人APOEε2及ε4等位基因修饰的神经干细胞的建立  

Establishment of human apolipoprotein E ε2 and ε4 modified neural stem cells

作　　者：江涌[1,2] 吴海涛[1] 孙晓川[1] 陈礼刚 郐莉[1] 郭宗铎[1] 顾应江[2] 刘洛同[2] 

机构地区：[1]重庆医科大学附属第一医院神经外科,重庆400016 [2]泸州医学院附属第一医院神经外科,四川泸州646000

出　　处：《第三军医大学学报》2010年第4期334-337,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30973087);重庆市卫生局重点项目(05-1-017);四川省教育厅青年基金(08zb049)~~

摘　　要：目的构建APOEε2和APOEε4的真核表达载体,转染APOE敲除鼠神经干细胞,分别建立转染APOEε2和ε4的神经干细胞系。方法以前期克隆得到的APOEε3cDNA为基础,通过定点突变技术得到APOEε2和APOEε4cDNA,亚克隆入表达载体pEGFP-N1,构建pEGFP-N1-APOEε2及pEGFP-N1-APOEε4的真核表达载体,经双酶切和测序确定序列及阅读框正确。以脂质体转染法转染APOE敲除鼠神经干细胞,通过G418筛选,建立转染APOEε2和ε4的神经干细胞系。采用RT-PCR、Westernblot及免疫荧光法检测APOE的表达。结果APOE敲除鼠NSCs转染6h后发出绿色荧光,48h达到高峰,荧光显微镜下观察转染率约(40.0±2.3)%,转染pEGFP-N1-APOE的NSCs在筛选至第10天可见细胞克隆形成。经鉴定保持了自我更新、多向分化保持性,其中分化为神经元的比例为(32.15±2.61)%,各组之间无明显差异。结论成功构建了稳定表达源性APOEε2及ε4的神经干细胞克隆,相应等位基因修饰的神经干细胞能高效表达目的基因蛋白并保留其干细胞特性。Objective To construct the eukaryotic expression vector of apolipoprotein E （APOE） ε2 and ε4 and establish APOE subtype gene-modified neural stem cells （NSCs） based on the clone of human APOE ε3 gene. Methods APOE ε3 cDNA was cloned in our previous work. Site-directed mutagenesis was used to obtain cDNA encoding APOE ε2 and ε4 isforms. The APOE ε2 and ε4 isforms were inserted in the eu- karyotic expression vector pEGFP-N1. Primary NSCs were isolated, cultivated and purified from rat embryo pallium of APOE knock-out mice. Recombinant plasmid was transfected into NSCs with Lipofectamine 2000 and selectively cultured with G418. After NSCs expressed the EGFP gene and APOE, they were amplified. Expression of target gene in the amplified NSCs was analyzed by RT-PCR, Western blotting and laser confocal microscopy, respectively. Results The eukaryotic expression vectors of APOEε2 and APOEε4 were constructed. The plasmids were transfected into NSCs by Lipofectamine 2000 （40.0 ± 2.3 ）%. The NSCs could express EG- FP after transfection. Some clones expressing EGFP were formed after selective culture with G418 at 10 day. The differented rate of neuronis （32.15 ±2.61 ）%. RT-PCR, Western blotting and laser confocal microscopy demonstrated that the human APOE gene isoforms （ε2, ε4） were expressed in NSCs. Conclusion Eukaryotic expression vector of APOE （ε2, ε4） can be transfected into NSCs and muhipotent NSCs expressing target gene can be obtained and are able to renew, thus laying a foundation for further studies on the function of APOE subtype.

关 键 词：载脂蛋白E类 等位基因 真核表达载体 神经干细胞 基因表达 

分 类 号：R322.8[医药卫生—人体解剖和组织胚胎学] R329.2[医药卫生—基础医学]