The Study Of Technetium-99m Labeled Anti-α7nAchRs Monoclonal Antibody For SPECT Imaging Of Cholangiocarcinoma Model In Nude Mice

ObjectiveTo develop a novel molecular probe using ^99mTc labeled CHRNA7 m Ab and to investigate its ability to targetα7n ACh Rs in vivo,further set the foundation for clinical SPECT imaging of cholangiocarcinoma.Methods:1.The expression level ofα7n ACh Rs in Hu CCT-1 cells and Hep G2 cells were detected by cellular immunofluorescence assay.2.SHNH and CHRNA7 m Ab was connected and ^99mTc was labeled to prepare radiolabeled probe^99mTc-SHNH-CHRNA7 m Ab,then identified the chemical properties of the ^99mTc labeled radiomarker.3.Cellular uptake test and block test were carried out to detect the affinity and specificity of the radiolabeled probes to the target.4.Tumor bearing nude mice model of cholangiocarcinoma was established and studies of in vivo SPECT imaging and distribution were performed.Results:1.Cellular immunofluorescence assay verifyα7-n ACh Rs expression levelThe results of cellular immunofluorescence assay showed that Hu CCT-1 cells were deeply stained andα7n ACh Rs were highly expressed,while it was poorly expressed on Hep G2 cells.2.Antibody radiolabeling and radiochemical determinationSHNH-CHRNA7 m Ab was purified several times in the synthetic process,and its chemical properties remained unchanged;the radiochemical purity of^99mTc-SHNH-CHRNA7 m Ab was about 97.40±2.71%;after 24 hours at 37℃,the radiochemical purity of the radiomarker was 76.05±2.30%,The^99mTc labeled probe showed good stability in mouse serum.3.In vitro cell uptake and blocking experimentsThe uptake rate of ^99mTc-SHNH-CHRNA7 m Ab in Hu CCT-1 cells gradually increased over time,and the uptake rate of Hu CCT-1 group was significantly higher than that of Hep G2 group at 4 h（16.31±2.36%vs.5.60±0.71%;P<0.05）.The uptake rate of Hu CCT-1 blocking group was decreased to 7.36±1.67%（P<0.05）.4.In vivo SPECT imaging and distribution analysisSPECT scan results showed that the tumor of Hu CCT-1 group began to accumulate radioactivity at 6 h after injection of ^99mTc-SHNH-CHRNA7 m Ab,and gradually concentrated with time,and the radioactivity uptake reached its peak at24h.However,the radioactive uptake of tumor tissues in the Hu CCT-1 blocking group was significantly reduced at 24h,which was similar to background muscle uptake.In vivo distribution study showed that the radioactive uptake of tumor tissues in the Hu CCT-1 group was significantly higher than that in the blocking group（13.04±1.30%ID/g v 1.80±0.75%ID/g;P<0.05）,tumor to muscle ratio（T/NT）was 8.51±3.04 and 2.63±1.72 respectively（P<0.05）.ConclusionOur study showed^99mTc labeled CHRNA7 m Ab has high radiochemical purity and specific activity with high affinity and specificity for targetingα7-n ACh Rs.It exhibited high tumor uptake inα7n ACh Rs positive tumors.The high specificity and excellent tumor targeting properties of^99mTc labeled CHRNA7 m Ab.This probe has a good application prospect in the tracer and location diagnosis of tumor-specific targets.