【作者】 张超；

【导师】 韩建奎；

【作者基本信息】 山东大学 ， 影像医学与核医学， 2008， 博士

【摘要】 为了探讨以MIF为靶向进行炎症显像的可行性,本研究所自行制备了放射性碘化标记的抗MIF单克隆抗体（anti-MIF McAb）及其对照抗体IgG,并在金黄色葡萄球菌、大肠杆菌和医用松节油诱导建立的三种小鼠炎症模型体内进行了生物学分布和显像的实验研究。研究内容分为以下三部分:第一部分:放射性碘化标记anti-MIF McAb和IgG的制备及生物学活性鉴定目的采用固相Iodogen法碘化标记anti-MIF McAb和IgG,对其标记率、放射化学纯度、稳定性及生物学活性进行系列研究,为下一步的研究提供实验基础。方法采用固相Iodogen法,¹²⁵I和/或¹³¹I分别碘化标记了anti-MIF McAb和IgG。检测放射性碘化标记anti-MIF McAb及其对照抗体在室温储存不同时间后的放射化学纯度,观察其在体外的稳定性。以酶联免疫吸附法检测放射性碘化标记anti-MIF McAb的免疫学活性。结果1.¹²⁵I-anti-MIF McAb的标记率为82.35%,放化纯为98.25%,放射性比活度为29.56 GBq/μmol。2.¹³¹I-anti MIF McAb的标记率为87.25%,放化纯为97.10%,放射性比活度为29.56 GBq/μmol。3.¹³¹I-IgG的标记率为81.56%,放化纯为97.17%,放射性比活度为30.12GBq/μmol。结论三种标记物的稳定性和免疫活性良好,达到了用于进一步实验研究的要求标准。第二部分¹²⁵I—anti—MIF McAb在小鼠炎症模型体内的生物学分布研究目的建立金黄色葡萄球菌、大肠杆菌和医用松节油诱导的三种小鼠炎症模型,观察¹²⁵I-anti-MIF McAb在三种小鼠炎症模型的体内生物学分布,同时利用磷屏进行放射自显影炎症显像研究。方法1.建立小鼠炎症模型:BALB/c小鼠60只随机分为3组,每组20只。利用大肠杆菌、金葡萄球菌、松节油诱导建立小鼠炎症模型。2.¹²⁵I-anti-MIF McAb在炎症模型小鼠的体内分布:对每种炎症模型的小鼠采用腹腔注射法注射¹²⁵I-anti-MIF McAb 0.2ml约3.7MBq。在完成注射后第30min、4h、24h、48h和72h,从3组炎症小鼠中随机各取出3只脱臼处死,解剖取出全部炎症组织、心、肝、脾、肺、肾、骨、甲状腺、胸腺和左侧对照肌肉组织的样本,称重,利用γ计数器测定各器官或组织的放射性计数（cpm）,并计算其平均cpm/g;测定¹²⁵I-antiMIF McAb注入量标准源的放射性计数,计算各器官或组织内放射性占标准源放射性的百分比（%ID）、T/NT。3.金黄色葡萄球菌组、大肠杆菌组、松节油组炎症模型小鼠各5只尾静脉注射3.7MBq 0.2mL¹²⁵I-anti-MIF McAb,在注射后24h、48h、72h分别用0.06%戊巴比妥0.2ml麻醉炎症模型小鼠,俯卧在磷屏上进行放射自显影。4.以RT-PCR、免疫组化法检测小鼠炎症模型炎症组织MIF基因及蛋白表达水平的变化,以证实放射性碘化标记的anti-MIF McAb在炎症部位的浓聚是由局部高表达的MIF基因及蛋白引起的。结果1.三种小鼠炎症模型在注射¹²⁵I-anti-MIFMcAb后30min,血液和肾、肝、脾等器官组织内的放射性计数均迅速增高,持续增高至4h后迅速降低,24h以后下降缓慢。在各器官组织中以肌肉和股骨内的放射性计数在各时间点为最低。2.炎症部位的放射性摄取在注射¹²⁵I-anti-MIF McAb 4h后开始明显增加,金葡菌组炎症部位24h的摄取最高,48h炎症部位摄取迅速跌落形成峰状;而松节油组4h炎症部位摄取即达较高值并持续至48h,72h迅速跌落;大肠杆菌组从4h开始炎症部位的放射性积聚一直持续缓慢增加,但其摄取均低于金葡菌组和松节油组。各时间点T/NT比值随时间的增加而逐渐升高:金葡菌组在30min即可升至4,24h金葡菌组、大肠杆菌组和松节油组分别为9.65、3.46和5.37,以后T/NT比值继续升高,48h三组小鼠炎症模型的T/NT比值均达到了7以上。炎症部位的放射自显影结果与体内生物学分布观察到的T/NT变化相一致。3.RT-PCR、免疫组化结果表明:小鼠金葡菌炎症组织中MIF mRNA、MIF蛋白高表达,与炎症显像浓聚程度正相关。结论¹²⁵I-anti-MIF McAb能够靶向性聚集于炎症病灶,且在靶组织中清除缓慢,放射自显影与体内生物学分布观察到的T/NT变化相一致。第三部分¹³¹I-anti-MIF McAb和¹³¹I-IgG在小鼠炎症模型体内生物学分布的对比研究目的建立金黄色葡萄球菌小鼠炎症模型,对比研究¹³¹I-anti-MIF McAb和¹³¹I—IgG在小鼠炎症模型体内的生物学分布及磷屏显像特点方法1.建立BALB/c小鼠金葡菌炎症模型。2.40只模型鼠随机分为两组,采用尾静脉注射法,每只模型小鼠注射¹³¹I-anti-MIF McAb或¹³¹I-IgG各0.2ml约3.7 MBq。3.在注射后24h、48h和72h,从两组炎症小鼠模型中随机各取出5只脱臼处死,解剖取出全部炎症组织、心、肝、脾、肺、肾、骨、甲状腺、胸腺和左侧对照肌肉组织的样本,称重,利用γ单道能谱仪测定各器官或组织的放射性计数（cpm）,并计算其平均cpm/g;测定¹³¹I-IgG注入量标准源的放射性计数,计算各器官或组织内放射性占标准源放射性的百分比（%ID）。4.从两组炎症小鼠模型中随机各取出5只,分别在24h、48h、72h用0.06%戊巴比妥0.2ml麻醉,进行磷屏自显影显像。结果1.¹³¹I-anti-MIF McAb和¹³¹I-IgG在炎症模型鼠体内的分布各自有明显不同的特点。¹³¹I-IgG组血液放射性明显高于¹³¹I-anti-MIF McAb,且消减缓慢,而¹³¹I-anti-MIF McAb组肝、脾及肾的放射性在各时相均较¹³¹I-IgG组高,差异有显著性（p＜0.05）。2.注射¹³¹I-anti-MIF McAb和¹³¹I-IgG 24h后,炎症部位均有较多的放射性浓聚。¹³¹I-anti-MIF McAb的炎症部位积聚量在注射后24h、48h、72h均明显高于¹³¹I-IgG组（p＜0.05）,表明¹³¹I-anti-MIF-McAb在炎症部位有比¹³¹I-IgG更特异的浓聚。3.炎症部位的放射自显影显像结果与体内生物学分布观察到的生物学分布变化相一致。结论与¹³¹I-IgG相比,¹³¹I-anti-MIF-McAb在炎症部位更具有靶向的特异性浓聚。¹³¹I的半衰期8天,比较适合于临床显像。本研究结果表明:放射性碘化标记的抗MIF单克隆抗体（anti-MIF McAb）制备简便、性能稳定,能够靶向性聚集于炎症病灶,且在靶组织中清除缓慢,放射自显影图像质量好,为其临床应用研究提供了可靠的实验依据。更多还原

