靶向肝细胞癌LAPTM4B受体的新型荧光及PET分子探针的构建及显像研究

发布时间：2018-03-11 12:42

本文选题：恶性肿瘤　切入点：肝细胞癌　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:靶向治疗有望成为晚期肝细胞癌的重要治疗手段。溶酶体跨膜蛋白4β受体(lysosomal protein transmembrane 4 beta,LAPTM4B)在肝细胞癌细胞表面高表达而在正常肝细胞表面不表达,有望成为肝细胞癌的靶向治疗靶点。多肽AP2H(氨基酸序列为:IHGHHIISVG)是针对LAPTM4B受体的细胞膜外片段EL2设计的特异性反义靶向肽。采用5-羧基荧光素(5-carboxyfluorescein,FAM)和正电子核素18氟(18F)、68镓(68Ga)标记该靶向多肽制备分子探针,有望实现在活体上将该靶点的表达可视化,从而用于诊断并指导相应的靶向治疗。研究目的:1.合成多肽AP2H并用FAM标记制备荧光探针(FAM-AP2H),从细胞学、组织学及动物显像等论证该探针的LAPTM4B受体靶向特异性。2.研究AP2H的18F、68Ga标记方法学,研制PET分子探针,通过micro PET/CT显像无创性地将肿瘤的LAPTM4B受体表达可视化。3.探索如何减少肝胆排泄以提高肿瘤显像效果。研究方法:一、采用固相合成法合成AP2H,FAM-AP2H并对多肽进行分离纯化及分析鉴定。二、采用免疫荧光方法确认肿瘤细胞高表达LAPTM4B。通过孵育,研究FAM-AP2H与肝癌细胞的结合情况。并采用CCK-8法,对AP2H与FAM-AP2H是否存在细胞毒性进行确认。三、建立HepG2、BEL-7402、U87MG裸鼠肿瘤模型,通过组织免疫病理检查确认其LAPTM4B表达。四、进行肝细胞癌肿瘤模型的FAM-AP2H荧光显像研究。五:制备受体靶向肽探针:[18F]-FP-AP2H、[18F]AlF-NODA-MP-C6-AP2H、[68Ga]Ga-NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2H并研究肿瘤模型的microPET/CT 显像结果:一、成功合成了多肽AP2H和FAM-AP2H,纯度分别为98.89%和98.64%,主峰分子量(m/z:Da)分别为1069.7和1427.9,与理论分子量1069.24和1427.56相符,满足实验要求。二、Cy3体外免疫荧光实验,肝细胞癌细胞系BEL-7402及HepG2的细胞膜及细胞浆内均见到浓染的红色荧光,提示这两种细胞均有LAPTM4B受体大量表达。人正常肝细胞L-02仅见到少许红色荧光,提示L-02细胞的LAPTM4B受体表达量极低。三、体外细胞摄取及抑制实验,细胞浓度为1×104个/ml,多肽浓度从OμM、2.5μM、5μM、10μM、20μM、40μM、80μM逐渐升高,荧光显微镜下发现肝癌细胞的荧光强度依次增强,多肽主要定位于细胞膜,当浓度为40μM时细胞摄取荧光多肽的相对荧光强度基本达到饱和。抑制实验:当抑制多肽浓度达到荧光多肽浓度的10倍以上,HepG2细胞摄取荧光探针明显降低。通过流式细胞学定量测定,HepG2细胞荧光结合率从0%逐渐上升,在40μM时候荧光结合强度接近100%。其细胞摄取特点符合受体-配体结合的饱和曲线。而人正常肝细胞L-02未见到明显的荧光结合,符合受体-配体结合的特异性。四、CCK-8法研究AP2H,FAM-AP2H两种多肽对HepG2的毒性,细胞的存活率均在80%以上。五、采用尾静脉注射给药法,将FAM-AP2H注入荷瘤裸鼠体内,5h后处死,取各脏器荧光计数值的平均数进行计算,荷HepG2裸鼠的靶/非靶比值依次为:肿瘤/正常肝脏=1.68±0.48,肿瘤/脑=1.36±0.35,肿瘤/心脏=2.07±0.67,肿瘤/肺=1.75±0.40,肿瘤/脾脏=2.18±0.64,肿瘤/肾脏=0.93±0.08,肿瘤/小肠=0.51±0.28,肿瘤/肌肉=1.63±0.42。BEL-7402裸鼠的靶/非靶比值依次为:肿瘤/正常肝脏=1.36±0.21,肿瘤/脑=1.28±0.15,肿瘤/心脏=1.75±0.54,肿瘤/肺=1.61±0.18,肿瘤/脾脏=1.96±0.30,肿瘤/肾脏=0.96±0.17,肿瘤/小肠=0.39±0.20,肿瘤/肌肉=1.91 ±0.73。从体内分布看,该荧光探针主要通过胆道系统代谢。六、成功合成[18F]-FP-AP2H、[18F]AlF-NODA-MP-C6-AP2H、68Ga-NODA-MP-C6-AP2H,[18F]-FP-GGGRDN-AP2H。经衰减校正的产率依次约为15%,30%-40%,90%,15%。产物峰单一,与未标记前的多肽出峰位置基本相同。在室温下、生理盐水中静置4h后通过HPLC测定,产物稳定。七、荷HepG2和BEL-7402肿瘤模型的裸鼠经尾静脉注射放射性药物60min后进行 micro PET/CT 显像,[18F]-FP-AP2H、[18F]AlF-NODA-MP-C6-AP2H、[68Ga]Ga-NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2H 在 HepG2 肿瘤内百分注射剂量(%ID/g)依次为 3.70±0.56%ID/g,1.33±0.12%ID/g,2.20±0.55%ID/g,1.70±0.27%ID/g。其肿瘤/肝比值依次为 0.81 ±0.06,2.00±0.52,1.10±0.07,1.71 ± 0.33。经尾静脉注射[18F]AlF-NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2H 60min 后 BEL-7402 裸鼠肿瘤内的百分注射剂量(%ID/g)为 1.00±0.26,2.20±0.61。其肿瘤/肝比值为 2.33±1.15,1.60±0.13。八、荷U87MG裸鼠肿瘤模型经尾静脉注射[18F]AlF-NODA-MP-C6-AP2H约60min后进行micro PET/CT显像,肿瘤的百分注射剂量(%ID/g)为1.50±0.53。其肿瘤/脑比值为20.74±5.94。结论:1.成功合成了 AP2H、FAM-AP2H、NODA-MP-C6-AP2H,纯度达到 98.89%、98.23%和 95.8807%。2.体外实验FAM-AP2H能与肝癌细胞结合,其结合符合受体-配体结合规律。3.荷瘤鼠体内分布显示FAM-AP2H能特异性靶向肿瘤病灶。4.成功合成了[18F]-FP-AP2H、[18F]AlF-NODA-MP-C6-AP2H、[68Ga]Ga-NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2H,通过 micro PET/CT 显像证明该探针可以特异性靶向肝细胞癌病灶。
