神经干细胞特异性调控启动子研究

Type-specific regulation promoter in neural stem cells

目的研究神经干细胞特异性调控启动子。方法 PCR扩增出小鼠nestin基因启动子全序列(4 000 bp)、核心序列(400 bp)和内含子-2;以pcDNA3.1为模板,构建6种重组质粒,分别用CMV、CMV+内含子-2、nestin基因启动子全序列、nestin基因启动子全序列+内含子-2、nestin基因启动子核心序列、nestin基因启动子核心序列+内含子-2作为启动子调控报告基因EGFP表达;6种重组质粒分别转染nestin^+、nesti^-细胞,荧光显微镜下观察转染后细胞内EGFP表达,同时采用流式细胞仪法测定表达EGFP细胞表达率。结果 nestin基因启动子全序列及核心序列都能非特异性调控EGFP基因在细胞内表达,并且具有较强调控能力,与内含子-2融合后,只能特异性调控EGFP基因在nestin^+细胞表达,而CMV启动子与内含子-2序列融合只具有非特异性调控能力。结论 nestin基因启动子全序列与内含子-2协同调控外源基因在nestin^+细胞特异性表达。

Objective To study the type-specific regulation promoter in neural stem cells.Methods The mouse nestin promoter sequence（4 000 bp）,core sequence（400 bp） and intron-2 were amplified with PCR.With pcDNA3.1 as a template,CMV,CMV ＋ intron-2,nestin promoter sequence,nestin promoter sequence ＋ intron-2,nestin promoter core sequence,and nestin promoter core sequence ＋ intron-2 were constructed and used as promoters to regulate expression of enhanced green fluorecence protein（EGFP）.These six kinds of recombinant plasmids were transfected into nestin~＋ and nestin ^-cells.Then EGFP expression was observed under a fluorescence microscope and determined by flow cytometry.Results Both nestin promoter sequence and nestin promoter core sequence,with strong regulatory capacity, could non-specifically regulate expression of the EGFP gene in nestin~＋ and nestin^- cells.Once constructed with intron-2, they could only regulate expression of the EGFP gene in nestin~＋ cells.The CMV promoter sequence had nonspecific regulatory capacity after being fused with intron-2.Conclusion Nestin promoter sequences and intron-2 could collaboratively,type-specifically regulate expression of foreign genes in nestin~＋ cells.