人类GAD67基因转染骨髓基质源性神经干细胞及初步检测  

Transfection and initial detection of human GAD67 gene into bone marrow stromal cells-derived neural stem cells

作　　者：马宏伟[1,5] 徐如祥[1] 姜晓丹[1] 刘智良[2] 张雪[3] 陈镇洲[1] 郭爱林[4] 苏健[4] 王娆[1] 张冉[1] 黄伟[1] 黄永安[5] 

机构地区：[1]南方医科大学珠江医院神经外科、神经医学研究所,广州510282 [2]中山大学佛山市第一人民医院博士后科研工作站,佛山528000 [3]南方医科大学珠江医院药剂科,广州510282 [4]广东省人民医院医学研究中心,广州510080 [5]解放军空军总医院神经外科,北京100036

出　　处：《中华神经医学杂志》2007年第3期228-232,共5页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（30400464）;广东省自然科学基金（06301027）

摘　　要：目的利用转基因技术将人类谷氨酸脱羧酶（GAD）67基因转染骨髓基质源性神经干细胞仍MSCs-D-NSCs），获得GAD67修饰的成体专能干细胞。方法以PCR、双酶切及测序鉴定pEGFP-C2-GAD67和pcDNA3.1-GAD67重组子；利用密度梯度离心法分离培养大鼠BMSCs并促其向NSCs方向分化，将编码人类GAD67基因的真核表达质粒pEGFP-C2-GAD67及pcDNA3．1-GAD67分别转染BMSCs-D-NSCs，荧光显微镜下观察并用流式细胞仪检测转染效率。结果pEGFP-C2-GAD67及pcDNA3.1-GAD6，均可扩增出约1800bp目的条带，经双酶切和基因测序证实。荧光显微镜下观察可见大量绿色荧光细胞，两种载体转染效率分别为39.3％和40.5％。结论pEGFP-C2-GAD67及pcDNA3.1-GAD67真核表达载体重组正确，利用脂质体介导人类GAD6，基因能有效转染BMSCs-D-NSCs，为进一步观察其GAD67基因和蛋白的表达提供了前提条件。Objective To transfect the eukaryotic expression vectors encoding human glutamic acid decarboxylase （GAD）67 into rat bone marrow stromal cells-derived neural stem cells （BMSCs-D-NSCs）, and to construct genetically engineered GAD67-modified stem cells. Methods pEGFP-C2-GAD67 and pcDNA3.1-GAD67 were identified by PCR, and confirmed by double restriction enzyme digestion and sequencing. BMSCs were isolated using density gradient centrifugation, cultured, and then transfected with the recombinant plasmids pEGFP-C2-GAD67, pcDNA3.1-GAD67, respectively. The cells were observed by fluorescence microscope. The efficiency of transfection was detected by flow cytometry. Results The eDNA fragments of about 1 800 bp were amplified by PCR from pEGFP-C2-GAD67 and pcDNA3.1-GAD67, respectively, which were confirmed by double restriction enzyme digestion and DNA sequencing. Green fluorocytes were shown under fluorescence microscope. The efficiency of transfection was 39.3% and 40.5% , respectively. Conclusion The pEGFP-C2-GAD67 and pcDNA3.1-GAD67 recombinant eukaryotic expression vectors can be successfully constructed and effectively transfected into BMSCs-D-NSCs, which provides a precondition for detecting the mRNA and protein expression of human GAD67.

关 键 词：谷氨酸脱羧酶67 转染 骨髓基质细胞 神经干细胞 

分 类 号：R686[医药卫生—骨科学]