小鼠脑白质DISC1真核表达质粒载体构建及其体外-第三军医大学学报.DOC

DISC1基因过表达对星形胶质细胞质膜伸展的影响 刘淑宝，田衍平，陈显军，肖岚 第三军医大学基础部组织学与胚胎学教研室，重庆，400038 [摘要] 目的 构建DISC1蛋白表达质粒，检测其过表达后对星形胶质细胞（Astrocytes，ASTs）质膜伸展的影响。方法 针对小鼠DISC1基因mRNA编码序列，设计合成全长扩增引物；以小鼠脑白质cDNA为模板进行PCR扩增，构建全长表达质粒，命名为pEGFP-n1-DISC1。分别将过表达组pEGFP-n1-DISC1和空载体对照组pEGFP-n1电转染原代培养ASTs， 以Western blot及免疫荧光染色检测DISC1-GFP融合蛋白的表达，利用Image-Pro Plus 5.0软件分析对照组与DISC1过表达组星形胶质细胞质膜伸展面积的大小。结果 成功从cDNA中扩增出DISC1编码序列并插入pEGFP-n1载体，双酶切和测序结果证实，DISC1蛋白编码序列插入载体，并能正确读码翻译。构建质粒转染ASTs 60h后，可见DISC1-GFP融合蛋白表达；与对照组相比，DISC1过表达组ASTs的质膜伸展面积明显增加（P0.05）。结论 成功构建小鼠DISC1全长基因表达载体，DISC1基因过表达后可促使ASTs质膜伸展面积增加。 [关键词] 精神分裂症断裂基因1（Disrupted-In-Schizophrenia 1，DISC1）；真核表达质粒；星形胶质细胞（AST）；质膜伸展 [中图法分类号] R363.15；R338；R34 Effect of DISC1 overexpression on membrane expansion of astrocytes Liu Shubao, Tian Yanping, Chen Xianjun, Xiao Lan Department of Histology and Embryology, College of Basic Medical Sciences，Third Military Medical University, Chongqing, 40038, China [Abstract] Objective To construct the expression plasmid vector carrying DISC1 gene, and to determine the effect of membrane expansion area after overexpression of DISC1 in astrocytes. Methods Primers for full-length of mouse DISC1 was designed and synthetized. The coding sequence of DISC1 was amplified by PCR from cDNA reversed from mouse white matter RNA, and then inserted into vector pEGFP-n1using recombinant DNA technique. The expression plasmid was verified by enzyme digestion and DNA sequencing, named pEGFP-n1-DISC1. The expression plasmid and the empty control pEGFP-n1 vector were transfected into astrocytes by electroporation relatively. The expression of DISC1-GFP fusion protein in astrocytes was detected by Western blot and immunofluorescence staining. The membrane expansion of control (pEGFP-n1) and overexpression (pEGFP-n1-DISC1) groups were determined by analysising extended area of astrocytes with software Image-Pro Plus 5.0. Results The enzyme digestion and DNA sequencing confirmed that the coding sequence of DISC1 from mouse was consistant with that in G