| The preparation and preliminary application of 99 m Tc-labeled VCAM-1 sc Fv antibodyObjective: To prepare and identify 99 m Tc-labeled VCAM-1 single-chain antibody, and apply the synthesized 99 m Tc-labeled VCAM-1 single-chain antibody for the imaging of mouse tumor models.Methods:(1) Preparation and identification of 99 m Tc-HYNIC-VCAM-1-sc Fv: HYNIC and VCAM-1 were first acted in acid environment for 2h; and then the products were filtered, and acted with fresh 99 m Tc O4 leacheate under the condition of reducing agent for 30 min. A small amount of reaction products was taken for radioactive thin-layer chromatography to measure the labeled rate; PD-10 column was used for purification, and radioactive thin-layer chromatography was used to measure the radiochemical purity.(2) Three kinds of tumor cells(786-O, SK-OV-3 and HT-1080) with different expressions of VCAM-1 were cultivated, and Western blot and immunohistochemical method were adopted to detect the expressions of VACM-1 in the three kinds of cells.(3) Cell tests: Cell uptake test and cell efflux test.(4) Biological distribution and SPECT imaging: Images of the mice were observed at the set time point and compared, after administration of drugs via caudal vein. Percentage injection dose rates(%ID/g) were measured in various tissues and organs(including blood, brain, heart, lungs, liver, kidneys, spleen, stomach, intestine, bones, and muscles) and tumor in mice, after administration of drugs via caudal vein.Results: 1. The labeled rate of 99 m Tc-HYNIC-VCAM-1-sc Fv was 78.79%±5.70%(n=5), and the radiochemical purity was 92.17±3.32%(n=5). 2. Western Blot results showed that VCAM-1 was highly expressed in HT-1080 and SK-OV-3 cells, while lowly expressed in 786-O cells. Immunohistochemical results showed expressions of VCAM-1 inthe HT-1080 cells are the hightest, SK-OV-3 cells are medium, and 786-O cells are the lowest. 3.The uptake of 99 m Tc-HYNIC-VCAM-1-sc Fv in the 786-O cells, SK-OV-3 cells and HT-1080 cells increase rapidly within 2 hours, after 2 hour the increase of cell uptake becomes much slower. 2 hour-cell-uptake of 786-O cells, SK-OV-3 cells and HT-1080 cells are 3.54%, 5.04%and 8.5%. The efflux test suggested high stability and longer stays of imaging agent in cells, which were suitable for imaging. 4. Blood clearance of 99 m Tc-VCAM-1-sc Fv was slow in nude mice, and higher uptake rates in the liver and kidneys suggested it was mainly metabolized by liver and kidneys. 5. SPECT imaging: Comparison on SPECT images of mice with labeled drugs and 99 m Tc O4-showed that: In the latter ones, image of thyroid gland was not obvious, and the concentration of imaging agent was reduced in bladder while increased in the liver. 2h after injection of labeled drugs, obvious images of tumor tissues could be seen in HT-1080 tumor-bearing mice, the rough images of tumor tissues could be seen in SK-OV-3 tumor-bearing mice, while the obvious image of tumor tissues could not be seen in 786-O tumor-bearing mice. 4 h after injection, images of tumors were further clear in HT-1080 tumor-bearing mice, and the images of tumors were more obvious in SK-OV-3 tumor-bearing mice, while images of tumors were still not seen in 786-O tumor-bearing mice.Conclusion:99m Tc-HYNIC-VCAM-1-sc Fv is easy to prepare, with mild reaction and stable yield. The labeled rate of labeled products and radiochemical purity were higher. Cell test, biological distribution and SPECT imaging all show that 9m Tc-HYNIC-VCAM-1-sc Fv has the potential to become a new type of tumor imaging agent.Preliminary experimental study of 99 m Tc-labeled VCAM-1 sc Fv antibody in mice inflammatory models imagingObjective: To establish the inflammation model in mice and apply the synthesized Tc-labeled VCAM-1 single-chain antibody in the SPECT imaging and biological distribution test of mice inflammation model, and verify the expressions of VCAM-1 in inflammation from the another aspect according to the SPECT imaging and biological distribution results.Methods: 1. Preparation of inflammation models in mice: 0.1m L turpentine oil was intramuscularly injected to the mice via right hind limbs. 2. SPECT imaging: Labeled drugs were administrated to normal C57 mice via caudal vein and the SPECT images were observed after 1h, 2h and 4h. 3. Biological distribution: Percentage injection dose rates(%ID/g) were measured in various tissues and organs(including blood, brain, heart, lungs, liver, kidneys, spleen, stomach, small and large intestine, muscles of left and right hind limbs and bone) in mice at different time points after administration of drugs.Results:1. It is easy to establish inflammation model in C57 mice, in a short time. 2.Biodistribution: Blood clearreace is slow. The uptake of liver, spleen, and kidney is the highest among all organs. The 2 hour-muscle-uptake of inflammation model mice's right and left hind limbs are 1.32±0.63% and 0.35±0.07%; as the 4 hour-muscle-uptake of inflammation model mice's right and left hind limbs are 0.84±0.15% and 0.19±0.08%. 3. SPECT imaging clearly shows that the images of the right hind limb were more obvious than the left hind limb, and the image reached the highest at 4 h. Concentration of imaging agent in right hind of model mice was increased at 1 week, but only slightly image left at 2 weeks.Conclusion: With the new synthesized molecular probes, changes of images within two weeks in mouse inflammation models can be seen. And the next research direction should focus on blocking imaging. |
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