本文選題：惡性腫瘤　切入點(diǎn)：肝細(xì)胞癌　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景:靶向治療有望成為晚期肝細(xì)胞癌的重要治療手段。溶酶體跨膜蛋白4β受體(lysosomal protein transmembrane 4 beta,LAPTM4B)在肝細(xì)胞癌細(xì)胞表面高表達(dá)而在正常肝細(xì)胞表面不表達(dá),有望成為肝細(xì)胞癌的靶向治療靶點(diǎn)。多肽AP2H(氨基酸序列為:IHGHHIISVG)是針對(duì)LAPTM4B受體的細(xì)胞膜外片段EL2設(shè)計(jì)的特異性反義靶向肽。采用5-羧基熒光素(5-carboxyfluorescein,FAM)和正電子核素18氟(18F)、68鎵(68Ga)標(biāo)記該靶向多肽制備分子探針,有望實(shí)現(xiàn)在活體上將該靶點(diǎn)的表達(dá)可視化,從而用于診斷并指導(dǎo)相應(yīng)的靶向治療。研究目的:1.合成多肽AP2H并用FAM標(biāo)記制備熒光探針(FAM-AP2H),從細(xì)胞學(xué)、組織學(xué)及動(dòng)物顯像等論證該探針的LAPTM4B受體靶向特異性。2.研究AP2H的18F、68Ga標(biāo)記方法學(xué),研制PET分子探針,通過micro PET/CT顯像無創(chuàng)性地將腫瘤的LAPTM4B受體表達(dá)可視化。3.探索如何減少肝膽排泄以提高腫瘤顯像效果。研究方法:一、采用固相合成法合成AP2H,FAM-AP2H并對(duì)多肽進(jìn)行分離純化及分析鑒定。二、采用免疫熒光方法確認(rèn)腫瘤細(xì)胞高表達(dá)LAPTM4B。通過孵育,研究FAM-AP2H與肝癌細(xì)胞的結(jié)合情況。并采用CCK-8法,對(duì)AP2H與FAM-AP2H是否存在細(xì)胞毒性進(jìn)行確認(rèn)。三、建立HepG2、BEL-7402、U87MG裸鼠腫瘤模型,通過組織免疫病理檢查確認(rèn)其LAPTM4B表達(dá)。四、進(jìn)行肝細(xì)胞癌腫瘤模型的FAM-AP2H熒光顯像研究。五:制備受體靶向肽探針:[18F]-FP-AP2H、[18F]AlF-NODA-MP-C6-AP2H、[68Ga]Ga-NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2H并研究腫瘤模型的microPET/CT 顯像結(jié)果:一、成功合成了多肽AP2H和FAM-AP2H,純度分別為98.89%和98.64%,主峰分子量(m/z:Da)分別為1069.7和1427.9,與理論分子量1069.24和1427.56相符,滿足實(shí)驗(yàn)要求。二、Cy3體外免疫熒光實(shí)驗(yàn),肝細(xì)胞癌細(xì)胞系BEL-7402及HepG2的細(xì)胞膜及細(xì)胞漿內(nèi)均見到濃染的紅色熒光,提示這兩種細(xì)胞均有LAPTM4B受體大量表達(dá)。人正常肝細(xì)胞L-02僅見到少許紅色熒光,提示L-02細(xì)胞的LAPTM4B受體表達(dá)量極低。三、體外細(xì)胞攝取及抑制實(shí)驗(yàn),細(xì)胞濃度為1×104個(gè)/ml,多肽濃度從OμM、2.5μM、5μM、10μM、20μM、40μM、80μM逐漸升高,熒光顯微鏡下發(fā)現(xiàn)肝癌細(xì)胞的熒光強(qiáng)度依次增強(qiáng),多肽主要定位于細(xì)胞膜,當(dāng)濃度為40μM時(shí)細(xì)胞攝取熒光多肽的相對(duì)熒光強(qiáng)度基本達(dá)到飽和。抑制實(shí)驗(yàn):當(dāng)抑制多肽濃度達(dá)到熒光多肽濃度的10倍以上,HepG2細(xì)胞攝取熒光探針明顯降低。通過流式細(xì)胞學(xué)定量測(cè)定,HepG2細(xì)胞熒光結(jié)合率從0%逐漸上升,在40μM時(shí)候熒光結(jié)合強(qiáng)度接近100%。