【作者】 刘小海；

【导师】 王任直；

【作者基本信息】 北京协和医学院 ， 外科学， 2012， 博士

【摘要】 垂体腺瘤(pituitary adenoma)是发生在腺垂体的良性肿瘤,约占颅内肿瘤的20%,且近年来有逐渐增加的趋势。系统放射学检查和尸检结果显示,垂体腺瘤占普通人群的17-25%左右。无功能垂体腺瘤(nonfunctional pituitary adenoma, NFPA)是垂体腺瘤中最常见的类型,约占垂体腺瘤总数的30%。目前针对NFPA首选的治疗方式是手术切除,但约有40%的NFPA表现为恶性肿瘤的侵袭生长习性：向蝶鞍上、前生长,侵犯下丘脑、第三脑室及／或视神经；向两侧生长,侵犯双侧海绵窦、包绕颈内动脉等重要结构；向下、后生长,破坏鞍底甚至斜坡骨质。此时,无论经口鼻蝶窦手术,抑或经额下、翼点入路手术,均难以做到全切肿瘤,且手术后约有20%的病例发生肿瘤复发。而常用的药物(例如多巴胺受体激动剂及生长抑素类似物)均对NFPA疗效甚微。放疗只能控制部分垂体肿瘤的生长,而且鞍区放疗容易导致正常垂体、下丘脑及视神经等重要结构的损伤,引发垂体、下丘脑功能低下和视力受损等并发症,严重时,患者因严重放疗并发症而死亡。因此,临床上亟待寻找针对NFPA,特别是侵袭性NFPA的特异性的分子标志物和临床靶向诊疗的靶点。新近,针对叶酸受体(Folate Receptor,FR或folate-binding proteins, FBP)的研究给NFPA的靶向诊断及药物治疗带来了新希望。FR是一种能与叶酸特异性结合的N端糖基化蛋白,包括四种异构体：α、β、γ／γ’和δ。其中,FR α在正常组织中不表达或极低表达,而在卵巢癌、肾细胞癌和非小细胞肺癌等上皮来源的恶性肿瘤中特异性高表达,并通过介导细胞对叶酸的摄取而促进肿瘤细胞生长及分裂增殖。鉴于FR α这种表达模式,越来越多的基于FR α的分子靶向诊断和治疗药物被开发出来,已有大量实验研究据此展开,针对卵巢癌、非小细胞肺癌等恶性肿瘤的FR α靶向诊断制剂及药物不断被开发,EC145等FR α靶向药物已被用于卵巢癌和非小细胞肺癌的II期临床试验。在垂体腺瘤方面,Oyesiku等首次发现FR α在NFPA中特异性高表达,而在其它亚型垂体腺瘤和正常腺垂体中不表达或极低表达。但是,FR α与NFPA肿瘤临床特征之间的关系、FR α在NFPA发生和侵袭性发展的过程中扮演的作用、FRα能否作为NFPA靶向诊治的分子靶点等间题尚无报道。本实验研究首先在76例人垂体腺瘤组织标本和7例正常腺垂体组织中,利用免疫组织化学、免疫荧光和qRT-PCR证实了：FR α仅在NFPA中特异性高表达,而在其他类型肿瘤和正常腺垂体中不表达；并在国际上首次发现FR α的表达水平与NFPA的肿瘤大小、侵袭性及Ki-67存在正相关关系。进一步,我们以人垂体腺瘤原代细胞为平台,探讨了叶酸靶向阿霉素脂质体(F-L-DOX)对垂体腺瘤的疗效及相关分子生物学机制,首次证实了FR α的这种表达特异性为NFPA靶向诊断和治疗提供了良好的分子靶点。最后,因为目前尚无荷人NFPA肿瘤裸鼠模型,我们利用荷人宫颈癌的裸鼠为动物模型,初步评价了叶酸靶向阿霉素脂质体(F-L-DOX)对人宫颈癌的疗效及相关分子生物学机制,在动物体内水平,证实了F-L-DOX可以特异性地通过肿瘤细胞表面的FR α对肿瘤做到“增效减负”的治疗效果。因此,FR α在NFPA中的表达模式不仅可以作为NFPA诊断及判断侵袭性的分子标志物之一,而且可以作为肿瘤的临床靶向诊疗靶点之一。第一部分叶酸受体在垂体腺瘤中的表达及其与肿瘤临床特征之间的关系目的：检测叶酸受体α(FR α)在各型垂体腺瘤和正常腺垂体中的表达情况；探讨叶酸受体表达水平与无功能垂体腺瘤(NFPA)各临床特征之间的相关性,寻找对NFPA诊断及其侵袭性判断具有重要意义的分子标志物。方法：收集散发性垂体腺瘤手术标本76例和正常腺垂体组织7例,利用免疫组织化学、免疫荧光共聚焦、实时定量逆转录PCR(quantitative RT-PCR)等方法检测FR α在各组织标本中的表达情况。探讨FR α表达水平与NFPA各临床特征(病人性别、年龄、肿瘤大小、肿瘤侵袭性和KI-67等)之间的相关性。结果：FR α仅在NFPA中特异性高表达,在其他类型的垂体腺瘤和正常腺垂体中不表达,具有显著性差异(P=0.001<0.05)；FR α的表达水平与NFPA侵袭性、Ki-67、肿瘤大小存在显著相关性(P<0.05),与年龄、性别等因素没有显著相关性(P>0.05)。结论： FR α可能在NFPA的发生和侵袭性发展中起到重要的作用；同时,FR α可作为NFPA特异性的诊断和判断侵袭性的指标,并为肿瘤的靶向诊治提供了理论基础。第二部分叶酸受体靶向阿霉素脂质体对人原代垂体腺瘤细胞的效用及机制目的：常规手术、化疗及放疗多难以治愈侵袭性无功能垂体腺瘤,新的替代疗法亟待研发。基于FR α的特异性表达模式,临床已尝试将FR α靶向的显影剂和治疗药物用于非小细胞肺癌、肾细胞癌和卵巢癌等FR α过表达的肿瘤,在药物的临床前研究和临床试验研究中均取得良好效果。本实验首次研究了一种新型的脂溶性阿霉素脂质体叶酸衍生物(F-L-DOX)对人原代无功能垂体腺瘤细胞的疗效及可能机制。方法：临床选取25例垂体腺瘤进行原代细胞培养,与叶酸靶向显影剂F-L-calcein、非叶酸靶向显影剂L-calcein、游离calcein及F-L-calcein加1mM游离叶酸共孵育,观察各类型肿瘤细胞摄取各型calcein的能力。利用梯度浓度的F-L-DOX. L-DOX. free DOX及F-L-DOX加1mM游离叶酸孵育细胞72h后,CCK-8法检测细胞活力,并依据细胞生长抑制曲线获得IC50值。收集IC50浓度各型阿霉素制剂处理的细胞样本行凋亡检测、TUNEL染色等,对细胞凋亡程度进行统计学分析。使用Caspase-Gl(?)试剂盒检测肿瘤细胞caspase-3/7,-8及-9的活性改变。Western blotting法检测凋亡相关蛋白Bax、Bcl-2及PARP的表达情况,并利用CytoSelect96-well invasion kit检测各型药物对肿瘤侵袭力的影响,Human MMP-2and MMP-9ELISA kits检测药物影响细胞侵袭力的可能机制。结果：在高表达FR α的NFPA中,较L-calcein、游离calcein相比,叶酸靶向显影剂F-L-calcein可以通过FR α更好地被NFPA细胞内吞,而且这种靶向内吞作用能被1mM游离叶酸阻断。在高表达FR α的NFPA中,F-L-DOX、L-DOX、free DOX和F-L-DOX+1mM FA的IC50分别为5.3μM,40.2μM,0.8μM和20.3μM。