【作者】 张峰；

【导师】 张贺龙； 刘理礼；

【作者基本信息】 第四军医大学 ， 肿瘤学， 2013， 博士

【摘要】 肺癌是目前世界上最常见的恶性肿瘤之一，其发病率和死亡率在许多国家都位居前列。肺癌骨转移是肺癌患者中经常出现的严重并发症。伴随着骨转移的发生，肺癌患者的生存质量和生存时间都将受到严重威胁。然而，目前对肺癌骨转移的研究相对较少，尚不能阐明肺癌骨转移发生与发展的机制。因此，临床上可用于肺癌骨转移治疗的药物和手段都非常有限。在本实验室以往的蛋白质组学研究中，我们发现膜联蛋白A1在高骨转移小细胞肺癌细胞中呈高表达，提示膜联蛋白A1可能与肺癌骨转移相关。为了探明膜联蛋白A1在肺癌骨转移发生发展中的作用，在国家自然科学基金的资助下，我们通过一系列实验对本课题进行了研究。目的：应用细胞与动物模型，分别在体外和体内两方面观察膜联蛋白A1在小细胞肺癌细胞中的生物学作用；揭示膜联蛋白A1与小细胞肺癌骨转移的关系；初步探讨膜联蛋白A1在小细胞肺癌骨转移中作用的分子机制；为临床开发新的防治肺癌骨转移的药物与方法提供理论依据。方法：1、应用Real-time PCR和Western blot方法检测相似遗传背景不同骨转移能力的小细胞肺癌细胞株中膜联蛋白A1的表达差异；2、构建膜联蛋白A1过表达和siRNA慢病毒表达载体，并分别建立膜联蛋白A1表达上调或抑制的小细胞肺癌细胞株，通过MTT、平板克隆形成、流式细胞检测细胞周期、Transwell检测细胞体外侵袭与转移、体外骨黏附实验等方法在体外观察膜联蛋白A1在小细胞肺癌细胞株中的生物学作用；3、应用NOD/SCID鼠作为动物模型，观察不同膜联蛋白A1表达水平的小细胞肺癌细胞骨转移形成能力，揭示膜联蛋白A1与小细胞肺癌骨转移的关系；4、通过Real-time PCR、Western blot和Elisa检测等手段验证膜联蛋白A1与小细胞肺癌细胞中甲状旁腺素相关蛋白（PTHrP）合成与分泌的关系，并通过TGF-β诱导，观察膜联蛋白A1对小细胞肺癌细胞TGF-β/Smad信号通路的影响，初步阐明膜联蛋白A1在小细胞肺癌骨转移中作用的分子机制。结果：1、膜联蛋白A1在高骨转移潜能小细胞肺癌细胞株SBC-5中的表达高于其在相似遗传背景的低骨转移潜能细胞株SBC-3中的表达，提示膜联蛋白A1可能是一个新的肺癌骨转移相关蛋白；2、体外实验证实膜联蛋白A1具有促进小细胞肺癌细胞增殖、侵袭与转移以及黏附骨片的作用；3、在NOD/SCID鼠平台上证实膜联蛋白A1具有促进小细胞肺癌细胞在体内形成骨转移灶的作用；4、膜联蛋白A1可促进小细胞肺癌细胞中PTHrP的合成与分泌。在体外TGF-β诱导下，膜联蛋白A1可通过促进Smad2的磷酸化来增强TGF-β诱导细胞分泌PTHrP的能力，从而增强肺癌骨转移中PTHrP-TGF-β正反馈恶性循环的作用。结论：膜联蛋白A1是一个新的肺癌骨转移相关蛋白，它可以增强小细胞肺癌细胞的增殖、侵袭与迁移以及骨黏附能力，并能促进肿瘤细胞在NOD/SCID鼠中形成骨转移。膜联蛋白A1可促进小细胞肺癌细胞合成与分泌PTHrP，并可以通过增加细胞中Smad2的磷酸化来上调TGF-β诱导下细胞分泌PTHrP的能力，从而增强肺癌骨转移过程中的PTHrP-TGF-β正反馈恶性循环的作用，这可能是膜联蛋白A1促进小细胞肺癌骨转移的分子机制之一。更多还原

【Abstract】 Globally, Lung cancer is the major cause of malignancy-related mortality, and itsincidence is on the rise in many countries. Bone Metastasis is one of the most seriouscomplications of lung cancer. Bone metastases are often associated with skeletal-relatedevents, which may decrease the quality of life for patients. Due to less known about themolecular mechanism of bone metastasis, little progress has been made in treating skeletalmetastasis, currently. In previous proteomic study, we found that Annexin A1wasoverexpressed in bone-metastatic small cell lung cancer (SCLC) cells. It indicated thatAnnexin A1might play roles in osteotropism metastasis of SCLC. In our present study, weperform a list of experiments to confirm this hypothesis.PurposeTo observe the biofunction of Annexin A1in SCLC cells in vitro and in vivo, prove that the Annexin A1is closely related to the bone metastasis of SCLC, and explore themolecular mechanism of Annexin A1related bone metastases.Methods1. Using Real-time PCR and Western blot assays to demonstrate the expression ofAnnexin A1in SCLC cell lines with different capability of bone metastasis.2. Constructing the Lentiviral vector mediated overexpression or siRNA targeteddownregulation of Annexin A1in SCLC cells, and observing the cell behavior includingproliferation, cell cycle, invasion and migration and the ability of bone adhesion in vitro.3. Using NOD/SCID mouse as bone metastasis model to detect the bone metastaticability of SCLC cells with different Annexin A1expression level.4. In order to explore the molecular mechanism of Annexin A1related bonemetastases, Real-time PCR, Western blot and Elisa assays were performed to detect theexpression of PTHrP induced by Annexin A1in different SCLC cells, and observe theeffection of Annexin A1on TGF-β/Smad signal passway in SCLC cells with or withoutTGF-β incubation.Results1. Annexin A1is overexpressed in bone-metastatic SCLC cell line SBC-5. Itindicates that the Annexin A1maybe associated with bone metastases in SCLC.2. Annexin A1is able to enhance SCLC cells proliferation，invasion, migration andbone adhesion in vitro.3. Annexin A1has the potential to promote the bone-metastatic ability of SCLCcells in NOD/SCID mouse.4. Annexin A1has ability to upregulate the synthesis and secretion of PTHrP inSCLC cells. With TGF-β incubation, Annexin A1is able to increase the secretion ofPTHrP through enhancing the phosphorylation of Smad2in SCLC cells.ConclusionAnnexin A1has the potential to promote the SCLC cells proliferaton, invasion,migration and bone adhesion in vitro and bone metastases in vivo. Annexin A1induced overexpression of PTHrP is involved in Annexin A1related bone metastases in SCLCcells. Annexin A1is able to enhance the PTHrP-TGF-β vicious cycle in tumor bonemetastasis though TGF-β/Smad passway.更多还原