【Abstract】 BackgroundTo quickly and exactly detect acute or subacute inflammatory lesions is a difficult subject in clinical. In most cases to determine the anatomical location of the inflammatory lesions is even more important than to identify of the type of infection pathogen. The fast and accurate positioning inflammatory lesions are very helpful to clarify the cause of inflammation and to make rapid effective treatment. Traditional diagnostic imaging technologies such as X-ray, CT and ultrasound imaging can not make accurate identification when the abscess had not yet formed. The radioisotope inflammatory imaging can detect and locate the inflammatory lesions, especially deep inflammatory lesions which is its advantage. So radioisotope inflammatory imaging is a most effective method of early diagnosis of inflammation or infection.The commonly used inflammatory imaging agents are ⁶⁷Ga-citrate, ¹¹¹In-WBC, ^99mTc-HMPAO labeled leukocytes, ^99mTc-or ¹¹¹In-IgG, radiolabeled liposome, and so on, but all of them have some shortcomings and deficiencies. For example: the commonly used radiolabeled-WBC imaging need a large number of human blood, that may have the risk of blood pollution and cross infection during isolation ,which limits its use in clinical. The radiolabeled non-specific immunoglobulin imaging is easily to use, but there are non-specific deposition in blood, liver, spleen, and kidney. In recent years, there are many new types of inflammatory imaging agent research reported along with the medicine and pharmaceutical development. For example: the radiolabeled anti-granulocyte monoclonal antibody or antibody fragment, radiolabeled IL-1, 8, radiolabeled the chemical synthesized peptides of WBC receptor and ciprofloxacin. However, some kinds of specific and sensitive deficiencies still existed, which limit their clinical application. Therefore, seeking a new, specific and sensitive inflammatory imaging agent is currently urgent in clinical.With the rapidly development of immunology and other basic medical research, people begin to know more and more about the effect of cytokines during inflammation developing. The significance of cytokines in diagnostic and treatment inflammation has been cause a highly attention. In recent years, macrophage migration inhibitory factor （macrophage migration inhibitory factor, MIF） has been confirmed to be an important inflammatory response regulator. MIF can promote macrophage accumulation, proliferation, activation, adhesion, and phagocytosis in the local of inflammation, it also can promote the formation of a variety of cytokines, can offset the immune inhibition by corticosteroid hormones for other inflammatory cytokines. MIF plays an important role in the local or systemic inflammation and immune responses, and is a key media in systemic inflammatory response syndrome. The study of anti-inflammatory drugs targeted MIF has become a hot spot in medical research at present. The purpose of this study is to provide experimental basis for clinical application of the MIF targeted inflammation imaging agent by investigating the synthesis, preparation, biodistribution and dynamic imaging using radioisotope labeled anti-MIF monoclonal antibody.Method1 .Radiolabeled anti-MIF McAb was oxidized by Iodogen with 125-I （or 131 -I） . The stability was detected by investigating the radiochemical purity