食管鳞癌中藻蓝蛋白受体的分离和鉴定及其在食管鳞癌中的功能

藻蓝蛋白(phycocyanin，PC)是从海水蓝藻中提取的海洋生物活性物质，是当今海洋医药发展中研究的前沿和热点，我们前期研究结果证明藻蓝蛋白具有从体外抑制食管鳞癌细胞增殖的能力，但其确切的分子作用机制尚不清楚。本研究分别构建GST-PC/α链/β链的融合蛋白原核表达载体，同时提取不同食管鳞癌细胞株的细胞膜蛋白，采用GST-pull down研究食管鳞癌中特异性的PC受体蛋白，利用蛋白质测序技术和生物信息学手段对分离的PC受体蛋白进行分析和鉴定。进一步利用PCR分段缺失所鉴定的PC受体蛋白，构建不同区段的表达载体，研究其与PC相互作用的位点。最后从体内外不同层面阐明PC受体蛋白在食管鳞癌发生、发展中的作用，并探讨其可能的分子作用机制，从而建立食管鳞癌中PC和PC受体相互作用所介导的信号转导途径。该课题最终为阐明藻蓝蛋白抗肿瘤作用的分子机制以及发现新的肿瘤分子靶点提供理论依据。

Phycocyanin (PC) is a kind of marine bioactive substances extracted from the marine cyanobacteria, and is currently a hot topic in marine medicine development. Our preliminary results demonstrated that PC significantly inhibited the proliferation of esophageal squamous cell carcinoma (ESCC) in vitro, but its functional mechanism remains to be elucidated. In the present study, GST-PC/αchain/β chain fusion protein prokaryotic expression vectors will be constructed, respectively, meanwhile, cell membrane proteins will be isolated from a panel of ESCC cell lines, subsequently, GST-pull down method will be utilized to detect specific PC receptor proteins in different ESCC cell lines, and PC related receptor proteins will be analyzed and identified by protein sequencing technology and bioinformatics tools. Further, a series of different PC receptor proteins fragments were prepared by PCR method, and recombinant expression vectors with different PC receptor fragments will be constructed, interaction sites of PC receptor and PC will be investigated. Finally, the roles of PC receptors in ESCC will be clarified in vitro and in vivo,and the molecular mechanisms will be explored, which will establish the interactions of PC and PC receptors mediated signaling transduction pathway in ESCC. The study will ultimately provide a theoretical basis for elucidation of the molecular mechanism of anti-tumor effect of phycocyanin and discovery of new tumor molecular targets.