【作者】 董孟杰；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 影像医学与核医学， 2002， 硕士

【摘要】 目的:本课题致力于研究一种更为简便的、快速的、不改变生物活性的99mTc标记EGF的方法；探索99mTc标记表皮生长因子（99mTc-EGF）对肿瘤显像诊断作用。方法:首先运用还原剂巯基乙醇使EGF分子的二硫键还原，然后用 Sephadex G50柱去掉多余的还原剂，由MDP药盒提供的Sn2+将高价锝还原使之与还原的EGF的巯基结合，通过内皮细胞的培养鉴定99mTc-EGF的生物活性。体外实验：1）C6细胞放射性摄取实验：24孔板接种C6细胞2×105个，移入细胞培养箱培养，当细胞数量达到50%左右时，4℃分别与不同浓度的99mTc-EGF孵育进行细胞摄取实验,EGF终浓度分别为0.0165nM（0.208KBq）、 0.165nM(2.08KBq)、1.65nM(20.8KBq)、16.5nM(208KBq)、165nM(2080KBq)，同时以胶质细胞作为对照；2）C6细胞放射性滞留实验：24孔板接种C6细胞2×105个，移入细胞培养箱培养，当细胞数量达到50%左右时，与EGF终浓度为165nM的99mTc-EGF(2080KBq)分别孵育不同时间（5min 、10min 、30min 、 1h 、2h 、3h 、6h）进行细胞的放射性滞留实验，同时用神经胶质细胞作为对照。32只体重为100-150克的Wistar大鼠右股内侧皮下接种C6肿瘤细胞数约1.5×106左右建立鼠胶质瘤模型，2周后肿瘤长至0.5-1cm时随机分为四组，每只尾静脉<WP=5>注射3.7MBq的 99mTc-EGF后分别在30分钟、2小时、4小时及6小时眼静脉取血，解剖动物，观察99mTc-EGF 在鼠血液、心、肝、脾、肾、胃、肠、肺和瘤组织放射性分布。每只荷瘤鼠与正常对照鼠尾静脉注射3.7MBq 99mTc-EGF，给药后4小时置Siemens Diacam型SPECT下进行显像。结果：99mTc -EGF标记率为76.29%，99mTc -EGF生物活性与EGF对照组比较无明显变化(P>0.05)。摄取实验中C6细胞摄取99mTc -EGF的cpm值随着99mTc -EGF浓度的升高而升高，在165nM达到最高值，而神经胶质细胞对照组摄取99mTc -EGF的cpm值无明显的变化；滞留实验显示C6细胞随着与99mTc -EGF孵育时间的延长而有增加的趋势。体内放射性分布实验：99mTc -EGF在正常组织中主要分布于肝脾肾等组织，瘤区放射性分布较高，脑组织最低，其他组织均无明显的放射性浓聚。SPECT显像清晰，瘤组织放射性分布明显，肝脾组织放射性分布较高,轮廓完整。结论：本方法标记方法简便，标记率76.29%，具有良好的稳定性，瘤区放射性摄取高，除肝、脾、肾外T/TN比值高，图像清晰。99mTc直接标记EGF有望为临床神经胶质瘤的诊断及其为高表达EGFR的肿瘤提供一种新的、特异性的、分子水平的核医学诊断方法。更多还原

【Abstract】 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 injected<WP=7>subcutaneously 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 the<WP=8>radiopharmaceutials .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.更多还原