of radioiodine labeled anti-MIF McAb and its control antibody in the 37℃storage at different times. The immunological activity of radioiodine labeled anti-MIF McAb was measured by ELISA.2. Mice were induced inflammation by injecting of S.aurous, E.coli and turpentine oil. When the swelling of the muscle was apparent, the radiopharmaceuticals were injected .The mice of each group were sacrificed at different time after the injection. A sample of 1ml blood was obtained. The whole infected tissue, the control lateral muscle, heart, liver, spleen, kidney, lung and the sample of bone were taken, weighted, counted for radioactivity in a gamma counter and then calculated the tissue concentrations which expressed as percentage injected dose per gram %ID/g,T/NT.3. In order to make sure that the high intake of radiolabeled anti-MIF antibody in inflammatory tissue is caused by high expression of MIF in locus; the expression of MIF in locus was analyzed by RT-PCR and immunohistochemistry.4. Whole-body images were obtained with storage phosphor screen. Serial images were performed at 24h, 48h, and 72h after injection. The anesthetized mice were placed on the storage phosphor screen plate with the ventral side facing the plate, in subdued light. The plate was exposed to a mouse for 45 minutes. At cessation of exposure, the plate was immediately covered with an opaque plastic sheet, then transferred to the scanner, and scanned by typhoon trio + （laser red 633 nm, pixel size 200 mcrons, phosphor mode: best sensitivity）.Results1. Radioiodine labeled antibody was separated from free iodine using a size exclusion column （Sephadex G-25, Pharmacia）. The specific activity of ¹²⁵I-anti-MIF McAb is 29.56 GBq/μmol. The radiochemical purity is 98.25% （paper chromatography）. The specific activity of ¹³¹I-anti-MIF McAb is 29.56 GBq/μmol, of ¹³¹I-IgG is 30.12 GBq/μmol. The radiochemical purity of ¹³¹I-anti-MIF McAb is 97.10% （paper chromatography）, and that of ¹³¹I-IgG is 97.17% （paper chromatography）.The immunological activities of radioiodine labeled anti MIF McAb was proved through ELISA （Enzyme-linked immune -sorbent assay）.2. ¹²⁵I-anti-MIF McAb shortly transited from the peritoneal cavity to the circulation after intraperitoneal injection, it went up and reached a zenith at 4 hours post injection, and then the activity in the blood went down quickly. The change of activity in the heart and lung was the same as blood. The biodistribution of ¹²⁵I-anti-MIF McAb showed the highest uptake and the lowest decrease in the inflammatory tissue. The activity in the blood was higher than the kidney, liver, spleen, heart, and lung. Peak uptake in the kidney, liver, spleen occurred around 30 minutes, followed by gradual clearance over time. This indicated that the product of ¹²⁵I-anti-MIF McAb was excreted from kidney or swallowed by reticuloendothelium of liver and spleen, resulting in deiodination; the activity of ¹²⁵I-anti-MIF McAb in the inflammatory tissue increased gradually for three inflammation models. The highest uptake was S. aureus group and the lowest was E. coli group. In all three groups, T/NT was >3 at 4h post