摘要ErbB受体信号通路在神经系统发育、疾病发生，以及肿瘤形成与发展中起着重要的作用。ErbB分子磷酸化后会激活下游信号通路，促进细胞增殖以及转移或改变细胞功能，是重要的疾病靶点。目前已经在临床上使用的抑制ErbB信号通路的癌症疗法主要以抗体和小分子的酪氨酸激酶抑制剂（TKIs）为主，更多的药物还在研发中。利用基因工程技术获得的dnEGFR（EGFR显性负性突变体）对于ErbB受体信号通路有广谱抑制作用，可以作为研究ErbB信号通路的有力工具，甚至开发一个新的治疗癌症或神经系统疾病的方法。61367

本文将对dnEGFR这个分子工具在体外细胞中与体内的功能进行验证。通过分子克隆将dnEGFR的cDNA序列连接到质粒上，然后用重组质粒进行细胞转染并对其表达的蛋白通过Western Blot检测对比，验证其在体外细胞中的功能。另一方面通过提取dnEGFR的基因工程小鼠的皮层下白质区的蛋白，并通过Western Blot检测对比，验证其在体内细胞中的功能。

实验结果表明dnEGFR对ErbB家族蛋白的磷酸化水平具有抑制作用，它也许可以作为一个分子工具辅助研发新的治疗方法，从而有效治疗由ErbB分子异常表达导致的神经系统疾病以及癌症。

Abstract ErbB receptor signaling pathways play important roles in the process of tumor formation and neural development. Phosphorylated ErbB receptors activate multiple downstream signaling pathways. Nowadays the main cancer therapies we have used in clinic to inhibit ErbB signaling pathways are antibodies and small molecule tyrosine kinase inhibitors (TKIs).There are more drugs in development. DnEGFR (dominant negative EGFR mutant) that engineered by genetic technology has potential to inhibit all endogenous ErbB receptors, and it can be used as a new method for the treatment of neural disorders or cancer.

We validated here the function of molecular tool dnEGFR in cells in vitro and in vivo. Plasmid containing dnEGFR was cloned from its cDNA sequence in a transgenic mouse. The recombinant plasmid was then transfected into cells and its inhibitory function was verified in vitro by Western blotting. On the other hand, total proteins of subcortical white matter in genetically engineered mice containing dnEGFR transgene were extracted and the inhibitory function of the protein in vivo was verified by Western blotting.

The experimental results showed that dnEGFR has inhibitory effect on phosphorylated level of ErbBs in vitro and in vivo. It may help to develop new treatments as a molecular tool so that humen can treat neural disorders and cancer caused by abnormal expression of ErbBs effectively.

关键词：ErbB； 显性负性突变体； 信号通路；广谱抑制

Keyword: ErbB； Dominant negative mutant； Signaling pathways；Pan-ErbB Inhibitor

目录

目录 3

引言 4

1.材料与方法 5

1.1实验材料 5

1.1.1实验小鼠 5

1.1.2载体质粒 5

1.1.3实验试剂 5

1.2实验方法 7

1.2.1提基因组DNA 7

1.2.2 PCR扩增dnEGFR cDNA序列 8

1.2.3重组质粒的获得 9

1.2.4 dnEGFR序列在体外的表达验证 10

1.2.5 dnEGFR序列在体外的功能验证 12

1.2.6 dnEGFR序列在体内的表达验证 13

2.结果与分析