重组小鼠pEGFP-GDNF质粒的构建及其在骨髓基质干细胞中的表达

目的 克隆小鼠胶质细胞源性神经营养因子(GDNF)并转染骨髓基质于细胞，制备转基因工程细胞。方法 应用逆转录聚合酶链反应(RT-PCR)方法从新生小鼠大脑皮层细胞克隆出GDNFcDNA片断，以pEGFP-C1质粒为载体导入骨髓基质于细胞，用流式细胞仪检测转染率，用荧光显微镜和免疫细胞化学方法检测蛋白质的表达。结果 流式细胞仪检测转染率约50％，荧光显微镜下见转染pEGFP／GDNF质粒的骨髓基质干细胞(MSC)发出明亮的绿色荧光，免疫细胞化学方法检测发现转基因MSC抗GDNF蛋白染色成强阳性，正常MSC染色成阴性。结论 成功制备了高效表达外源性基因GDNF的MSC。绿色荧光蛋白作为报告基因可反应GDNF表达情况。

Cloning of Glial Cell Line-derived Neurotrophic Factor Gene and its Expression in Bone Marrow Mesenchymal Stem Cell in Mice

Objective To prepare a kind of gene engineering cells secreting glial cell line-derived neurotrophic factor (GDNF). Methods Mouse GDNF cDNA was amplified from newly-born mouse cortex cell of cerebrum by RT-PCR, and merged into the pEGFP-C1 vector. Then the recombined plasmid was transfected into bone marrow mesenchymal stem cell (MSC). The transfection rate was determined by flow cytometry. Expressions of enhanced green fluorescent protein (EGFP) and GDNF were detected by fluorescence microscope and immunohistochemical technique. Results Plasmid pEGFP/GDNF which was mediated via lipofectamine 2 000 could delivered both EGFP and GDNF gene with high efficiency to MSC. The rate of the transfection of the recombined plasmid into MSC, which was determined by flow cytometry, was 50%. The blight green fluorescence could been observed with fluorescence microscope in pEGFP/GDNF-transfected MSC.GDNF in the engineering cells was strongly positive and it in the others was negative. Conclusion The recombinant eukaryotic expression vector, pEGFP/GDNF can be constructed and expressed efficiently in MSC. EGFP, as a report gene, may reflect expression of GDNF in the engineering cells.