Tα1启动子驱动的荧光报告系统在人胚胎干细胞神经分化示踪中的应用  

Application of α-tubulin promoter-driven fluorescence reporter system in the neural differentiation studies of human embryonic stem cells

作　　者：陈京[1] 余伟华[2] 李付贵[1] 

机构地区：[1]中山大学附属中山医院肿瘤研究所,广东中山528403 [2]中山大学干细胞与组织工程研究中心,广东广州510080

出　　处：《临床检验杂志》2016年第9期657-661,共5页Chinese Journal of Clinical Laboratory Science

基　　金：广东省自然科学基金(2014A030310013);中国博士后基金(2014M562244)

摘　　要：目的观察携带Tα1(α-tubulin)基因启动子驱动的荧光报告系统在人胚胎干细胞(h ESCs)神经分化示踪中的应用。方法胎鼠成纤维细胞(MEF)培养h ESCs,免疫荧光染色鉴定其生物学标记。将携带p LV/Final-puro-Tα1(α-tubulin)-hr GFP载体的慢病毒感染人胚胎干细胞(h ESCs),嘌呤霉素(puromycin)筛选14 d后获得纯化h ESCs,并诱导其向神经元样细胞分化,同时显微镜观察细胞绿色荧光变化。第23天对表达绿色荧光的细胞行免疫荧光染色鉴定。结果 h ESCs细胞的OCT-4、SSEA-4及TRA-1-60表达呈强阳性。慢病毒感染并筛选14 d后得到具有puromycin抗性的纯化h ESCs。该细胞经神经分化后,显微镜下观察到绿色荧光表达,且α-tubulin、βⅢ-tubulin、nestin表达均呈阳性。结论成功获得了表达Tα1(α-tubulin)-hr GFP载体的h ESCs,该细胞可以示踪Tα1的表达。Objective To investigate the application of α-tubulin （Tα1 ） promoter-driven fluorescence reporter system in tracing the neural differentiation of human embryonic stem cells （hESCs）. Methods Mouse embryonic fibroblasts （MEF） were used as feeder cells to culture hESCs, and the immunofluorescence staining was used to identify the biological characteristics of hESCs, hESCs were infected with lentivirus carrying pLV/Final-puro-Tα1-hrGFP vectors, and purified hESCs were obtained 14 days after puromycin screening. Then, the purified hESCs were induced to differentiate into neuron-like cells. During the process, the changes of green fluo- rescence in hESCs were observed by a microscope. Results There were strong expressions of OCT-d-, SSEA-4 and TRA-1-60 in hESCs. The purified hESCs resistant puromycin were obtained 14 days after lentivirus infection. After hESCs were induced to differen- tiate into neuron-like cells, the expressions of GFP, Tα1 , βⅢ-tubulin and nestin were all observed. Conclusion hESCs expressing Tαl and hrGFP are successfully obtained, which may be used to trace the expression of Tαl.

关 键 词：人胚胎干细胞 慢病毒 绿色荧光蛋白 

分 类 号：R394.2[医药卫生—医学遗传学]