相比非靶向的L-DOX, F-L-DOX通过激活caspase-8的和caspase-9和caspase-3/7,更有效地抑制NFPA肿瘤的生长和促进细胞的凋亡。此外,F-L-DOX通过抑制MMP-2和MMP-9分泌,更有效地拮抗肿瘤的侵袭能力。相反,当添加1mM游离叶酸,F-L-DOX的对NFPA细胞的促凋亡和抗侵袭作用均显著减弱。结论：叶酸靶向阿霉素脂质体(F-L-DOX)能更好抑制人原代NFPA细胞活力,并通过调控死亡受体及线粒体凋亡信号通路促进肿瘤细胞凋亡,同时可以通过抑制MMP-2和MMP-9的分泌拮抗肿瘤的侵袭能力。我们的研究结果表明,FR α在此过程中发挥了关键作用,这可能是治疗NFPAs的一个良好的替代治疗策略。第三部分叶酸靶向阿霉素脂质体对裸鼠移植性人宫颈癌疗效研究目的：目前尚不能建立裸鼠移植性人无功能垂体腺瘤模型,我们在裸鼠移植性人宫颈癌的动物平台上,探讨叶酸靶向阿霉素脂质体(F-L-DOX)和阿霉素脂质体(L-DOX)对FR α过表达肿瘤---人宫颈癌的治疗作用。方法：建立荷人宫颈癌的裸鼠为动物模型,随机分为对照组(NS组)、游离DOX组、L-DOX组和F-L-DOX组,进行瘤旁注射给药,观察各组药物对肿瘤的生长抑制情况和动物本身一般状况。结果：叶酸靶向阿霉素脂质体组(F-L-DOX)对人宫颈癌的移植瘤有显著抑制作用,瘤重抑瘤率为72.33%,显著高于非靶向阿霉素脂质体组(53.40%)(P<0.05)、游离阿霉素组(42.72%)(P<0.001),叶酸靶向阿霉素脂质体组凋亡率(71.80%)均显著高于非靶向阿霉素脂质体组(51.40%)(P<0.05)、游离阿霉素组(42.30%)和空白组(P<0.001)o实验期间，DOX组和非靶向阿霉素脂质体组小鼠体重一直未见增加，而靶向叶酸阿霉素脂质体组小鼠体重平稳增加，且饮食、活动未见明显变化。结论：叶酸靶向阿霉素脂质体在治疗人宫颈癌的移植瘤过程中初步做到了“增效减负”，作为一种新型抗FR过表达肿瘤的靶向药物显示了良好的应用前景。更多还原

【Abstract】 Pituitary adenomas (PAs) are the second most common intracranial neoplasms, accounting for approximately20%of primary brain tumors, with an increaing morbidity. Autopsy studies demonstrate a higher incidence of PAs at25%in population. PAs are associated with significant morbidity and mortality due to hormone hypersecretion and mass effects. Nonfunctional pituitary adenomas (NFPAs) account for approximately30%of PAs and are the most common type. Because of their hormonal inactivity, NFPAs are often difficult to diagnose until they are large enough to cause tumor mass effects, such as hypopituitarism, visual field defects, or headaches. The first-line treatment is surgery, but more than40%of NFPAs are locally invasive, and infiltrate the dura, bones, and sinuses. Consequently, total resection may not be possible which leads to a high recurrence rate with a poor clinical outcome. Although postoperative radiotherapy may be effective in preventing tumor regrowth, it is associated with severe complications and has been frequently withheld by some clinicians in the past decade. Furthermore, present chemotherapy for treating NFPAs is mainly based on dopamine agonists (DA) and somatostatin (SRIF) analogs. Unfortunately, these strategies all harbor clinical limitations. As a result, there is an urgent need to determine new diagnositic and therapeutic biomarkers and construct novel antitumor agents to combat these refractory NFPAs.Recent research into folate receptors (FRs) has opened up new perspectives on the targeted imaging, diagnosis and therapy of NFPAs. Membrane FRs, including FRa and FRβ,are glycosylphosphatidylinositol (GPI)-anchored glycoproteins that recognize and internalize folate via endocytosis. FRa expression is amplified in over90%of ovarian carcinomas and at varying frequencies in other epithelial cancers. Meanwhile, FRβ is expressed in a non-functional form in neutrophils, and in a functional form in activated