[Abstract]:Background: targeted therapy is expected to become an important means of treatment of advanced hepatocellular carcinoma. Lysosomal transmembrane protein 4 beta (lysosomal protein transmembrane 4 beta, LAPTM4B) was highly expressed on the surface of hepatocellular carcinoma cells in normal liver cell surface expression of hepatocellular carcinoma, is expected to become the target of targeted therapy. Polypeptide AP2H (amino acid sequence: IHGHHIISVG) on cell membrane LAPTM4B receptor fragments designed EL2 specific antisense targeting peptides. Using 5- carboxyfluorescein (5-carboxyfluorescein, FAM) and positron nuclide 18 (18F), 68 gallium fluoride (68Ga) labeled the targeting peptide preparation of molecular probe, is expected to achieve in the expression in vivo visualization of the target, thus, for the diagnosis and guidance of the corresponding targeted therapy. Objective: 1. synthetic peptide AP2H and preparation of fluorescent probe labeled with FAM system (FAM-AP2H), from cytology, and animal tissue imaging demonstration LAPTM4B receptor target of the probe to the specific.2. of AP2H 18F, 68Ga labeling method, developed by micro PET probe, PET/CT imaging and noninvasive expression of tumor LAPTM4B receptor.3. to explore how to reduce visual hepatobiliary excretion to improve tumor imaging effect. Research methods: first, using solid phase synthesis of AP2H FAM-AP2H, and the peptide was identified and purified. Two, using immunofluorescence method to confirm the high expression of LAPTM4B. in tumor cells by incubation with FAM-AP2H and liver cancer cells. And using the method of CCK-8, AP2H and FAM-AP2H to confirm whether there is cytotoxicity. Three, HepG2, BEL-7402, U87MG tumor model in nude mice through the organization, by pathological examination to confirm the LAPTM4B expression. Four of hepatocellular carcinoma model FAM-AP2H fluorescence imaging. Five: preparation of receptor targeting peptide probe: [[18F]-FP-AP2H 18F]AlF-NODA-MP-C6-AP2H, [68Ga]Ga-NODA-MP-C6-AP2H, [18F]-FP-GGGRDN-AP2H and microPET/CT on tumor model imaging results: a successful synthesis of a polypeptide of AP2H and FAM-AP2H, the purity were 98.89% and 98.64%, the peak molecular weight (m/z:Da) were 1069.7 and 1427.9, with a theoretical molecular weight of 1069.24 and 1427.56 line to meet the experimental requirements. Two, Cy3 in vitro immunofluorescence experiment. The cell membrane in hepatocellular carcinoma cell lines BEL-7402 and HepG2 and cytoplasm were seen red fluorescent stain, suggesting that the two kinds of cells have a large number of people. The expression of LAPTM4B receptor in normal liver cells L-02 only a few red fluorescent cells, suggesting that L-02 LAPTM4B receptor expression was very low. Three, the in vitro cell uptake and inhibition the cell concentration of 1 * 104 /ml from O, peptide concentration M, 2.5 M, 5 M, 10 M, 20 M, 40 M, 80 M increased gradually, liver cancer cells under the fluorescence microscope found The fluorescence intensity increased gradually, the polypeptide mainly located in cell membrane, when the concentration is 40 M relative fluorescence intensity of cellular uptake of fluorescent peptide reached saturation. The inhibition when the concentration reached 10 times the inhibitory polypeptide fluorescent peptide concentration, cell uptake of HepG2 fluorescent probes were significantly reduced. Through flow cytometric quantitative determination, the rate of gradually