构建睫状神经营养因子绿色荧光蛋白融合表达载体电穿孔转染雪旺细胞的研究  

Construction of CNTF-GFP plasmid and transfection of Schwann cells by electroporation

作　　者：方媛[1] 莫晓芬[1,2] 孙兴怀[1,2] 郭文毅[1] 田洁[1] 孔德升[3] 马端[3] 张萌[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院,上海200031 [2]复旦大学脑科学研究院 [3]复旦大学上海医学院教育部分子医学重点实验室

出　　处：《中华眼科杂志》2009年第6期544-549,共6页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金资助项目（30300379）;上海市自然科学基金资助项目（034119807）;国家重点基础研究发展计划（2007CB512204）;教育部新世纪优秀人才计划（NCET-05-0370）;复旦大学脑科学研究院开放研究课题基金资助项目

摘　　要：目的构建睫状神经营养因子-绿色荧光蛋白（CNTF-GFP）融合表达质粒，以电穿孔法辅助转染培养的雪旺细胞，为优化细胞移植、促进视神经再生提供效果更好且便于观察的方法。方法实验研究。构建重组质粒pcDNA3．1／CNTF-GFP；经测序鉴定后，电穿孔法辅助转染培养的雪旺细胞；荧光显微镜下动态观察转染效果，流式细胞计数；转染24h后，用RT—PCR及免疫组化法检测基因转染及蛋白表达情况。结果重组质粒经测序鉴定无误；在3h即可见部分荧光，12h逐渐增多，24—72h到达高峰，持续至7d仍有表达；流式细胞计数得转染率为44．93％±12．92％，转染24h后，RT-PCR示目的基因有较高转录，免疫组化示有CNTF蛋白的高表达，并与GFP蛋白表达部位完全重合。结论成功构建了CNTF-GFP融合表达质粒；电穿孔法转染雪旺细胞后，表达良好，为制备转基因的种子细胞从而促进视神经再生的研究奠定了基础。Objective To enhance the neurotrophic effect of Schwann cells, promote optic nerve regeneration, and simplify the means of observation, the CNTF-GFP fusion plasmid was constructed and transferred into Schwann cells by electroporation. Methods It was an experimental study. Plasmid peDNA3.1/ CNTF-GFP was constructed and verified by auto-sequence. Cultured Schwann cells were transfected by electroporation. The transfection efficiency was counted by flow eytometry, the transcription of the gene was evaluated by RT-PCR, and the expression of protein was observed by fluorescence mierosphere and cell immunofluorescenee. Results CNTF-GFP plasmid was verified correctly. After electroporation, the green fluorescence of gene-transfected Schwann cells can be seen at 3 hours later, increased at 12 hours later, reached the peak during 24 h to 72 h later, and still could be seen at 7 days later. The transfeetion efficiency was evaluated at 44. 93% ± 12. 92% by flow cytometry. The transcription of CNTF gene and the expression of CNTF protein have been proven by RT-PCR and cell immunofluorescence. Condusions CNTF-GFP plasmid was constructed correctly and Schwann cells were transfected by electroporation successfully and CNTF-GFP fusion protein can be expressed well, which became a good foundation for our future＇s study on the transplantation of gene-modified Sehwann cells to promote optic nerve regeneration.

关 键 词：睫状神经营养因子 绿色荧光蛋白质类 视神经 神经再生 电穿孔 

分 类 号：R686[医药卫生—骨科学]