摘要
第一部分Folate-BSA-Gd-DTPA的制备
目的：制备BSA-Gd-DTPA和folate-BSA-Gd-DTPA，测定成分并作叶酸受体磁共振显像和化学荧光显像。方法：DTPA与乙酸酐形成的DTPA双酸酐，以摩尔比50∶1与BSA合成BSA-DTPA，后者再与GdCl_3螯合，透析分离游离的Gd~(3+)，得到BSA-Gd-DTPA。叶酸与EDC反应，再以摩尔比10∶1共价耦联上BSA-Gd-DTPA。采用电感耦合等离子体原子发射光谱仪测量BSA-Gd-DTPA中Gd~(3+)的含量。采用紫外可见光分光光度仪测量folate-BSA-Gd-DTPA中叶酸的含量。人肝癌细胞SMMC-7721与folate-BSA-Gd-DTPA、BSA-Gd-DTPA和Gd-DTPA结合后行磁共振T1加权扫描，对其信号作定性分析。将BSA和folate-BSA标记上FITC，与SMMC-7721结合后作荧光显像。结果：成功制备了BSA-Gd-DTPA和folate-BSA-Gd-DTPA。BSA-Gd-DTPA中Gd~(3+)的含量为11.01mg，Gd~(3+)的联接率为70％，装载量为8.47％。Folate-BSA-Gd-DTPA中叶酸的含量为63.27mg。T1加权图像示SMMC-7721结合folate-BSA-Gd-DTPA产生的信号高于BSA-Gd-DTPA和Gd-DTPA，并且，结合folate-BSA-Gd-DTPA产生的信号随SMMC-7721浓度的减小而减小。FITC-folate-BSA可使SMMC-7721细胞膜荧光染色，而FITC-BSA则无明显荧光染色。结论：Folate-BSA-Gd-DTPA可作为叶酸受体靶向磁共振对比剂应用。
第二部分Folate-BSA-Gd-DTPA对正常小鼠的磁共振显像
目的：研究folate-BSA-Gd-DTPA对正常小鼠的磁共振显像表现、体内分布及代谢特点。方法：将30只小鼠随机分为三组，分别给予Gd-DTPA、BSA-Gd-DTPA和folate-BSA-Gd-DTPA。采用1.5T MRI行T2加权和T1加权平扫，以及注射这三种对比剂后15min、30min、1h、2h、4h、6h、8h、12h、18h和24h的T1加权增强扫描，测量平扫与增强后各个时间点下腔静脉、肝脏、肌肉和肾脏皮质的T1加权信号，比较上述部位的相对强化率，并观察胆囊腔内对比剂的显示情况。结果：三组下腔静脉的相对强化率在增强后15min、30min、1h、2h、4h、6h、8h和12h差异有统计学意义。三组肝脏的相对强化率在增强后15min、30min、1h、2h、4h、6h、8h、12h、18h和24h差异有统计学意义。三组肌肉的相对强化率在增强后30min和1h差异有统计学意义。三组肾脏皮质的相对强化率在增强后15min、30min、1h、2h、4h、6h、8h、12h、18h和24h差异有统计学意义。Folate-BSA-Gd-DTPA组肾脏皮质的相对强化率在增强后4h、6h、8h、12h、18h和24h高于BSA-Gd-DTPA组。BSA-Gd-DTPA组和folate-BSA-Gd-DTPA组在增强后18h和24h，分别有80％和90％在胆囊腔内显示高信号对比剂。结论：BSA-Gd-DTPA和folate-BSA-Gd-DTPA可使血管、肝脏和肾脏皮质明显持续地强化，部分可经肝胆途径排泄。Folate-BSA-Gd-DTPA可实现肾脏皮质的磁共振靶向显像。
第三部分Folate-BSA-Gd-DTPA对肝癌原位移植裸鼠的磁共振显像
目的：研究folate-BSA-Gd-DTPA对肝癌原位移植裸鼠的磁共振显像表现，探讨其对肝癌靶向诊断的价值。方法：人肝癌细胞SMMC-7721培养扩增，将含有1.0×10~7个活细胞的悬液0.2ml接种于裸鼠腋部背侧皮下。当皮下肿瘤形成后行手术切除，剪成0.5mm~3大小瘤块，接种于裸鼠肝脏。将肝癌原位移植术后3-4周的20只裸鼠随机分为两组，分别给予BSA-Gd-DTPA和folate-BSA-Gd-DTPA。采用1.5T MRI行T2加权和T1加权平扫，以及注射这两种对比剂后15min、30min、1h、2h、4h、6h、8h、12h、18h和24h的T1加权增强扫描，测量平扫与增强后各个时间点肿瘤、肝脏和肾脏皮质的T1加权信号，比较肿瘤的相对强化率，并观察肿瘤—肝脏对比噪声比的变化规律。分析肿瘤在注射这两种对比剂后的强化表现，与病理结果作对照。结果：裸鼠肝癌原位移植均获得成功。所有20个肿瘤均被MRI检出，大小7.4mm×6.8mm-14.8mm×12.1mm。增强后2h、4h、6h、8h、12h、18h和24h，folate-BSA-Gd-DTPA组肿瘤的相对强化率高于BSA-Gd-DTPA组。Folate-BSA-Gd-DTPA组的肿瘤—肝脏对比噪声比在增强后8h达到高峰。Folate-BSA-Gd-DTPA组肿瘤内部强化超过肝脏实质，而BSA-Gd-DTPA组则始终低于肝脏实质。肿瘤的强化方式与其大小和病理有关。结论：Folate-BSA-Gd-DTPA可实现肝癌的磁共振靶向显像，具有定性诊断的价值。
PartⅠPreparation and evaluation of BSA-Gd-DTPA and folate-BSA-Gd-DTPAPurpose: To synthesize BSA-Gd-DTPA and folate-BSA-Gd-DTPA, determine the pharmaceutical properties and study in vitro magnetic resonance imaging and cytochemical fluorescence imaging of folate receptor. Methods: DTPA was transformed to cDTPAa and conjugated to bovine serum albumin (BSA) at the molar ratio of 50:1．GdCl was reacted with BSA-DTPA and the unreacted Gd ion was dialyzed and separated from BSA-Gd-DTPA. Using EDC as linker, folate was covalently coupled to BSA-Gd-DTPA at the molar ratio of 10:1．