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该研究成功地构建了C/EBP β过表达慢病毒载体,在体外人胚肾细胞(HEK293FT)进行病毒包装并感染小鼠海马神经元细胞(HT22)。通过荧光素酶报告基因实验(luciferase assay)检测C/EBPβ对淀粉样前体蛋白(amyloid precursor protein,APP)启动子活性的影响;通过实时荧光定量PCR(Q-PCR)来检测C/EBPβ对APP和Sp1在转录水平上的表达;通过蛋白免疫印迹分析(Western blot assay)检测C/EBPβ对APP和Sp1蛋白表达的作用。萤火虫荧光素酶分析结果显示,C/EBPβ对APP启动子的表达有正调控作用;Western blot和Q-PCR分析的结果表明,C/EBPβ对APP和Sp1基因的表达有正调控作用。C/EBPβ对APP基因表达的调控作用的机制可能在于C/EBPβ上调了内源性转录因子Sp1的基因表达,而Sp1基因表达的增强直接导致了APP基因表达的上调。
The study successfully constructed C / EBP β over-expressed lentiviral vector, virus-encapsulated in vitro human embryonic kidney cells (HEK293FT) and infected mouse hippocampal neurons (HT22). The effect of C / EBPβ on the promoter activity of amyloid precursor protein (APP) was detected by luciferase assay and the C / EBPβ pair was detected by real-time fluorescence quantitative PCR (Q-PCR) The expression of APP and Sp1 at the transcriptional level; the effect of C / EBPβ on the expression of APP and Sp1 protein was detected by Western blot assay. The results of firefly luciferase assay showed that C / EBPβ had a positive regulatory effect on the expression of APP promoter. Western blot and Q-PCR analysis showed that C / EBPβ positively regulated the expression of APP and Sp1 genes. The possible mechanism of C / EBPβ regulating APP gene expression may be that C / EBPβ up-regulates the gene expression of Sp1, whereas the increase of Sp1 gene directly leads to the up-regulation of APP gene expression.