其細(xì)胞攝取特點(diǎn)符合受體-配體結(jié)合的飽和曲線。而人正常肝細(xì)胞L-02未見到明顯的熒光結(jié)合,符合受體-配體結(jié)合的特異性。四、CCK-8法研究AP2H,FAM-AP2H兩種多肽對(duì)HepG2的毒性,細(xì)胞的存活率均在80%以上。五、采用尾靜脈注射給藥法,將FAM-AP2H注入荷瘤裸鼠體內(nèi),5h后處死,取各臟器熒光計(jì)數(shù)值的平均數(shù)進(jìn)行計(jì)算,荷HepG2裸鼠的靶/非靶比值依次為:腫瘤/正常肝臟=1.68±0.48,腫瘤/腦=1.36±0.35,腫瘤/心臟=2.07±0.67,腫瘤/肺=1.75±0.40,腫瘤/脾臟=2.18±0.64,腫瘤/腎臟=0.93±0.08,腫瘤/小腸=0.51±0.28,腫瘤/肌肉=1.63±0.42。BEL-7402裸鼠的靶/非靶比值依次為:腫瘤/正常肝臟=1.36±0.21,腫瘤/腦=1.28±0.15,腫瘤/心臟=1.75±0.54,腫瘤/肺=1.61±0.18,腫瘤/脾臟=1.96±0.30,腫瘤/腎臟=0.96±0.17,腫瘤/小腸=0.39±0.20,腫瘤/肌肉=1.91 ±0.73。從體內(nèi)分布看,該熒光探針主要通過膽道系統(tǒng)代謝。六、成功合成[18F]-FP-AP2H、[18F]AlF-NODA-MP-C6-AP2H、68Ga-NODA-MP-C6-AP2H,[18F]-FP-GGGRDN-AP2H。經(jīng)衰減校正的產(chǎn)率依次約為15%,30%-40%,90%,15%。產(chǎn)物峰單一,與未標(biāo)記前的多肽出峰位置基本相同。在室溫下、生理鹽水中靜置4h后通過HPLC測(cè)定,產(chǎn)物穩(wěn)定。七、荷HepG2和BEL-7402腫瘤模型的裸鼠經(jīng)尾靜脈注射放射性藥物60min后進(jìn)行 micro PET/CT 顯像,[18F]-FP-AP2H、[18F]AlF-NODA-MP-C6-AP2H、[68Ga]Ga-NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2H 在 HepG2 腫瘤內(nèi)百分注射劑量(%ID/g)依次為 3.70±0.56%ID/g,1.33±0.12%ID/g,2.20±0.55%ID/g,1.70±0.27%ID/g。其腫瘤/肝比值依次為 0.81 ±0.06,2.00±0.52,1.10±0.07,1.71 ± 0.33。經(jīng)尾靜脈注射[18F]AlF-NODA-MP-C6-AP2H、[18F]-FP-GGGRDN-AP2H 60min 后 BEL-7402 裸鼠腫瘤內(nèi)的百分注射劑量(%ID/g)為 1.00±0.26,2.20±0.61。其腫瘤/肝比值為 2.33±1.15,1.60±0.13。八、荷U87MG裸鼠腫瘤模型經(jīng)尾靜脈注射[18F]AlF-NODA-MP-C6-AP2H約60min后進(jìn)行micro PET/CT顯像,腫瘤的百分注射劑量(%ID/g)為1.50±0.53。其腫瘤/腦比值為20.74±5.94。結(jié)論:1.成功合成了 AP2H、FAM-AP2H、NODA-MP-C6-AP2H,純度達(dá)到 98.89%、98.23%和 95.8807%。2.體外實(shí)驗(yàn)FAM-AP2H能與肝癌細(xì)胞結(jié)合,其結(jié)合符合受體-配體結(jié)合規(guī)律。3.荷瘤鼠體內(nèi)分布顯示FAM-AP2H能特異性靶向腫瘤病灶。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,通過 micro PET/CT 顯像證明該探針可以特異性靶向肝細(xì)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7;R730.44

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本文編號(hào)：1598218