The Gd ion conjugation rate of BSA-Gd-DTPA was measured using ICP-AES and the folate in folate-BSA-Gd-DTPA was detected by spectrophotometer. MR T1W scan was performed to qualitatively determine the uptake of folate-BSA-Gd-DTPA, BSA-Gd-DTPA and Gd-DTPA by the human hepatocelluar carcinoma cell line SMMC-7721 at different concentration. BSA and folate-BSA were labeled with fluorescein isothiocyanate (FITC) and fluorescent staining was performed to determine the uptake of FITC-BSA and FITC-folate-BSA by SMMC-7721．Results: BSA-Gd-DTPA and folate-BSA-Gd-DTPA was successfully synthesized. The Gd ion in BSA-Gd-DTPA was 11．01mg, the conjugation rate was 70% and the loading capacity was 8．47%. The folate in folate-BSA-Gd-DTPA was 63．27mg. The T1W signal of folate-BSA-Gd-DTPA binding to SMMC-7721 was higher than that of BSA-Gd-DTPA binding and Gd-DTPA binding, and the T1W signal of folate-BSA-Gd-DTPA binding decreased with the concentration of SMMC-7721．FITC-folate-BSA was observed to bind to the cell surface of SMMC-7721, while little fluorescent binding was found with FITC-BSA. Conclusions: Folate-BSA-Gd-DTPA could be used as the potential MR contrast agent targeted to the folate receptor.PartⅡMagnetic resonance imaging of folate-BSA-Gd-DTPA in normal micePurpose: To study in vivo MR imaging, distribution and excretion of folate-BSA-Gd-DTPA in normal mice. Methods: 30 Balb/c mice were radomly divided into three groups and administered with three contrast agents: Gd-DTPA, BSA-Gd-DTPA and folate-BSA-Gd-DTPA. A 1．5 Tesla Siemens MR scanner and the head coil were used. The plain T2W FSE and T1W SE sequences were performed. The repeated T1W SE sequences were then performed 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 18h and 24h after the intravenous injection of Gd-DTPA, BSA-Gd-DTPA and folate-BSA-Gd-DTPA. The signal intensity (SI) of the inferior vena cana, liver, muscle and renal cortex in the pre-and post-contrast T1WI was measured using a circular region of interesting with 5 pixels and the relative enhancement (RE) was calculated as (SI-SI) / SI×100%. The appearance of the contrast agent in the gallbladder was observed. Results: The RE of the inferior vena cana was statistically different at the time point of 15min, 30min, 1h, 2h, 4h, 6h, 8h and 12h among three groups, the RE of the liver at the time point of 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 18h and 24h, the RE of the muscle at the time point of 30min and 1h, and the RE of the renal cortex at the time point of 15min, 30min, 1h, 2h, 4h, 6h, 8h, 12h, 18h and 24h. The RE of the renal cortex after folate-BSA-Gd-DTPA enhancement was higher than the RE after BSA-Gd-DTPA enhancement at the time point of 4h, 6h, 8h, 12h, 18h and 24h. 8 mice administered with BSA-Gd-DTPA and 9 mice with folate-BSA-Gd-DTPA had high signal intensity caused by contrast agent in the gallbladder at the time point of 18h and 24h, while all 10 mice with Gd-DTPA did not have the contrast agent in the gallbladder. Conclusions: BSA-Gd-DTPA and