重组腺病毒介导的RNA干扰对EGFR高表达肿瘤细胞及肿瘤的干预实验研究
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摘要
背景与目的：表皮生长因子受体(EGFR)是原癌基因erbB-1的表达产物，属Ⅰ型跨膜酪氨酸激酶糖蛋白生长因子受体，分子量约为170KD。表皮生长因子等配体与EGFR结合后，启动其信号转导，引起下游一系列的级联反应，主要通过两条途径将信号传递至细胞核，一条是Ras→Raf→MAPK(有丝分裂原活化蛋白激酶)途径，通过c-jun、c-fos将信号传导至核内激活AP-1；另一条是PI3K(磷脂酰肌醇3激酶)→PKC→IKK途径，使IκBα磷酸化后导致NF—κB移位至核内，最终将胞外信号传导至内。促使细胞增殖、血管形成及抑制调亡，当EGFR过渡表达或表达失调时会导致肿瘤形成，尤其是上皮源性的人类肿瘤与之关系密切。阻断EGFR信号转导通路是开发新的肿瘤治疗药物及治疗方法的较新思路。尽管人类已经在使用EGFR单克隆抗体及小分子酪氨酸抑制剂治疗肿瘤方面取得了较大的进步，但是这些药剂仍有不足之处，关于抑制EGFR表达治疗肿瘤的方法仍需改进。RNA干扰是用与目的基因mRNA相同序列的21-23nt双链小分子干扰RNA来特异性高效率的抑制目的基因蛋白的表达，有研究表明，带有shRNA(小发夹RNA)表达框架的载体可以取得较高的RNA干扰效应，然而其作为抗肿瘤药物的潜能仍需实验验证。本实验的目的：
1．构建表达shRNA质粒载体，通过对A431细胞(人上皮肿瘤细胞)体外RNAi实验，筛选一条对EGFR基因沉默效率高的RNA干扰序列。
Background and Objective: Epidermal growth factor receptor (EGFR or erbB1) is a glycoprotein with a molecular weight of 170 000 with an intrinsic tyrosinespecific protein kinase, which is stimulated upon EGF binding. Activation of EGF receptor tyrosine kinase results in the generation of a number of intracellular signals that culminate in cell proliferation. The known downstream effectors of EGF receptor include PI3-K, RAS-RAF-MAPK signal pathways, and protein kinase C signaling pathways. EGF receptor signaling involves in cell growth, angiogenesis and DNA repair. Deregulated and excessive expression of EGFR, the transmembrane receptor tyrosine kinase , is a feature and/or cause of a wide range of human cancers, especially in epithelium cell originated carcinomas. Blockade of EGF receptor signaling pathway represents a new perspective on the development of novel and selective anticancer therapies. Although considerable progress has been made in the application of EGF receptor —targeted antibodies and
1．构建表达shRNA质粒载体，通过对A431细胞(人上皮肿瘤细胞)体外RNAi实验，筛选一条对EGFR基因沉默效率高的RNA干扰序列。
Background and Objective: Epidermal growth factor receptor (EGFR or erbB1) is a glycoprotein with a molecular weight of 170 000 with an intrinsic tyrosinespecific protein kinase, which is stimulated upon EGF binding. Activation of EGF receptor tyrosine kinase results in the generation of a number of intracellular signals that culminate in cell proliferation. The known downstream effectors of EGF receptor include PI3-K, RAS-RAF-MAPK signal pathways, and protein kinase C signaling pathways. EGF receptor signaling involves in cell growth, angiogenesis and DNA repair. Deregulated and excessive expression of EGFR, the transmembrane receptor tyrosine kinase , is a feature and/or cause of a wide range of human cancers, especially in epithelium cell originated carcinomas. Blockade of EGF receptor signaling pathway represents a new perspective on the development of novel and selective anticancer therapies. Although considerable progress has been made in the application of EGF receptor —targeted antibodies and
引文
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