling rate of ¹²⁵I-Liraglutide was approximately 95%.The equilibrium dissociation constant K_d between¹²⁵I-Liraglutide and the GLP-1 receptor was 128.8±30.4 nmol/L,and the half-inhibition concentration IC₅₀ was 542.4±187.5 nmol/L.The ¹²⁵I-Liraglutide SPECT/CT imaging successfully monitored the transplanted INS-1 insulinoma in vivo.Finally,the labelling rate of ¹²⁵I-Irisin was approximately 95%.The small-animal SPECT/CT imaging revealed the highest level of radioactivity in the gallbladder followed by the liver and kidney,which is consistent with the radioactive distribution in the various organs.Conclusion:This study revealed the feasibility of Bac-NIS-SB-NeoR system in long-term monitoring transplanted therapeutic cells as well as the feasibility of Liraglutide labelled with radionuclide in long-term and real-time monitoring transplanted isletβcells,which provides the multimodal imaging means for monitoring transplanted therapeutic cells.