【作者】 韩慧兰；

【导师】 陆伟跃；

【作者基本信息】 复旦大学 ， 药剂学， 2004， 博士

【摘要】 叶酸(Folic acid)是一种分子结构中含有喋呤环的小分子维生素,为真核细胞单碳代谢和核苷合成所必需。动物细胞缺乏叶酸生物合成的关键酶,它们只能依赖细胞表面的叶酸特异结合蛋白(即叶酸受体,Folate receptors)主动摄取外源性叶酸来维持正常的生命活动。近 50 年来的叶酸受体的研究表明,叶酸受体既广泛分布在正常组织,又分布在肿瘤组织中,其不同点在于,多数肿瘤细胞叶酸受体的数量和活性远远超过正常细胞。目前,肿瘤细胞叶酸受体受到广泛关注,已成为肿瘤诊断和治疗研究的新靶点。肿瘤细胞表面的叶酸受体及叶酸受体介导的叶酸高分子复合物的肿瘤靶向性是本研究的主题。 本研究的主要目的在于结合体内外抗肿瘤活性试验,从组织、细胞和分子水平探讨并初步确立叶酸-右旋糖酐复合物类药物靶向抗肿瘤机理,为叶酸-右旋糖酐复合物作为抗肿瘤药物的后续研究提供理论依据。 本研究的主要内容包括:常规细胞培养基和实验动物饲料中叶酸含量的调整;叶酸高分子复合物抗肿瘤谱的筛选;叶酸-右旋糖酐复合物抗肿瘤活性初步研究;叶酸-右旋糖酐复合物靶向抗肿瘤机理的初步探讨;其它叶酸高分子复合物作为介导叶酸受体靶向肿瘤细胞药物或药物载体的可能性探讨;防御素对肿瘤细胞作用的初步探讨。 集落形成法考察不同浓度叶酸培养液对肿瘤细胞生长的影响结果表明,在一定浓度范围内,Hela229 细胞集落形成率随叶酸浓度的增加而急剧增加,当叶酸浓度达到 453.1nmol/L 时逐渐趋于饱和。使用人体生理条件下叶酸浓度的培养液时的集落形成率为 73%左右,不影响细胞维持正常生理功能。这为后续的叶酸受体分析和药效学研究中低叶酸培养液,提供了依据。通过放射免疫法测定市售实验动物饲料中的叶酸含量,调整饲料中的叶酸含量,并考察其对小鼠血清浓度和生长的影响。结果表明,与普通饲料相比,使用低叶酸饲料后,动物的血清叶酸浓度虽然降低显著(p<0.001),仍能维持在正常范围内,且不影响动物的正常生长。因此,为低叶酸饲料替代普通高压料用于饲养与叶酸受体研究有关实验动物提供了依据。 叶酸高分子复合物抗肿瘤谱的筛选结果表明,鼻咽癌(KB)、子宫颈癌(HeLa,HeLa229)、肝癌(SMMC7721, Bel7402)、胃癌(SGC-7901)和肺癌(SPC-A-1)等肿瘤细胞表面叶酸结合位点均高于 60fmol/106 cells, 叶酸与叶酸受体复合物的解离常数 Kd <5.06×10-10mol/L。其中, SGC-7901、SMMC7721、Bel7402、HeLa229、SPC-A-1 细胞和人正常肝细胞 L02 表面的叶酸受体表达数量和活性未见国内任 1<WP=10>中 文 摘 要何研究报道。肝癌细胞、胃癌细胞、肺癌细胞和子宫颈癌 HeLa229 细胞的叶酸受体被首次检测, 拓展了富含叶酸受体的人类肿瘤细胞谱。 SGC-7901 裸鼠肿瘤组织冷冻切片、KB 裸鼠肿瘤组织膜蛋白提取液与 I- 125叶酸均有较高的特异性结合量,提示叶酸受体在多数肿瘤细胞和肿瘤组织中均有较高的表达,因而以叶酸为靶向头基的叶酸复合物对体内外肿瘤细胞均能寻找到特异性的靶标。 体内外抗肿瘤活性试验和安全性试验结果表明,叶酸-右旋糖酐复合物能杀伤体内外肿瘤细胞、抑制肿瘤组织生长,同时未见对正常组织的明显损伤,也未引起动物明显的过敏反应,故有可能开发成一种低毒安全的抗癌药物。 从组织、细胞和分子水平初步探讨叶酸-右旋糖酐复合物靶向抗肿瘤机理的试验结果表明,叶酸右旋糖酐复合物与叶酸受体结合也具有高特异性和高亲和性,其抗肿瘤作用可能是介导肿瘤细胞表面的叶酸受体靶向并内化入肿瘤细胞内,诱导肿瘤细胞分化或凋亡,从而起到靶向抗肿瘤作用,研究结果为叶酸-右旋糖酐复合物作为抗肿瘤药物的后续研究提供依实验依据。 本研究中的叶酸高分子复合物也对过度表达叶酸受体的肿瘤细胞有特异性亲和力。羧甲基化右旋糖酐复合物在小鼠体内组织分布、叶酸-羧甲基化右旋糖酐复合物在荷瘤裸鼠体内组织分布和竞争抑制 125 I-叶酸与肿瘤细胞结合等试验结果表明,右旋糖酐经羧甲基化后, 其被肝脏所摄取的量显著降低,对叶酸受体表达丰富的体外培养肿瘤细胞 HeLa 229 和 KB 细胞也具有特异和亲和作用,并对体内肿瘤显现出良好的靶向性。叶酸-聚乙二醇复合物中 F-PEG-DTPA 和MeO-PEG-F 对 KB 细胞也具有特异性亲和作用,并对 HeLa 229 和 KB 细胞具有一定的促进凋亡作用。 形态学观察和流式细胞仪法测试结果表明,10-6mol/L 防御素 ABPD2、ABPD4 和 ABPD6 对 HeLa229 细胞周期 DNA 含量和凋亡有一定影响,这为防御素和叶酸右旋糖酐复合物构成药物组合提供了实验依据。本研究的创新点在于: 1) 首次探讨原本无抗肿瘤活性和无明显毒副作用的右旋糖酐介导叶酸受体靶向肿瘤细胞后的抗肿瘤机理。 2) 首次从检测到胃癌(SGC-7901)、 肝癌(SMMC7721、Bel7402)、肺癌(SPC-A-1)等国内建株的肿瘤细胞和人正常肝细胞 L02 表面的叶酸受体表达数量和活性。 3) 首次评价叶酸-羧甲基化右旋糖酐复合物、叶酸-聚乙二醇复合物对叶酸受体的特异性亲和作用。更多还原

