FasL相互作用蛋白的筛选及其在大肠癌研究中的意义

目的建立FasL相互作用蛋白的酵母双杂交系统，筛选与FasL相互作用的蛋白质，探讨FasL及其相互作用蛋白在大肠癌发生与发展中的意义，为筛选大肠癌早期诊断标志物和治疗靶标提供理论依据。方法以FasL作为诱饵蛋白，在人胎肝cDNA文库中筛选与FasL相互作用的蛋白质；将FasL相互作用蛋白的基因转染人直肠癌HR-8348细胞，通过细胞学实验（四唑蓝比色法），研究FasL及相互作用蛋白对直肠癌细胞耐药性的影响；应用免疫组织化学方法检测FasL及相互作用蛋白在大肠癌标本中表达情况，比较在大肠癌不同分期中这些蛋白的表达差异，研究他们在大肠癌发生与发展中的作用机制。结果筛选出10个与FasL特异性相互作用的蛋白，包括金属硫蛋白1K、1G、2A，组织蛋白酶B，脂肪酸合成酶，干扰素d诱导蛋白27，磷脂清除酶，丝氨酸／苏氨酸样激酶，锚着黏附蛋白以及纤维微丝蛋白一5等。细胞耐药实验证实，FasL与金属硫蛋白和组织蛋白酶之间的相互作用能够增强人直肠癌HR-8348细胞的耐药能力。免疫组织化学结果表明，癌变的大肠癌组织中FasL、组织蛋白酶、金属硫蛋白表达明显增高，高于正常大肠黏膜（P〈O．05）。结论FasL与组织蛋白酶、金属硫蛋白、锚着黏附蛋白等之间的相互作用与大肠癌的侵袭力及癌细胞产生耐药性密切相关：FasL、组织蛋白酶、金属硫蛋白可能为大肠癌诊断的标志物；FasL及其相互作用蛋白可作为治疗大肠癌新的分子标靶。

Screening of proteins interact with Fasl and its significance in colorectal carcinoma metastasis

Objective] To construct the yeast two-hybrid system, and screen the proteins which interact with FasL, and investigate the relationship of FasL and hepatic metastasis of colorectal carcinoma. Method] We have cloned the FasL gene into the pGBKT7 vector as the bait, then screened the fetal liver eDNA library, and have got a series Of specific proteins that interact with FasL protein. Using the bioinformatics, we analyzed the interacting proteins in the mechanism of colorectal carcinoma metastasis. Results] We have screened several proteins that interacted with FasL protein, including metallothionein 1K, 1G, 2A, cathepsin B, fatty acid synthase, interferon alphainducible protein 27, phospholipid scramblase, Ser/Thr-like kinase, anchor attachment protein, fibulin-5. Conclusions] We have successfully constructed the yeast two-hybrid system, and preliminary identified that the interaction between FasL, metallothionein, cathepsin and anchor attachment protein is radically closely to the hepatic colorectal carcinoma metastasis.