macrophages and myeloid leukemias. In contrast, most normal tissues do not express either FR isoform. Folic acid (folate) is a high affinity ligand for the FRs and retains high FR affinity after derivatization via one of its carboxyl groups. Because of its small size and bioavailability, folate has become one of the most investigated targeting ligands for tumor-specific drug delivery. Since targeted delivery via selective cellular markers can potentially increase the efficacy and reduce the toxicity of therapeutic agents, a variety of molecules and drug carriers, including chemotherapeutic agents, imaging agents, oligonucleotides, proteins, haptens, gene transfer vectors, nanoparticles and liposomes, have been conjugated to folate and evaluated for FRα-targeted diagnosis and therapy. These FRα-targeted agents have shown promising efficacy in preclinical models and have significant potential for future clinical application in a wide range of cancers, such as ovarian carcinomas and non-small cell lung cancer.Previous studies have demonstrated that FRα was uniquely overexpressed in NFPAs but not in functional pituitary adenomas (FPAs) or normal pituitary glands. However, whether the expression of FRα in NFPAs was positively correlated with tumor invasiveness and proliferation, whether FRα plays a important role in the tumorigenesis and progression of NFPA and whether FRα could be used as the molecular target for targeted diagnosis and therapy are unclear. In this study, we evaluated differential expression of folate receptor alpha in pituitary adenomas and its relationship to tumor behavior of NFPA. Then we assessed the targeting ability and cytotoxic efficacy of a novel lipophilic folate derivative containing liposomes loaded with doxorubicin (F-L-DOX); We further evaluated the mechanisms involved in the anti-tumor effect of F-L-DOX on the treatment of NFPAs. At last, we studied the effect and involved mechanisms of F-L-DOX on human cervical cancer xenografts in nude mice.Section One Differential Expression of Folate Receptor Alpha in Pituitary Adenomas and Its Relationship to Tumor BehaviorObjective:To identify a possible biomarker for the diagnosis of nonfunctional pituitary adenomas (NFPAs) that could also be used to assess tumor behavior.Methods:Sporadic pituitary tumor specimens (n=76) and normal pituitary glands (n=7) were examined. FRa protein and mRNA expression were quantified by immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction, respectively. We verified the differential expression of FRa in pituitary adenomas and evaluated the associations of FRa expression with Ki-67labeling index (LI) and clinicopathologic characteristics of NFPAs. Statistical significance was determined by using the Student t test or one-way analysis of variance.Results:FRa mRNA and protein was uniquely