| Objectives: We attempted to find a method for direct radiolabeling of EGF with technetium-99(99mTc),and the biological activity was remained; Another purpose was to explore a simple and available method for the diagnosis of tumor expressing high level EGFR.Methods:Following reduction of EGF with 2-ME and purification on a sepadex G50 column ,EGF was labeled with 99mTc in the presence of Sn2+ (provided by MDP kit),and the biological activity was verified by the growth of endothelial cells.1)In vitro celluar uptake experiment :C6 cell(about 2×105),grown in 24-well plates, were incubated at 4℃for 90 minutes in cell culture medium and the final concentration of EGF was 0.0165nM(0.208KBq), 0.165nM(2.08KBq),1.65nM(20.8KBq),16.5nM(208KBq),165nM(2080KBq ) .2)Celluar retention of 99mTc-EGF radioactivity :C6 cell ,grown in 24-well plates(about 2×105), were incubated at 37℃ with medium containing final concentration of EGF being 165nM for periods of time ranging from 5min to 6hour.Thirty two Wistar rats weighing 150-200g were injectedsubcutaneously in the right hind leg with 1.5×106 C6 cells.Radiodistribution was carried out after injection of 99mTc-EGF into 2- week-old tumor bearing rats when the tumor reached a diameter of 0.5-1cm at 30 minute,2 hour,4 hour and 6hour,including blood,heart,liver,spleen, kidney,stomach,intestines,lung and tumor.SPECT images was carried out after 4 hours of intravenous injection of 99mTc-EGF in 2- week-old tumor and non-tumor -bearing rats.Results: Radiolabeling efficiency was 76.29%,and stability of 99mTc-EGF was well so it could meet experimental and clinical needs. 99mTc-EGF was almost keep the same ability to promote the growth of endothelial cells as EGF .In vitro celluar uptake experiment showed that 99mTc-EGF radioactivity in C6 cells was obviously increasing as a function of concentration of 99mTc-EGF,but control group was not;Celluar rentention experiment of cell-bound radioactivity following inculation with 99mTc-EGF showed that continuous inculation with 99mTc-EGF resulted in an increase of cell-associated radioactivity proportional to the time of incubation.The normal tissues that accumulated the highest concentration of the radiopharmaceutials were liver ,kidney and spleen,and brain took lowest concentration of the radiopharmaceutials. Although accumulated lower concentration of the radiopharmaceutials than liver ,kidney and spleen.,Tumor also took high concentration of theradiopharmaceutials .From images of SPECT,tumor tissues were clearly seen,the liver and spleen were the major normal organs visualized on the images ,and the other tissues didn't have obvious accumulation of radiopharmaceutials .Conclusions:The method of direct radiolabeling was simple and took few minutes and radiolabelling efficiency was 76.29% ;Tumor accumulated relatively high radioactivity, T/TN was very high except liver,spleen and kidney; 99mTc-EGF had characteristic of specificity and stability ,so radiolabeling EGF with direct labeling could offer a new and specfic approach to diagnize tumor such as glioma and other tumors expressing high level EGFR from molecular level. |
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