Notch通路及其阻断剂对无功能垂体腺瘤发生、发展的调控作用及机制的研究

Non-functioning pituitary adenomas(NFPAs) are relatively common. Clinically recognized non-functioning pituitary adenomas constitute approximately one-third of all tumours of the anterior pituitary. Non-functioning pituitary tumours are mostly of gonadotroph cell origin and are devoid of humoral hypersecretory syndromes. They are usually large at the time of diagnosis, commonly presenting with headaches, visual ?eld defects and hypopituitarism. The best treatment options remain neurosurgery and radiation therapy. Unfortunately, although several clinical studies have been carried out, no clear effective medical therapy for these tumours has emerged. Therefore, more speci?c and effective drugs must be developed for the medical treatment of non-functioning pituitary adenomas. The fundamental mechanisms of NFPAs tumorigenesis involve the cell cycle regulation, oncogene, tumor suppressor gene et.c. Recently, three further oncogenic pathways have been implicated in NFPAs tumorigenesis: the Ras-BRAF-mitogen-activated protein kinase (MAPK) pathway, the Wnt signalling pathway and the maternally expressed gene 3 (MEG3). Recent study has showed that Notch pathway was overexpressed in NFPAs other than in normal human pituitary tissue and hormone-secreting pituitary adenomas. The fundamental mechanisms of Notch proteins involve the regulation of various aspects of cell differentiation, proliferation and apoptosis. And Notch pathway is proved to be has crosstalk with MAPK pathway and Wnt pathway. Increasing evidence suggests that abnormal Notch signalling also contributes to the pathogenesis of various human tumors, such as pancreatic, ovarian, and lungcancers. Gamma-secretase inhibitor (GSI) is a inhibitor of Notch signalling pathway. GSI can reduce tumor cell proliferation, inhibite serum independence, and induce apoptosis in various tumors through inhibiting of Notch signalling pathway. Although several studies have suggested that Notch pathway may play a important role in the tumorigenesis of NFPAs, further studies are needed to elucidate the function of Notch pathway in the pathogenesis of human non-functioning pituitary adenomas and whether GSI can reduce tumor cell proliferation and induce apoptosis in NFPAs. Our study provide the ?rst comprehensive analysis of the expression of Notch signaling pathways in NFPAs and for the first time determine the effects of GSI on tumor cell growth, proliferation, apoptosis and hormone secreting of NFPAs in vitro and in vivo. Our study will provides new insights into the pathogenesis of human non-functioning pituitary adenomas and implicates the Notch pathway and its antagonist as a molecular therapeutic target for the treatment of NFPAs.

无功能腺瘤是一种常见的垂体腺瘤，严重危害患者的健康及生活质量，目前缺乏有效的药物治疗，针对其发病机制进行深入研究寻找理想的治疗靶点具有重要的临床意义。Notch通路和多种肿瘤的发生发展密切相关，其阻断剂γ分泌酶抑制剂可以在体内外试验中抑制多种Notch通路相关肿瘤的生长、增殖。我们在既往的研究中证明无功能腺瘤中Notch3及其配体Jagged-1表达升高，结合既往大量研究提示Notch信号通路在无功能腺瘤发生、发展中可能有重要作用。但目前的研究仅限于Notch受体，缺乏通路中配体、靶基因及阻断剂在无功能腺瘤中作用及其机制的阐述。本研究拟对Notch通路在无功能腺瘤中的作用做一系统性研究，阐明Notch通路在无功能腺瘤发生发展中的作用并探讨可能的机制；同时在体内、外试验中研究其阻断剂对无功能腺瘤的调节作用并探索可能的机制，旨在为无功能腺瘤的药物治疗提供理论依据。

无功能腺瘤是一种常见的垂体腺瘤，严重危害患者的健康及生活质量，目前缺乏有效的药物治疗。Notch通路和多种肿瘤的发生发展密切相关，其阻断剂γ分泌酶抑制剂可以在体内外试验中抑制多种Notch通路相关肿瘤的生长、增殖。本研究以无功能腺瘤为研究对象，同时以泌乳素腺瘤、生长激素腺瘤及正常垂体组织为对照组，对它们Notch通路的表达情况进行系统检测。同时在体外实验（细胞系）和体内实验（动物模型）中研究Notch通路抑制剂γ分泌酶抑制剂对肿瘤生长、增殖、凋亡及Notch信号通路表达的影响。.基因芯片筛查发现Notch通路为垂体腺瘤发生、发展的关键通路，Notch1、2和4在各亚型差异表达。PCR实验显示Notch通路中17个分子的mRNA水平有显著性差异；用312例病人标本构建组织芯片，免疫组化染色结果显示Notch2在无功能腺瘤中为高表达， DLL3的表达水平和肿瘤复发正相关，DLL4的表达水平和肿瘤复发负相关。在无功能腺瘤中Notch1的水平和DLL3的表达水平负相关，和DLL4的表达水平呈正相关，Notch4表达缺失；在生长激素腺瘤中Notch1、4和DLL4明显高于其他亚型；Notch2在不同亚型垂体腺瘤中与侵袭性密切相关。Notch通路抑制剂DAPT能够抑制原代无功能腺瘤肿瘤细胞分泌FSH和LH，显著抑制GH3细胞和生长激素腺瘤原代肿瘤细胞分泌生长激素的能力；Notch通路阻断剂GSI和DAPT能明显降低无功能腺瘤原代肿瘤细胞和GH3细胞的侵袭能力。在裸鼠移植瘤中应用DAPT能明显减少肿瘤的体积和重量，平均抑瘤率49.8%，同时降低Notch2、DLL3和VEGF的表达水平。在原代肿瘤细胞和GH3细胞中干扰Notch2的表达能降低细胞的细胞活力和侵袭能力的同时，显著降低DLL3和Cyclin D1的水平。.本研究发现Notch通路在不同亚型垂体腺瘤中呈差异表达，明确Notch2和垂体腺瘤侵袭密切相关，DLL3和肿瘤复发具有相关性；Notch抑制剂DAPT能抑制原代肿瘤细胞和GH3细胞的激素释放和侵袭能力，体内外实验均证实DAPT的肿瘤抑制作用，为垂体腺瘤的药物治疗提供了新的选择。