【作者】 任新玲；

【导师】 钱桂生；

【作者基本信息】 第三军医大学 ， 内科学， 2006， 博士

【摘要】 肺癌死亡率在世界范围内居恶性肿瘤首位,虽然近年来一些新化疗药物的出现,其治愈率仍然<15%。HER2/neu(erbB2)基因,为表皮生长因子受体(EGFR)家族成员,其编码蛋白为185KDa,具有酪氨酸蛋白激酶活性,与EGFR高度同源。HER2/neu受体可以与家族其他成员形成异源二聚体,从而发挥强有力的肿瘤信号传导作用。研究显示,HER2/neu介导的信号传导与肿瘤的形成、增值、转移、侵袭及放化疗耐药有关。约1/3非小细胞肺癌(NSCLC)存在HER2/neu过表达,HER2/neu过表达在NSCLC形成、增值、转移、侵袭等方面的作用尚需进一步研究证实。RNA干涉(RNAi)是双链RNA(dsRNA)介导的、序列特异的转录后基因沉默((PTGS )现象,广泛存在于多种自然界。RNAi的核心功能是抗病毒感染免疫,维持基因组中转座子的稳定性,清除异常RNA,同时参与基因表达调控。目前,RNAi技术己发展成为一种新型的基因阻断技术,用于高效特异地关闭或降低特定基因的表达,广泛用于新基因筛选、基因功能鉴定以及基因治疗等方面,在肿瘤、病毒感染等治疗方面已取得积极进展,显示了非常广阔的应用前景。但是,针对HER2/neu的RNAi对NSCLC的研究报道少见。研究目的本研究利用siRNA沉默肺癌细胞HER2/neu表达后,通过体外体内系列实验观察肿瘤细胞生物学行为的改变,了解HER2/neu基因在肺癌发生、发展中的作用,检测RNA干涉效果并探讨该方法应用于肺癌基因治疗的潜在药用价值,为肺癌的治疗提供一条新思路。研究内容1.根据人HER2/neu mRNA基因编码区序列,设计并合成针对HER2/neu的4条64nt的小发夹RNA(shRNA)模板脱氧寡核苷酸序列,首先构建针对HER2/neu的siRNA表达质粒pSUPER-siHER1和pSUPER-siHER2,并进行酶切鉴定和DNA测序;然后将构建正确的pSUPER-siHER2 H1启动子连同编码siRNA的模板序列用EcoR I和Xho I切出,再连接入去除CMV启动子的pcDNA3.0载体,构建带有新霉素抗性筛选标志的pcDNA3.0-siHER2表达质粒,并进行酶切鉴定。更多还原

【Abstract】 Lung cancer is the leading cause of cancer death in the world. The cure rate remains <15% despite some advances in chemotherapeutic agents [2]. HER-2/neu gene also called erbB2,is a member of the epidermal growth factor receptor (EGFR)family and encodes a 185 kDa protein with tyrosine kinas activity and shows partial homology to epidermal growth factor receptor.The HER2/neu protein can dimerize with other members of the EGFR family and serves as strong oncogenic signals.The HER2/neu oncogene is over expressed in 30% of Non small cell lung cancer (NSCLC), and its signaling pathways play critical roles in tumor occurring, cancer cell growth, differentiation, metastasis, anti-apoptosis and resistance to chemotherapeutic agents.RNA interference (RNAi) is the phenomenon of sequence-specific, post- transcriptional gene silencing (PTGS) triggered by double-stranded RNA (dsRNA). RNAi exists in extensive organisms such as Caenorhabditis elegans, plants, mammalian, and is regarded as the genome’s immune system. RNAi is now recognized as a mechanism for cellular protection and cleansing: it defends the genome against molecular parasites such as viruses and transposes, while removing abundant but aberrant nonfunctional messenger RNAs, as well as regulate developmental programs in a sequence specific manner. Now RNAi has developed into a novel gene-blocking tool and used to knockdown specific gene expression efficiently in various species. Its application includes screening new genes, functional genomics and virus and cancer gene therapy in which great achievement has been made. However, there is little application report about RNAi targeting HER2/neu gene in NSCLC. AimTo construct RNA interference vector to down-regulate HER2/neu gene and study the siRNA against HER2/neu effect on the cell cycle, tumor growth, and colony forming capability of lung adenocarcinoma cell line SPC-A-1 in vitro and tumor growth in athymic更多还原