| BackgroundAllogeneic organ transplantation plays an important role in treating cancers, blood system diseases and organ failure, however acute rejection (AR, Acute rejection) occurred during this process may cause organ damage, seriously affect the prognosis of organ recipients. Acute rejection occurs within the few weeks or months post transplantation. To find a new noninvasive technique that can be validly real-time monitoring, is very urgent for the treatment of disease and prolonging the life of an organ. Biopsy was well known as the gold standard for diagnosis of acute rejection, however,invasive damage and side effects limited its the broad clinical application.Molecular imaging which is applied as a new technique of medical imaging, proceeded the qualitative and quantitative study of cell and molecular level in vivo state of biological processes. It makes radiographic imaging from gross morphology important step forward microscopic morphology, biological metabolism, gene imaging and other aspects.It is necessary for the new technology of molecular and genetic level access to clinical therapy. Radionuclide-molecular imaging is the most advancing technique among molecular imaging with high sensitivities and easy application However, disadvantage of application of nonspecific tracer,such as 18F-labeled FDG,a molecules similar as glucose,made it non-specific targeting to transplanted organ. So, it is urgent to find a high-selective target molecule to image transplanted organ with radi-nuclide labeled in clinic.Research has been shown that Toll-like receptor family (Toll-like receptor, TLRs) that specifically recognizes pathogen-associated molecular patterns (pathogen-associated molecular pattern, PAMPs) plays an important role in the activation of innate immunity and adaptive immunity, which connected innate with adaptive immunity. TLR5,the only negative regulator of immune molecules in TLRs family, can selectively high-expressed on the surface of regulatory T cells (Treg). Bacterial flagellum (flagellin, Flic), as a TLR5 specific activator, can bind and enhance the effect of Treg. Flic which is the only particularly exogenous TLR5 ligand, can specifically bind to TLR5, recognize and promote cellular and humoral immune responses. Given TLR5 can regulate the effect of Treg cells, Treg cells played an important part in the allograft rejection. so TLR5 may be a key molecule in allograft rejection. This research was performed with radioactive iodine 125 to label recombinant flagellin (rFlic) and its fragments rFlic△180-400, to study the biodistribution and targeting on acute allograft rejection with non-invasive real-time monitoring to provide new allograft rejection monitoring target.Methods1. The establishment of mouse skin transplantation model:C57BL/6 female mice were used as donors and BALB/c female mice as recipients, estabolished the allograft model; BALB/c, female mice both as donor and recipient, estabolished a isograft transplantation model. Prepared a skin-graft from donor mice firstly;then, recipient mice were anesthetized,by intraperitoneal injection of 0.6% sodium pentobarbital solution (50μg/g); prepared a transplant bed on.its right backside and a skin graft was transplanted.7 days after transplantation, unpacking, skin graft we observed daily, grafts growth conditions were recorded. When graft necrosis was more than 90%, it was defined as graft rejection.2. The purification, expression and identification of rFlic and rFlic∞180-400 were done through IPTG inducing expression and SDS-PAGE/Western Blot identification with BL21 E.Coli containing specific plasmids-rFlic and rFlic△180-400.3. Preparation of 125I-rFlic and rFlic△180-400:to lable rFlic and rFlic△180-400 with prepared Iodogen tube and reserve eluated protein peak specimens for subsequent experiments.4. Radiochemical purity and stability:radioactivity counting and calculating radiochemical purity with TLC method.5. Radioligand binding experiments:mice were sacrificed on day 8 after transplantation, spleen cells were used in receptor-ligand binding experiments to determine the affinity and specificity of prepared 125I-rFlic and rFlicA180-400.6. Pharmacokinetic experiments:model mice thyroid were blocked and then injected with 125I-rFlic and 125I-rFlic△180-400 at day 8 after transplantation.Blood was taken and radioactivity was counted at different time point to investigate pharmacokinetic of 125I-rFlic and rFlic△180-400.7. Biodistribution experiments:after thyroid blocked and 125I-rFlic and 125I-rFlic△180-400 injected, various organs of model mice were taken, weighed and radioactivity was measured to detect the biodistribution of 125I-rFlic and rFlic△180-400.8. Whole-body autoradiography:at day 8 after transplantation, the mice were injected intravenously with 125I-rFlic and 125I-rFlic△180-400 and then anaesthesiaed. Images were acquired at 6,24,48,72 h.Results1. Successfully constructed allogeneic/isotype skin graft model:The skin grafts were soft and moist at the day 7 after transplantation,grafted- skin without induration and granular sensation, as well as surrounding wound healed well, no swelling and oozing pus.2. rFlic and rFlic△180-400 was successfully expressed, extracted and purified.3. Successfully prepared both radio-labeled rFlic and rFlic△180-400:125I-rFlic labeled rate was (93.5±0.15)%,125I-rFlic△180-400 labeled rate was (94± 0.25)%. The radiochemical purity of both labeled tracers were still higher than 93% 72h later.4. Radioligand binding results showed that both tracers with high affinities to TLR5, while the affinity of 125I-rFlic△180-400 is higher than the affinity of 125I-rFlic (P<0.05).5. Pharmacokinetics analysis showed that pharmacokinetics of 125I-rFlic and 125I-Flic△180-400 could be divided into two stages:a rapid distribution phase and a slow decline phase.Data demonstrated that the spread of 125I-Flic△180-400 was faster than125I-rFlic throughout the organ.6. The biodistribution results showed:in addition to skin grafts, the distribution of radioactivity trend in other tissues and organs for two group of tracers, was consistent and mainly gathered in the liver and kidney. T/NT was 2.5±0.34 (125I-rFlic) and 3.5±0.25 (125I-rFlic△180-400) at 24h after injected in two tracers groups separately, showed significant differences (P<0.05). The control group showed no significant radioactivities in skin grafts and the T/NT ratio was 1.45± 0.54 at 24h. Compared with isograft group, the radioactivity in allograft group significantly increased.7. The whole-body phosphor-screen autoradiography imaging showed that 6,24,48h after two tracers injection, the control group of skin grafts and other various parts no significant radioactivity concentration. The allograft imaging was shown much clearer in group of 125I-Flic△180-400 than the 125I-rFlic group at 6 hours after injection of tracers.However, at 48h,the radioimage clarity decreased. The blocking experiments showed no significant radioactivity concentrated within 48h in skin grafts.ConclusionSuccessfully expressed recombinant Flagellin and its fragment. Successfully labeled these proteins with radioiodine 125.125I-rFlic and 125I-Flic△180-400 could image allograft of acute rejection clearly,while 125I-Flic△180-400 faster metabolized and earlier focused on allograft than 125I-rFlic which indicated its better potential clinical application. |
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