抗癌胚抗原免疫毒素生物学功能的研究

为了研究免疫毒素发挥生物学作用的全过程，构建了两种不同形式的基于假单胞菌外毒素(Pseudomonas exotoxin A,PE)的抗癌胚抗原（carcinoembryonic antigen, CEA）免疫毒素CEA(Fv)/PE38/KDEL和PE35/CEA(Fv)/KDEL.用流式细胞术经间接免疫荧光法测定免疫毒素的抗原结合活性.免疫毒素的内化(internalization)通过间接免疫荧光标记的方法在激光共聚焦显微镜下进行检测，采用流式细胞术进行免疫毒素内化的定量分析.细胞凋亡采用FITC-annexin V/PI双荧光染色方法进行分析.用噻唑蓝(MTT)法测定免疫毒素的靶细胞杀伤功能.对两种形式的免疫毒素在各个功能阶段的性质进行研究和比较，全面展现了免疫毒素发挥生物学功能的过程及各个生物作用过程之间的相互影响，为详尽的机制研究和免疫毒素的进一步优化改造提供了重要的依据.

Biological Functions of Anti-CEA Immunotoxins

In order to reveal the events during the course of killing target cells of immunotoxins, two forms of Pseudomonas exotoxin A based immunotoxins against carcinoembryonic antigen (CEA) were constructed and named CEA(Fv)/PE38/KDEL and PE35/CEA(Fv)/KDEL. Antigen binding activity of the immunotoxins were assessed by flow cytometry, internalization of immunotoxins was detected by optical confocal microscopy and flow cytometry quantitatively. Cell apoptosis and necrosis were analyzed by staining the cells with FITC-annexin V/PI. The MTT method was used to assay the cytotoxicity of the immunotoxins. The biological functions of the two forms of immunotoxins in the different functional phases were compared. And the interaction between different functional phases was analyzed. The results revealed the whole functional process from the cell binding to the cytotoxicity of the immunotoxins, which provided more details about the biological functions of immunotoxins and could be valuable in the mechanism research and the optimization of immunotoxins.