increased from 0% HepG2 cell fluorescence combined with saturation curve of receptor ligand binding to 40 M when the fluorescence binding strength close to 100%.. The cellular uptake and human normal liver cell line L-02 showed no obvious fluorescence combined with specific receptor ligand binding. Four, CCK-8 of AP2H, the toxicity of FAM-AP2H two polypeptide of HepG2, cell survival rate was above 80%. Five, the tail intravenous injection, FAM-AP2H was injected into the nude mice were sacrificed after 5h, the average organs of fluorescence count The number is calculated, the nude mice bearing HepG2 target / non target ratio is as follows: the tumor / normal liver tumor / brain =1.68 + 0.48, =1.36 + 0.35, tumor / heart / lung cancer =2.07 + 0.67, =1.75 + 0.40, =2.18 + 0.64 tumor / spleen, kidney neoplasms / =0.93 + 0.08, =0.51 + / small intestine tumor 0.28, tumor / muscle =1.63 + 0.42.BEL-7402 in target / non target ratio is as follows: the tumor / normal liver tumor / brain =1.36 + 0.21, =1.28 + 0.15, tumor / heart / lung cancer =1.75 + 0.54, =1.61 + 0.18, tumor / spleen =1.96 + 0.30, =0.96 + 0.17 / kidney tumor, tumor / intestinal =0.39 + 0.20, =1.91 + 0.73. from tumor / muscle distribution in vivo, the fluorescent probe mainly through the biliary system metabolism. Six, the successful synthesis of [18F]-FP-AP2H, [18F]AlF-NODA-MP-C6-AP2H, 68Ga-NODA-MP-C6-AP2H, [18F]-FP-GGGRDN-AP2H. by the attenuation correction yields are about 15%, 90%, 30%-40%, 15%. and unlabeled single product peak, The peptide peak positions are basically the same. At room temperature, saline static 4H measured by HPLC, the product stability. Seven, bearing HepG2 and BEL-7402 tumor model in nude mice by intravenous injection of radioactive drugs after the 60min micro PET/CT [18F]-FP-AP2H, [18F] imaging, AlF-NODA-MP-C6-AP2H, [68Ga]Ga-NODA-MP-C6-AP2H, [18F]-FP-GGGRDN-AP2H in HepG2 tumor% injection dose (%ID/g) was 3.70 + 0.56%ID/g, 1.33 + 0.12%ID/g, 2.20 + 0.55%ID/g, 1.70 + 0.27%ID/g. and the tumor / liver ratio were 0.81 + 0.06,2.00 + 0.52,1.10 + 0.07,1.71 + 0.33. by intravenous injection of [18F]AlF-NODA-MP-C6-AP2H, [18F]-FP-GGGRDN-AP2H 60min BEL-7402 in the tumor of nude mice per cent injection dose (%ID/g) was 1 + 0.26,2.20. 0.61. the tumor / liver ratio was 2.33 + 1.15,1.60 + 0.13. eight, U87MG tumor in nude mice by tail vein injection of [18F]Al model About F-NODA-MP-C6-AP2H 60min after micro PET/CT imaging, percent injected dose of tumor (%ID/g) was 1.50 + 0.53. and the ratio of tumor to brain was 20.74 + 5.94. conclusion: AP2H, the successful synthesis of 1. FAM-AP2H, NODA-MP-C6-AP2H, the purity reached 98.89%, 98.23% and 95.8807%.2. in vitro experiments FAM-AP2H can bind with hepatoma cells, combined with its receptor ligand binding of.3. in nude mice distribution showed that FAM-AP2H can specifically target tumor.4. were successfully synthesized [18F]-FP-AP2H, [18F]AlF-NODA-MP-C6-AP2H, [68Ga]Ga-NODA-MP-C6-AP2H, [18F]-FP-GGGRDN-AP2H, micro by PET/CT imaging show that the probe can be specifically targeted for hepatocellular carcinoma lesions.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7;R730.44

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