folate-BSA-Gd-DTPA could result in more and persistent enhancement of the blood, liver and renal cortex and excreted partially through the biliary route. Folate-BSA-Gd-DTPA could target to the renal cortex and be imaged by MR.PartⅢMagnetic resonance imaging of folate-BSA-Gd-DTPA with orthotopic transplantation of human hepatocellular carcinoma in nude micePurpose: To study in vivo MR imaging of folate-BSA-Gd-DTPA with orthotopic transplantation of human hepatocellular carcinoma in nude mice. Methods: The human hepatocellular carcinoma cell line SMMC-7721 was cultured and 0．2 ml suspended cells with the concentration of 1．0×10 /ml were inoculated into the subcutaneous tissue of the dorsal subscapular region of 4 athymic nude mice. After the subcutaneous tumor developed into the bulky mass, the tumor was excised and cut into the fragments with the diameter of 0．5 mm. The fragments were then implanted through the envelope into the liver parenchyma of 20 nude mice. Three to four weeks after the liver transplantation, 20 nude mice were randomly divided into two groups and administered with BSA-Gd-DTPA and folate-BSA-Gd-DTPA. MR T2W FSE sequence and T1 W SE sequences before and after the intravenous injection of two contrast agents were performed. The scan parameters and the time points were the same as partⅡ. The T1 W SI of the tumor, liver and renal cortex was measured and the RE was calculated. The value of tumor-to-liver contrast to noise ratio (CNR), which corresponds to the conspicuity score, was obtained with the fomular of (SI-SI) / background noise and the relation of CNR to the time point was observed. The MR imaging features of the tumor after BSA-Gd-DTPA and folate-BSA-Gd-DTPA enhancement were analyzed and compared with the pathologic results. Results: The orthotopic transplantation model of the human hepatocellular carcinoma was successfully obtained in 20 nude mice. All the hepatocellular carcinoma were detected by MR and the range of the tumor size was from 7．4mm×6．8mm to 14．8mm×12．1mm. The RE of the tumor after folate-BSA-Gd-DTPA enhancement was higher than the RE after BSA-Gd-DTPA enhancement at the time point of 2h, 4h, 6h, 8h, 12h, 18h and 24h. The CNR of tumor-to-liver reached peak at the time point of 8h. The tumor is exhibited as hyper-intensity compared with the liver parenchyma after folate-BSA-Gd-DTPA enhancement, while exhibited as hypo-intensity after BSA-Gd-DTPA enhancement. The enhancement mode of the tumor correlated with the size and pathological structure. Conclusions: Folate-BSA-Gd-DTPA could target to the hepatocellular carcinoma and be imaged by MR. Folate-BSA-Gd-DTPA could be of qualitative value in the diagnosis of hepatocellular carcinoma.
引文
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1 丁建辉，曾蒙苏，薛琼，等．叶酸受体型MR对比剂对荷人胃癌裸鼠诊断价值的实验研究．中华放射学杂志．2005，39(8)：882-886．
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