【作者】 李学农；

【导师】 丁彦青；

【作者基本信息】 第一军医大学 ， 病理学， 2002， 博士

【摘要】 一、背景、目的和意义 肿瘤转移(tumor metastasis)是一个多步骤、多因素、多基因的复杂过程，基本过程包括脱离(detachment)、侵入(intravasation)、循环(circulation)、附着(arrest)、侵出(extravasation)、血管生成(angiogenesis)及生长(growth)等。而所有步骤均可归结为癌细胞与宿主细胞及细胞外基质(Extracellular Matrix，ECM)之间的相互作用——粘附、浸润、移动、蛋白酶分泌以及复杂的信号转导过程，基于细胞信息传递和调控在细胞生命活动中处于主导地位，癌细胞信号转导系统的作用及相关机制对于揭示肿瘤转移的本质，探索新的抗肿瘤转移途径具有十分重要的作用。我们发现胚胎组织形态分化过程中干细胞及其原始分化细胞一系列迁移演化的过程与肿瘤转移过程极其相似。从这一新思路出发，通过文献分析得知，HGF/SF-c-met信号通路是目前诱导细胞迁移的关键信号通路。HGF/SF-c-met信号通路是由配体分子HGF/SF(hepatocyte growth factor/scatter factor，以下简作HGF)作用于其特异性受体c-met，引起一系列信号转导蛋白的酶促反应，促发相应生物学效应的信号传递路径。HGF是由间质细胞(如纤维母细胞、神经胶质细胞、肝贮脂细胞、巨噬细胞等)产生的具有多种生物学效应的多能性细胞生长因子，是目前所发现具有最强活性的刺激肝细胞再生的细胞因子，曾误以为它就是特异性肝细胞生长因子，事实上它对肾小管上皮、肺泡上皮、胃肠道粘膜上皮等许多上皮细胞均具有促有丝分裂(mitogen)、促运动(motogen)及促形态发生 。oz。。。。。。y。帅orphogen）活性，llGF书F能促进癌细胞生长、粘附、移动及浸润，并能刺激血管内皮细胞生长，己列为重要的促血管生成因子。c仰o t是HGF”F－－met信号通路的第一进门户，它仅存在于上皮细胞细胞膜，属于生长因子酪氨酸激酶跨膜受体家族成员。IIGF／SF、e t信号通路异常激活是肿瘤恶性生长、演进、浸润及转移的重要因素，并可能发挥关键性因果性作用（critical causal role）。但HGF／SF－。1et信号通路的作用靶分子及其与增殖、浸润、转移的联系分子机理尚不清楚。HGF书F、et信号通路的一系列生物学效应是以启动相应基因功能为基础的，但有关HGF书F、et信号通路转录诱导基因表达谱的研究尚未见报道。 为此，本研究以人大肠癌细胞为研究对象，利用基因芯片技术能同时大规模地检测成千上万个基因的特点，探索HGF亿F1et信号转导通路转录诱导基因表达谱，试图从基因水平探讨HGF书Fmet信号通路在大肠癌中的作用机制。并应用先进的反义基因治疗技术，首次构建旷met侣 联合反义真核表达载体，探索阻断HGF／SF、e土信号传导通路的抗浸润、抗转移效应，摸索抗转移治疗的新途径。并以期从正反两方面报诱导和阻断）探讨HGF书F、et信号传导通路在大肠癌转移中的作用及其分子机理。主要研究内容如下： 1．检测HGP、。－met在人大肠癌中的表达状态，通过体外粘附性、移 动性及浸润力等细胞生物学实验初步确定HGF对人大肠癌细胞的 刺激效应，以探讨HGF沾F1et信号通路在大肠癌转移中的作用。 2．采用基因芯片技术，比较HGF刺激前后的基因表达谱改变，以探索 HGF书F－－met信号通路相关诱导转录基因表达谱，从基因水平探讨 其作用机制。 3．构建阻断HGP／SF－－met信号通路的。、et／S100A6联合反义基因真核 表达载体，探讨联合反义真核表达载体对人大肠癌细胞的体外生 物学效应及抗转移作用，探索抗信号转导的反义基因治疗新策略。 4．探讨我室发现的具有抗转移作用的中药有效成分3，4，5－三羟基 蔑－3－p－单o－葡萄糖昔（3，4，5，－trihydroxystibene－3－P 1。n。“0“gin。。sNe，＊删G）对Hm丫即－me亡信号通路的作用及抗转 移作用机理。 二、材料和方洁 。oz。。。。。。h。 二．设立HGF梯度浓度处理组及空白对照组（D－Hanks液），对HGP刺激激 活HGF书F1et信号通路的体外生物学效应，包括生长曲线、粘附率、 移动性、浸润力、细胞周期、。－inn，MMP－2的蛋白表达进行系列分析， 采用国际通用的Trans。elf浸润小室模型法研究细胞移动性和浸润力， 应用PI染色流式细胞术（FCM）检测细胞周期改变，应用SABC免疫荧光 技术及FCM定性与定量相结合研究相关蛋白表达。采用D－actin内参 照半定量RT－－PCR技术研究大肠癌细胞的。－met，HGF的表达状态。 2．采用含有4096条人类基因CDNA的HGEC－405芯片，从三次重复的HGF 刺激实验大肠癌细胞中提取RNA，逆转录合成CDNA，应用双荧光探针 标记法，Cy3－dUTP标记对照组细胞mRNA，用心－dUTP标记HGF刺激细 胞的 mRNA，通过竞争性芯片杂交，用 GenePix 4000B芯片扫描仪（Axon） 扫描芯片，GenePix Pro 3．0分析Cy3?更多还原

