【摘要】 目的探讨Hes1基因高表达对神经前体细胞分化的影响。方法从大鼠脑组织中提取总RNA,用RT-PCR方法获得Hes1基因的全长cDNA,插入pGEM-T-Easy克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pCDNA3.1,用脂质体将重组质粒转染原代培养的神经前体细胞,经G418筛选获得抗性细胞克隆,用RT-PCR方法鉴定Hes1基因在神经前体细胞基因组中的存在,实时荧光定量PCR检测Hes1基因在转染细胞中的过表达情况。结果经限制性内切酶酶切图谱分析和DNA序列测定,证实目的基因已插入重组质粒,RT-PCR证明,经G418筛选得到的转基因神经前体细胞克隆的基因组DNA中存在Hes1基因;实时荧光定量PCR进一步证明,转基因神经前体细胞Hes1基因的mRNA高表达。结论构建了大鼠Hes1基因的真核表达载体,获得了高表达Hes1基因的神经前体细胞克隆。Hes1基因高表达可抑制神经前体细胞Ngn1的表达,并促进神经前体细胞向神经胶质细胞的分化。更多还原

【Abstract】 Objective To clone rat Hes1 gene cDNA,construct its eukaryotic expression vector and obtain positive neural precursor cell clones expressing Hes1 gene stably,Then to study their effects to the differentiation of neural precursor cells. Methods The total RNA was extracted from 14 days Wistar rat brain. The full-length cDNA encoding Hes1 gene was obtained using RT-PCR method and inserted into pGEM T Easy cloning vector. After the sequencing was confirmed,the gene was subcloned to pCDNA3.1 to construct recombinant eukaryotic expression vectors of pCDNA3.1-Hes1. The recombinant plasmid was transfected into neural precursor cells from cortex of 14 days Wistar rat brain by lipofectamine method and positive cell clones were screened with G418. The existence of Hes1 gene in the transfected cells genomic DNA,and the over-expression of their mRNA in the transfected cells were confirmed with Fluorescent quantitative real-time RT-PCR(FQ-PCR). Results Enzyme digestion analysis and sequencing showed that the target genes were cloned into recombinant vector. The existence and over-expression of Hes1 gene in the transfected neural precursor cells was identified with FQ-PCR and it display that HES1 mRNA have high expressed in the transfected neural precursor cells.Conclusion The eukaryotic expression plasmid containing Hes1 genes were successfully constructed. The positive neural precursor cell clones expressing Hes1 gene stably were obtained,which may be a promising cell model for studying the biological function of Hes1 genes and the role of Hes1 genes in the differentiation of neural precursor cells. The experiment manifested that the high expression of Hes1 can restrain the expression of Ngn1 gene,and enhance the neural precursor cells differentiation to glial cells.更多还原