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目的研究量子点标记靶向探针对人肝癌裸鼠模型的体内成像技术。方法将巯基乙酸修饰的水溶性量子点结合鼠抗人甲胎蛋白(AFP)单克隆抗体制备成水溶性量子点-AFP-Ab复合物探针。荧光、紫外光光谱分析及透射电镜研究其特性。通过直接免疫荧光法,用该复合物探针特异性识别肝癌细胞株HCCLM6 AFP抗原。将体外培养的肝癌细胞株HCCLM6通过皮下接种和尾静脉注射裸鼠分别建立人肝癌裸鼠模型和肺转移模型。尾静脉注射量子点-AFP-Ab探针,用蓝光二极管照射获得活体荧光成像;用掺Ti蓝宝石激光器照射,对肿瘤部位和正常部位进行光谱分析。取血清检测丙氨酸转氨酶、天冬氨酸转氨酶、尿素氮和肌苷水平。取裸鼠肝、脾、肾、肺、心和脑6种主要实质性器官行振荡切片,共聚焦显微镜观察,研究量子点-AFP-Ab探针在裸鼠体内的非特异性摄取.结果量子点-AFP-Ab复合物探针具有激发光谱宽、荧光强度高的特点,能特异性与肝癌细胞AFP抗原高亲和力结合,在体内能特异性靶向肿瘤组织进行活体成像,无明显急性毒性。光谱分析显示量子点- AFP-Ab复合物探针主要分布于肿瘤的外周部位,少数该探针被肝、脾和肺非特异性摄取。结论量子点-AFP-Ab复合物探针具有优良的光学特性和生物相容性,能够进行肝癌体内靶向成像,将有助于肝癌的分子靶向研究。
Objective To study the in vivo imaging of human hepatocellular carcinoma (HCC) nude mice by using quantum dot-labeled probes. Methods A water-soluble QD-AFP-Ab complex probe was prepared by combining thioglycolic acid-modified water-soluble quantum dots with mouse monoclonal anti-human alpha-fetoprotein (AFP) monoclonal antibody. Fluorescence, UV spectroscopy and transmission electron microscopy were used to study their properties. HCCLM6 AFP antigen was specifically recognized by this complex probe by direct immunofluorescence. The human hepatoma cell line HCCLM6 cultured in vitro was inoculated subcutaneously and injected into the tail vein of nude mice to establish human hepatocellular carcinoma nude mice model and lung metastasis model respectively. AFP-Ab probe was injected through the tail vein, and fluorescence imaging was performed by using blue light diode. The Ti-sapphire laser was irradiated to analyze the tumor site and the normal site. Serum alanine aminotransferase, aspartate aminotransferase, urea nitrogen and inosine levels. Six major kinds of essential organs such as liver, spleen, kidney, lung, heart and brain in nude mice were obtained and examined by confocal microscopy to investigate the nonspecific uptake of QD-AFP-Ab probe in nude mice.Results Quantum dot- The AFP-Ab complex probe has the characteristics of wide excitation spectrum and high fluorescence intensity, and can specifically bind to AFP antigen of hepatoma cells with high affinity, and can specifically target tumor tissues in vivo to perform in vivo imaging without obvious acute toxicity. Spectral analysis showed that the QDs-AFP-Ab complex probes were mainly distributed in the periphery of tumor, and a few of them were nonspecifically up-regulated by liver, spleen and lung. Conclusion The quantum dot-AFP-Ab complex probe has excellent optical properties and biocompatibility, and can be used for targeted imaging of hepatocellular carcinoma in vivo. It will be helpful for the molecular targeted research of hepatocellular carcinoma.