摘要
目的 探索增强型绿色荧光蛋白 (EGFP)能否作为标记基因追踪胶质源性神经生长因子(GDNF)基因修饰的骨髓基质干细胞 (MSC)在体内外的存活、生长、分化和表达外源目的基因的情况。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质细胞克隆出GDNFcDNA片断 ,连入pEGFP C1载体 ,构建表达EGFP和GDNF融合蛋白的质粒并转染MSC ,将稳定表达EGFP GDNF基因的细胞株植入小鼠纹状体。用荧光显微镜和免疫组织化学的方法在体内外观察MSC。结果 成功制备EGFP GDNF融合基因修饰的MSC工程细胞 ,免疫组织化学方法检测显示GDNF呈强阳性 ,在体内外MSC工程细胞均发出明亮的绿色荧光 ,并且绿色荧光可反映细胞形态学变化。结论在EGFP GDNF融合基因修饰的MSC工程细胞中EGFP可作为标记基因同时标记GDNF和MSC。
Objective To investigate whether the enhanced green fluorescent protein (EGFP) gene could be used as a marker to trace the surviving, growth, differention and expression of exogenetic genes of the bone marrow mescenchymal stem cell(MSC) which was modified by the glial cell lined derived neurotrophic factor ( GDNF ) fused with EGFP gene.Methods Mouse GDNF cDNA was amplified from newly born mouse cortex cells of cerebrum by reverse transcription polymerase chain reaction(RT PCR),and was ligated into the pEGFP C1 vector to construct recombinant plasmid pEGFP GDNF which expressed EGFP and GDNF in a form of fused protein, then the recombined plasmid was transferred into MSC.The MSC which stably expressed EGFP GDNF fused protein was implanted into the striatum of adult mouse. The station of MSC and expression of EGFP GDNF were detected with fluorescence microscopy and immunohistochemistry straning.Results MSC which modified by the GDNF fused with EGFP gene was constructed successfully. Immuno staining with GDNF in engineered cell was strong positive, while the others were negative. The bright green fluorescence could be observed with fluorescence microscopy in pEGFP/GDNF transfected MSC both in vivo and in vitro, and green fluorescence also could reflect morphologic change of MSC.Conclusion EGFP, as a marker, can trace MSC itself which is modified by the GDNF fused with EGFP gene and reflects its expression of GDNF at the same time.
出处
《中华神经外科疾病研究杂志》
CAS
2004年第6期529-532,共4页
Chinese Journal of Neurosurgical Disease Research
关键词
胶质细胞源性神经营养因子
骨髓
干细胞
增强型绿色荧光蛋白
基因
转染
Glial cell lined derived neurotrophic factor
Bone marrow
Stem cell
Enhanced green fluorescent protein
Gene
Transfection