【Abstract】 BACKGROUND/OBJECTIVESTumor metastasis is an extremely complex process involving multi-genes, multi-factors and multi-steps, in which neoplastic cells must escape from the primary tumor, degrade the extracellular matrix, migrate to distant sites, arrest in the capillaries, and migrate through the basement membrane and underlying connective tissue to the metastatic site. The essential events of the process are detachment, intravasation, circulation, arrest, extravasation, angiogenesis and proliferation, which come down to interactions of neoplastic cells with host cells and extracellular matrix -governed by adhesion, migration, invasion, proteaselysis, and the complicated signal transduction. In view of signaling play the leading roles in the cell function, signal transduction system and its mechanisms in the tumor metastasis are the key to understanding of essence of the process and exploring new anti-metastasis techniques. Considering similarities between tumor metastasis and the migration of progenitor cells in embryonic tissue development, HGF/SF-c-met signaling pathway may be very important to the tumor metastasis just as it to the migration of embryo primitive cells. HGF/SF-c-met signaling is a pathway by stimulation of c-Met via its ligand hepatocyte growth factor /scatter factor (HGF/SF), leads toa plethora of biological and biochemical effects in the cell. HGF/SF, a multifunction cell growth factor yielded by interstitial cell (fibroblasts, neuroglial cells, glial cells fat-storing cells, macrophages), primarily as specific to the liver, proved to be have mitogen, motogen, morphogen activity for nearly all epithelial cells. HGF/SF, having a magic scatter effect to tumor cells, named as scatter factor, promotes adhesiveness, invasiveness, motion and metastasis and stimulates blood vessel growth for cancer cells. c-Met, only presented in the epithelial cells, is a receptor tyrosine kinase shown to be over-expressed and mutated in a variety of malignancies. HGF/SF-c-met signaling may play an important or critical causal role in tumor progression and invasion and metastasis. However, little is known about the target molecules, molecules links to invasion, migration and metastasis, and gene expression profiles by which it exerts biological function through start-up transcriptional gene expression.This paper aims to explore the transcriptional gene expression profiles by activating HGF/SF-c-met signaling in colorectal carcinoma cells by using the microarray technology which can detect thousands of genes in the same time, and to construct c-met and S100A6 antisense eukaryotic expression vector for blockage of HGF/SF-c-met signaling and to find a new way for antisense gene therapy, and to probe into anti-metastasis effect and mechanisms of 3 , 4 , 5-trihydroxystibene-3- P -mono-D-glucoside (THMG, a Chinese drug extract).METHODS1. Cell biological techniques to detect cell growth curve, adhesion rate, migration, invasiveness, cell cycle, gene and protein expression were used for estimation of in vitro effects by stimulation of HGF/SF, Coastar Transwell Culture System for migration and invasiveness, PI stain flow cytometry for cellcycle analysis, and SABC Immune fluorescence technique and FCM for protein expression detection, and P -actin semi-quantitative RT-PCR for analysis of gene expression of c-met and HGF/SF.2. HGEC-40S microarray (Biostar, Shanghai) containing 4096 cDNA spots, fluorescent probe Labelled by Cy3-dUTP and Cy5-dUTP, derived from cDNA reverse-transcripted from RNA, extracted from human colorectal adenocarcinoma cell LoVo by HGF/SF stimulating experiments, were applied to exploring differential gene expression profiles between with and without HGF/SF stimulation. GenePix 4000B scanner was for scanning the microarray, and GenePix Pro 3. 0 software for analysis of intensity and ratio of Cy5/Cy3.3. Bioinformatic technology was used to optimize object gene sequences for antisense therapy. The c-met and S100A6 sequences was amplified from Hela cells by RT-PCR,更多还原