【Abstract】 Folates are low molecular weight pterin-based vitamins involved in one-carbontransfer reactions during the synthesis of nucleic acids and metabolism of amino acids,and the form of folate added to fortified foods and vitamin supplements is folic acid.Because humans and other mammal cells lack key enzymes of the folate biosyntheticpathway, they cannot synthesize folate, and must obtain it from exogenous sources forsurvival and proliferation.There are two mechanisms for the cellular internalization of the vitamin. The firstmechanism involves a low-affinity (Kd~5×10-6mol/L) membrane-spanning proteinthat transports reduced folates directly into the cell cytosol. The second mechanismuses a high affinity (Kd~10-10mol/L) glycosylphosphatidylinositol-anchoredglycoprotein receptor, generally referred to as the folate receptor, which preferentiallymediates the uptake of oxidized forms of folate (e.g. folic acid) into the cell byendocytosis. The folate transporter is present on virtually all cells, whereas thehigh-affinity folate receptor is expressed at high levels mainly on cancer cells.Overexpression of folate receptor by cancer cells might provide the cells with agrowth advantage relative to neighbouring normal tissue, and highly undifferentiatedmetastatic cancers express considerably more folate receptor than their localized,low-grade counterparts.The discovery of high-levels expression of folate receptor on many human cancercells has rendered the folate receptor an attractive candidate for development of tumorspecific therapeutics. In this role, the folate receptor can effectively serve either oftwo distinct functions: one as a vehicle for the non-destructive trafficking ofextracellular diagnostic agents into the cytoplasm of targeted tumor cells, and theother as a simple tumor marker that allows ligand-mediated enrichment of therapeuticmacromolecules including toxins, polymers, gene therapy vectors, andliposome-encapsulating drugs on tumor cell surfaces.The main object for this study is also folate receptors overexpressed in tumor cells, 4<WP=13>英 文 摘 要and to evaluate the antitumor activity and mechanism of some folate- macromolecules(synthesized by our lab ) by the point of view of tissue, cellular and molecular level.The main molecule used in the study was folate-dextran conjugate which was whichhas been authorized a patent by State Intellectual Property office of P. R. China.Thefolate –macromolecules including folate-carboxymethylized dextran conjugates andfolate-PEG conjugate.The first step for the study was to determine folate receptor expression in 18kinds ofcell lines like KB、HeLa229、HeLa、SGC-7901、SMMC7721、Bel7402、SPC-A-1、SK-BR-3、Raji、CNE2、 L02;SCI-375、SK-OV-3 、MCF 7 by determine itsmaximum binding sites and affinity by performing the classic radioligand bindinganalysis. Our results show that KB、HeLa229、HeLa、SGC-7901、SMMC7721、Bel7402、SPC-A-1、overexpresses folate receptors and its radioligand bindingbehavior was shown to be of high affinity, satuarable competitable and reversible. The125I-folate binding sites on the surface of these tumor cells were all> 60fmol/106cell-1;The KD values of folate receptors of different cells were all<5.06×10-10mol/l. Thetumor cells expressed more folate receptors than those normal hepatic cells, such asL02. Up to now, there isn’t any report about folate receptor overexpressed on cells ofSMMC7721, Bel7402 and SPC-A-1. The results obtained in our study expanded thetumor cell lineage which overexpress folate receptors and these cells can be used asnew targets for non-destructive diagnosis and tumor-specific drug and gene delivery.Folate receptor bidnding studies indicated that folate-dextran conjugate,folate-carboxymethylized dextran conjugates and folate-PEG conjugate.couldspecifically binding to folate receptor bearing tumor cells which described above, andcould competitivel更多还原