细胞生物学的研究思路.ppt

细胞生物学研究思路的探讨;研究背景;神经嵴发育不全：由于神经嵴细胞（neural crest cells，NCC）发育缺陷或障碍而导致神经嵴出现异常的增殖、生存、迁徙和分化。由于NCC发育不良导致的这些组织和细胞的病变统称为神经嵴病。 ;研究背景;;研究背景;基因组水平：WS致病基因突变检测;研究内容; 课程提纲 ;蛋白质加上标签 (Tag); 课程提纲 ;定点突变 site-directed mutagenesis;突变引物设计;; 课程提纲 ;转染-transfection;Transfection methods;DEAE-dextran Diethylaminoethyl (DEAE)-dextran is one of the oldest methods for introducing nucleic acids into cultured mammalian cells. The positively charged DEAE-dextran molecule interacts with the negatively charged phosphate backbone of the nucleic acid. The DNA–DEAE dextran complexes appear to adsorb onto the cell surface and be taken up by endocytosis. 优缺点： 仅适合瞬转 （transient transfection），不适合稳转； 细胞毒较高； 重复性较好。 ;Cationic polymer;Calcium Phosphate磷酸钙 The principle involves mixing DNA in a phosphate buffer with calcium chloride. The resulting calcium-phosphate–DNA complexes adhere to the cell membrane and enter the cytoplasm by endocytosis. 优缺点： 适合稳转； 重复性较差，主要影响因素包括溶液的pH值； 一些细胞不适合磷酸钙转染。;Cationic Liposome 脂质体 The liposomes currently in use typically contain a mixture of cationic and neutral lipids organized into lipid bilayer structures. Transfection-complex formation is based on the interaction of the positively charged liposome with the negatively charged phosphate groups of the nucleic acid. The uptake of the liposome–DNA complexes may be mediated by endocytosis. 品牌：Lipofectamine (Invitrogen)。 优缺点： 转染效率高； 重复性好； 转染时需降低血清浓度，从而提高了细胞毒性； ;Electroporation Cells in a suitable cuvette are subjected to a short high-voltage pulse that causes the membrane potential of the cells to break down. As a result, pores are formed through which macromolecules such as DNA can enter. 优缺点： 适合几乎所有的细胞； 瞬转、稳转； 需要DNA量大； 高达50–70%细胞死亡；;Microinjection;研究结果; 课程提纲 ;Reporter Assay: for transcriptional factors (TF);Reporter genes;CAT： chloramphenicol acetyltransferase 氯霉素乙酰转移酶;CAT assay： acetylated non-acetylated chloremphenicol are cheched by TLC;CAT assay： ELISA;?-gal (?-galactosidase):;;Luciferase:;GFP: Green Fluores