injection and increased continually in the whole observed period: T/NT ratio was >7 at 48h and >9 at 72h Mice with the left thigh inflammation were injected intravenously via the tail vein by ¹³¹I-anti-MIF McAb or ¹³¹I IgG, respectively. It shows that the ¹³¹I-anti-MIF McAb group T/NT ratio was > 6 at 24h, and increased continually, T/NT ratio was > 9 until 72h, but the ¹³¹I-IgG group T/NT ratio was > 6 at 48h and > 8 at 72h. The highest uptake happened in the ¹³¹I-anti-MIF McAb group （p<0.05）. The T/B ratios for the ¹³¹I-anti-MIF McAb group werel.41±0.031, 1.53±0.018, 2.58±0.025 at 24h, 48h and 72h after injection, respectively. These ratios were significantly higher than that of ¹³¹I-IgG group （p<0.05）.3. Result of RT-PCR showed that the expression of MIF mRNA in normal tissues were little changes at 24h, 48h and 72h. However, there was a 3-fold increase in MIF mRNA expression in inflammatory tissues at 24h compared with normal tissues （p<0.05）. There was a 2-fold increase in MIF mRNA levels in inflammatory tissues at 48h compared with normal tissues （p<0.05）. Immunohistochemistry result showed that the localization of MIF protein in tissues specimens from inflammatory mouse at 24h, 48h, and 72h after focal inflammation occurred. MIF was negative expressed in normal tissues. However, there was a significant increase in MIF expression in inflammatory specimens according to the time after focal inflammation occurred in the first 48h and then decreased at 72h. MIF-expression localized among the inflammatory cells strongly after focal inflammation occurred.4. After ¹²⁵I-anti-MIF McAb were injected intravenously via the tail vein. Whole-body autoradiography showed that all inflammation foci could be visualized clearly from 24h after injection, but after 48h images were much clearer in accordance with the high T/NT ratio. The radioactivity was the highest in S. aureus lesion, average in turpentine lesion, and the lowest in E.coli lesion. The whole-body autoradiography images of the ¹³¹I-anti-MIF McAb and ¹³¹I-IgG at 24h, 48h and 72h after injection showed that both radiotracers focally increased uptake in the inflammatory muscles beginning at 24h. Compared analysis of the scintigrams of three times points showed that the ¹³¹I-anti-MIF McAb group had much more clear images than the ¹³¹I-IgG groups which is in accordance with the high T/NT ratio （p<0.05）.Conclusion1. Radioiodine labeled anti-MIF McAb made from Iodogen method is of high radiochemical purity and with high stability.2. Radioiodine labeled anti-MIF McAb achieve very high target to background ratio and has relatively low level of accumulation in non-target tissues, it possess a high relations with local expression of MIF.3. Whole-body autoradiography showed that all inflammation foci could be visualized clearly by storage phosphor screen. This study demonstrates the ability of radioiodinated anti-MIF McAb to measure in vivo inflammatory events represented by high expression of MIF and suggests that radiolabeled anti-MIF McAb warrants further investigation as a potential inflammation-seeking agent for imaging to detect inflammatory disorders.更多还原