overexpressed in NFPAs but not in functional adenomas (adrenocorticotropic hormone, growth hormone, and prolactin) or normal adenohypophysial tissues (P<.001). The expression of FRa was positively correlated with tumor invasiveness, size and Ki-67LI in NFPAs.Conclusion:FRa may play an important role in the development and progression of NFPAs. Therefore, FRa may be useful as a molecular biomarker for the diagnosis of NFPAs and assessment of tumor invasiveness. Section two Effects of a novel lipophilic folate derivative containing liposomes loaded with doxorubicin on cell viability in nonfunctional pituitary adenomasObjective:To determine whether the novel lipophilic folate derivative containing liposomes loaded with doxorubicin (F-L-DOX) could be a useful therapy for nonfunctional pituitary adenomas(NFPAs).Methods:the targeting ability, cytotoxic effect and the anti-invasive ability of F-L-DOX which targeted the overexpressed FRa on the cell membrane of primary human NFPA cells were evaluated in25cases of pituitary adenomas.Results:FRa-targeted liposomes could effectively target the NFPA cells through the FRa and the endocytosis was blocked by1mM free folic acid. F-L-DOX more effectively inhibited tumor growth and promoted the apoptosis of NFPA cells through activating caspase-8, caspase-9and caspase-3/7, compared to non-targeted liposomal doxorubicin (L-DOX). Furthermore, F-L-DOX also exerted greater anti-invasive ability by suppressing the secretion of MMP-2and MMP-9from NFPA cells compared to L-DOX. In contrast, when1mM free folic acid was added, the pleotropic effects of F-L-DOX on NFPA cells were significantly blunted.Conclusion:Our findings suggest that FRa may play a critical role in mediating the antitumor effect of F-L-DOX which could be an excellent alternative therapeutic strategy to treat NFPAs. Section Three Effect of F-L-DOX on human cervical cancer xenografts in nude miceObjective:To study the effect of F-L-DOX on human cervical cancer xenografts in nude mice.Methods:Human cervical cancer cell line Hela was implanted into36nude mice. Different kinds of Doxorubicin formulations were injected around the tumor bearing nude mice divided into4groups. The tumor growth, morphological changes of tumor tissues, apoptosis rate of tumor cells were determined and compared.Results:The growth speed of tumor in the group of F-L-DOX significantaly slowed down than other groups. The rate of tumor restrain in tumor weight and tumor apoptosis of F-L-DOX group were72.33%and71.80%, which were remarkably higher than those of the DOX (53.40%,51.40%) and L-DOX group(42.72%,42.30%)(all P<0.05). The body weight of nude mouse in DOX group failed to increase, while that in F-L-DOX group increased steadly.Conclusion:F-L-DOX as a new FRa targeted anti-tumor drug may have a favorable clinical prospect.更多还原